Pineal-adrenal gland interactions in search of an anti-stressogenic role for melatonin
- Authors: Van Wyk, Elizabeth Joy
- Date: 1993
- Subjects: Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4054 , http://hdl.handle.net/10962/d1004115 , Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Description: The multiple functions of the pineal gland have been collectively interpreted as constituting a general anti-stressogenic role. The adrenal glands play a central role in maintaining homeostasis. The major neuroendocrine consequence of long-term stress is elevated circulating glucocorticoid levels. In this study, the effect of chronic, oral hydrocortisone treatment on pineal biochemistry was investigated in male Wi star rats of the albino strain. The results show that seven days of oral hydrocortisone treatment endows the pineal gland with the ability to increase melatonin synthesis in organ culture. The increase is accompanied by a rise in NAT activity, cyclic AMP levels and enhanced specific binding to the pineal B-adrenergic receptors. It appears that hydrocortisone sensitizes the pineal gland to stimulation by B-adrenergic agonists. thus rendering the pineal more responsive to B-adrenergic agonists. Further studies were directed at demonstrating an anti-stressogenic function for the pineal gland by investigating whether the principal pineal indole, melatonin. could protect against the deleterious effects of elevated. circulating drocortisone levels. The results show that chronic, oral hydrocortisone treatment significantly increases liver tryptophan pyrrolase activity. The catabolism of tryptophan by tryptophan pyrrolase is an important determinant of tryptophan availability to the brain, and therefore, brain serotonin levels. The findings show that melatonin inhibits basal and hydrocortisone-stimulated liver tryptophan pyrrolase apoenzyme activity in a dose-dependent manner. This inhibition suggests that melatonin may protect against excessive loss of tryptophan from circulation and against deficiencies in the cerebral serotinergic system which are associated with mood and behavioural disorders. It was shown that another deleterious effect of chronic hydrocortisone treatment is a significant increase in the number of glutamate receptors in the forebrain of male Wistar rats. The increase in receptor number observed in this study is probably due to an increase in the synthesis of glutamate receptors and is associated with a marked reduction in the affinity of the glutamate receptors for glutamate. possible to demonstrate an receptor number or the For practical reasons, it was not effect of melatonin on either glutamate affinity of glutamate receptors for glutamate in rat forebrain membranes. In view of the neurotoxic effect of glutamate in the eNS, the functional significance of recently described glutamate receptors in the pineal gland was investigated. The results show that 10-4 M glutamate significantly inhibits the isoprenaline-stimulated synthesis of N-acetylserotonin and melatonin in organ culture when the pineal glands were pre-incubated with glutamate for 4 hours prior to stimulation with isoprenalin and when glutamate and isoprenaline were administered together in vitro. GABA, a glutamate metabolite could not mimic the decrease in isoprenalinestimulated melatonin, and it is likely that the observed effects were directly attributed to glutamate. Incubation of the pineal gland with 10-4 M glutamate in organ culture did not affect HIOMT activity in pineal homogenates, but significantly elevated both basal and isoprenaline-stimulated NAT activity. It was concluded that glutamate only inhibits melatonin synthesis in intact pineal glands and not in pineal homogenates. The present study has provided further support for an interaction between the pineal and the adrenal glands. There is an ever increasing likelihood that melatonin is an anti-stressogenic hormone and that the pineal gland may have a protective role to play in the pathology of stress-related diseases.
- Full Text:
- Date Issued: 1993
- Authors: Van Wyk, Elizabeth Joy
- Date: 1993
- Subjects: Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4054 , http://hdl.handle.net/10962/d1004115 , Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Description: The multiple functions of the pineal gland have been collectively interpreted as constituting a general anti-stressogenic role. The adrenal glands play a central role in maintaining homeostasis. The major neuroendocrine consequence of long-term stress is elevated circulating glucocorticoid levels. In this study, the effect of chronic, oral hydrocortisone treatment on pineal biochemistry was investigated in male Wi star rats of the albino strain. The results show that seven days of oral hydrocortisone treatment endows the pineal gland with the ability to increase melatonin synthesis in organ culture. The increase is accompanied by a rise in NAT activity, cyclic AMP levels and enhanced specific binding to the pineal B-adrenergic receptors. It appears that hydrocortisone sensitizes the pineal gland to stimulation by B-adrenergic agonists. thus rendering the pineal more responsive to B-adrenergic agonists. Further studies were directed at demonstrating an anti-stressogenic function for the pineal gland by investigating whether the principal pineal indole, melatonin. could protect against the deleterious effects of elevated. circulating drocortisone levels. The results show that chronic, oral hydrocortisone treatment significantly increases liver tryptophan pyrrolase activity. The catabolism of tryptophan by tryptophan pyrrolase is an important determinant of tryptophan availability to the brain, and therefore, brain serotonin levels. The findings show that melatonin inhibits basal and hydrocortisone-stimulated liver tryptophan pyrrolase apoenzyme activity in a dose-dependent manner. This inhibition suggests that melatonin may protect against excessive loss of tryptophan from circulation and against deficiencies in the cerebral serotinergic system which are associated with mood and behavioural disorders. It was shown that another deleterious effect of chronic hydrocortisone treatment is a significant increase in the number of glutamate receptors in the forebrain of male Wistar rats. The increase in receptor number observed in this study is probably due to an increase in the synthesis of glutamate receptors and is associated with a marked reduction in the affinity of the glutamate receptors for glutamate. possible to demonstrate an receptor number or the For practical reasons, it was not effect of melatonin on either glutamate affinity of glutamate receptors for glutamate in rat forebrain membranes. In view of the neurotoxic effect of glutamate in the eNS, the functional significance of recently described glutamate receptors in the pineal gland was investigated. The results show that 10-4 M glutamate significantly inhibits the isoprenaline-stimulated synthesis of N-acetylserotonin and melatonin in organ culture when the pineal glands were pre-incubated with glutamate for 4 hours prior to stimulation with isoprenalin and when glutamate and isoprenaline were administered together in vitro. GABA, a glutamate metabolite could not mimic the decrease in isoprenalinestimulated melatonin, and it is likely that the observed effects were directly attributed to glutamate. Incubation of the pineal gland with 10-4 M glutamate in organ culture did not affect HIOMT activity in pineal homogenates, but significantly elevated both basal and isoprenaline-stimulated NAT activity. It was concluded that glutamate only inhibits melatonin synthesis in intact pineal glands and not in pineal homogenates. The present study has provided further support for an interaction between the pineal and the adrenal glands. There is an ever increasing likelihood that melatonin is an anti-stressogenic hormone and that the pineal gland may have a protective role to play in the pathology of stress-related diseases.
- Full Text:
- Date Issued: 1993
Heterologous expression of the helicoverpa armigera stunt virus in Saccharomyces cerevisiae
- Authors: Venter, Philip Arno
- Date: 2002
- Subjects: Helicoverpa armigera Saccharomyces cerevisiae
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3895 , http://hdl.handle.net/10962/d1003811
- Description: Lepidopteran insects like Helicoverpa armigera, more commonly known as the cotton bollworm, are economically important pests of a wide variety of crops throughout the world. The Helicoverpa armigera stunt virus (HaSV), a tetravirus with a bipartite single-stranded positive-sense RNA genome, has great potential as a biological pesticide against H. armigera. The larger genomic strand of this virus (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71 kDa capsid protein precursor (p71). 240 copies of p71 assemble into a procapsid with the concomitant encapsidation of the viral RNA. This is followed by a complex maturation event that is characterized by the autoproteolytic cleavage of p71 into the 64 kDa capsid protein (P64) and a 7 kDa peptide (p7). The rearrangements that occur during maturation results in the formation of mature HaSV capsids that can thereupon deliver RNA to other susceptible host cells. The principal objective of the research described in this study was to demonstrate that this virus could be assembled in Saccharomyces cerevisiae. S. cerevisiae expression vectors were constructed for the production of p71. This protein was detected in cell lysates from two different strains of S. cerevisiae, both containing either chromosomal or episomal copies of an expression cassette for P71. A number of factors relating to the expression of P71 (e.g. strains used, expression loci and expression rate) and the preparation of protein extracts from S. cerevisiae (e.g. the presence of various protease inhibitors and salt concentrations) were examined to attain optimal levels of soluble p71. A small fraction of the optimized soluble p71 was shown to be in the form of virus-like particles (VLPs), with a yield of ≤10⁷ VLPs from a 1.5l culture of P71⁺ cells. These particles were exclusively in the procapsid form, had a similar buoyant density to that of wild-type HaSV and could undergo maturation when the pH was reduced to 5. S. cerevisiae vectors were constructed for the episomal expression of the HaSV genomic RNAs. These vectors directed the transcription of RNA1 and RNA2 transcripts, which had similar sizes to those of the HaSV genomic RNAs. Mature HaSV particles were purified from cells, transgenic for P71, RNA1 and RNA2, by way of two different virus purification protocols that were developed during this study. RT-PCR analyses on RNA-extracts from these particles demonstrated that RNA transcripts, which were produced in trans with p71, could be encapsidated by HaSV capsids in S. cerevisiae. A droplet-feed bioassay on H. armigera larvae demonstrated that the S. cerevisiae-derived HaSV particles caused impaired larval development. This response was correlated with the detection of HaSV RNA2 in RNA extractions from larvae that were used in this bioassay. The results that were generated through the course of this study, provided proof for the concept of the non-host production of infectious HaSV particles from S. cerevisiae. This work could serve as a foundation for future research on the development of an expression system for the large-scale production of this virus as a biopesticide.
- Full Text:
- Date Issued: 2002
- Authors: Venter, Philip Arno
- Date: 2002
- Subjects: Helicoverpa armigera Saccharomyces cerevisiae
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3895 , http://hdl.handle.net/10962/d1003811
- Description: Lepidopteran insects like Helicoverpa armigera, more commonly known as the cotton bollworm, are economically important pests of a wide variety of crops throughout the world. The Helicoverpa armigera stunt virus (HaSV), a tetravirus with a bipartite single-stranded positive-sense RNA genome, has great potential as a biological pesticide against H. armigera. The larger genomic strand of this virus (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71 kDa capsid protein precursor (p71). 240 copies of p71 assemble into a procapsid with the concomitant encapsidation of the viral RNA. This is followed by a complex maturation event that is characterized by the autoproteolytic cleavage of p71 into the 64 kDa capsid protein (P64) and a 7 kDa peptide (p7). The rearrangements that occur during maturation results in the formation of mature HaSV capsids that can thereupon deliver RNA to other susceptible host cells. The principal objective of the research described in this study was to demonstrate that this virus could be assembled in Saccharomyces cerevisiae. S. cerevisiae expression vectors were constructed for the production of p71. This protein was detected in cell lysates from two different strains of S. cerevisiae, both containing either chromosomal or episomal copies of an expression cassette for P71. A number of factors relating to the expression of P71 (e.g. strains used, expression loci and expression rate) and the preparation of protein extracts from S. cerevisiae (e.g. the presence of various protease inhibitors and salt concentrations) were examined to attain optimal levels of soluble p71. A small fraction of the optimized soluble p71 was shown to be in the form of virus-like particles (VLPs), with a yield of ≤10⁷ VLPs from a 1.5l culture of P71⁺ cells. These particles were exclusively in the procapsid form, had a similar buoyant density to that of wild-type HaSV and could undergo maturation when the pH was reduced to 5. S. cerevisiae vectors were constructed for the episomal expression of the HaSV genomic RNAs. These vectors directed the transcription of RNA1 and RNA2 transcripts, which had similar sizes to those of the HaSV genomic RNAs. Mature HaSV particles were purified from cells, transgenic for P71, RNA1 and RNA2, by way of two different virus purification protocols that were developed during this study. RT-PCR analyses on RNA-extracts from these particles demonstrated that RNA transcripts, which were produced in trans with p71, could be encapsidated by HaSV capsids in S. cerevisiae. A droplet-feed bioassay on H. armigera larvae demonstrated that the S. cerevisiae-derived HaSV particles caused impaired larval development. This response was correlated with the detection of HaSV RNA2 in RNA extractions from larvae that were used in this bioassay. The results that were generated through the course of this study, provided proof for the concept of the non-host production of infectious HaSV particles from S. cerevisiae. This work could serve as a foundation for future research on the development of an expression system for the large-scale production of this virus as a biopesticide.
- Full Text:
- Date Issued: 2002
Isolation and evolution of novel nucleoside phosphorylases
- Authors: Visser, Daniel Finsch
- Date: 2010
- Subjects: AIDS (Disease) -- Treatment -- Africa HIV Infections -- Treatment -- Africa AIDS (Disease) -- Patients -- Africa HIV-Positive persons -- Africa Antiretroviral agents Pyrimidine nucleotides
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3972 , http://hdl.handle.net/10962/d1004031
- Description: Approximately 33.4 million people are living with HIV/AIDS. Of those, 97% live in low and middle income countries, with 22.4 million in sub-Saharan Africa. Only 42% of the people who require anti-retrovirals (ARVs) in low to middle income countries are receiving anti-retroviral therapy (ART). There is a need to develop novel and cost effective methods for producing antiretroviral drugs. Stavudine and azidothymidine (AZT) were identified as potential targets because they could both be produced through a common intermediate – 5 methyluridine (5-MU). It has been established that the biocatalytic production of 5-methyluridine is possible through a reaction known as transglycosylation, in a process which has not previously been demonstrated as commercially viable. A selection of biocatalysts were expressed either in recombinant E. coli strains or in the wild type organisms, purified and then screened for their ability to produce 5-MU. A combination of Bacillus halodurans purine nucleoside phosphorylase 1 (BHPNP1) and E. coli uridine phosphorylase (EcUP) gave the highest 5-MU yield (80%). This result represents the first combination of free enzymes from different organisms, giving high yields of 5-MU under high substrate conditions. Both enzymes were purified and successfully characterised. The established pH optimum was pH 7.0 for both enzymes. Temperature optima and stability data for BHPNP1 (70 C and t1/2 at 60 C of 20.8 h) indicated that the biocatalytic step was operating within the capabilities of this enzyme and would operate well at elevated temperatures (up to 60 C). Conversely, the temperature optimum and stability data for EcUP (optimum of 40 C and t1/2 at 60 C of 9.9 h) indicated that the enzyme remained active at 40 C for the duration of a 25 h biotransformation, but at 60 C would only be operating at 20% of its optimum activity and would lose activity rapidly. BHPNP1 and EcUP were used in a bench scale (650 ml) transglycosylation for the production of 5-MU. A 5-MU yield of 79.1% was obtained at this scale with a reactor productivity of 1.37 g.l-1.h-1. Iterative saturation mutagenesis was used to rapidly evolve EcUP for improved thermostability. A moderately high throughput colorimetric method was developed for screening the mutants based on the release of p-nitrophenol upon phosphorolysis of a pyrimidine nucleoside analogue. By screening under 20 000 clones the mutant UPL8 was isolated. The mutant enzyme showed an optimum temperature of 60 C and improved stability at 60 C (t1/2 = 17.3 h). The increase in stability of UPL8 is due to only 2 mutations (Lys235Arg, Gln236Ala). These mutations may have caused an increase in stability due to interactions with other structural units in the protein, stabilization of the entrance to the binding pocket, or by decreasing the flexibility of the α-helix at the N-terminus. Transglycosylation experiments showed that the mutant enzyme UPL8 is a superior catalyst for the production of 5-MU. A 300% increase in reactor productivity was noted when free enzyme preparations of UPL8 was combined with BHPNP1 at 1.5% m.m-1 substrate loading. The high yield of 5-MU (75-80% mol.mol-1) was maintained at 9% m.m-1 substrate loading. A commercially viable productivity of 31 g.l-1.h-1 was thus realised. Further optimisation of the process could produce still higher productivities. Future work in directed evolution of nucleoside phosphorylases is envisaged for improved stability and enhanced substrate range for application to other commercially relevant transglycosylation reactions.
- Full Text:
- Date Issued: 2010
- Authors: Visser, Daniel Finsch
- Date: 2010
- Subjects: AIDS (Disease) -- Treatment -- Africa HIV Infections -- Treatment -- Africa AIDS (Disease) -- Patients -- Africa HIV-Positive persons -- Africa Antiretroviral agents Pyrimidine nucleotides
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3972 , http://hdl.handle.net/10962/d1004031
- Description: Approximately 33.4 million people are living with HIV/AIDS. Of those, 97% live in low and middle income countries, with 22.4 million in sub-Saharan Africa. Only 42% of the people who require anti-retrovirals (ARVs) in low to middle income countries are receiving anti-retroviral therapy (ART). There is a need to develop novel and cost effective methods for producing antiretroviral drugs. Stavudine and azidothymidine (AZT) were identified as potential targets because they could both be produced through a common intermediate – 5 methyluridine (5-MU). It has been established that the biocatalytic production of 5-methyluridine is possible through a reaction known as transglycosylation, in a process which has not previously been demonstrated as commercially viable. A selection of biocatalysts were expressed either in recombinant E. coli strains or in the wild type organisms, purified and then screened for their ability to produce 5-MU. A combination of Bacillus halodurans purine nucleoside phosphorylase 1 (BHPNP1) and E. coli uridine phosphorylase (EcUP) gave the highest 5-MU yield (80%). This result represents the first combination of free enzymes from different organisms, giving high yields of 5-MU under high substrate conditions. Both enzymes were purified and successfully characterised. The established pH optimum was pH 7.0 for both enzymes. Temperature optima and stability data for BHPNP1 (70 C and t1/2 at 60 C of 20.8 h) indicated that the biocatalytic step was operating within the capabilities of this enzyme and would operate well at elevated temperatures (up to 60 C). Conversely, the temperature optimum and stability data for EcUP (optimum of 40 C and t1/2 at 60 C of 9.9 h) indicated that the enzyme remained active at 40 C for the duration of a 25 h biotransformation, but at 60 C would only be operating at 20% of its optimum activity and would lose activity rapidly. BHPNP1 and EcUP were used in a bench scale (650 ml) transglycosylation for the production of 5-MU. A 5-MU yield of 79.1% was obtained at this scale with a reactor productivity of 1.37 g.l-1.h-1. Iterative saturation mutagenesis was used to rapidly evolve EcUP for improved thermostability. A moderately high throughput colorimetric method was developed for screening the mutants based on the release of p-nitrophenol upon phosphorolysis of a pyrimidine nucleoside analogue. By screening under 20 000 clones the mutant UPL8 was isolated. The mutant enzyme showed an optimum temperature of 60 C and improved stability at 60 C (t1/2 = 17.3 h). The increase in stability of UPL8 is due to only 2 mutations (Lys235Arg, Gln236Ala). These mutations may have caused an increase in stability due to interactions with other structural units in the protein, stabilization of the entrance to the binding pocket, or by decreasing the flexibility of the α-helix at the N-terminus. Transglycosylation experiments showed that the mutant enzyme UPL8 is a superior catalyst for the production of 5-MU. A 300% increase in reactor productivity was noted when free enzyme preparations of UPL8 was combined with BHPNP1 at 1.5% m.m-1 substrate loading. The high yield of 5-MU (75-80% mol.mol-1) was maintained at 9% m.m-1 substrate loading. A commercially viable productivity of 31 g.l-1.h-1 was thus realised. Further optimisation of the process could produce still higher productivities. Future work in directed evolution of nucleoside phosphorylases is envisaged for improved stability and enhanced substrate range for application to other commercially relevant transglycosylation reactions.
- Full Text:
- Date Issued: 2010
An investigation into the bacterial diversity associated with South African latrunculid sponges that produce bioactive secondary metabolites
- Authors: Walmsley, Tara Aisling
- Date: 2014
- Subjects: Sponges -- South Africa -- Algoa Bay , Sponges -- Classification , Metabolites -- South Africa -- Algoa Bay , Marine metabolites -- South Africa -- Algoa Bay , PQQ (Biochemistry) , Bacterial diversity
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4109 , http://hdl.handle.net/10962/d1012943
- Description: Algoa Bay Latrunculid sponges are well known for their production of cytotoxic pyrroloiminoquinones with speculation that these secondary metabolites may have a microbial origin. This study describes a thorough investigation into the bacterial community associated with Tsitsikamma favus, Tsitsikamma scurra a newly described Latrunculia sp. and a yellow encrusting sponge associated with T. scurra. Molecular and chemical characterisation were used in conjunction with traditional taxonomy in identification of the sponge specimens. The 28S rRNA and COX1 analysis confirmed the traditional taxonomy with T. favus and T. scurra being very closely related. Chemical analysis revealed that T. favus and T. scurra shared the discorhabdins 2,4-debromo-3-dihydrodiscorhabdin C, 7,8-dehydro-3-dihydrodiscorhabdin C and 14-bromo-1-hydroxy-discorhabdin V in common with each other and Tsitsikamma pedunculata indicating that these pyrroloiminoquinones are common to Tsitsikamma sponges in general. The bacterial community associated with T. favus was explored using 16S rRNA molecular techniques including DGGE, clonal libraries of full length 16S rRNA genes, as well as 454 pyrosequencing. DGGE analysis revealed that the bacterial community associated with T. favus appeared to be highly conserved, which was confirmed by both the clone library and 454 pyrosequencing, with the Betaproteobacteria as the most dominant class. Further exploration into T. favus, as well as T. scurra, Latrunculia sp. and the yellow encrusting sponge indicated that the bacterial populations associated with each of these sponge species were conserved and species specific. OTU analysis to the species level revealed that T. favus and T. scurra shared an abundant Spirochaete species in common while the most abundant species in the Latrunculia sp. and the yellow encrusting sponge belonged to the class Betaproteobacteria. The exclusivity of the tsitsikammamines to T. favus precipitated attempts to culture the T. favus associated bacteria, with a focus on the dominant betaproteobacterium as indicated by the 16S rRNA clone library. Actinobacteria associated with the Algoa Bay sponge specimens were also cultured and the actinobacterial isolates were sent for screening against Mycobacterium aurum with two Kocuria kristinae isolates and a Streptomyces albdioflavus isolate showing good antimycobacterial activity.
- Full Text:
- Date Issued: 2014
- Authors: Walmsley, Tara Aisling
- Date: 2014
- Subjects: Sponges -- South Africa -- Algoa Bay , Sponges -- Classification , Metabolites -- South Africa -- Algoa Bay , Marine metabolites -- South Africa -- Algoa Bay , PQQ (Biochemistry) , Bacterial diversity
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4109 , http://hdl.handle.net/10962/d1012943
- Description: Algoa Bay Latrunculid sponges are well known for their production of cytotoxic pyrroloiminoquinones with speculation that these secondary metabolites may have a microbial origin. This study describes a thorough investigation into the bacterial community associated with Tsitsikamma favus, Tsitsikamma scurra a newly described Latrunculia sp. and a yellow encrusting sponge associated with T. scurra. Molecular and chemical characterisation were used in conjunction with traditional taxonomy in identification of the sponge specimens. The 28S rRNA and COX1 analysis confirmed the traditional taxonomy with T. favus and T. scurra being very closely related. Chemical analysis revealed that T. favus and T. scurra shared the discorhabdins 2,4-debromo-3-dihydrodiscorhabdin C, 7,8-dehydro-3-dihydrodiscorhabdin C and 14-bromo-1-hydroxy-discorhabdin V in common with each other and Tsitsikamma pedunculata indicating that these pyrroloiminoquinones are common to Tsitsikamma sponges in general. The bacterial community associated with T. favus was explored using 16S rRNA molecular techniques including DGGE, clonal libraries of full length 16S rRNA genes, as well as 454 pyrosequencing. DGGE analysis revealed that the bacterial community associated with T. favus appeared to be highly conserved, which was confirmed by both the clone library and 454 pyrosequencing, with the Betaproteobacteria as the most dominant class. Further exploration into T. favus, as well as T. scurra, Latrunculia sp. and the yellow encrusting sponge indicated that the bacterial populations associated with each of these sponge species were conserved and species specific. OTU analysis to the species level revealed that T. favus and T. scurra shared an abundant Spirochaete species in common while the most abundant species in the Latrunculia sp. and the yellow encrusting sponge belonged to the class Betaproteobacteria. The exclusivity of the tsitsikammamines to T. favus precipitated attempts to culture the T. favus associated bacteria, with a focus on the dominant betaproteobacterium as indicated by the 16S rRNA clone library. Actinobacteria associated with the Algoa Bay sponge specimens were also cultured and the actinobacterial isolates were sent for screening against Mycobacterium aurum with two Kocuria kristinae isolates and a Streptomyces albdioflavus isolate showing good antimycobacterial activity.
- Full Text:
- Date Issued: 2014
Regulation of tryptophan-2,3-dioxygenase and pineal indoleamines by selected tryptophan derivatives and antidepressants
- Authors: Walsh, Harold Archibold
- Date: 1997
- Subjects: Antidepressants Tryptophan -- Physiological effect Tryptophan -- Therapeutic use Depression, Mental -- Treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4017 , http://hdl.handle.net/10962/d1004077
- Description: The regulation of tryptophan-2,3-dioxygenase (TDO) (EC 1.13.1.12) and, to a lesser extent, pineal indoleamines, both in vitro and in vivo, is examined in this study. Rat liver TDO is a cytosolic enzyme which plays a crucial role in the regulation of circulating tryptophan (TRP) levels. Stimulation of this enzyme by heme enhances the catabolism of TRP, making less TRP available for uptake into the brain and other tissues, and for protein synthesis. At pH 7, the enzyme has an approximate Km of 100μM, is subject to substrate inhibition immediately beyond Sopt([S] at Vmax), and response of the enzyme is cooperative in both uninhibited and inhibited regions. Hill analysis of the uninhibited region reveals a biphasic plot and two classes of binding sites. Negative cooperativity is brought about through deprotonation of the enzyme. Substrate iphibition also occurs at both acidic and basic pH values with concomitant shifts in Sopt. The results obtained indicate that substrate inhibition could be an additional mechanism whereby the flux through the TRP-kynurenine pathway is regulated. TDO is subject to a diurnal rhythm, with peak activity during the pre-dark period and the loweSt activity towards the end of the dark period. It is possible that the enzyme controls the synthesis of the neurotransmitter serotonin (5-HT), and that the circadian rhythm in TDO activity is due to the endogenous rhythm of melatonin (aMT) production by the pineal gland. In the present study, aMT displaces TRP from bovine serum albumin (BSA) in vitro, and it is therefore possible for the indoleamine to regulate the availability of TRP for uptake into the brain for conversion to its derivatives. Chronic intraperitoneal administration of aMT affects physiological hepatic parameters in rats, such as TDO activity and stromal fatty acid composition, whilst no observable effect is demonstrable with respect to protein synthesis, nucleic acid metabolism, membrane fatty acid composition and pineal indole biosynthesis. On the other hand, chronic treatment of rats with antidepressants, the tricyclic desmethylimipramine (DMI) and the selective serotonin reuptake inhibitor (SSRI), fluoxetine, reveals significant negative alterations in TDO concentrations and pineal indole amine synthesis. Combining aMT with any of these two drugs normalises the activity of the hepatic enzyme. DMI is found to be an effective inhibitor of TDO in the micromolar range in vitro, and also affects total enzyme concentrations in vivo. Fluoxetine has no effect on TDO in vitro, but in vivo also reduces total enzyme levels in the liver. However, the SSRI does not affect conjugation between apo- and holoenzyme. Instead, it decreases extant holoenzyme levels. Indoleamine synthesis by the pineal gland, in organ culture, is altered by both antidepressants, although in different ways. DMI increases N-acetylserotonin levels and reduces the output of methoxyindole acetic acid and meth6xytryptophol. Fluoxetine treatment markedly reduces aMT concentrations and also brings about high levels of the 5-HT catabolites, 5-hydroxytryptophol and 5-hydroxyindole acetic acid. Insulin also lowers aMT synthesis significantly in pineal organ cultures, via a mechamsm that involves inhibition of the enzyme, N-acetyl transferase, that regulates aMT synthesis. The effects of insulin on pineal indole metabolism are due to the observation that a carbohydrate rich diet which induces insulin release elevates plasma TRP and brain 5-HT, but has no effect on pineal TRP and indole amine synthesis. It could thus be possible for insulin to have an effect on the pineal, since the latter is outside the blood brain barrier. The finilings of this study support the biogenic amine deficiency hypothesis, implicating some of the major biogenic amines such as noradrenaline (NA), 5-HT and aMT in depression. There is believed to be a deficiency of NA and 5-HT at their respective synapses in the depressed state. The drug DMI could act, firstly, by inhibiting TDO and thus increasing plasma TRP levels, and could, secondly, stimulate NA release and inhibit NA reuptake at the pineal membrane. The combined effect would be to enhance aMT synthesis, with eventual remission. Fluoxetine, on the other hand, appears to utilize a slightly different mode of action to DMI, which seems to focus on the preservation of 5-HT. The fact that aMT counteracts the effects of both antidepressants, and restores the activity of TDO to that of the controls, is also consistent with the observation that the therapeutic action of drugs such as these coincides willi the restoration of normal plasma levels of the neurohormone in depressives. In view of the biogenic amine deficiency hypothesis of depression and the contentious claim that TDO is the major peripheral determinant of brain TRP, brain 5-HT and ultimately aMT, the regulation of TDO is investigated and discussed. The study concludes that TDO activity is regulated by a number of endogenous compounds which are mainly derivatives of TRP, such as aMT and oxidized nicotinamide adenine dinucleotide and exogenous substances, of which DMI and fluoxetine are but two. In addition, modulation of IDO activity in depression appears to be an important aspect of antidepressant action. aMT, the product of the pineal gland, also has the potential to increase plasma TRP and hence forebrain TRP levels, and ultimately 5-HT concentrations, firstly by displacing TRP from serum albumin and secondly by inhibiting TDO.
- Full Text:
- Date Issued: 1997
- Authors: Walsh, Harold Archibold
- Date: 1997
- Subjects: Antidepressants Tryptophan -- Physiological effect Tryptophan -- Therapeutic use Depression, Mental -- Treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4017 , http://hdl.handle.net/10962/d1004077
- Description: The regulation of tryptophan-2,3-dioxygenase (TDO) (EC 1.13.1.12) and, to a lesser extent, pineal indoleamines, both in vitro and in vivo, is examined in this study. Rat liver TDO is a cytosolic enzyme which plays a crucial role in the regulation of circulating tryptophan (TRP) levels. Stimulation of this enzyme by heme enhances the catabolism of TRP, making less TRP available for uptake into the brain and other tissues, and for protein synthesis. At pH 7, the enzyme has an approximate Km of 100μM, is subject to substrate inhibition immediately beyond Sopt([S] at Vmax), and response of the enzyme is cooperative in both uninhibited and inhibited regions. Hill analysis of the uninhibited region reveals a biphasic plot and two classes of binding sites. Negative cooperativity is brought about through deprotonation of the enzyme. Substrate iphibition also occurs at both acidic and basic pH values with concomitant shifts in Sopt. The results obtained indicate that substrate inhibition could be an additional mechanism whereby the flux through the TRP-kynurenine pathway is regulated. TDO is subject to a diurnal rhythm, with peak activity during the pre-dark period and the loweSt activity towards the end of the dark period. It is possible that the enzyme controls the synthesis of the neurotransmitter serotonin (5-HT), and that the circadian rhythm in TDO activity is due to the endogenous rhythm of melatonin (aMT) production by the pineal gland. In the present study, aMT displaces TRP from bovine serum albumin (BSA) in vitro, and it is therefore possible for the indoleamine to regulate the availability of TRP for uptake into the brain for conversion to its derivatives. Chronic intraperitoneal administration of aMT affects physiological hepatic parameters in rats, such as TDO activity and stromal fatty acid composition, whilst no observable effect is demonstrable with respect to protein synthesis, nucleic acid metabolism, membrane fatty acid composition and pineal indole biosynthesis. On the other hand, chronic treatment of rats with antidepressants, the tricyclic desmethylimipramine (DMI) and the selective serotonin reuptake inhibitor (SSRI), fluoxetine, reveals significant negative alterations in TDO concentrations and pineal indole amine synthesis. Combining aMT with any of these two drugs normalises the activity of the hepatic enzyme. DMI is found to be an effective inhibitor of TDO in the micromolar range in vitro, and also affects total enzyme concentrations in vivo. Fluoxetine has no effect on TDO in vitro, but in vivo also reduces total enzyme levels in the liver. However, the SSRI does not affect conjugation between apo- and holoenzyme. Instead, it decreases extant holoenzyme levels. Indoleamine synthesis by the pineal gland, in organ culture, is altered by both antidepressants, although in different ways. DMI increases N-acetylserotonin levels and reduces the output of methoxyindole acetic acid and meth6xytryptophol. Fluoxetine treatment markedly reduces aMT concentrations and also brings about high levels of the 5-HT catabolites, 5-hydroxytryptophol and 5-hydroxyindole acetic acid. Insulin also lowers aMT synthesis significantly in pineal organ cultures, via a mechamsm that involves inhibition of the enzyme, N-acetyl transferase, that regulates aMT synthesis. The effects of insulin on pineal indole metabolism are due to the observation that a carbohydrate rich diet which induces insulin release elevates plasma TRP and brain 5-HT, but has no effect on pineal TRP and indole amine synthesis. It could thus be possible for insulin to have an effect on the pineal, since the latter is outside the blood brain barrier. The finilings of this study support the biogenic amine deficiency hypothesis, implicating some of the major biogenic amines such as noradrenaline (NA), 5-HT and aMT in depression. There is believed to be a deficiency of NA and 5-HT at their respective synapses in the depressed state. The drug DMI could act, firstly, by inhibiting TDO and thus increasing plasma TRP levels, and could, secondly, stimulate NA release and inhibit NA reuptake at the pineal membrane. The combined effect would be to enhance aMT synthesis, with eventual remission. Fluoxetine, on the other hand, appears to utilize a slightly different mode of action to DMI, which seems to focus on the preservation of 5-HT. The fact that aMT counteracts the effects of both antidepressants, and restores the activity of TDO to that of the controls, is also consistent with the observation that the therapeutic action of drugs such as these coincides willi the restoration of normal plasma levels of the neurohormone in depressives. In view of the biogenic amine deficiency hypothesis of depression and the contentious claim that TDO is the major peripheral determinant of brain TRP, brain 5-HT and ultimately aMT, the regulation of TDO is investigated and discussed. The study concludes that TDO activity is regulated by a number of endogenous compounds which are mainly derivatives of TRP, such as aMT and oxidized nicotinamide adenine dinucleotide and exogenous substances, of which DMI and fluoxetine are but two. In addition, modulation of IDO activity in depression appears to be an important aspect of antidepressant action. aMT, the product of the pineal gland, also has the potential to increase plasma TRP and hence forebrain TRP levels, and ultimately 5-HT concentrations, firstly by displacing TRP from serum albumin and secondly by inhibiting TDO.
- Full Text:
- Date Issued: 1997
Establishing experimental systems for studying the replication biology of Providence virus
- Authors: Walter, Cheryl Tracy
- Date: 2009
- Subjects: Insects -- Viruses Insects -- Diseases Insects -- Parasites Host-virus relationships RNA viruses DNA Insects as carriers of disease
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3928 , http://hdl.handle.net/10962/d1003987
- Description: Providence virus (PrV) is a member of the Tetraviridae, a family of small, positive sense, single-stranded RNA viruses, which characteristically infect the midgut tissue of heliothine larvae. PrV is the only known tetravirus that replicates in cultured insect cells. The virus comprises a monopartite genome resembling members of the genus Betatetravirus with the capsid precursor protein undergoing autoproteolytic cleavage at its C-terminus consistent with other tetravirus capsid precursor proteins. Analysis of viral cDNA predicted the presence of three potential overlapping gene products (from 5` to 3`): (1) p130, a protein of unrecognized nucleotide or amino acid homology with a 2A-like processing site at its N-terminus; (2) p104, the replicase ORF, which was found to be phylogenetically related to tombus-and umbraviruses replicases. The presence of a read-through stop signal in the p104 ORF was proposed to produce and amino terminal product with a predicted MW of 40 kDa (p40) and (3) the capsid protein precursor (81 kDa) which has two 2A-like processing sites at its N-terminus. Metabolic radiolabelling of viral translation products in persistently infected MG8 cells and in vitro translation of the individual ORFs were performed in order to analyse the expression of PrV gene products. p130 was translated with no evidence of 2A-like processing. Two products of 40 kDa and 104 kDa were translated from the p104 ORF, indicating that the read-through stop signal was likely to be functional. Finally, the capsid protein precursor ORF produced a major translation product of 68 kDa corresponding to the capsid protein precursor as well a peptide of 15 kDa that was attributed to the activity of the second 2A-like site at the N-terminus of the p81 ORF. The subcellular distribution of viral RNA (vRNA) and p40 in MG8 cells was investigated using immunofluorescence and biochemical fractionation. The results showed that p40/p104 and vRNA accumulated in polarized, punctate structures in some but not all MG8 cells and in some cases, co-localization was observed. This thesis concludes that PrV is a novel tetravirus with significant similarities plant carmolike viruses that should be re-classified at the family level.
- Full Text:
- Date Issued: 2009
- Authors: Walter, Cheryl Tracy
- Date: 2009
- Subjects: Insects -- Viruses Insects -- Diseases Insects -- Parasites Host-virus relationships RNA viruses DNA Insects as carriers of disease
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3928 , http://hdl.handle.net/10962/d1003987
- Description: Providence virus (PrV) is a member of the Tetraviridae, a family of small, positive sense, single-stranded RNA viruses, which characteristically infect the midgut tissue of heliothine larvae. PrV is the only known tetravirus that replicates in cultured insect cells. The virus comprises a monopartite genome resembling members of the genus Betatetravirus with the capsid precursor protein undergoing autoproteolytic cleavage at its C-terminus consistent with other tetravirus capsid precursor proteins. Analysis of viral cDNA predicted the presence of three potential overlapping gene products (from 5` to 3`): (1) p130, a protein of unrecognized nucleotide or amino acid homology with a 2A-like processing site at its N-terminus; (2) p104, the replicase ORF, which was found to be phylogenetically related to tombus-and umbraviruses replicases. The presence of a read-through stop signal in the p104 ORF was proposed to produce and amino terminal product with a predicted MW of 40 kDa (p40) and (3) the capsid protein precursor (81 kDa) which has two 2A-like processing sites at its N-terminus. Metabolic radiolabelling of viral translation products in persistently infected MG8 cells and in vitro translation of the individual ORFs were performed in order to analyse the expression of PrV gene products. p130 was translated with no evidence of 2A-like processing. Two products of 40 kDa and 104 kDa were translated from the p104 ORF, indicating that the read-through stop signal was likely to be functional. Finally, the capsid protein precursor ORF produced a major translation product of 68 kDa corresponding to the capsid protein precursor as well a peptide of 15 kDa that was attributed to the activity of the second 2A-like site at the N-terminus of the p81 ORF. The subcellular distribution of viral RNA (vRNA) and p40 in MG8 cells was investigated using immunofluorescence and biochemical fractionation. The results showed that p40/p104 and vRNA accumulated in polarized, punctate structures in some but not all MG8 cells and in some cases, co-localization was observed. This thesis concludes that PrV is a novel tetravirus with significant similarities plant carmolike viruses that should be re-classified at the family level.
- Full Text:
- Date Issued: 2009
Development of experimental systems for studying the biology of Nudaurelia capensis ß virus
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Date Issued: 2005
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Date Issued: 2005
Tertiary treatment in integrated algal ponding systems
- Authors: Wells, Charles Digby
- Date: 2005
- Subjects: Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4068 , http://hdl.handle.net/10962/d1006162 , Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Description: Inadequate sanitation is one of the leading causes of water pollution and consequently illness in many underdeveloped countries, including South Africa and, specifically, the Eastern Cape Province, where cholera has become endemic. As modern wastewater treatment processes are often energy intensive and expensive, they are not suitable for use in these areas. There is thus a need to develop more sustainable wastewater treatment technologies for application in smaller communities. The integrated algal ponding system (IAPS) was identified as a possible solution to this wastewater management problem and was investigated for adaptation to local conditions, at the Rhodes University Environmental Experimental Field Station in Grahamstown, South Africa. The system was monitored over a period of nine years, with various configuration adjustments of the high rate algal pond (HRAP) unit operation investigated. Under standard operating conditions, the system was able to achieve levels of nutrient and organic removal comparable with conventional wastewater treatment works. The mean nitrate level achieved in the effluent was below the 15mg.l-1 South African discharge standard, however, nitrate removal in the IAPS was found to be inconsistent. Although the system was unable to sustain chemical oxygen demand (COD) removal to below the 75mg.l-1 South African discharge standard, a removal rate of 87% was recorded, with the residual COD remaining in the form of algal biomass. Previous studies in the Eastern Cape Province have shown that few small wastewater treatment works produce effluent that meets the microbial count specification. Therefore, in addition to the collation of IAPS data from the entire nine year monitoring period, this study also investigated the use of the HRAP as an independent unit operation for disinfection of effluent from small sewage plants. It was demonstrated that the independent high rate algal pond (IHRAP) as a free standing unit operation could consistently produce water with Escherichia coli counts of 0cfu.100ml-1. The observed effect was related to a number of possible conditions prevailing in the system, including elevated pH, sunlight and dissolved oxygen. It was also found that the IHRAP greatly enhanced the nutrient removal capabilities of the conventional IAPS, making it possible to reliably and consistently maintain phosphate and ammonium levels in the final effluent to below 5mg.l-1 and 2mg.l-1 respectively (South African discharge standards are 10mg.l-1 and 3mg.l-1 in each case). The quality of the final effluent produced by the optimisation of the IAPS would allow it to be used for irrigation, thereby providing an alternative water source in water stressed areas. The system also proved to be exceptionally robust and data collected during periods of intensive and low management regimes were broadly comparable. Results of the 9 year study have demonstrated reliable performance of the IAPS and its use an appropriate, sustainable wastewater treatment option for small communities.
- Full Text:
- Date Issued: 2005
- Authors: Wells, Charles Digby
- Date: 2005
- Subjects: Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4068 , http://hdl.handle.net/10962/d1006162 , Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Description: Inadequate sanitation is one of the leading causes of water pollution and consequently illness in many underdeveloped countries, including South Africa and, specifically, the Eastern Cape Province, where cholera has become endemic. As modern wastewater treatment processes are often energy intensive and expensive, they are not suitable for use in these areas. There is thus a need to develop more sustainable wastewater treatment technologies for application in smaller communities. The integrated algal ponding system (IAPS) was identified as a possible solution to this wastewater management problem and was investigated for adaptation to local conditions, at the Rhodes University Environmental Experimental Field Station in Grahamstown, South Africa. The system was monitored over a period of nine years, with various configuration adjustments of the high rate algal pond (HRAP) unit operation investigated. Under standard operating conditions, the system was able to achieve levels of nutrient and organic removal comparable with conventional wastewater treatment works. The mean nitrate level achieved in the effluent was below the 15mg.l-1 South African discharge standard, however, nitrate removal in the IAPS was found to be inconsistent. Although the system was unable to sustain chemical oxygen demand (COD) removal to below the 75mg.l-1 South African discharge standard, a removal rate of 87% was recorded, with the residual COD remaining in the form of algal biomass. Previous studies in the Eastern Cape Province have shown that few small wastewater treatment works produce effluent that meets the microbial count specification. Therefore, in addition to the collation of IAPS data from the entire nine year monitoring period, this study also investigated the use of the HRAP as an independent unit operation for disinfection of effluent from small sewage plants. It was demonstrated that the independent high rate algal pond (IHRAP) as a free standing unit operation could consistently produce water with Escherichia coli counts of 0cfu.100ml-1. The observed effect was related to a number of possible conditions prevailing in the system, including elevated pH, sunlight and dissolved oxygen. It was also found that the IHRAP greatly enhanced the nutrient removal capabilities of the conventional IAPS, making it possible to reliably and consistently maintain phosphate and ammonium levels in the final effluent to below 5mg.l-1 and 2mg.l-1 respectively (South African discharge standards are 10mg.l-1 and 3mg.l-1 in each case). The quality of the final effluent produced by the optimisation of the IAPS would allow it to be used for irrigation, thereby providing an alternative water source in water stressed areas. The system also proved to be exceptionally robust and data collected during periods of intensive and low management regimes were broadly comparable. Results of the 9 year study have demonstrated reliable performance of the IAPS and its use an appropriate, sustainable wastewater treatment option for small communities.
- Full Text:
- Date Issued: 2005
A study of the molecular variation between orbivirus proteins
- Authors: Whistler, Toni
- Date: 1985 , 2013-03-13
- Subjects: Proteins -- Analysis , Polypeptides , Bluetongue virus , Orbivirus infections
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3892 , http://hdl.handle.net/10962/d1003290 , Proteins -- Analysis , Polypeptides , Bluetongue virus , Orbivirus infections
- Description: The aim of this study was to initiate a structural analysis of the capsid polypeptides from several serotypes of bluetongue virus in order to provide insight into the relatedness and possible origins of the different serotypes. Tryptic peptide mapping of ¹²⁵I-labelled group antigen by ion exchange chromatography was used to assess the structural relatedness of seven BTV serotypes from Southern Africa, North America and Australia. Each serotype had several tyrosine containing tryptic peptides which were unique, but approximately 35% of the peptides analyzed were found to be highly conserved between all 7 serotypes. BTV-20 appeared to be closely related to BTV-B and these two serotypes with BTV-4 and BTV-17 appeared to form a closely knit central cluster. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 1985
- Authors: Whistler, Toni
- Date: 1985 , 2013-03-13
- Subjects: Proteins -- Analysis , Polypeptides , Bluetongue virus , Orbivirus infections
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3892 , http://hdl.handle.net/10962/d1003290 , Proteins -- Analysis , Polypeptides , Bluetongue virus , Orbivirus infections
- Description: The aim of this study was to initiate a structural analysis of the capsid polypeptides from several serotypes of bluetongue virus in order to provide insight into the relatedness and possible origins of the different serotypes. Tryptic peptide mapping of ¹²⁵I-labelled group antigen by ion exchange chromatography was used to assess the structural relatedness of seven BTV serotypes from Southern Africa, North America and Australia. Each serotype had several tyrosine containing tryptic peptides which were unique, but approximately 35% of the peptides analyzed were found to be highly conserved between all 7 serotypes. BTV-20 appeared to be closely related to BTV-B and these two serotypes with BTV-4 and BTV-17 appeared to form a closely knit central cluster. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 1985
Sulphide-enhanced hydrolysis of primary sewage sludge : implications for the bioremediation of sulphate-enriched wastewaters
- Whittington-Jones, Kevin John
- Authors: Whittington-Jones, Kevin John
- Date: 2000
- Subjects: Bioremediation Sewage sludge Hydrolysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3910 , http://hdl.handle.net/10962/d1003969
- Description: The potential application of sulphate reducing bacteria for the bioremediation of acid mine drainage has already been recognised, and offers significant financial advantages over conventional chemical treatment approaches. Although the technology has been demonstrated successfully on both small- and large-scale, it’s extensive implementation has been constrained by the provision of suitable and cost effective electron donor and carbon sources. Primary sewage sludge is readily available in large quantities, but the slow rate of solubilization and low yield of soluble products do not apparently favour its use for this application. A number of pre-treatment steps have been introduced in an attempt to improve the yield and rates under methanogenic conditions. However, although early work suggested that degradation of lignocellulose and proteins may be more rapid under sulphate reducing conditions, the fate of primary sewage sludge under these conditions has been ignored. It was proposed that by combining the hydrolysis of primary sewage sludge and biological sulphate reduction, in a settling sludge bed, both processes would be enhanced. The aim of this study was to test this hypothesis on laboratory- and pilot-scale, and attempt to elucidate the underlying mechanism involved. The solubilization of primary sewage sludge was enhanced in the presence of sulphate reduction in continuous laboratory-scale reactors. Particulate matter accumulated in the bed of non-sulphidogenic systems, but not in sulphidogenic ones. This was attributed to increased solubilization and the smaller average floc size in the latter. Solubilization occurred within the settling sludge bed of the reactors, and offered a possible explanation for the better performance of the multiple- over single-stage reactor. A pilot-scale Falling Sludge Bed Reactor was constructed at Grootvlei Gold Mine, Springs, South Africa, and resulted in the solubilization of more than 70% of the influent primary sewage sludge. The system was also found to be highly resilient to severe perturbations, and returned rapidly to steady-state. Flask studies revealed that the hydrolysis of both proteins and complex carbohydrates was accelerated in the presence of biological sulphate reduction or sulphide. A study of the enzymology of sludge digestion revealed that sulphate reduction had little direct effect on the activity of the hydrolytic enzymes, but that reactor design was critical in the prevention of washout of these enzymes. Finally, a descriptive model was developed to explain the enhanced hydrolysis of primary sewage sludge. The model incorporated the effect of sulphidogenesis on floc fracture and reflocculation, and likely implications for mass transfer limitations.
- Full Text:
- Date Issued: 2000
- Authors: Whittington-Jones, Kevin John
- Date: 2000
- Subjects: Bioremediation Sewage sludge Hydrolysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3910 , http://hdl.handle.net/10962/d1003969
- Description: The potential application of sulphate reducing bacteria for the bioremediation of acid mine drainage has already been recognised, and offers significant financial advantages over conventional chemical treatment approaches. Although the technology has been demonstrated successfully on both small- and large-scale, it’s extensive implementation has been constrained by the provision of suitable and cost effective electron donor and carbon sources. Primary sewage sludge is readily available in large quantities, but the slow rate of solubilization and low yield of soluble products do not apparently favour its use for this application. A number of pre-treatment steps have been introduced in an attempt to improve the yield and rates under methanogenic conditions. However, although early work suggested that degradation of lignocellulose and proteins may be more rapid under sulphate reducing conditions, the fate of primary sewage sludge under these conditions has been ignored. It was proposed that by combining the hydrolysis of primary sewage sludge and biological sulphate reduction, in a settling sludge bed, both processes would be enhanced. The aim of this study was to test this hypothesis on laboratory- and pilot-scale, and attempt to elucidate the underlying mechanism involved. The solubilization of primary sewage sludge was enhanced in the presence of sulphate reduction in continuous laboratory-scale reactors. Particulate matter accumulated in the bed of non-sulphidogenic systems, but not in sulphidogenic ones. This was attributed to increased solubilization and the smaller average floc size in the latter. Solubilization occurred within the settling sludge bed of the reactors, and offered a possible explanation for the better performance of the multiple- over single-stage reactor. A pilot-scale Falling Sludge Bed Reactor was constructed at Grootvlei Gold Mine, Springs, South Africa, and resulted in the solubilization of more than 70% of the influent primary sewage sludge. The system was also found to be highly resilient to severe perturbations, and returned rapidly to steady-state. Flask studies revealed that the hydrolysis of both proteins and complex carbohydrates was accelerated in the presence of biological sulphate reduction or sulphide. A study of the enzymology of sludge digestion revealed that sulphate reduction had little direct effect on the activity of the hydrolytic enzymes, but that reactor design was critical in the prevention of washout of these enzymes. Finally, a descriptive model was developed to explain the enhanced hydrolysis of primary sewage sludge. The model incorporated the effect of sulphidogenesis on floc fracture and reflocculation, and likely implications for mass transfer limitations.
- Full Text:
- Date Issued: 2000
The removal and recovery of toxic and valuable metals from aqueous solutions by the yeast Saccharomyces cerevisiae
- Authors: Wilhelmi, Brendan Shane
- Date: 1998
- Subjects: Saccharomyces cerevisiae , Yeast fungi -- Biotechnology , Sewage -- Purification -- Heavy metals removal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4002 , http://hdl.handle.net/10962/d1004062 , Saccharomyces cerevisiae , Yeast fungi -- Biotechnology , Sewage -- Purification -- Heavy metals removal
- Description: This project considered the use of the yeast Saccharomyces cerevisiae as a biosorbent for the removal and recovery of a range of metals from contaminated waters. S. cerevisiae, as a biosorbent, has the potential to provide a cost effective, selective and highly efficient purification system. Initial studies focused on metal accumulation by an immobilized baker's S. cerevisiae biosorbent. The parameters affecting metal uptake were investigated, these included metal concentration, time and solution pH. Metal uptake was rapid. Gold and cobalt reached saturation within 5 min of contact with the biosorbent in batch reactors. Copper, zinc, nickel, cadmium and chromium reached saturation within 30 min of contact. Metal accumulation was pH dependent and was generally unaffected at a solution pH ≥ 4, and was substantially decreased at pH ≤ 2. The exception was gold which was preferentially accumulated at a solution pH of 2. The immobilized baker's yeast accumulated metals with maximum binding capacities in the order of gold > cadmium > cobalt > zinc > copper > chromium > nickel. A rapid method to assess metal recovery was developed. Bioaccumulated metal was efficiently recovered using dilute mineral acids. Copper recovery of ≥ 80 % was achieved by decreasing the solution pH of the reaction mixture to 2 with the addition of nominal quantities of HCl, H₂SO₄ or RNO₃. Adsorption-desorption over 8 cycles had no apparent adverse effect on metal uptake or recovery in batch reactors. Transmission electron microscopy showed no evidence of damage to cells used in copper adsorption-desorption investigations. Biosorption columns were investigated as bioreactors due to their application potential. The metals investigated were effectively removed from solution. At a saturation threshold, metal uptake declined rapidly. Most metals investigated were desorbed from the columns by eluting with 0.1 M HCl. Initially recoveries of copper, cobalt and cadmium were as high as 100%. Desorbed copper, zinc, cadmium, nickel and cobalt were concentrated in 10 to 15 ml of eluent, representing up to a 40 fold decrease in solution volume. Cadmium, nickel and zinc uptake increased with the second application to the columns. Initial accumulation of gold and chromium was 42.2 μmol/g and 28.6 μmol/g, however, due to the low recoveries of these two metals, a second application was not investigated. Copper was applied to a single column for 8 consecutive adsorption-desorption cycles. Uptake increased from an initial 31.3 μmol/g to 47.8 μmol/g at cycle 7. The potential for selective metal recovery was demonstrated using two biosorption columns in series. Copper was accumulated and recovered most efficiently. Zinc, cobalt and cadmium were displaced to the second column. Copper bound preferentially to zinc at a ratio of 6:1. Copper bound preferentially to cobalt at a ratio of 4:1. Cadmium was only displaced at a ratio of 2:1. The successful transfer of the bioremediation technology from the laboratory to an industrial application has yet to be realized. Bioremediation of a Plaatjiesvlei Black Mountain mine effluent, which contained copper, zinc, lead and iron, was investigated in this project. The removal of the metals was most effective at pH 4. A combined strategy of pH adjustment and bioremediation using immobilized S. cerevisiae decreased the copper concentration by 92.5%, lead was decreased by 90% and zinc was decreased by 60%. Iron was mostly precipitated from solution at pH ≥ 4. An ageing pond at the mine with conditions such as; pH, water volume and metal concentration, which were more conducive to biological treatment was subsequently identified. The investigation indicated a possible application of the biomass as a supplement to chemical remediation. The metal removal capability of a waste brewer's yeast was subsequently investigated. A yeast conditioning step increased metal uptake up to 100% and enhanced reproducibility. Metal removal from solution was rapid and pH dependent. The metals were efficiently removed from solution at pH ≥ 4. Uptake was substantially inhibited at pH ≤ 3. The waste brewer's yeast accumulated metals with maximum binding capacities in the order of copper (25.4 μmol/g) > lead (19.4 μmol/g) > iron (15.6 μmol/g) > zinc (12.5 μmol/g). No correlation between cell physiology and metal uptake was observed. Uptake of the four metals was confirmed by energy dispersive X-ray microanalysis. The interference of lead, zinc and iron on copper uptake by the waste brewer's yeast, and the interference of copper on the uptake of lead, zinc and iron was investigated. Maximum copper uptake was not decreased in the presence of lead. The Bmax remained constant at approximately 25 μmol/g. The dissociation constants increased with increasing lead concentrations. Lead bioaccumulation was significantly decreased in the presence of copper. The type of inhibition was dependent on the initial copper concentrations. Zinc had a slight synergistic effect on copper uptake. The copper Bmax increased from 30.8 μmol/g in a single-ion system to 34.5 μmol/g in the presence of 200 μmol/l of zinc. Zinc uptake was severely inhibited in the presence of copper. The maximum uptake and dissociation constant values were decreased in the presence of copper, which suggested an uncompetitive inhibition. The affinity of copper was substantially higher than zinc. The presence of higher levels of copper than zinc in the yeast cells was confirmed by energy dispersive microanalysis. Copper uptake was decreased in the presence of iron, with the copper Bmax being decreased from 25.4 μmol/g in a single-ion system to 20.1 μmol/g in the presence of 200 μmol/l iron. Iron Bmax values remained constant at 16.0 μmol/g. Combined biosorption and EDXA results suggested the iron bound at a higher affinity than copper to the cell wall. Total copper removal was higher as larger quantities of copper were deposited in the cell cytoplasm. Metal removal from the Plaatjiesvlei effluent by free cell suspensions of the waste brewer's yeast was satisfactory. Copper levels were decreased by 96%, iron by 42%, lead 25% and zinc 2%. Waste brewer's yeast is a cheap source of biomass in South Africa, and could potentially provide the basis for the development of an innovative purification system for metal-contaminated waters.
- Full Text:
- Date Issued: 1998
- Authors: Wilhelmi, Brendan Shane
- Date: 1998
- Subjects: Saccharomyces cerevisiae , Yeast fungi -- Biotechnology , Sewage -- Purification -- Heavy metals removal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4002 , http://hdl.handle.net/10962/d1004062 , Saccharomyces cerevisiae , Yeast fungi -- Biotechnology , Sewage -- Purification -- Heavy metals removal
- Description: This project considered the use of the yeast Saccharomyces cerevisiae as a biosorbent for the removal and recovery of a range of metals from contaminated waters. S. cerevisiae, as a biosorbent, has the potential to provide a cost effective, selective and highly efficient purification system. Initial studies focused on metal accumulation by an immobilized baker's S. cerevisiae biosorbent. The parameters affecting metal uptake were investigated, these included metal concentration, time and solution pH. Metal uptake was rapid. Gold and cobalt reached saturation within 5 min of contact with the biosorbent in batch reactors. Copper, zinc, nickel, cadmium and chromium reached saturation within 30 min of contact. Metal accumulation was pH dependent and was generally unaffected at a solution pH ≥ 4, and was substantially decreased at pH ≤ 2. The exception was gold which was preferentially accumulated at a solution pH of 2. The immobilized baker's yeast accumulated metals with maximum binding capacities in the order of gold > cadmium > cobalt > zinc > copper > chromium > nickel. A rapid method to assess metal recovery was developed. Bioaccumulated metal was efficiently recovered using dilute mineral acids. Copper recovery of ≥ 80 % was achieved by decreasing the solution pH of the reaction mixture to 2 with the addition of nominal quantities of HCl, H₂SO₄ or RNO₃. Adsorption-desorption over 8 cycles had no apparent adverse effect on metal uptake or recovery in batch reactors. Transmission electron microscopy showed no evidence of damage to cells used in copper adsorption-desorption investigations. Biosorption columns were investigated as bioreactors due to their application potential. The metals investigated were effectively removed from solution. At a saturation threshold, metal uptake declined rapidly. Most metals investigated were desorbed from the columns by eluting with 0.1 M HCl. Initially recoveries of copper, cobalt and cadmium were as high as 100%. Desorbed copper, zinc, cadmium, nickel and cobalt were concentrated in 10 to 15 ml of eluent, representing up to a 40 fold decrease in solution volume. Cadmium, nickel and zinc uptake increased with the second application to the columns. Initial accumulation of gold and chromium was 42.2 μmol/g and 28.6 μmol/g, however, due to the low recoveries of these two metals, a second application was not investigated. Copper was applied to a single column for 8 consecutive adsorption-desorption cycles. Uptake increased from an initial 31.3 μmol/g to 47.8 μmol/g at cycle 7. The potential for selective metal recovery was demonstrated using two biosorption columns in series. Copper was accumulated and recovered most efficiently. Zinc, cobalt and cadmium were displaced to the second column. Copper bound preferentially to zinc at a ratio of 6:1. Copper bound preferentially to cobalt at a ratio of 4:1. Cadmium was only displaced at a ratio of 2:1. The successful transfer of the bioremediation technology from the laboratory to an industrial application has yet to be realized. Bioremediation of a Plaatjiesvlei Black Mountain mine effluent, which contained copper, zinc, lead and iron, was investigated in this project. The removal of the metals was most effective at pH 4. A combined strategy of pH adjustment and bioremediation using immobilized S. cerevisiae decreased the copper concentration by 92.5%, lead was decreased by 90% and zinc was decreased by 60%. Iron was mostly precipitated from solution at pH ≥ 4. An ageing pond at the mine with conditions such as; pH, water volume and metal concentration, which were more conducive to biological treatment was subsequently identified. The investigation indicated a possible application of the biomass as a supplement to chemical remediation. The metal removal capability of a waste brewer's yeast was subsequently investigated. A yeast conditioning step increased metal uptake up to 100% and enhanced reproducibility. Metal removal from solution was rapid and pH dependent. The metals were efficiently removed from solution at pH ≥ 4. Uptake was substantially inhibited at pH ≤ 3. The waste brewer's yeast accumulated metals with maximum binding capacities in the order of copper (25.4 μmol/g) > lead (19.4 μmol/g) > iron (15.6 μmol/g) > zinc (12.5 μmol/g). No correlation between cell physiology and metal uptake was observed. Uptake of the four metals was confirmed by energy dispersive X-ray microanalysis. The interference of lead, zinc and iron on copper uptake by the waste brewer's yeast, and the interference of copper on the uptake of lead, zinc and iron was investigated. Maximum copper uptake was not decreased in the presence of lead. The Bmax remained constant at approximately 25 μmol/g. The dissociation constants increased with increasing lead concentrations. Lead bioaccumulation was significantly decreased in the presence of copper. The type of inhibition was dependent on the initial copper concentrations. Zinc had a slight synergistic effect on copper uptake. The copper Bmax increased from 30.8 μmol/g in a single-ion system to 34.5 μmol/g in the presence of 200 μmol/l of zinc. Zinc uptake was severely inhibited in the presence of copper. The maximum uptake and dissociation constant values were decreased in the presence of copper, which suggested an uncompetitive inhibition. The affinity of copper was substantially higher than zinc. The presence of higher levels of copper than zinc in the yeast cells was confirmed by energy dispersive microanalysis. Copper uptake was decreased in the presence of iron, with the copper Bmax being decreased from 25.4 μmol/g in a single-ion system to 20.1 μmol/g in the presence of 200 μmol/l iron. Iron Bmax values remained constant at 16.0 μmol/g. Combined biosorption and EDXA results suggested the iron bound at a higher affinity than copper to the cell wall. Total copper removal was higher as larger quantities of copper were deposited in the cell cytoplasm. Metal removal from the Plaatjiesvlei effluent by free cell suspensions of the waste brewer's yeast was satisfactory. Copper levels were decreased by 96%, iron by 42%, lead 25% and zinc 2%. Waste brewer's yeast is a cheap source of biomass in South Africa, and could potentially provide the basis for the development of an innovative purification system for metal-contaminated waters.
- Full Text:
- Date Issued: 1998
The role of Hsp90/Hsp70 organising protein (Hop) in the Proliferation, Survival and Migration of Breast Cancer Cells.
- Authors: Willmer, Tarryn
- Date: 2012
- Subjects: Cancer -- Treatment , Heat shock proteins , Cancer cells , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4130 , http://hdl.handle.net/10962/d1015720
- Description: Hop (the Hsp90/Hsp70 organising protein) is a co-chaperone that acts as an adapter between the major molecular chaperones Hsp90 and Hsp70 during the cellular assembly of the Hsp90 complex. The Hsp90 complex regulates the stability and conformational maturation of a range of important cellular proteins, many of which are deregulated in cancer. In this study, we hypothesised that Hop knockdown inhibits proliferation and migration of cancer cells. We characterised the expression of Hop in cell models of different cancerous status, and provided evidence that Hop was upregulated in tumour cells compared to normal cell counterparts. Using an RNA interference approach, a 60-90% knockdown of Hop was achieved for up to 144 hours in the MDA-MB-231 and Hs578T breast cancer cell lines. Hop knockdown resulted in downregulation of the Hsp90 client proteins, Akt and Stat3, as well as a change in the expression of other Hsp90 co-chaperones, p23, Cdc37 and Aha1, while no change in the levels of Hsp90 or Hsp70 was observed. Silencing of Hop impaired cell proliferation in Hs578T cells but an increase in proliferation in MDA-MB-231, suggesting that the role of Hop in cancer cell proliferation was dependent on type of cancer cell. Hop knockdown in Hs578T and MDA-MB- 231 cells did not lead to any significant changes in the half maximal inhibitory concentrations (IC50) of selected small molecule inhibitors (paclitaxel, geldanamycin and novobiocin) in these cell lines after 72 hours. Hop knockdown cells were however, more sensitive than control cells to the Hsp90 inhibitors geldanamycin and novobiocin at earlier time points and in the presence of the drug transporter inhibitor, verapamil. Hop knockdown caused a decrease in cell migration as measured by the wound healing assay in both Hs578T and MDA-MB-231 cells. Hop was present in purified pseudopodia fractions of migrating cells, and immunofluorescence analysis showed that Hop colocalised with actin at the leading edges of pseudopodia, points of adhesion and at intercellular junctions of cells that have been stimulated to migrate with the chemokine stromal derived factor-1. Hop was able to bind to actin in vitro using actin cosedimentation assays, and silencing of Hop dramatically reduced the capacity of Hs578T cells to form pseudopodia. These results establish a correlation between Hop and actin dynamics, pseudopodia formation and migration in the context of Hop silencing, and collectively suggest that Hop plays a role in cancer cell migration. This study presents experimental evidence for a promising alternative to targeting Hsp90 and Hsp70 chaperones, a novel drug target in cancer therapy.
- Full Text:
- Date Issued: 2012
- Authors: Willmer, Tarryn
- Date: 2012
- Subjects: Cancer -- Treatment , Heat shock proteins , Cancer cells , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4130 , http://hdl.handle.net/10962/d1015720
- Description: Hop (the Hsp90/Hsp70 organising protein) is a co-chaperone that acts as an adapter between the major molecular chaperones Hsp90 and Hsp70 during the cellular assembly of the Hsp90 complex. The Hsp90 complex regulates the stability and conformational maturation of a range of important cellular proteins, many of which are deregulated in cancer. In this study, we hypothesised that Hop knockdown inhibits proliferation and migration of cancer cells. We characterised the expression of Hop in cell models of different cancerous status, and provided evidence that Hop was upregulated in tumour cells compared to normal cell counterparts. Using an RNA interference approach, a 60-90% knockdown of Hop was achieved for up to 144 hours in the MDA-MB-231 and Hs578T breast cancer cell lines. Hop knockdown resulted in downregulation of the Hsp90 client proteins, Akt and Stat3, as well as a change in the expression of other Hsp90 co-chaperones, p23, Cdc37 and Aha1, while no change in the levels of Hsp90 or Hsp70 was observed. Silencing of Hop impaired cell proliferation in Hs578T cells but an increase in proliferation in MDA-MB-231, suggesting that the role of Hop in cancer cell proliferation was dependent on type of cancer cell. Hop knockdown in Hs578T and MDA-MB- 231 cells did not lead to any significant changes in the half maximal inhibitory concentrations (IC50) of selected small molecule inhibitors (paclitaxel, geldanamycin and novobiocin) in these cell lines after 72 hours. Hop knockdown cells were however, more sensitive than control cells to the Hsp90 inhibitors geldanamycin and novobiocin at earlier time points and in the presence of the drug transporter inhibitor, verapamil. Hop knockdown caused a decrease in cell migration as measured by the wound healing assay in both Hs578T and MDA-MB-231 cells. Hop was present in purified pseudopodia fractions of migrating cells, and immunofluorescence analysis showed that Hop colocalised with actin at the leading edges of pseudopodia, points of adhesion and at intercellular junctions of cells that have been stimulated to migrate with the chemokine stromal derived factor-1. Hop was able to bind to actin in vitro using actin cosedimentation assays, and silencing of Hop dramatically reduced the capacity of Hs578T cells to form pseudopodia. These results establish a correlation between Hop and actin dynamics, pseudopodia formation and migration in the context of Hop silencing, and collectively suggest that Hop plays a role in cancer cell migration. This study presents experimental evidence for a promising alternative to targeting Hsp90 and Hsp70 chaperones, a novel drug target in cancer therapy.
- Full Text:
- Date Issued: 2012
Development of a novel in situ CPRG-based biosensor and bioprobe for monitoring coliform β-D-Galactosidase in water polluted by faecal matter
- Authors: Wutor, Victor Collins
- Date: 2008
- Subjects: Biosensors Molecular probes Enterobacteriaceae Feces -- Microbiology Water -- Pollution -- Environmental aspects Environmental monitoring Chromogenic compounds
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3944 , http://hdl.handle.net/10962/d1004003
- Description: The ultimate objective of this work was to develop a real-time method for detecting and monitoring β-D-galactosidase as a suitable indicator of the potential presence of total coliform bacteria in water environments. Preliminary comparison of the chromogenic substrate, chlorophenol red β-D-galactopyranoside and the fluorogenic substrate, MuGAL, revealed unreliable results with the fluorogenic technique due to interference from compounds commonly found in environmental water samples. Thus, the chromogenic assay was further explored. Hydrolysis of the chromogenic substrate chlorophenol red β-D-galactopyranoside by β-D-galactosidase to yield chlorophenol red was the basis of this assay. Fundamental studies with chlorophenol red β-Dgalactopyranoside showed that β-D-galactosidase occurs extracellularly and in low concentrations in the polluted water environment. A direct correlation between enzyme activity and an increase in environmental water sample volume, as well as enzyme activity with total coliform colony forming unit counts were observed. Spectrophotometric detection was achieved within a maximum period of 24 h with a limit of detection level of 1 colony forming unit 100 ml[superscript -1]. This enzyme also exhibited physical and kinetic properties different from those of the pure commercially available β-D-galactosidase. Cell permeabilisation was not required for releasing enzymes into the extracellular environment. PEG 20 000 offered the best option for concentrating β-D-galactosidase. The source of β-D-galactosidase in the polluted environmental water samples was confirmed as Escherichia coli through SDS-PAGE, tryptic mapping and MALDI-TOF, thus justifying the further use of this method for detecting and/or monitoring total coliforms. Several compounds and metal ions commonly found in environmental water samples (as well as those used in water treatment processes) did have an effect on β-D-galactosidase. All the divalent cations except Mg [superscript 2+], at the concentrations studied, inhibited the relative activity of β-D-galactosidase in both commercial β-D-galactosidase and environmental samples. Immobilisation of chlorophenol red β-D-galactopyranoside onto a solid support material for the development of a strip bioprobe was unsuccessful, even though the nylon support material yielded some positive results. A monthly (seasonal) variation in β-Dgalactosidase activity from the environmental water samples was observed, with the highest activity coinciding with the highest monthly temperatures. Electro-oxidative detection and/or monitoring of chlorophenol red was possible. Chlorophenol red detection was linear over a wide range of concentrations (0.001-0.01 μg ml[superscript -1]). Interference by chlorophenol red β-D-galactopyranoside in the reduction window affected analysis. A range of phthalocyanine metal complexes were studied in an attempt to reduce fouling and/or increase the sensitivity of the biosensor. The selected phthalocyanine metal complexes were generally sensitive to changes in pH with a reduction in sensitivity from acidic pH to alkaline pH. The tetrasulphonated phthalocyanine metal complex of copper was, however, more stable with a minimum change of sensitivity. The phthalocyanine metal complexes were generally stable to changes in temperature. While only two consecutive scans were possible with the unmodified glassy carbon electrode, 77 consecutive scans were performed successfully with the CuPc-modified glassy carbon electrode. Among the phthalocyanine metal complexes studied, the CuPc-modified glassy carbon electrode therefore provided excellent results for the development of a biosensor. The CuPc modified-glassy carbon electrode detected 1 colony forming unit 100 ml[superscript -1] in 15 minutes, while the plain unmodified glassy carbon electrode required 6 hours to detect the equivalent number of colony forming units. CoPc, ZnPc and CuTSPc required 2, 2.25 and 1.75 h, respectively, to detect the same numbers of colony forming units. The CuPcmodified glassy carbon electrode detected 40 colony forming units 100 ml[superscript -1] instantly. In general, a direct correlation between colony forming units and current generated in the sensor was observed (R2=0.92). A higher correlation coefficient of 0.99 for 0-30 coliform colony forming units 100 ml[superscript -1] was determined. Current was detected in some water samples which did not show any colony forming units on the media, probably due to the phenomenon of viable but non-culturable bacteria, which is the major disadvantage encountered in the use of media for detecting indicator microorganisms. This novel biosensor therefore presents a very robust and sensitive technique for the detection and/or monitoring of coliform bacterial activity in water.
- Full Text:
- Date Issued: 2008
- Authors: Wutor, Victor Collins
- Date: 2008
- Subjects: Biosensors Molecular probes Enterobacteriaceae Feces -- Microbiology Water -- Pollution -- Environmental aspects Environmental monitoring Chromogenic compounds
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3944 , http://hdl.handle.net/10962/d1004003
- Description: The ultimate objective of this work was to develop a real-time method for detecting and monitoring β-D-galactosidase as a suitable indicator of the potential presence of total coliform bacteria in water environments. Preliminary comparison of the chromogenic substrate, chlorophenol red β-D-galactopyranoside and the fluorogenic substrate, MuGAL, revealed unreliable results with the fluorogenic technique due to interference from compounds commonly found in environmental water samples. Thus, the chromogenic assay was further explored. Hydrolysis of the chromogenic substrate chlorophenol red β-D-galactopyranoside by β-D-galactosidase to yield chlorophenol red was the basis of this assay. Fundamental studies with chlorophenol red β-Dgalactopyranoside showed that β-D-galactosidase occurs extracellularly and in low concentrations in the polluted water environment. A direct correlation between enzyme activity and an increase in environmental water sample volume, as well as enzyme activity with total coliform colony forming unit counts were observed. Spectrophotometric detection was achieved within a maximum period of 24 h with a limit of detection level of 1 colony forming unit 100 ml[superscript -1]. This enzyme also exhibited physical and kinetic properties different from those of the pure commercially available β-D-galactosidase. Cell permeabilisation was not required for releasing enzymes into the extracellular environment. PEG 20 000 offered the best option for concentrating β-D-galactosidase. The source of β-D-galactosidase in the polluted environmental water samples was confirmed as Escherichia coli through SDS-PAGE, tryptic mapping and MALDI-TOF, thus justifying the further use of this method for detecting and/or monitoring total coliforms. Several compounds and metal ions commonly found in environmental water samples (as well as those used in water treatment processes) did have an effect on β-D-galactosidase. All the divalent cations except Mg [superscript 2+], at the concentrations studied, inhibited the relative activity of β-D-galactosidase in both commercial β-D-galactosidase and environmental samples. Immobilisation of chlorophenol red β-D-galactopyranoside onto a solid support material for the development of a strip bioprobe was unsuccessful, even though the nylon support material yielded some positive results. A monthly (seasonal) variation in β-Dgalactosidase activity from the environmental water samples was observed, with the highest activity coinciding with the highest monthly temperatures. Electro-oxidative detection and/or monitoring of chlorophenol red was possible. Chlorophenol red detection was linear over a wide range of concentrations (0.001-0.01 μg ml[superscript -1]). Interference by chlorophenol red β-D-galactopyranoside in the reduction window affected analysis. A range of phthalocyanine metal complexes were studied in an attempt to reduce fouling and/or increase the sensitivity of the biosensor. The selected phthalocyanine metal complexes were generally sensitive to changes in pH with a reduction in sensitivity from acidic pH to alkaline pH. The tetrasulphonated phthalocyanine metal complex of copper was, however, more stable with a minimum change of sensitivity. The phthalocyanine metal complexes were generally stable to changes in temperature. While only two consecutive scans were possible with the unmodified glassy carbon electrode, 77 consecutive scans were performed successfully with the CuPc-modified glassy carbon electrode. Among the phthalocyanine metal complexes studied, the CuPc-modified glassy carbon electrode therefore provided excellent results for the development of a biosensor. The CuPc modified-glassy carbon electrode detected 1 colony forming unit 100 ml[superscript -1] in 15 minutes, while the plain unmodified glassy carbon electrode required 6 hours to detect the equivalent number of colony forming units. CoPc, ZnPc and CuTSPc required 2, 2.25 and 1.75 h, respectively, to detect the same numbers of colony forming units. The CuPcmodified glassy carbon electrode detected 40 colony forming units 100 ml[superscript -1] instantly. In general, a direct correlation between colony forming units and current generated in the sensor was observed (R2=0.92). A higher correlation coefficient of 0.99 for 0-30 coliform colony forming units 100 ml[superscript -1] was determined. Current was detected in some water samples which did not show any colony forming units on the media, probably due to the phenomenon of viable but non-culturable bacteria, which is the major disadvantage encountered in the use of media for detecting indicator microorganisms. This novel biosensor therefore presents a very robust and sensitive technique for the detection and/or monitoring of coliform bacterial activity in water.
- Full Text:
- Date Issued: 2008
Synthesis of silver nanoparticles and their role against a thiazolekinase enzyme from Plasmodium falciparum
- Yao, Jia
- Authors: Yao, Jia
- Date: 2014
- Subjects: Silver , Nanoparticles , Thiazoles , Plasmodium falciparum , Antimalarials , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4168 , http://hdl.handle.net/10962/d1020894
- Description: Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.
- Full Text:
- Date Issued: 2014
- Authors: Yao, Jia
- Date: 2014
- Subjects: Silver , Nanoparticles , Thiazoles , Plasmodium falciparum , Antimalarials , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4168 , http://hdl.handle.net/10962/d1020894
- Description: Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.
- Full Text:
- Date Issued: 2014
The spatial evolution of the chemotaxis proteins of the Bacillus subtilis group
- Yssel, Anna Elizabeth Johanna
- Authors: Yssel, Anna Elizabeth Johanna
- Date: 2011
- Subjects: Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4027 , http://hdl.handle.net/10962/d1004087 , Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Description: The aim of this work was to study spatial evolution of the chemotaxis proteins of a group of plant-associated soil-dwelling bacteria vernacularly referred to as the B. subtilis group. This was achieved by creating homology models for the chemotaxis proteins if a suitable template was available, and by analysing the selective forces (positive, purifying or neutral) acting upon the chemotaxis proteins. Chemotaxis is the phenomenon in which bacteria direct their movement towards more favourable conditions, and is critical for processes such as obtaining nutrients, escaping toxic compounds, host colonization and bio-film formation. Members of the B. subtilis group exhibit different preferences for certain host plants, and it is therefore feasible that their chemotactic machinery are fine-tuned to respond optimally to the conditions of the various niches that the strains inhabit. Homology models were inferred for the plant growth promoting B. amyloliquefaciens FZB42 proteins CheB, CheC, CheD, CheR, CheW and CheY. The interactions between: CheC-CheD, the P1 and P2 domains of CheA with CheY and CheB, and the P4 and P5 domains of CheA with CheW were also modelled. The hydrophobic interactions contributing to intra- and inter-protein contacts were analysed. The models of the interactions between CheB and the various domains of CheA are of particular interest, because to date no structures have been solved that show an interaction between a histidine kinase (such as CheA) and a multidomain response regulator (such as CheB). Furthermore, evidence that phospho-CheB may inhibit the formation of phospho-CheY by competitively binding to the P2 domain of CheA is also presented. Proteins were analysed to determine if individual amino acid sites are under positive, neutral or purifying selection. The Methyl Accepting Chemotaxis Proteins (MCPs), CheA and CheV were also analyzed, but due to a lack of suitable templates, no homology models were constructed. Site-specific positive and purifying selection were estimated by comparing the ratios of non-synonymous to synonymous substitutions at each site in the sequences for the chemotaxis proteins as well as for the receptors McpA, McpB, and McpC. Homology models were coloured according to intensity of selective forces. It was found that the chemotaxis proteins of member of the B. subtilis group are under strong evolutionary constraints, hence it is unlikely that positive selection in these proteins are responsible for the differences in habitat preference that these organism exhibit.
- Full Text:
- Date Issued: 2011
- Authors: Yssel, Anna Elizabeth Johanna
- Date: 2011
- Subjects: Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4027 , http://hdl.handle.net/10962/d1004087 , Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Description: The aim of this work was to study spatial evolution of the chemotaxis proteins of a group of plant-associated soil-dwelling bacteria vernacularly referred to as the B. subtilis group. This was achieved by creating homology models for the chemotaxis proteins if a suitable template was available, and by analysing the selective forces (positive, purifying or neutral) acting upon the chemotaxis proteins. Chemotaxis is the phenomenon in which bacteria direct their movement towards more favourable conditions, and is critical for processes such as obtaining nutrients, escaping toxic compounds, host colonization and bio-film formation. Members of the B. subtilis group exhibit different preferences for certain host plants, and it is therefore feasible that their chemotactic machinery are fine-tuned to respond optimally to the conditions of the various niches that the strains inhabit. Homology models were inferred for the plant growth promoting B. amyloliquefaciens FZB42 proteins CheB, CheC, CheD, CheR, CheW and CheY. The interactions between: CheC-CheD, the P1 and P2 domains of CheA with CheY and CheB, and the P4 and P5 domains of CheA with CheW were also modelled. The hydrophobic interactions contributing to intra- and inter-protein contacts were analysed. The models of the interactions between CheB and the various domains of CheA are of particular interest, because to date no structures have been solved that show an interaction between a histidine kinase (such as CheA) and a multidomain response regulator (such as CheB). Furthermore, evidence that phospho-CheB may inhibit the formation of phospho-CheY by competitively binding to the P2 domain of CheA is also presented. Proteins were analysed to determine if individual amino acid sites are under positive, neutral or purifying selection. The Methyl Accepting Chemotaxis Proteins (MCPs), CheA and CheV were also analyzed, but due to a lack of suitable templates, no homology models were constructed. Site-specific positive and purifying selection were estimated by comparing the ratios of non-synonymous to synonymous substitutions at each site in the sequences for the chemotaxis proteins as well as for the receptors McpA, McpB, and McpC. Homology models were coloured according to intensity of selective forces. It was found that the chemotaxis proteins of member of the B. subtilis group are under strong evolutionary constraints, hence it is unlikely that positive selection in these proteins are responsible for the differences in habitat preference that these organism exhibit.
- Full Text:
- Date Issued: 2011
Removal and recovery of heavy metals from synthetic solutions and electroplating effluents using yeast and the water fern Azolla filiculoides
- Authors: Zhao, Ming
- Date: 1998
- Subjects: Heavy metals -- Environmental aspects Azolla filiculoides -- Biological control Aquatic weeds -- Biological control Yeast Metal ions Yeast fungi -- Biotechnology Cations
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4001 , http://hdl.handle.net/10962/d1004061
- Description: The aims of the project were twofold. The initial objective of the study, based on previous results, was to develop an economically viable methodology for immobilizing yeast cells for the treatment of heavy metal-laden waste water. The non-viable yeast cross-linked by 13% (w/v) formaldehyde/1N HNO₃ exhibited satisfactory mechanical strength and rigidity in a continuous-flow column operation. No apparent disruption of the biomass after repeated use was observed. The cost of immobilizing 1kg dry yeast pellets was estimated at less than US$I. Zn uptake capacity of FA-cross-linked pellets, on batch trials, remained similar to that of raw yeast, reflecting that the immobilizing procedure did not hinder its metal removing capacity. In column studies, cation metals were effectively removed by the yeast pellets from aqueous solution at natural pHs, and then recovered completely by washing the pellets in situ with O.1M HCl. The recovered metals were concentrated in such small volumes that recycling or precipitation of them was facilitated. The metal uptake capacity of the regenerated biomass remained constant in comparison with cycle 1, indicating that reuse of the yeast would be possible. In the case of Cr⁶⁺, a gradual breakthrough curve of Cr in the column profile was noted, with a simultaneous reduction of Cr⁶⁺ to Cr³⁺. However, Cr⁶⁺ in the effluent can be markedly minimised either by accumulation onto the biomass or reduction to its trivalent form. Desorption of bound Cr⁶⁺ with either alkali or salt could not accomplish the regeneration of the biomass. A combination of reduction and desorption with FA/HNO₃ appeared promising in regeneration of the saturated biomass at 4°C. The metal sorption capacities of the yeast pellets, on a batch or a fixed-bed system are relatively lower than that of documented sorbents. Apparently more of the yeast pellets would be required for treating a certain volume of waste effluent, than with other sorbents. Therefore Azolla filiculoides was examined as a suitable sorbent for this purpose. This constitutes the second part of the project. Azolla filiculoides, a naturally-abundant water fern, was screened for its metal sorption and recovering capacities, mechanical stability, flow-permeability and reusability. The azolla biomass appeared to have fulfilled the required mechanical criteria during the repeated sorption-desorption column operations. It is water-insoluble and appears flexible under pressure when rinsed with water. These characters are of crucial importance in a continuous-flow system since a column can be operated at high flow rates without apparent compact of the biomass and pressure loss. Therefore, immobilization of the biomass can be avoided. The sorption isotherm data, obtained from batch removal of Cr⁶⁺, showed that the sorption process was effective, endothermic and highly pH dependent. Considerable amounts of Cr⁶⁺ were accumulated at the optimum pHs of 2-2.5. Column sorption of Cr⁶⁺ at a low flow rate and pH of 2.5 showed optimum performance with a total Cr uptake of 50.4mg/g at 60% saturation of the biomass. Removal of Cr⁶⁺ from an electroplating effluent using an azolla column was deemed reasonably satisfactory, although the uptake declined slightly. Desorption of bound Cr⁶⁺ with various desorbents was incomplete, which resulted in a low regeneration efficiency of about 50%. However, removal and recovery of Cr³⁺ using the azolla column was than that of Cr⁶⁺. Desorption of Cr³⁺ from the spent biomass column was accomplished with the recovery of 80% using O.5N H₂SO₄, The regeneration efficiencies for Cr³⁺ removal were up to 90% and demonstrated that the biomass is reusable. Cation metal uptake capacities of azolla, obtained either from batch or column experiments, are reasonably high in comparison with other sorbents. The uptake of Ni or Zn ions from solution is pH dependent showing the optimum pH of around 6 to 6.5, under the current experimental conditions. The sorption kinetics for cation metals was rapid with about 80% of the bound Ni ions being taken up in the first 10 min. The character of rapid binding is extremely important in a column sorption process, especially on a large scale since it favours an optimum uptake of metals at high flow rates. The Ni or Zn uptakes in column sorption were not markedly affected when the flow rates were increased from 80mllh up to 800ml/h for the 5g biomass used. The cation heavy metals removed from waste effluents were recovered in a concentrated solution of small volume. The desorption of bound Ni and Zn ions from the saturated biomass was accomplished with either O.2N HCl or H₂SO₄ that resulted in recoveries of more than 95%. The metals recovered, in the case of Ni and Zn, are identical to that of plating agents ego nickel sulphate or chloride, so that recycling of the metals is possible. An effluent-free, closed loop of Ni or Zn treatment system was proposed, whereby the Ni or Zn ions can be recycled to the plating bath whilst the purified water is fed back to the rinse tanks. Ca and Mg ions, commonly present in the electroplating effluents, appeared to affect sorption of heavy metals by azolla when metal concentrations were relatively low, presumedly through its competitive binding for the shared sites on surfaces of azolla. The data obtained from column sorption of Ni and Zn follows the BDST model well, enabling the application of the model to predicting design parameters for scale-up of the biosorption column system. It is interesting that the values of metal uptake, expressed in molar quantities, obtained on respective single-metal solutions and the multiple metal system, are similar, implying that the mechanisms involved in the sorption of all metal cations are similar and that the binding sites on surfaces of azolla are probably shared by all cation metals. The surface of the biomass provides sites for metal binding estimated in the range of 0.45-0.57mmol/g, based on the current experiments. The biomass has a surface area of 429 m²/g and water retention of 14.3 ml/g. The functional groups on the surface of azolla were partially identified using chemical modification and metal binding comparison. Among the functional groups examined, carboxyl groups, provided by amino acids and polysaccharides, appeared to play an important role in metal cation binding. The infrared spectra of the samples support this conclusion.
- Full Text:
- Date Issued: 1998
- Authors: Zhao, Ming
- Date: 1998
- Subjects: Heavy metals -- Environmental aspects Azolla filiculoides -- Biological control Aquatic weeds -- Biological control Yeast Metal ions Yeast fungi -- Biotechnology Cations
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4001 , http://hdl.handle.net/10962/d1004061
- Description: The aims of the project were twofold. The initial objective of the study, based on previous results, was to develop an economically viable methodology for immobilizing yeast cells for the treatment of heavy metal-laden waste water. The non-viable yeast cross-linked by 13% (w/v) formaldehyde/1N HNO₃ exhibited satisfactory mechanical strength and rigidity in a continuous-flow column operation. No apparent disruption of the biomass after repeated use was observed. The cost of immobilizing 1kg dry yeast pellets was estimated at less than US$I. Zn uptake capacity of FA-cross-linked pellets, on batch trials, remained similar to that of raw yeast, reflecting that the immobilizing procedure did not hinder its metal removing capacity. In column studies, cation metals were effectively removed by the yeast pellets from aqueous solution at natural pHs, and then recovered completely by washing the pellets in situ with O.1M HCl. The recovered metals were concentrated in such small volumes that recycling or precipitation of them was facilitated. The metal uptake capacity of the regenerated biomass remained constant in comparison with cycle 1, indicating that reuse of the yeast would be possible. In the case of Cr⁶⁺, a gradual breakthrough curve of Cr in the column profile was noted, with a simultaneous reduction of Cr⁶⁺ to Cr³⁺. However, Cr⁶⁺ in the effluent can be markedly minimised either by accumulation onto the biomass or reduction to its trivalent form. Desorption of bound Cr⁶⁺ with either alkali or salt could not accomplish the regeneration of the biomass. A combination of reduction and desorption with FA/HNO₃ appeared promising in regeneration of the saturated biomass at 4°C. The metal sorption capacities of the yeast pellets, on a batch or a fixed-bed system are relatively lower than that of documented sorbents. Apparently more of the yeast pellets would be required for treating a certain volume of waste effluent, than with other sorbents. Therefore Azolla filiculoides was examined as a suitable sorbent for this purpose. This constitutes the second part of the project. Azolla filiculoides, a naturally-abundant water fern, was screened for its metal sorption and recovering capacities, mechanical stability, flow-permeability and reusability. The azolla biomass appeared to have fulfilled the required mechanical criteria during the repeated sorption-desorption column operations. It is water-insoluble and appears flexible under pressure when rinsed with water. These characters are of crucial importance in a continuous-flow system since a column can be operated at high flow rates without apparent compact of the biomass and pressure loss. Therefore, immobilization of the biomass can be avoided. The sorption isotherm data, obtained from batch removal of Cr⁶⁺, showed that the sorption process was effective, endothermic and highly pH dependent. Considerable amounts of Cr⁶⁺ were accumulated at the optimum pHs of 2-2.5. Column sorption of Cr⁶⁺ at a low flow rate and pH of 2.5 showed optimum performance with a total Cr uptake of 50.4mg/g at 60% saturation of the biomass. Removal of Cr⁶⁺ from an electroplating effluent using an azolla column was deemed reasonably satisfactory, although the uptake declined slightly. Desorption of bound Cr⁶⁺ with various desorbents was incomplete, which resulted in a low regeneration efficiency of about 50%. However, removal and recovery of Cr³⁺ using the azolla column was than that of Cr⁶⁺. Desorption of Cr³⁺ from the spent biomass column was accomplished with the recovery of 80% using O.5N H₂SO₄, The regeneration efficiencies for Cr³⁺ removal were up to 90% and demonstrated that the biomass is reusable. Cation metal uptake capacities of azolla, obtained either from batch or column experiments, are reasonably high in comparison with other sorbents. The uptake of Ni or Zn ions from solution is pH dependent showing the optimum pH of around 6 to 6.5, under the current experimental conditions. The sorption kinetics for cation metals was rapid with about 80% of the bound Ni ions being taken up in the first 10 min. The character of rapid binding is extremely important in a column sorption process, especially on a large scale since it favours an optimum uptake of metals at high flow rates. The Ni or Zn uptakes in column sorption were not markedly affected when the flow rates were increased from 80mllh up to 800ml/h for the 5g biomass used. The cation heavy metals removed from waste effluents were recovered in a concentrated solution of small volume. The desorption of bound Ni and Zn ions from the saturated biomass was accomplished with either O.2N HCl or H₂SO₄ that resulted in recoveries of more than 95%. The metals recovered, in the case of Ni and Zn, are identical to that of plating agents ego nickel sulphate or chloride, so that recycling of the metals is possible. An effluent-free, closed loop of Ni or Zn treatment system was proposed, whereby the Ni or Zn ions can be recycled to the plating bath whilst the purified water is fed back to the rinse tanks. Ca and Mg ions, commonly present in the electroplating effluents, appeared to affect sorption of heavy metals by azolla when metal concentrations were relatively low, presumedly through its competitive binding for the shared sites on surfaces of azolla. The data obtained from column sorption of Ni and Zn follows the BDST model well, enabling the application of the model to predicting design parameters for scale-up of the biosorption column system. It is interesting that the values of metal uptake, expressed in molar quantities, obtained on respective single-metal solutions and the multiple metal system, are similar, implying that the mechanisms involved in the sorption of all metal cations are similar and that the binding sites on surfaces of azolla are probably shared by all cation metals. The surface of the biomass provides sites for metal binding estimated in the range of 0.45-0.57mmol/g, based on the current experiments. The biomass has a surface area of 429 m²/g and water retention of 14.3 ml/g. The functional groups on the surface of azolla were partially identified using chemical modification and metal binding comparison. Among the functional groups examined, carboxyl groups, provided by amino acids and polysaccharides, appeared to play an important role in metal cation binding. The infrared spectra of the samples support this conclusion.
- Full Text:
- Date Issued: 1998