The aim of this thesis was to gain further insight into the biology of normal human skin and basal cell carcinoma (BCC). Morphology in combination with microdissection was used as primary tool for sampling. Using the X-chromosome inactivation assay, we found normal human skin to consist of a mosaic of cells, with either the maternal or the paternal X-chromosome inactivated. We believe that each tile is made up of several epidermal proliferative units with identical X-chromosome inactivation patterns. Using the same method, we found BCC to be a monoclonal neoplasm imbedded in polyclonal stroma. However, one tumor displayed clear evidence of being composed of two intermingled monoclonal tumors. To better enable molecular analysis of defined cells from tissue sections, we investigated a zinc-based fixative as alternative to neutral-buffered formalin. Zinc-based fixative preserves good quality of genomic DNA, with only slight impairment of morphology. In addition, it partly abrogates the need for antigen retrieval. The patched gene is involved in BCC development. We analyzed the distribution of a coding polymorphism (Pro/Leu) at codon 1315 in populations with different skin types. We found a reduced Pro/Pro genotype frequency in populations with lighter pigmentation. This in combination with genotype analyses of patients with multiple BCCs, showed that failure to lose the Pro allele during a shift towards lighter pigmented skin may be associated with an increased risk of developing BCC. We compared the expression profile of BCC cells with putative progenitor cells in the basal layer of epidermis. In addition to discovering several unknown genes, we found the Wnt signaling pathway to upregulated. Furthermore, differentiation markers were downregulated together with proteins important for scavenging of oxygen radicals. In conclusion, the combination of morphology, microdissection and subsequent molecular applications provided valid information deepening our understanding of normal skin and BCC.

In this study photocatalytic antibacterial and self-cleaning activities of TiO2-SiO2 thin films as a function of TiO2/SiO2 ratios were investigated. TiO2-SiO2 mixed oxides were synthesized by sol-gel method and coated over soda-lime glass plates by dip coating technique. Escherichia coli was used as a model microorganism for the photocatalytic antibacterial tests. Degradation rate of methylene blue (MB) molecules was used to characterize photocatalytic self-cleaning activities of thin film surfaces. The maximum antibacterial activity was achieved over 92 wt% SiO2 containing thin films. However, when the SiO2 content exceeds 92 wt%, photocatalytic antibacterial activity decreased considerably, which was explained by the dilution of TiO2 phase and inaccessibility of TiO2. Increase in photocatalytic antibacterial activity was attributed to increases in the relative surface area, roughness, hydroxyl (OH-) groups and bacterial adhesion. The favored bacterial adhesion enhanced direct contact of bacteria with TiO2 particles and surface reactive oxygen species. The highest initial decomposition rate of MB was obtained for 60 wt% SiO2 and the activity decreases as SiO2 concentration increases. The increase in photocatalytic activity by the SiO2 addition can be explained by the increase of the amount of MB per unit area of TiO2-SiO2 thin films. Different adsorption capability of thin films against MB molecule and E. coli cell was explained as the first reason why the antibacterial and self-cleaning activities reached their maximum values at different SiO2 ratios. The second reason could be related with the different control mechanisms of self-cleaning and antibacterial activities by different textural and surface properties.

Voltage-gated ion channels play fundamental roles in neural excitability, they are for instance responsible for every single heart beat in our bodies, and dysfunctional channels cause disease that can be even lethal. Understanding how the voltage sensor of these channels function is critical for drug design of compounds targeting neuronal excitability. The opening and closing of the pore in voltage-gated potassium (Kv) channels is caused by the arginine-rich S4 helix of the voltage sensor domain (VSD) moving in response to an external potential. In fact, VSDs are remarkably efficient at turning membrane potential into conformational changes, which likely makes them the smallest existing biological engines. Exactly how this is accomplished is not yet fully known and an area of hot debate, especially due to the lack of structures of the resting and intermediate states along the activation pathway. In this thesis I study how the VSD activation works and show how toxic compounds modulate channel gating through direct interaction with these quite unexplored drug targets. First, I show that a secondary structure transition from alpha- to 3(10)-helix in the S4 helix is an important part of the gating as this helix type is significantly more favorable compared to the -helix in terms of a lower free energy barrier. Second, I present new models for intermediate states along the whole voltage sensor cycle from closed to open and suggest a new gating model for S4, where it moves as a sliding 3(10)-helix. Interestingly, this 3(10)-helix is formed in the region of the single most conserved residue in Kv channels, the phenylalanine F233. Located in the hydrophobic core, it directly faces S4 and creates a structural barrier for the gating charges. Substituting this residue alters the deactivation free energy barrier and can either facilitate the relaxation of the voltage sensor or increase the free energy barrier, depending on the size of the mutant. These results are confirmed by new experimental data that supports that a rigid ring at the phenylalanine position is the rate-limiting factor for the deactivation gating process, while the activation is unaffected. Finally, we study how the activation can be modulated for pharmaceutical reasons. Neurotoxins such as hanatoxin and stromatoxin push S3b towards S4 helix limiting S4's flexibility. This makes it harder for the VSD to activate and might explain the stronger binding affinities in resting state. All these results are highly important both for the general topic of biological macromolecules undergoing functionally critical conformational transitions, as well as the particular case of voltage-gated ion channels where understanding of the gating process is probably the key step to explain the effects of mutations or drug interactions. / <p>QC 20121115</p>

Choline oxidase from Arthrobacter globiformis is a flavin-dependent enzyme that catalyzes the oxidation of choline to betaine aldehyde through two sequential hydride-transfer steps. The study of this enzyme is of importance to the understanding of glycine betaine biosynthesis found in pathogenic bacterial or economic relevant crop plants as a response to temperature and salt stress in adverse environment. In this study, chemical modification of choline oxidase using two irreversible inhibitors, tetranitromethane and phenylhydrazine, was performed in order to gain insights into the active site structure of the enzyme. Choline oxidase can also be inactivated irreversibly by freezing in 20 mM sodium phosphate and 20 mM sodium pyrophosphate at pH 6 and -20 oC. The results showed that enzyme inactivation was due to a localized conformational change associated with the ionization of a group in close proximity to the flavin cofactor and led to a complete lost of catalytic activity.

L'inactivation du chromosome X est la réponse trouvée par l'évolution pour pallier à la divergence gonosomique entre mâle (XY) et femelle (XX). Ce phénomène sert donc à mettre les deux sexes sur un pied d'égalité en limitant la quantité de transcrits provenant des chromosomes X présents dans les cellules femelles. Au cours de mon doctorat, j'ai tenté de contribuer à l'étude des mécanismes de régulation transcriptionnelle, notamment l'activation, des deux acteurs principaux de l'inactivation: Xist et Tsix, son transcrit antisens. Pendant ces 4 anne��es, j'ai entrepris de cartographier le profil de fixation de plusieurs protéines le long du locus Xist/Tsix, dans le but de comprendre les mécanismes permettant une surexpression de Xist lors de la disparition de ses facteurs répressifs en cours de différenciation. J'ai donc pu établir un modèle de régulation transcriptionnelle de l'ARN non-codant Xist, impliquant plusieurs protéines connues pour leur rôle dans la régulation transcriptionnelle (CTCF et YY1) aussi bien que dans la formation de structures tridimensionnelles (la cohésine). La pertinence de ce modèle est renforcée par nos études montrant que de nombreux aspects de ce modèle sont conservés à travers l'évolution (notamment chez l'homme). J'ai également pu contribuer à la découverte de nouveaux activateurs de Tsix, certains facteurs de pluripotence se fixant au minisatellite DxPas34 afin de réguler l'élongation de la transcription de l'antisens. Ces résultats apportent donc d'importantes informations concernant les mécanismes régulant la mise en place du phénomène d'inactivation du chromosome X au cours du développement précoce de l'embryon.

<p>PIWI proteins belong to an evolutionary conserved protein family as the sister sub-family of ARGONAUTE (AGO) proteins. While AGO proteins are functionally well-characterized and shown to mediate small-RNA guided gene regulation, the function of PIWI proteins remain elusive. Here we pursued functional characterization of PIWI proteins by studying MILI and MIWI, two PIWI proteins in the mouse.</p><p>We first show that both MIWI and MILI co-immunoprecipitate with a novel class of non-coding small RNAs from the post-natal mouse testis extract, which are named Piwi-interacting RNAs (piRNAs). Our cloning efforts identified thousands of different piRNA sequences, mostly derived from intergenic regions. Interestingly, both MILI and MIWI piRNAs correspond to the same regions on the genome and differ primarily in length. We propose piRNAs in the adult testis are produced by the processing of long, single stranded RNA precursors, based on the observation that piRNAs originate in clusters from a number of sites on the genome in a head-to-tail homology. In support, we bioinformatically predicted putative promoters, and yeast one hybrid analysis on two such regions found out that they interact with Krueppel C2H2 type zinc finger transcription factors. We did not observe the features of the "ping-pong" mechanism in their biogenesis: Both MILI and MIWI piRNAs are biased for 5` Uracil without an Adenine bias on the 10th nucleotide position, and do not significantly consist of sequences complementary to each other along their first 10nt. Moreover, MILI piRNAs are not down-regulated in Miwi-/- testis. These results indicate that the post-natal testicular piRNAs are produced independent of the ping-pong mechanism. </p><p>Although piRNAs are highly complex, PAGE and in situ analyses showed that piRNAs are germ cell-specific with predominant expression in spermatocytes and round spermatids, suggestive of a meiotic function. Correspondingly, we found that Miwi-/-; Mili-/- mice undergo only male infertility with terminal spermatogenic arrest during meiosis. piRNAs show a nucleo-cytoplasmic distribution, with enrichment in the chromatoid and dense bodies, two male germ cell-specific structures. The dense body has been implicated in synapsis and in the heterochromatinization of the sex chromosomes during male meiosis, a process known as meiotic sex chromosome inactivation (MSCI). Our histological analysis on Miwi-/-; Mili-/- testes showed that, while the overall synapsis is not affected, the sex chromosomes retain the euchromatin marker acetyl-H4K16 and lacks the heterochromatin marker H3K9-dimethyl. These observations indicate that murine PIWI proteins are necessary for MSCI. Moreover, we identified piRNA production from the X chromosome before MSCI, and propose PIWI proteins utilize piRNAs to target and silence unpaired chromosomal regions during meiosis.</p> / Dissertation

Reproduction de : Thèse de doctorat : Sciences de la vie et de la santé : Lille 1 : 2004. / N° d'ordre (Lille 1) : 3505. Résumé en français et en anglais. Articles en anglais non reproduits dans la version électronique. Titre provenant de la page de titre du document numérisé. Bibliogr. f. 156-182.

HIV infection has risen to pandemic proportions. Interleukin-10 (IL-10), a potent antiinflammatory cytokine has been shown to enhance the establishment and persistence of chronic viral infections through inactivation of effector antiviral immune responses and it may also directly influence HIV-1 replication in cells of diverse lineages. IL-10 promoter polymorphisms have been shown to affect HIV-1 susceptibility and pathogenesis. However, the underlying mechanisms are poorly understood. We investigated the relationship between IL-10 promoter variants, plasma IL-10 levels, and markers of disease outcome in chronically HIV-1-infected individuals. To investigate the mechanistic role of IL-10 and its genetic variants on HIV pathogenesis, we studied markers of activation on B cells, CD4+ and CD8+ T cells, and assessed effects on CD4+ T cell proliferation with and without blockade of the IL- 10 pathway. We used Taqman genotyping assays to genotype three IL-10 promoter single nucleotide polymorphisms (SNPs) in our study cohort. Baseline plasma IL-10 levels were measured using Luminex technology for 112 individuals. Viral load, CD4+ T cell counts and cytotoxic T lymphocyte (CTL) immune responses were measured at baseline. The rate of CD4+ T cell decrease was calculated in 300 individuals with a median follow-up of 25 months. CD38, CD95, Ki67, IgG and PD-1, markers of activation or exhaustion were measured on B cells, and CD38, CD95, Ki67, HLA-DR and PD-1 were measured on CD4+ and CD8+ T cells in a subset of 63 individuals. CD4+ T cell proliferation was measured using Carboxyfluorescein succinimidyl ester (CFSE) assays, following IL-10 receptor blockade in a subset of 31 individuals. The IL-10 -1082G, -592A and -3575 variants were observed at frequencies of 0.3, 0.34 and 0.23 respectively, in our study cohort. Plasma IL-10 levels were significantly higher in the - 1082GG group than in the combined AA/AG group (p=0.0006). There was a significant association between the 592AA genotype and a greater breadth of CTL responses compared to the CC and CA (p= 0.002 and 0.004 respectively). The -592AA genotype associated significantly with an attenuated loss of CD4 cells (p= 0.0496), with -592AA having the least change in CD4 cells per year. The median expression of HLA-DR, a marker of T cell activation was significantly higher in the-1082AA group for CD8 cells (p= 0.047), and the - 592AA group for CD4 T cells (p= 0.01). The median expression of IgG on the surface of B cells was significantly higher in the -1082GG genotype and the -592CC genotype (p=0.0183 and 0.0659 respectively). Overall, IL-10 variants correlated with IL-10 expression and CD4 decline during chronic HIV-1 infection. IL-10 promoter variants may influence the rate of HIV-1 disease progression by regulating IL-10 levels, which in-turn, may affect the breadth of CTL responses. Furthermore, the increased expression of HLA-DR and PD-1 on CD8+ and CD4+ T cells, indicates that lower IL-10 levels are associated with increased immune activation and immune exhaustion. The increased expression of IgG on B cells, suggests that in a setting of lower IL-10, there is possibly a bias towards a Th2 immune response. These data suggest a significant role for IL-10 genetic variants and IL-10 in HIV pathogenesis. Further studies to determine whether and how the IL-10 pathway may be manipulated for therapeutic or vaccine strategies for HIV are warranted. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2012.

Le développement embryonnaire précoce des mammifères femelles s'accompagne de l'inactivation transcriptionnelle d'un de leurs deux chromosomes X. Cet évènement est initié suite à l'expression mono-allélique de l'ARN non codant Xist, qui est contrôlée par de nombreux éléments cis-régulateurs présents dans le centre d'inactivation du chromosome X (Xic) - tel son anti-sens répresseur Tsix. Mon travail de thèse a consisté à développer des approches permettant d'appréhender le paysage structural dans lequel s'exerce cette régulation. La caractérisation de l'architecture tridimensionnelle du Xic, par des techniques basées sur la capture de conformation chromosomique (3C) et l'hybridation in situ en fluorescence (FISH), m'a permis de mettre en évidence que les promoteurs respectifs de Xist et Tsix sont engagés dans des interactions physiques intimes avec des loci distaux, localisés au sein du Xic, et de montrer qu'au moins certaines de ces régions exercent un effets régulateurs à longue-distance. Les éléments du Xic contactés par les régions promotrices de Xist et de Tsix sont en outre fondamentalement différents, chacune engageant des associations chromosomiques sur plusieurs centaines de kilobases dans leur direction 5' respective.Ce travail a également permis de révéler des propriétés insoupçonnées de l'architecture chromosomiques. En effet, le Xic apparaît scindé en plusieurs sous-régions, couvrant chacune entre 200kb et 1Mb, à l'intérieur desquelles les interactions chromosomiques sont préférentiellement établies. L'existence de ces domaines d'interaction s'intègre avec d'autres propriétés structurales du génome, tels la composition de la chromatine sous-jacente et l'association à la lamine nucléaire, mais n'apparaît pas en dépendre directement. En étudiant la dynamique de la conformation chromosomique du Xic au cours de la différenciation cellulaire, j'ai pu constater la robustesse de cette organisation, sauf sur le chromosome X inactif, qui se distingue par la perte des contacts chromosomiques préférentiels détectables sur son homologue actif.Enfin, j'ai pu mettre en évidence que la variabilité du repliement général du chromosome X amène à un instant donné chaque allèle de Tsix à contacter physiquement des jeux de séquences distales différents, suggérant que l'environnement structural instantané de chacun de ces allèles à l'orée de l'activation mono-allélique de Xist est différent. Ce travail, combinant des approches à l'échelle de la population cellulaire d'une part et de la fibre de chromatine unique d'autre part, apporte une nouvelle vision du paysage structural et régulateur dans lequel s'inscrit le contrôle de l'activité transcriptionnelle de Xist, et fourni de nouvelles perspectives concernant les principes fondamentaux de l'organisation topologique des chromosomes chez les mammifères.

Increasing consumer demand for new products with high nutritional qualities has spurred a search for new alternatives to food preservation. Pulsed electric field (PEF) is an emerging technology for non thermal food pasteurisation. Using this technology, enzymes, pathogenic and spoilage microorganisms can be inactivated without affecting the colour, flavour, and nutrients of the food. PEF treatment may be provided by applying pulsed electric field to a food product in a treatment zone between two electrodes at ambient , or slightly above ambient temperature. Exposure of microbial cells to the electric field induces a transmembrane potential in the cell membrane, which results in electroporation (the permeabilization of the membranes of cells and organelles) and/or electrofusion (the connection of two separate membranes into one) of the cells. An innovative pulsed electric field (PEF) unit was designed and constructed in the University of Auckland using modern IGBT technology. The system consists of main equipments, the high voltage pulse generator and the treatment chambers. The main focus of this work was to design an innovative PEF treatment chamber that provide uniform distribution of electric field, minimum increase in liquid temperature, minimum fouling of electrodes and an energy efficient system. Four multi pass treatment chambers were designed consisting of two stainless steel mesh electrodes in each chamber, with the treated fluid flowing through the openings of the mesh electrodes. The two electrodes are electrically isolated from each other by an insulator element designed to form a small orifice where most of the electric field is concentrated. Dielectric breakdown inside the chambers was prevented by removing the electrodes far from the narrow gap. The effect of the chambers different geometries on the PEF process in terms of electric parameters and microbial inactivation were investigated. Electric field intensity in the range of (17-43 kV/cm) was applied with square bipolar pulses of 1.7 µs duration. The effect of PEF treatment on the inactivation of gram-negative Escherichia coli ATCC 25922 suspended in simulated milk ultra-filtrate (SMUF) of 100%, 66.67% and 50% concentration was investigated. Treatments with the same electrical power input but higher electric field strengths provided larger degree of killing. The inactivation rate of E coli was significantly increased with increasing the electric field strength, treatment time and processing temperature. Morphological changes on E coli as a result of PEF treatment were studied under transmission electron microscopy (TEM). Significant morphological changes on E coli after PEF treatment were observed. The TEM studies suggested that the microbial inactivation was a consequence of electroporation and electrofusion mechanisms. Kinetic analysis of microbial inactivation due to PEF and thermal treatment of E coli suspended in SUMF were also studied. Comparison between measured (experimental) and predicted (theoretical) variation of E coli concentration with time following the PEF treatment was discussed, taking into consideration the recirculation mode of the PEF treatment. The treated liquid was circulated more than once through the treatment chamber to provide higher microbial inactivation. Arrhenius constants and activation energies of E coli inactivation using combined PEF and thermal treatment were calculated and generalized correlation for the inactivation rate constant as a function of electric field intensity and treatment temperature was developed. / Fonterra Research Institute (NZ) and the Foundation for Research Science and Technology (NZ)