The hemocompatibility of crystalline Silicon Carbide (SiC), in its cubic form (i.e., 3C-SiC), has been evaluated and compared to Silicon (Si), the leading material in biosensing applications. Silicon carbide (SiC) is a hard, chemically robust material, very well suited for harsh environment applications, and has been suggested to have very good biocompatibility. Additionally, SiC in its amorphous form, has been used as a coating for medical implantable devices such as bone prosthetics and cardiovascular stents. However, assessment of single crystal 3C-SiC for cardiovascular applications has not been reported. In this research we have studied the interactions of single crystal 3C-SiC with platelets and human microvascular endothelial cell (HMVEC) to assess the degree of hemocompatibility of 3C-SiC. The more hemocompatible a material is, the less platelet adhesion would be expected. Using fluorescence microscopy higher platelet adhesion was statistically observed on Si than on SiC. In addition 3C-SiC surfaces showed less platelet reactivity, measured by the degree of platelet adhesion, aggregation and activation, with mostly circular morphology of adhered platelets while Si showed an elevated presence of non-activated (Circular) platelet clumps. Additionally, HMVEC proliferation assessment suggest that 3C-SiC performs comparably to high attachment culture wells with enhanced proliferation, without affecting cell morphology. These results suggest that 3C-SiC is a promising candidate for applications in the blood stream due to its low thrombogenic characteristics and good hemocompatibility.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Environmental factors are known to have far reaching effects on nervous system function, and in the adult brain, it is clear that a wide range of environmental stimuli modulate cell proliferation and survival (e.g., neurogenesis). This project investigated whether social stimulation and concomitant changes in gonadal hormones can influence the proliferation of new cells in the adult brain. The adult green treefrog (Hyla cinerea)was used as the model system; studying the courtship behavior of the highly social treefrog affords a direct, quantifiable way to measure the effects of acoustic social cues and hormonal intervention on adult brain cell proliferation. Using immunohistochemistry techiques, endocrinological manipulations, and socially-relevant acoustic stimulus presentations, I report that social cues modulate cell proliferation in the brains of adult male and female H. cinerea. I first mapped the distribution of proliferative areas in the adult treefrog brain using 5-bromo-2′- deoxyuridine (BrdU) labeling. I then exposed naturally-cycling male and female treefrogs to random tones or a recording of a natural H. cinerea chorus for ten days during the breeding season. I found that male and female treefrogs that heard their conspecific chorus exhibited increased brain cell proliferation compared to animals that heard random tones. Moreover, this modulation was region-specific and occurred in those regions which reflected their presumed involvement in reproductive physiology and behavior: the preoptic area (POA) and the infundibular hypothalamus (IF). To determine the involvement of gonadal hormones in cell proliferation with and without social stimulation, I gonadectomized and implanted male and female H. cinerea with blank or steroid-filled implants. After exposing the treefrogs to the same acoustic conditions as above, I discovered that social modulation of adult cell proliferation can occur without the influence of gonadal hormones (i.e., androgens in the male and estrogen in the female). Furthermore, the results revealed that neither hormone was neurotrophic and in fact, chronically-elevated estrogen levels decreased cell proliferation in the female POA and IF. Together, these results indicate that the reception of acoustic social cues increases cell proliferation in brain regions mediating sexual behavior and endocrine regulation; furthermore, this modulation occurs in a sexually-differentiated fashion without gonadal hormone influence. / text

Cell proliferation is a process that is strictly regulated by a large number of proteins. An alteration in one of the encoding genes inserts an error into the regulative protein, which may result in uncontrolled cell growth and eventually tumor formation. Lymphoma is a cancer type originating in the lymphocytes, which are part of the body’s immune defence. In the present thesis, Znfn1a1, Notch1 and Bcl11b were studied; all involved in the differentiation of T lymphocytes. The three genes are located in chromosomal regions that have previously shown frequent loss of heterozygosity in tumor DNA. Ikaros is a protein involved in the early differentiation of T lymphocytes. In this thesis, mutation analysis of the Znfn1a1 gene in chemically induced murine lymphomas revealed point mutations and homozygous deletions in 13 % of the tumors. All of the detected deletions lead to amino acid substitutions or abrogation of the functional domains in the Ikaros protein. Our results support the role of Ikaros as a potential tumor suppressor in a subset of tumors. Notch1 is a protein involved in many differentiation processes in the body. In lymphocytes, Notch1 drives the differentiation towards a T-cell fate and activating alterations in the Notch1 gene have been suggested to be involved in T-cell lymphoma. We identified activating mutations in Notch1 in 39 % of the chemically induced murine lymphomas, supporting the involvement of activating Notch1 mutations in the development of T-cell lymphoma. Bcl11b has been suggested to be involved in the early T-cell specification, and mutations in the Bcl11b gene has been identified in T-cell lymphoma. In this thesis, point mutations and deletions were detected in the DNA-binding zinc finger regions of Bcl11b in 15 % of the chemically induced lymphomas in C57Bl/6×C3H/HeJ F1 mice. A mutational hotspot was identified, where four of the tumors carried the same mutation. Three of the identified alterations, including the hotspot mutation in Bcl11b, increased cell proliferation when introduced in a cell without endogenous Bcl11b, whereas cell proliferation was suppressed by wild-type Bcl11b in the same cell line. Mutations in Bcl11b may therefore be an important contributing factor to lymphomagenesis in a subset of tumors. A germ line point mutation was identified in BCL11B in one of 33 human B-cell lymphoma patients. Expression of BCL11B in infiltrating T cells was significantly lower in aggressive compared to indolent lymphomas, suggesting that the infiltrating T cells may affect the B-cell lymphomas.

The pathophysiology of asthma is characterized by airway inflammation, remodeling and hyper-responsiveness. Phenotypic changes in airway smooth muscle cells (ASM) play a pivotal role in the pathogenesis of asthma. ASM cells promote inflammation and are key drivers of airway remodeling. While airway hyper responsiveness and inflammation can be managed by bronchodilators and anti-inflammatory drugs, ASM remodeling is poorly managed by existing therapies. Therefore, targeting ASM remodeling remains a challenge, and a deeper understanding of the molecular mechanism that regulates ASM phenotypes in asthma pathogenesis will facilitate the search for next-generation asthma therapy. MicroRNAs are small yet versatile gene tuners that regulate a variety of cellular processes, including cell proliferation and inflammation - two phenotypes that are often altered in asthmatic ASM. We thus hypothesized that microRNAs regulate ASM phenotypes in asthma and represent new targets for future therapy. In this thesis, we used a genomic approach that combined next-generation sequencing with functional cellular assays to characterize the role of microRNAs in regulating airway smooth muscle function and drug response to conventional therapies. In Chapter 2, we identified miR-10a as the most abundant microRNA expressed in the primary human airway smooth muscle (HASM) cells. Using an unbiased target identification approach, we identified several novel potential targets of miR-10a, including the catalytic subunit alpha of PI3 kinase (PIK3CA)--the central component of the PI3K pathway. We demonstrated that miR-10a directly suppresses PIK3CA expression by targeting its 3' Untranslated region (3'-UTR). Inhibition of PIK3CA by miR-10a reduced AKT phosphorylation and blunted the expression of cyclins and cyclin-dependent kinases that are required for HASM proliferation. In Chapter 3, we examined the effect of conventional asthma therapies on miRNA expression. While we did not find significant changes in miRNA levels, it remains to be determined whether microRNAs play a role in ASM tissue response to asthma therapy. Our study is the first to examine the role of microRNAs in ASM proliferation. Results from our study identified a novel microRNA-mediated regulatory mechanism of PI3K signaling and ASM proliferation. They suggest further that miR-10a is a potential therapeutic target to treat airway remodeling in asthma.

published_or_final_version / abstract / toc / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Breast cancer is the most prevalent cancer type among Canadian women. Breast cancers originate from the malignant transformation of mammary epithelial cells, which causes them to adopt an uncontrolled cell proliferation phenotype. My research suggests that the activity of specific ion channels (KV10.1, KCa3.1 and TRPM8) contribute to the proliferation of MCF-7 cells, a cell line commonly used to study breast cancer in vitro. Pharmacologically inhibiting the activities of KV10.1 or KCa3.1 channels decreased basal, but not estrogen-stimulated [3H]-thymidine incorporation, demonstrating that these channels contribute to MCF-7 cell proliferation. One way K+ channel activity is hypothesized to control cell proliferation is via regulation of membrane potential-dependent Ca2+ influx. Inhibition of KCa3.1 but not KV10.1 channel activity resulted in a membrane potential-dependent decrease in basal Ca2+ influx, suggesting that the way in which KCa3.1 channels contribute to cell proliferation is via regulating Ca2+ influx. In addition, my research also demonstrated that TRAM-34 increased or decreased cell proliferation depending on the concentration used and mitogenesis by TRAM-34 was blocked by estrogen receptor antagonists. TRAM-34 increased progesterone receptor mRNA expression, decreased estrogen receptor-alpha mRNA expression and reduced the binding of radiolabelled estrogen to estrogen receptor protein, in each case mimicking the effects of estrogen. Our finding that TRAM-34 is able to activate the estrogen receptor suggests a novel action of this supposedly specific K+ channel inhibitor and raises concerns of interpretation in its use. TRPM8 channels were also identified in MCF-7 cells, where they appeared to be important Ca2+ entry pathways. Inhibiting the activity of TRPM8 pharmacologically, as well as knocking down TRPM8 mRNA expression decreased cell proliferation, indicating that TRPM8 also contributed to MCF-7 cell proliferation. In conclusion, my research demonstrates that the activities of KV10.1, KCa3.1 and TRPM8 channels contribute to basal breast cancer cell proliferation. These findings suggest that the activity of specific ion channels may be potential targets for future therapeutic agents to treat breast cancer.

Human Immunodeficiency Virus (HIV) infection is characterized by immune dysfunction that predisposes infected individuals to opportunistic infections such as Mycobacterium Tuberculosis (MTB). The result of this is an exacerbation of HIV-TB related deaths annually. Therefore there is an imperative need for HIV-TB focused research that aims to identify immunological factors that are involved in the control of MTB and HIV in both mono- and co-infected individuals. The CD161+ CD4+ T cell subset is linked to a distinct phenotypic and functional profile. Importantly, these CD161+ T cells may act as an important component of immunological defense and provide protection in infected tissues. CD161+ CD4+ T cells have also been identified as the precursor population of Th17 cells and it has been previously reported that reduction of CD161+ CD4+ T cells during HIV infection may limit Th17 reconstitution (Prendergast et al., 2010). This may ultimately contribute to impairment of mucosal immunity leading to the acquisition of opportunistic infections such as MTB and disease progression in HIV infected individuals. Our study aimed to comprehensively characterise the impact of HIV and MTB infection on the CD161+ CD4+ T cell subset and to assess the frequency, phenotype and function of these cells. The study also aimed to correlate the longitudinal variation in frequency, phenotype and function with markers of HIV disease progression. Methods The frequency, phenotype and function of the CD161+ CD4+ T cell subset was measured by flow cytometry. For the frequency and phenotypic assessment, whole blood was collected from HIV negative and HIV/MTB mono and co-infected subjects (n = 17 per patient group). Whole blood was surface stained with antibodies specific to CD3, CD4, CD8, CD161 and chemokine receptors CD103, CCR6, CXCR4, CCR5 and CXCR6. The percentage positive expression of CD161 on CD4+ T cells and chemokine receptor expression was measured. The functional assessment of CD161+ CD4+ T cells involved PBMC stimulation with antigenic stimulant, phorbol 12-myristate 13-acetate (PMA) and ionomycin or ESAT-6/CFP-10, GAG, TB10.4 and Ag85a followed by intracellular cytokine staining for IFN-γ, IL-17A, IL-22 and TNF-α. A subgroup of HIV negative (frequency and phenotype, n = 10, function n = 7) and HIV mono-infected subjects (frequency and phenotype, n = 10, function n = 7) were longitudinally followed to assess variations in the frequency, phenotype and function of CD161+ CD4+ T cells over time. Results The CD161+ CD4+ T cell subset demonstrated high-level expression of chemokine receptors CCR5, CCR6, CXCR4 and low-level expression of CD103 and CXCR6. The subset also demonstrated the ability to produce cytokines IFN-γ, IL17A, IL-22 and TNF-α in healthy subjects. Analysis of HIV infected samples revealed a significant reduction in the frequency of the CD161+ CD4+ subset (median = 06.86%, p < 0.0001) compared to that of healthy individuals (median = 14.75%). Correlation of the subset frequency to markers of disease progression revealed a positive trend to CD4 count (r = 0.2590, p = 0.0787) and a significant negative correlation to viral load (r = -0.3522, p = 0.0152). Unlike with HIV infection, no significant changes in CD161+ CD4+ T cell frequency was observed in individuals with LTBI (mono- or HIV co-infected) or active TB disease compared to that of the healthy patient group. However, the exception to this was HIV infected individuals with active TB disease (co-infected) (median = 03.80%, p < 0.0001). Decreased CCR6 expression on CD161+ CD4+ T cells was observed in HIV monoinfected (p = 0.0065) and HIV infected individuals with active TB disease (p = 0.007). No functional changes were observed in both the HIV and MTB mono- and co-infected cohorts following non-specific stimulation. An interesting positive trend in correlation between IFN-γ production and CD4 count (r = 0.2727, p = 0.0733) was demonstrated with a significant negative correlation between IFN-γ production and viral load observed following non-specific antigenic stimulation (r = -0.3705, p = 0.0133). CD161+ CD4+ T cells demonstrated antigen-specific T cell responses to peptides ESAT-6/CFP-10, TB10.4, Ag85a and GAG in a small proportion of 69 study participants with variable ranges in magnitude of the responses observed. The longitudinal assessment of CD161+ CD4+ T cell frequency and phenotype demonstrated low-level proportion of CD4+ T cells expressing CD161 and CCR6 expression longitudinally maintained in HIV mono-infected compared to that of healthy individuals. Conclusion The phenotypic and functional profile of the CD161+ CD4+ T cell population indicates that it may be an important component of immunological defense that may provide mucosal defense and protection at epithelial sites and tissues e.g. expression of tissue homing markers like CCR6 and the production of cytokines such as IL-17A and IL-22 (important in mucosal immunity). HIV infection is associated with a reduced frequency of CD161+ CD4+ T cells. The correlation between CD161+ CD4+ T cell frequency and markers of disease progression suggests that the observed low-level frequency in HIV infected individuals may in part be a result of non-specific HIV-mediated depletion of CD4+ T cells. However, lower levels of CD161+ CD4+ T cells in HIV infected individuals could also be a result of naturally lower levels being present in individuals prior to infection, thereby making these individuals more susceptible to HIV infection. The significantly reduced levels of CCR6 expression on CD161+ CD4+ T cells in HIV monoinfected individuals may also be an indication of cell subset migration to gut associated lymphoid tissue (GALT, target site of HIV replication) during HIV infection. Given their potential role in mediating signals that are essential for immune responses to microbes and microbial products, migration of CCR6+ CD161+ CD4+ T cells to target sites of HIV infection could serve as a protective measure in the fight against HIV infection. Although there were no observable changes in the functional capacity of the CD161+ CD4+ T subset in HIV infection, we believe that the reduction in frequency may contribute to HIV disease progression and susceptibility to opportunistic infections such as MTB or active TB disease. Unlike with HIV infection, infection with MTB appeared to have no significant impact on CD161+ CD4+ T cells as there were no observable differences in frequency or the functional capacity of the cell subset following PMA stimulation. However, MTB and HIV antigen-specific responses were observed in a small proportion of the total 69 subjects tested. This therefore indicates that a subset of CD161+ CD4+ T cells may act in an HIV and MTB-specific manner. Additional MTB and HIV-specific responses may be present in this CD161+ CD4+ population and may only be identified through stimulation with additional antigenic targets. Further investigation of CD161+ CD4+ T cells should be performed at the actual sites of infection to investigate if CD161+ CD4+ T cells are concentrated at sites of disease. Also it may be important to investigate the polyfunctionality of CD161+ CD4+ T cells to understand the multifunctional capacity of the cell subset in providing immunological defense to pathogens such as HIV and MTB. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.

Patients with type-1 diabetes lose their β-cells after autoimmune attack. Islet transplantation is a co-option for curing this disease, but survival of transplanted islets is poor. Thus, methods to enhance β-cell viability and function as well as methods to expand β-cell mass are required. The work presented in this thesis aimed to study the roles of neural crest stem cells or their derivatives in supporting β-cell proliferation, function, and survival. In co-culture when mouse boundary cap neural crest stem cells (bNCSCs) and pancreatic islets were in direct contact, differentiating bNCSCs strongly induced β-cell proliferation, and these proliferating β-cells were glucose responsive in terms of insulin secretion. Moreover, co-culture of murine bNCSCs with β-cell lines RIN5AH and β-TC6 showed partial protection of β-cells against cytokine-induced β-cell death. Direct contacts between bNCSCs and β-cells increased β-cell viability, and led to cadherin and β-catenin accumulations at the bNCSC/β-cell junctions. We proposed that cadherin junctions supported signals which promoted β-cell survival. We further revealed that murine neural crest stem cells harvested from hair follicles were unable to induce β-cell proliferation, and did not form cadherin junctions when cultured with pancreatic islets. Finally, we discovered that the presence of bNCSCs in co-culture counteracted cytokine-mediated insulin-producing human EndoC-βH1 cell death. Furthermore, these two cell types formed N-cadherin, but not E-cadherin, junctions when they were in direct contact. In conclusion, the results of these studies illustrate how neural crest stem cells influence β-cell proliferation, function, and survival which may improve islet transplantation outcome.

Mitochondrial mutations and heteroplasmy have been associated with disease states that result from inadequate cellular energy production. As mitochondrial DNA (mtDNA) encodes many of the polypeptides involved in oxidative phosphorylation (OXPHOS), mtDNA mutations may lower energy production which is required for cell division and sustained ATP synthesis. In order to test the relationship between mtDNA mutations and the rate of cell division, a mammary epithelial cancer cell line, MCF-7, is used as a model. Nine proliferate single cell clones have been isolated from MCF-7. Population doubling times of six single cell clones and the MCF-7 stock have been determined. Clones with distinctly different growth rates were selected for mutational analysis. Growth rates of these clones appeared to be different from each other. Using polymerase chain reaction (PCR) and DNA sequencing, three cases of heteroplasmy have been identified in the mitochondrial genes of the MCF-7 stock and four single cell clones (ATPase C9119T, ND6 T14300G, Cytb G15807A). Heteroplasmy present in the Cytb gene is differs between single cell clones. Differences between the growth rates may be indicative of metabolic variations in these single cell clones. The OXPHOS enzymes encoded by the mutated genes were quantified by standard enzymatic assays. The assays demonstrated significant differences in specific activity between the clones, but were not correlated with mitochondrial heteroplasmy. This thesis determines that the differences in specific activity observed between clones is of nuclear origin.