Arsenic is a universal environmental toxicant associated mostly with skin related diseases in people exposed to low doses over a long term. Low dose arsenic trioxide (ATO) with long exposure will lead to chronic exposure. Experiments were performed to provide new knowledge on the incompletely understood mechanisms of action of chronic low dose inorganic arsenic in keratinocytes. Cytotoxicity patterns of ATO on long-term cultures of HaCaT cells on collagen IV was studied over a time course of 14 days. DNA damage was also assessed. The percentages of viable cells after exposure were measured on Day 2, Day 5, Day 8, and Day 14. Statistical and visual analytics approaches were used for data analysis. In the result, a biphasic toxicity response was observed at a 5 μg/ml dose with cell viability peaking on Day 8 in both chronic and acute exposures. Furthermore, a low dose of 1 μg/ml ATO enhanced HaCaT keratinocyte proliferation but also caused DNA damage. Global gene expression study using microarray technique demonstrated differential expressions of genes in HaCaT cell exposed to 0.5 μg/ml dose of ATO up to 22 passages. Four of the up-regulated and 1 down-regulated genes were selected and confirmed with qRT-PCR technique. These include; Aldo-Keto Reductase family 1, member C3 (AKR1C3), Insulin Growth Factor-Like family member 1 (IGFL1), Interleukin 1 Receptor, type 2 (IL1R2) and Tumour Necrosis Factor [ligand] Super-Family, member 18 (TNFSF18), and down-regulated Regulator of G-protein Signalling 2 (RGS2). The decline in growth inhibiting gene (RGS2) and increase in AKR1C3 may be the contributory path to chronic inflammation leading to metaplasia. This pathway is proposed to be a mechanism leading to carcinogenesis in skin keratinocytes. The observed over expression of IGFL1 may be a means of triggering carcinogenesis in HaCaT keratinocytes. In conclusion, it was established that at very low doses, arsenic is genotoxic and induces aberrations in gene expression though it may appear to enhance cell proliferation. The expression of two genes encoding membrane proteins IL1R2 and TNFSF18 may serve as possible biomarkers of skin keratinocytes intoxication due to arsenic exposure. This research provides insights into previously unknown gene markers that may explain the mechanisms of arsenic-induced dermal disorders including skin cancer / Environmental Sciences / D. Phil. (Environmental science)

Arsenic is a universal environmental toxicant associated mostly with skin related diseases in people exposed to low doses over a long term. Low dose arsenic trioxide (ATO) with long exposure will lead to chronic exposure. Experiments were performed to provide new knowledge on the incompletely understood mechanisms of action of chronic low dose inorganic arsenic in keratinocytes. Cytotoxicity patterns of ATO on long-term cultures of HaCaT cells on collagen IV was studied over a time course of 14 days. DNA damage was also assessed. The percentages of viable cells after exposure were measured on Day 2, Day 5, Day 8, and Day 14. Statistical and visual analytics approaches were used for data analysis. In the result, a biphasic toxicity response was observed at a 5 μg/ml dose with cell viability peaking on Day 8 in both chronic and acute exposures. Furthermore, a low dose of 1 μg/ml ATO enhanced HaCaT keratinocyte proliferation but also caused DNA damage. Global gene expression study using microarray technique demonstrated differential expressions of genes in HaCaT cell exposed to 0.5 μg/ml dose of ATO up to 22 passages. Four of the up-regulated and 1 down-regulated genes were selected and confirmed with qRT-PCR technique. These include; Aldo-Keto Reductase family 1, member C3 (AKR1C3), Insulin Growth Factor-Like family member 1 (IGFL1), Interleukin 1 Receptor, type 2 (IL1R2) and Tumour Necrosis Factor [ligand] Super-Family, member 18 (TNFSF18), and down-regulated Regulator of G-protein Signalling 2 (RGS2). The decline in growth inhibiting gene (RGS2) and increase in AKR1C3 may be the contributory path to chronic inflammation leading to metaplasia. This pathway is proposed to be a mechanism leading to carcinogenesis in skin keratinocytes. The observed over expression of IGFL1 may be a means of triggering carcinogenesis in HaCaT keratinocytes. In conclusion, it was established that at very low doses, arsenic is genotoxic and induces aberrations in gene expression though it may appear to enhance cell proliferation. The expression of two genes encoding membrane proteins IL1R2 and TNFSF18 may serve as possible biomarkers of skin keratinocytes intoxication due to arsenic exposure. This research provides insights into previously unknown gene markers that may explain the mechanisms of arsenic-induced dermal disorders including skin cancer / Environmental Sciences / D. Phil. (Environmental science)

Le présent mémoire est subdivisé en deux principaux sujets. Le premier porte sur le développement d’une hydrolyse de thiazolidines assistée par micro-ondes en vue d’obtenir des cystéines a-substituées. Le second est axé sur le développement d’une méthodologie pour la synthèse catalytique énantiosélective d’alkylidènecyclopropanes 1,1-di-accepteurs. Dans un premier temps, les rôles et les utilités des acides aminés quaternaires, plus spécifiquement des cystéines a-substituées, seront abordés, puis une revue des différentes méthodes énantiosélectives pour accéder à ces unités sera effectuée. Par la suite, le développement d’une méthode rapide et efficace d’hydrolyse sous irradiation aux micro-ondes de thiazolines sera présenté. Finalement, les études menant à l’application de cette méthode à la synthèse de cystéines -substituées sur grande échelle au moyen de réacteurs en écoulement dynamique et à haut criblage seront détaillées. Dans la seconde partie, les applications ainsi que les synthèses générales des alkylidènecyclopropanes en synthèse organique seront décrites. Plus particulièrement, les applications spécifiques des alkylidènecyclopropanes 1,1-di-accepteurs ainsi que leurs synthèses seront traitées de manière exhaustive. Par la suite, le développement d’une méthodologie énantiosélective catalytique pour la synthèse d’alkylidènecyclopropanes 1,1-di-accepteurs sera présenté. L’extension de cette méthodologie à la synthèse de dérivés cyclopropanes et cyclopropènes, ainsi que l’application de réactions stéréospécifiques pour les alkylidènecyclopropanes 1,1-di-accepteurs seront brièvement discutées. / The present M.Sc. thesis will be divided into two main subjects. The first part will be focused on the development of an efficient microwave-assisted hydrolysis of thiazolidines for the synthesis of a-substituted cysteines. The second part will be on the development of methodology for the asymmetric synthesis of 1,1-di-acceptor alkylidenecyclopropanes. First, the role and the importance of quaternary amino acids, more specifically of a-substitued cysteines, will be described, then a review of the available enantioselective methods for their syntheses will be reported. Afterward, the development of a fast and efficient microwave-assisted hydrolysis of thiazolidines will be presented. Finally, the studies toward the applicability of the methodology for the multi-gram synthesis of -substituted cysteines using flow and high-throughput reactors will be detailed. In the second part, the general applications and syntheses of alkylidenecyclopropanes will be described. More specifically, the applications of 1,1-di-acceptor alkylidenecycloproanes as well as their syntheses will be exhaustively covered. Then, the development of a catalytic enantioselective methodology for the synthesis of di-acceptor alkylidenecyclopropanes will be presented. The extension of this methodology for the synthesis of cyclopropane and cyclopropene derivatives, as well as the applications of 1,1-diacceptor alkylidenecyclopropane in stereospecific derivatizations will be briefly discussed.

The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration.

Le présent mémoire est subdivisé en deux principaux sujets. Le premier porte sur le développement d’une hydrolyse de thiazolidines assistée par micro-ondes en vue d’obtenir des cystéines a-substituées. Le second est axé sur le développement d’une méthodologie pour la synthèse catalytique énantiosélective d’alkylidènecyclopropanes 1,1-di-accepteurs. Dans un premier temps, les rôles et les utilités des acides aminés quaternaires, plus spécifiquement des cystéines a-substituées, seront abordés, puis une revue des différentes méthodes énantiosélectives pour accéder à ces unités sera effectuée. Par la suite, le développement d’une méthode rapide et efficace d’hydrolyse sous irradiation aux micro-ondes de thiazolines sera présenté. Finalement, les études menant à l’application de cette méthode à la synthèse de cystéines -substituées sur grande échelle au moyen de réacteurs en écoulement dynamique et à haut criblage seront détaillées. Dans la seconde partie, les applications ainsi que les synthèses générales des alkylidènecyclopropanes en synthèse organique seront décrites. Plus particulièrement, les applications spécifiques des alkylidènecyclopropanes 1,1-di-accepteurs ainsi que leurs synthèses seront traitées de manière exhaustive. Par la suite, le développement d’une méthodologie énantiosélective catalytique pour la synthèse d’alkylidènecyclopropanes 1,1-di-accepteurs sera présenté. L’extension de cette méthodologie à la synthèse de dérivés cyclopropanes et cyclopropènes, ainsi que l’application de réactions stéréospécifiques pour les alkylidènecyclopropanes 1,1-di-accepteurs seront brièvement discutées. / The present M.Sc. thesis will be divided into two main subjects. The first part will be focused on the development of an efficient microwave-assisted hydrolysis of thiazolidines for the synthesis of a-substituted cysteines. The second part will be on the development of methodology for the asymmetric synthesis of 1,1-di-acceptor alkylidenecyclopropanes. First, the role and the importance of quaternary amino acids, more specifically of a-substitued cysteines, will be described, then a review of the available enantioselective methods for their syntheses will be reported. Afterward, the development of a fast and efficient microwave-assisted hydrolysis of thiazolidines will be presented. Finally, the studies toward the applicability of the methodology for the multi-gram synthesis of -substituted cysteines using flow and high-throughput reactors will be detailed. In the second part, the general applications and syntheses of alkylidenecyclopropanes will be described. More specifically, the applications of 1,1-di-acceptor alkylidenecycloproanes as well as their syntheses will be exhaustively covered. Then, the development of a catalytic enantioselective methodology for the synthesis of di-acceptor alkylidenecyclopropanes will be presented. The extension of this methodology for the synthesis of cyclopropane and cyclopropene derivatives, as well as the applications of 1,1-diacceptor alkylidenecyclopropane in stereospecific derivatizations will be briefly discussed.

The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration.

The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration. / Medicine, Faculty of / Graduate

A distinctive feature of host-pathogen interactions in the case of Mycobacterium tuberculosis is the asymptomatic latent phase of infection. The ability of the bacillus to survive for extended periods of time in the host suggests an adaptive mechanism in M. tuberculosis that can cope with a variety of environmental stresses and other host stimuli. Extensive genomic studies and analysis of knock-out phenotypes revealed elaborate cellular machinery in M. tuberculosis that ensures a rapid cellular response to host stimuli. Prominent amongst these are two-component systems and σ factors that exclusively govern transcription re-engineering in response to environmental stimuli. M. tuberculosis σK is a σ factor that was demonstrated to control the expression of secreted antigenic proteins. The study reported in this thesis was geared to understand the molecular basis for σK activity as well as to explore conditions that would regulate σK activity. Transcription in bacteria is driven by the RNA polymerase enzyme that can associate with multiple σ factors. σ factors confer promoter specificity and thus directly control the expression of genes. The association of different σ factors with the RNA polymerase is essential for the temporal and conditional re-engineering of the expression profile. Environment induced changes in expression rely on a subset of σ factors. This class of σ factors (also referred to as Class IV or Extra-cytoplasmic function (ECF) σ factors) is regulated by a variety of mechanisms. The regulation of an ECF σ factor activity at the transcriptional, translational or posttranslational steps ensures fidelity in the cellular concentration of free, active ECF σ factors. In general, ECF σ factors associate with an inhibitory protein referred to as an anti-σ factor. The release of a free, active σ factor from a σ /anti-σ complex is thus a mechanism that can potentially control the cellular levels of an active σ factor in the cell. M. tuberculosis σK is associated with a membrane bound anti-σK (also referred to as RskA) (Said-Salim et al., Molecular Microbiology 62: 1251-1263: 2006). The extracellular stimulus that is recognized by RskA remains unclear. However, recent studies have suggested the possibility of a regulated proteolytic cascade that can selectively degrade RskA and other membrane associated anti-σ factors. The goal of the study was to understand this regulatory mechanism with a specific focus on the M. tuberculosis σK/RskA complex. The structure of the cytosolic σK/RskA complex and the associated biochemical and biophysical characteristics revealed several features of this /anti-σ complex that were hitherto unclear. In particular, these studies revealed a redox sensitive regulatory mechanism in addition to a regulated proteolytic cascade. These features and an analysis of the M. tuberculosis σK/RskA complex vis-à-vis the other characterized σ/anti σfactor complexes are presented in this thesis. This thesis is organized as follows- Chapter 1 provides an overview of prokaryotic transcription. A brief description of the physiology of M. tuberculosis is presented along with a summary of characterized factors that contribute to the pathogenecity and virulence of this bacillus. The pertinent mechanistic issues of σ/anti-σ factor interactions are placed in the context of environment mediated changes in M. tuberculosis transcription. A summary of studies in this area provides a background of the research leading to this thesis. Chapters 2 and 3 of this thesis describe the structural and mechanistic studies on the σK/RskA complex. The crystal structure of the σK/RskA complex revealed a disulfide bond in domain 4 (σK4). σK4 interacts with the -35 element of the promoter DNA. The disulfide forming cysteines were seen to be conserved in more than 70% of σK homologs, across both gram-positive and gram-negative bacteria. The conservation of the disulfide-forming cysteines led us to further characterize the role of this disulfide in σK/RskA interactions. These were examined by several biochemical and biophysical experiments. The redox potential of these disulfide bond forming cysteine residues were consistent with the proposed role of a sensor. The crystal structure and biochemical studies thus suggest that M. tuberculosis σK is activated under reducing conditions. Chapter 4 of this thesis describes the progress made thus far in the structural and biochemical characterization of an intra-membrane protease, M. tuberculosis Rip1 (Rv2869c). This protein is an essential component of the proteolytic cascade that selectively cleaves RskA. The proteolytic steps that govern the selective degradation of an anti-σ factor were first characterized in the case of E. coli σE (Li, X. et al. Proc. Natl. Acad. Sci. USA, 106:14837-14842, 2009). This cascade is triggered by the concerted action of a secreted protease (also referred to as a site-1 protease) and a trans-membrane protease (also referred to as a site-2 protease). M. tuberculosis Rip1 was demonstrated to be bona-fide site 2 protease that acts on three anti-σ factors viz., RskA, RslA and RsmA (Sklar et al., Molecular Microbiology 77:605-617; 2010). To further characterize the role of Rip1 in the proteolytic cascade, this intra-membrane protease was cloned, expressed and purified for structural, biochemical and biophysical analysis. The preliminary data on this membrane protein is described in this chapter. The conclusions from the studies reported in this thesis and the scope for future work in this area is described in Chapter 5. Put together, the σK/RskA complex revealed facets of σ/anti-σ factor interactions that were hitherto unrecognized. The most prominent amongst these is the finding that an ECF σfactor can respond to multiple environmental stimuli. Furthermore, as seen in the case of the σK/RskA complex, the σ factor can itself serve as a receptor for redox stimuli. Although speculative, a hypothesis that needs further study is whether these features of the σK/RskA complex contribute to the variable efficacy of the M. bovis BCG vaccine. In this context it is worth noting that σK governs the expression of the prominent secreted antigens- MPT70 and MPT83. The studies reported in this thesis thus suggest several avenues for future research to understand mycobacterial diversity, immunogenicity and features of host-pathogen interactions. The appendix section is divided into two subparts- Appendix 1 of the thesis is a review on peptidase V. This is a chapter in The Handbook of Proteolytic enzymes (Elsevier Press, ISBN:9780123822192). Appendix 2 of the thesis includes technical details and an extended materials and methods section.