Analysis of the interaction of Hsp90 with the extracellular matrix protein fibronectin (FN)
- Authors: Hunter, Morgan Campbell
- Date: 2014
- Subjects: Heat shock proteins , Fibronectins , Extracellular matrix proteins , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4170 , http://hdl.handle.net/10962/d1020960
- Description: Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.
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- Date Issued: 2014
Fabrication, characterization and application of phthalocyanine-magnetite hybrid nanofibers
- Authors: Modisha, Phillimon Mokanne
- Date: 2014
- Subjects: Nanofibers , Nanoparticles , Magnetite
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4500 , http://hdl.handle.net/10962/d1013223
- Description: Magnetic nanoparticles comprising magnetite (Fe3O4) were functionalized with 3-aminopropyl-triethoxysilane forming amino functionalized magnetite nanoparticles (AMNPs). The amino group allows for conjugation with zinc octacarboxyphthalocyanine (ZnOCPc) or zinc tetracarboxyphthalocyanine (ZnTCPc) via the carboxyl group to form an amide bond. A reduced aggregation of ZnTCPc is observed after conjugation with AMNPs. The thermal stability, conjugation, morphology and the sizes of the nanoparticles and their conjugates were confirmed using thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and Powder X-ray diffractometry (PXRD), respectively. The covalent linkage of AMNPs to ZnOCPc or ZnTCPc resulted in improvement in the photophysical behavior of the phthalocyanines. Improvement in the triplet quantum yield (ΦT), singlet oxygen quantum yield (ΦΔ), triplet lifetime (τT) and singlet oxygen lifetime (τΔ) of the ZnOCPc or ZnTCPc were observed, hence improving the photosensitizers efficiency. The conjugates comprising of zinc octacarboxyphthalocyanine (ZnOCPc) and AMNPs were electrospun into fibers using polyamide-6 (PA-6). This was used for the photodegradation of Orange-G and compared with ZnOCPc-AMNPs in suspension. For ZnOCPc-AMNPs in suspension, it is noteworthy that the catalyst can be easily recovered using an external magnetic field. The singlet oxygen generation increases as we increase the fiber diameter by increasing the ZnOCPc concentration. The singlet oxygen quantum yield is higher for PA-6/ZnOCPc-AMNPs nanofibers when compared to PA-6/ZnOCPc. The rate of degradation of Orange-G increased with an increase in the singlet oxygen quantum yield. Moreover, the kinetic analysis showed that the photodecomposition of Orange-G is a first-order reaction according to the Langmuir-Hinshelwood model.
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- Date Issued: 2014
Petrography, geochemistry and origin of atypical sedimentary-igneous contact relationships at the base of the Hotazel Formation around Middelplaats, Northern Cape Province, RSA
- Authors: Terracin, Matthew Theodore
- Date: 2014
- Subjects: Petrology -- South Africa , Geochemistry -- South Africa , Igneous rocks -- South Africa , Manganese ores -- South Africa , Manganese ores -- Geology -- South Africa , Metasomatism (Mineralogy) , Potassium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5059 , http://hdl.handle.net/10962/d1012985
- Description: In the Middelplaats mine area of the Kalahari manganese field, two drill holes (MP53 and MP54) intersected anomalously high-grade manganese ore sitting stratigraphically just above an igneous body (likely a dike or sill). Manganese ore located within approximate 5 meters of the contact with the underlying igneous rocks has been substantially metasomatically upgraded from 25 percent manganese, to over 40 percent whilst the dominant manganese species within the ore has been altered to hausmannite. This report demonstrates the metasomatic alteration is related to devolatilization (removal and/or remobilization of H₂O, CO₂ and CaO) due to contact metamorphism caused by the underlying igneous rocks. The Middelplaats mine is situated in the southwest corner of the Kalahari manganese field where the paleo basin shallows out and ends. Within the mine area, several stratigraphic units pinch out or are truncated by the side of the basin. This pinching out of lithological formations has led to the underlying Ongeluk Formation being in contact with the much younger units of the Hotazel Formation. Therefore, geochemical investigation into the nature and source of the igneous rocks was also undertaken to see if the rocks from the two drill holes were related to one another and/or the underlying Ongeluk Formation. Results of these geochemical studies have demonstrated that the Middelplaats igneous rocks (dolerites) from the two drill holes (MP53 and MP54) share a co-genetic source region. There is also reasonable geochemical evidence that the source region of the Middelplaats igneous rocks was substantially similar to the source region of the Ongeluk Formation. This may indicate that the source region of the Ongeluk Formation was reactivated at some later stage resulting in the emplacement of doleritic dikes or sills in the Middelplaats mine area. The Middelplaats igneous rocks were also found to have undergone a slight but pervasive potassic alteration; with most of the original plagioclase feldspar showing some level of replacement by a potassium enriched feldspar. Although no source for this potassic fluid was found, the devolatilization reaction within the manganese ore appears to have released some potassium into the surrounding rocks. This additional potassium may be responsible for some localized potassic alteration.
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- Date Issued: 2014