Towards a biological profile for South African perinatal remains: osteological and genetic perspectives
- Authors: Thornton, Roxanne
- Date: 2019
- Subjects: Identification , Forensic osteology , Methylation , RNA , Autopsy
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/68102 , vital:29198 , DOI 10.21504/10962/68102
- Description: Forensic identification of abandoned and suspected infanticide cases admitted to the South African Forensic Pathology Services is often impossible due to decomposition of the remains. In these cases, investigation of suspected criminal activity is almost never pursued. Ancillary tests in the form of anthropological and molecular analyses can assist with the forensic identification of perinatal remains. To provide fundamental information about bone development of perinatal skeleton, osteological and genetic techniques focusing on the pars basilaris, pars lateralis, sternal rib and left femur were used. Samples were obtained from unidentified and unclaimed remains originating from the Johannesburg Forensic Pathology Service (JFPS). To provide a biological age to individuals in the collection, dental aging was used to categorize remains for comparisons with anthropological and molecular data. A molecular protocol was designed to sex individuals using the X-linked G6PD and Y-linked SRY genes. Bone development was studied using osteometric and morphological data of dry bone remains coupled with bone mineral density analysis (Micro-CT). The methylation levels of CpG rich sites within the promoter region of selected bone-associated genes were incorporated to examine silencing of genes during development. Osteological results support the use of the pars basilaris, pars lateralis and femur for age-at-death estimations as well as provide the foundation for dry bone aging criteria for South African individuals. Data compared with established skeletal aging standards indicated developmental differences between populations. Through the use of animal models and the perinatal sternal rib tissue, insights and precautions into the use of post mortem bone derived RNA for forensic applications is communicated. The methylation status of CpG rich sites within the promoter regions support the hypothesis for interdependent machinery involving selected genes during early bone development. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
- Authors: Thornton, Roxanne
- Date: 2019
- Subjects: Identification , Forensic osteology , Methylation , RNA , Autopsy
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/68102 , vital:29198 , DOI 10.21504/10962/68102
- Description: Forensic identification of abandoned and suspected infanticide cases admitted to the South African Forensic Pathology Services is often impossible due to decomposition of the remains. In these cases, investigation of suspected criminal activity is almost never pursued. Ancillary tests in the form of anthropological and molecular analyses can assist with the forensic identification of perinatal remains. To provide fundamental information about bone development of perinatal skeleton, osteological and genetic techniques focusing on the pars basilaris, pars lateralis, sternal rib and left femur were used. Samples were obtained from unidentified and unclaimed remains originating from the Johannesburg Forensic Pathology Service (JFPS). To provide a biological age to individuals in the collection, dental aging was used to categorize remains for comparisons with anthropological and molecular data. A molecular protocol was designed to sex individuals using the X-linked G6PD and Y-linked SRY genes. Bone development was studied using osteometric and morphological data of dry bone remains coupled with bone mineral density analysis (Micro-CT). The methylation levels of CpG rich sites within the promoter region of selected bone-associated genes were incorporated to examine silencing of genes during development. Osteological results support the use of the pars basilaris, pars lateralis and femur for age-at-death estimations as well as provide the foundation for dry bone aging criteria for South African individuals. Data compared with established skeletal aging standards indicated developmental differences between populations. Through the use of animal models and the perinatal sternal rib tissue, insights and precautions into the use of post mortem bone derived RNA for forensic applications is communicated. The methylation status of CpG rich sites within the promoter regions support the hypothesis for interdependent machinery involving selected genes during early bone development. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
A baculovirus-mediated expression system for the analysis of HaSV RNA packaging
- Authors: Mendes, Adriano
- Date: 2012
- Subjects: RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4025 , http://hdl.handle.net/10962/d1004085 , RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Description: The Helicoverpa armigera stunt virus (HaSV) is a member of a family of small nonenveloped (+) ssRNA insect viruses currently known as the Tetraviridae. This family is unique in terms of the T=4 quasi-symmetry of its capsid particles and the unusually narrow host range and tissue tropism. Assembly of tetraviral particles has been well characterised and involves the combination of 240 copies of a single capsid precursor protein (VCap) into a procapsid followed by autoproteolytic cleavage to yield the major (β) and minor (γ) capsid subunits within the mature particle. HaSV has two genomic RNAs, RNA 1 encoding the replicase and RNA 2 encoding VCap and p17, the ORF of which lies upstream of and overlaping with the 5’ end of the VCap ORF. Prior to this study, Vlok (2009) used a plasmid expression system to study RNA packaging in HaSV VLPs assembled in Spodoptera frugiperda 9 (Sf9) cells co-expressing p17 and VCap. The study showed that the p17 ORF was required for the packaging of RNA 2 during capsid assembly but it was unclear whether p17 expression was required for packaging. In addition, expression from the transfected plasmids was sub-optimal affecting both the yield of VLPs and the detection of p17. The aim of this study was to use the plasmid system to test whether p17 expression was required for plasmid-derived VLP RNA packaging and then develop a baculovirus-mediated system to test this hypothesis. By using a plasmid in which the start codon of p17 was mutated, it was shown that p17 expression was required for RNA 2 packaging into plasmid-VLPs. For the baculovirus system, four recombinant baculoviruses based upon the pFastBac Dual expression system, were constructed. These included Bac20, expressing wild type RNA 2, Bac21, RNA 2 with p17 silenced, Bac23, RNA 2 and p17 expressed on a separate transcript and Bac24, RNA 2 with p17 silenced plus p17 expressed on a separate transcript. Assembly of VLPs was more efficient using the baculovirus expression system and p17 expression was observed in cells infected with Bac20, Bac23 and Bac24, but not Bac21. In contrast to the plasmid-VLPs, bac-VLPs did not require p17 for the encapsidation of RNA 2. In addition to RNA 2, Bac23 and Bac24 packaged the p17 mRNA transcribed separately from RNA 2. This insinuated that bac-VLPs may be packaging RNA non-selectively. It was proposed that p17 may play a role in packaging in an RNA-limiting environment (plasmid system) but functioned differently when viral RNA was in excess (baculovirus system). This data points to the importance of developing a replication system for the analysis of the packaging pathways of these viruses and this study has laid down the foundations for such a system in which RNA 1 and RNA 2 can be introduced into a single cell by means of a single recombinant virus.
- Full Text:
- Date Issued: 2012
- Authors: Mendes, Adriano
- Date: 2012
- Subjects: RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4025 , http://hdl.handle.net/10962/d1004085 , RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Description: The Helicoverpa armigera stunt virus (HaSV) is a member of a family of small nonenveloped (+) ssRNA insect viruses currently known as the Tetraviridae. This family is unique in terms of the T=4 quasi-symmetry of its capsid particles and the unusually narrow host range and tissue tropism. Assembly of tetraviral particles has been well characterised and involves the combination of 240 copies of a single capsid precursor protein (VCap) into a procapsid followed by autoproteolytic cleavage to yield the major (β) and minor (γ) capsid subunits within the mature particle. HaSV has two genomic RNAs, RNA 1 encoding the replicase and RNA 2 encoding VCap and p17, the ORF of which lies upstream of and overlaping with the 5’ end of the VCap ORF. Prior to this study, Vlok (2009) used a plasmid expression system to study RNA packaging in HaSV VLPs assembled in Spodoptera frugiperda 9 (Sf9) cells co-expressing p17 and VCap. The study showed that the p17 ORF was required for the packaging of RNA 2 during capsid assembly but it was unclear whether p17 expression was required for packaging. In addition, expression from the transfected plasmids was sub-optimal affecting both the yield of VLPs and the detection of p17. The aim of this study was to use the plasmid system to test whether p17 expression was required for plasmid-derived VLP RNA packaging and then develop a baculovirus-mediated system to test this hypothesis. By using a plasmid in which the start codon of p17 was mutated, it was shown that p17 expression was required for RNA 2 packaging into plasmid-VLPs. For the baculovirus system, four recombinant baculoviruses based upon the pFastBac Dual expression system, were constructed. These included Bac20, expressing wild type RNA 2, Bac21, RNA 2 with p17 silenced, Bac23, RNA 2 and p17 expressed on a separate transcript and Bac24, RNA 2 with p17 silenced plus p17 expressed on a separate transcript. Assembly of VLPs was more efficient using the baculovirus expression system and p17 expression was observed in cells infected with Bac20, Bac23 and Bac24, but not Bac21. In contrast to the plasmid-VLPs, bac-VLPs did not require p17 for the encapsidation of RNA 2. In addition to RNA 2, Bac23 and Bac24 packaged the p17 mRNA transcribed separately from RNA 2. This insinuated that bac-VLPs may be packaging RNA non-selectively. It was proposed that p17 may play a role in packaging in an RNA-limiting environment (plasmid system) but functioned differently when viral RNA was in excess (baculovirus system). This data points to the importance of developing a replication system for the analysis of the packaging pathways of these viruses and this study has laid down the foundations for such a system in which RNA 1 and RNA 2 can be introduced into a single cell by means of a single recombinant virus.
- Full Text:
- Date Issued: 2012
Assembly of full-length cDNA, and heterologous expression, of Nudaurelia B virus RNA
- Authors: Luke, Gary Joseph
- Date: 2001
- Subjects: Imbrasia cytherea , RNA , Viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3913 , http://hdl.handle.net/10962/d1003972 , Imbrasia cytherea , RNA , Viruses , DNA
- Description: Nudaurelia beta virus (NβV) is a monopartite genome virus belonging to the family Tetraviridae. Its host range has been found to be limited to a single insect order, the Lepidoptera (moths and butterflies). The single-stranded positive-sense RNA genome consists of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerase. The 3' proximal ORF, of 1836 nucleotides, encodes the 66 kDa capsid precursor protein and overlaps the replicase gene by more than 99% and is in the +1 reading frame relative to the replicase reading frame. The full-length cDNA construct of the NβV genome was assembled using a homologous overlapping PCR linking method. The starting material consisted of seven overlapping pieces that were constructed for sequencing. Due to the degradation of the full-length RNA obtained from virus extracted from field-collected Nudaurelia cytherea capensis larvae other alternative methods needed to be applied. Sub-cloning using restriction enzyme sites also required an alternative method being used, due to the abundance of restriction sites of the same type in the NβV genome. This led to the use of a method similar to "DNA Shuffling" where overlapping pieces were connected using a modified PCR protocol. After the construction of the NβV genome, the full-length PCR product was cloned and checked for large insertion and deletions that could have resulted from the PCR amplification. The heterologous expression of the NβV capsid protein linked to a fusion protein (Glutathione S-transferase) in E.coli, confirmed the authenticity of the prescribed capsid gene ORF. The expression showed that the virus protein was subjected to protease digestion in DH5α E.coli, suggesting that the protein was insoluble in the cell cytoplasm. The capsid gene expression in a modified E.coli strain, Epicurian Coli BL21-CodonPlus (DE3)-RIL, resulted in high levels of the correct molecular weight protein with minimal degradation. The modified strain was designed for over-expression of eukaryotic protein with lowered protease activity. The above results have opened the way for further research that would yield valuable insight into the molecular biology and replication strategy of the NβV in cell cultures.
- Full Text:
- Date Issued: 2001
- Authors: Luke, Gary Joseph
- Date: 2001
- Subjects: Imbrasia cytherea , RNA , Viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3913 , http://hdl.handle.net/10962/d1003972 , Imbrasia cytherea , RNA , Viruses , DNA
- Description: Nudaurelia beta virus (NβV) is a monopartite genome virus belonging to the family Tetraviridae. Its host range has been found to be limited to a single insect order, the Lepidoptera (moths and butterflies). The single-stranded positive-sense RNA genome consists of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerase. The 3' proximal ORF, of 1836 nucleotides, encodes the 66 kDa capsid precursor protein and overlaps the replicase gene by more than 99% and is in the +1 reading frame relative to the replicase reading frame. The full-length cDNA construct of the NβV genome was assembled using a homologous overlapping PCR linking method. The starting material consisted of seven overlapping pieces that were constructed for sequencing. Due to the degradation of the full-length RNA obtained from virus extracted from field-collected Nudaurelia cytherea capensis larvae other alternative methods needed to be applied. Sub-cloning using restriction enzyme sites also required an alternative method being used, due to the abundance of restriction sites of the same type in the NβV genome. This led to the use of a method similar to "DNA Shuffling" where overlapping pieces were connected using a modified PCR protocol. After the construction of the NβV genome, the full-length PCR product was cloned and checked for large insertion and deletions that could have resulted from the PCR amplification. The heterologous expression of the NβV capsid protein linked to a fusion protein (Glutathione S-transferase) in E.coli, confirmed the authenticity of the prescribed capsid gene ORF. The expression showed that the virus protein was subjected to protease digestion in DH5α E.coli, suggesting that the protein was insoluble in the cell cytoplasm. The capsid gene expression in a modified E.coli strain, Epicurian Coli BL21-CodonPlus (DE3)-RIL, resulted in high levels of the correct molecular weight protein with minimal degradation. The modified strain was designed for over-expression of eukaryotic protein with lowered protease activity. The above results have opened the way for further research that would yield valuable insight into the molecular biology and replication strategy of the NβV in cell cultures.
- Full Text:
- Date Issued: 2001
Characterisation of the genome of Nudaurelia Omega Virus
- Authors: Cox, Dermot
- Date: 1995
- Subjects: Imbrasia cytherea , RNA , Insects -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4023 , http://hdl.handle.net/10962/d1004083 , Imbrasia cytherea , RNA , Insects -- Viruses
- Description: Nudaurelia co virus (Nco V) is a small RNA virus belonging to the Family Tetraviridae. Nco V was successfully isolated from field collected larvae of the pine emperor moth, Nudaurelia cytherea capensis. By polyacrylamide gel electrophoresis-it was possible Jo determine the size of the capsid proteins. Anti-NcoV antiserum was raised by inoculating a rabbit with purified virus. RNA was extracted from the purified virus using a phenol\chloroform extraction procedure. It was possible to separate the viral RNA into its constituent species using sucrose density gradient centrifugation. The sizes of both species of RNA was accurately determined by agarose gel electrophoresis. These sizes corresponded to the replicative form of the RNA which was extracted from infected host tissue. The absence of a poly(A) tract on the RNA was shown through poly(U) sepharose chromatography. Cell-free translation of the viral RNA elucidated the sizes of proteins encoded in vitro in a rabbit reticulocyte lysate system. Optimal conditions for in vitro translation of Nco V were determined for a range of conditions. Immunoprecipitaion of viral encoded proteins with anti-Nco V antiserum suggested that the putative coat protein of the virus was encoded by RNA 2, as a precursor polypeptide which underwent posttranslational cleavage. Reverse transcription - polymerase chain reaction (RT -PCR) was used to successfully produce a radiolabelled probe which could detect dot-blotted viral RNA. The efficacy of this probe in detecting the presence of Nco V RNA in infected tissue was also tested.
- Full Text:
- Date Issued: 1995
- Authors: Cox, Dermot
- Date: 1995
- Subjects: Imbrasia cytherea , RNA , Insects -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4023 , http://hdl.handle.net/10962/d1004083 , Imbrasia cytherea , RNA , Insects -- Viruses
- Description: Nudaurelia co virus (Nco V) is a small RNA virus belonging to the Family Tetraviridae. Nco V was successfully isolated from field collected larvae of the pine emperor moth, Nudaurelia cytherea capensis. By polyacrylamide gel electrophoresis-it was possible Jo determine the size of the capsid proteins. Anti-NcoV antiserum was raised by inoculating a rabbit with purified virus. RNA was extracted from the purified virus using a phenol\chloroform extraction procedure. It was possible to separate the viral RNA into its constituent species using sucrose density gradient centrifugation. The sizes of both species of RNA was accurately determined by agarose gel electrophoresis. These sizes corresponded to the replicative form of the RNA which was extracted from infected host tissue. The absence of a poly(A) tract on the RNA was shown through poly(U) sepharose chromatography. Cell-free translation of the viral RNA elucidated the sizes of proteins encoded in vitro in a rabbit reticulocyte lysate system. Optimal conditions for in vitro translation of Nco V were determined for a range of conditions. Immunoprecipitaion of viral encoded proteins with anti-Nco V antiserum suggested that the putative coat protein of the virus was encoded by RNA 2, as a precursor polypeptide which underwent posttranslational cleavage. Reverse transcription - polymerase chain reaction (RT -PCR) was used to successfully produce a radiolabelled probe which could detect dot-blotted viral RNA. The efficacy of this probe in detecting the presence of Nco V RNA in infected tissue was also tested.
- Full Text:
- Date Issued: 1995
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