The human skin provides us with an excellent protective barrier and possesses a remarkable ability of constant renewal. Basal cell carcinoma is the most common type of skin cancer. The aim of this project was to verify results from an earlier study investigating the molecular differences between basal cell carcinoma (BCC) and basal cells of normal human epidermis. In that study microdissection of cell populations from BCC and basal cells of normal epidermis respectively was performed in five cases of confirmed BCC. Following RNA extraction and amplification, a gene expression analysis was performed using a 46 k human cDNA microarray. Comparison of expression profiles showed a differential expression of approximately 300 genes in BCC. An upregulation of signaling pathways previously known to be of importance in BCC development could be observed, as well as a downregulation of differentiation markers, MHC class II molecules, and proteins active in scavenging of oxygen radicals. We wanted to confirm these findings for a number of selected genes, using real time PCR. The focal point of this project was microdissection of cells from BCC and subsequent isolation of RNA. Microdissection based methods offer a possibility of selecting well defined cell populations for further analysis by using a focused laser beam. Initially tests in order to optimize the method were also performed, concerning the dehydration process and choice of slides used in microdissection. Isolation of RNA may, as we experienced, be associated with problems due to destruction of RNA by degrading enzymes.

The purpose of this study was to identify and compare the specific writing assignments provided in the five basal readers, grades one through eight, adopted by the state of Texas. These seventy-eight basal reader's guides were first analyzed for statements indicating specific writing assignments. The total number of writing assignments in each of the teacher's guides were totaled for each publisher. The location of each writing assignment which supported the TABS categories was recorded. The writing assignments which did not support the TABS categories were assigned appropriate categories and recorded on a table. Another table compared the five publishers and the total number of writing assignments supporting the TABS categories. A fifth table compared the five publishers and the total number of writing assignments found in other categories not supporting the TABS categories.

Parmi les protéoglycannes à héparane sulphate (HSPGs) des lames basales, le perlécane présente un rôle majeur dans la morphogénèse de l'épiderme mais également dans la survie et la différenciation des kératinocytes. Celui‐ci régulerait ces processus en contrôlant la biodisponibilité des facteurs de croissance. Au cours de ce travail, nous avons étudié l'expression du perlécane dans la peau humaine au cours du vieillissement intrinsèque et son implication dans les défauts de l'épiderme liés à l'âge. Une analyse immunohistologique sur une cohorte de peau normale humaine provenant de donneurs allant de 22 à 73 ans, a révélé une diminution drastique du perlécane au niveau de la jonction dermo‐épidermique et des capillaires dermiques au cours de l'âge. Des études biochimiques combinées à des analyses génomiques ont pu démontrer que la diminution de l'expression du perlécane n'était pas due à une dégradation de la protéine mais à une diminution de sa régulation transcriptionnelle par les kératinocytes. Par ailleurs, la conception et la caractérisation de modèles de peaux reconstruites in vitro combinant des cellules provenant de différents âges a permis d'établir une corrélation entre le défaut de synthèse du perlécane et l'épaisseur de l'épiderme. Nous avons montré que l'addition de perlécane natif purifié dans le milieu de culture d'un modèle contenant des kératinocytes âgés a permis de restaurer l'architecture épidermique. L'ensemble de ces résultats démontre ainsi l'importance du perlécane dans l'homéostasie cutanée. Un criblage de composés a permis la sélection de molécules candidates capables de restaurer spécifiquement l'expression du perlécane dans des cultures de kératinocytes et de cellules endothéliales microvasculaires dermiques âgés. Des tests fonctionnels sur les modèles de peaux reconstruites précédemment mis au point, ont permis de démontrer leur capacité à améliorer la morphogénèse de l'épiderme en accord avec les fonctions du perlécane d'origine épithéliale. L'ensemble de nos résultats indique qu'une diminution significative de synthèse du perlécane par les kératinocytes au cours de l'âge participe à l'affinement et aux défauts de différenciation de l'épiderme observés durant le vieillissement cutané / Among basement membrane heparan sulphate proteoglycans (HSPGs), perlecan is known to be involved in epidermal formation and renewal by regulating keratinocyte survival and differentiation. HSPGs are known to be regulators of growth-factor signalling through different mechanisms. We have investigated the expression of perlecan during skin ageing and have examined whether its expression correlates with age-dependant modifications of the epidermal compartment. The immunohistochemical analysis of perlecan in a cohort of human skin samples from donors of ages ranging from 22 to 73 years revealed a strong decrease of its expression with age in good correlation with reduction of the epidermal thickness. Genomic analyses of keratinocytes isolated from these biopsies combined with further characterization of perlecan from aged donors revealed that the observed decreased expression is most likely a consequence of a reduced synthesis rather than a protein degradation or a deficient supramolecular exposure. The design and characterization of human skin models engineered with cells from donors of various ages confirmed this hypothesis and revealed that aged keratinocytes showing deficiency in perlecan production and deposit in the basement membrane, also displayed inability to form a multilayed epidermis. Addition of purified perlecan in the culture medium of these skin equivalents enhanced the epidermal thickness and restored a well-differentiated multilayed epithelium. Biological screening of a library compounds allowed the selection of candidates able to increase the expression of epithelial and endothelial-derived perlecan with high specificity. Skin engineering models mimicking ageing human skin demonstrates the capacity of one candidate to restore the perlecan functional failure in promoting epidermal renewal and morphogenesis. Our results suggest that the presence of perlecan in the skin basement membrane appears to be crucial for the maintenance of the epidermal integrity and that its decrease with age may contribute to the epidermal thinning

Les ganglions de la base (BG) sont un ensemble de noyaux qui contrôle des taches fondamentales de la vie quotidienne, notamment le control des mouvements, ainsi que l’apprentissage et le reward. En particulier, le striatum est le noyau principal des BG et le majeur relais d’input. Il est formé par deux sous-types de neurones de projection (SPN) qui modulent l’activité de sortie des BG directement (dSPN) ou indirectement (iSPN) via d’autres structures. Les deux populations sont intermelangés, ce qui permet l’activation parallèle des deux voies. Une perte d’équilibre entre l’activité des dSPN et des iSPN est partie de l’étiologie de plusieurs neuropathies des BG, y compris la maladie de Parkinson et celle de Huntington. Malgré l’importance fonctionnelle de ces neurones, on a une connaissance très incomplète de comment les deux sous-types sont spécifiés au cours du développement; de plus, la question de comment les deux sous-types se mélangent pour former l’architecture fonctionnelle du striatum reste à élucider. Utilisant une combinaison unique d’outils génétiques disponible dans la souris, j’ai montré que les dSPN et iSPN sont spécifiés dés très tôt et diffèrent dans leur distribution dans le striatum embryonnaire pour s’intermélanger progressivement. De plus, je montre que ce processus de mélange repose sur l’expression du facteur de transcription Ebf1, un gène qui est exprimé spécifiquement dans le dSPN et contrôle aussi l’intégration de ces derniers dans les circuits des BG. Mes résultats fournissent un nouveau contexte pour investiguer les mécanismes moléculaires qui contrôlent l’assemblage du striatum et donnent des informations essentielles pour la génération de neurones striataux in vitro. Une autre population des BG, les neurones du corridor, ont la même origine que les SPN; cependant, au lieu de migrer vers le striatum, ces neurones forment une structure provisoire qui est cruciale pour former la capsule interne, un des majeurs faisceaux d’axones dans le cerveau des mammifères. Malgré leur importance pour le développement de la connectivité cérébrale, on ne sait pas si ces neurones jouent aussi un rôle dans le cerveaux adulte. À travers une combinaison de fate mapping génétique et d’analyse moléculaire à différent stades du développement, je montre que ces neurones contribuent à des noyaux spécifiques de l’amygdale étendue, une structure impliquée dans le control de la peur et de l’anxiété. Ces résultats montrent que les neurones du corridor pourraient contribuer à la régulation de l’anxiété et améliorent notre connaissance sur la formation de ces structures, qui sont très conservés au cours de l’évolution et qui ont un grand intérêt pathologique. Pris dans l’ensemble, mes résultats fournissent non seulement des nouvelles et très importantes informations sur la façon dont les circuits des BG sont formés, mais déterminent un nouveau cadre conceptuel pour investiguer le développement et la connectivité du cerveau antérieur. / Basal ganglia (BG) are a set of brain nuclei that control crucial aspects of everyday life such as motor control, habit learning and reward. In particular, the striatum is the biggest nucleus and input station of BG. It is formed by two subsets of projection neurons (SPN) that modulate BG output activity either directly (dSPN) or indirectly via other BG structures (iSPN). The two populations are intermixed, allowing parallel activation of the two pathways. Impaired balance of dSPN and iSPN activity is part of the aetiology of many BG neuropathies, including Parkinson’s and Huntington’s diseases; however, to date we have poor knowledge on how the two subtypes are specified and how they intermix during development. Using a unique combination of mouse genetic tools, here I show that dSPN and iSPN are specified early as independent populations, have different early distribution and gradually intermix. Moreover, I show that the process of intermix relies on expression of transcription factor Ebf1 in dSPN, a gene that also controls dSPN ability to integrate in BG circuits. These findings provide a new framework to investigate the molecular mechanisms controlling striatal mosaic assembly and will provide instrumental to generate fully formed striatal neurons in vitro. Another BG population, corridor neurons, shares common origin with SPN; however, instead of migrating toward the striatum, these cells form a transient corridor (Co) that is crucial for the formation of the internal capsule, a major axonal pathway in mammals. Despite their importance for brain wiring, whether Co cells also play a role in the adult brain is unknown. Through a combination of genetic fate map and in vivo timecourse, I surprisingly show that these cells participate to specific nuclei of the central extended amygdala, a structure implicated in anxiety and fear response. This finding indicates that Co neurons might contribute to anxiety regulation and sheds new light on the formation of evolutionarily conserved structures of great behavioral and clinical interest. Taken together, my findings not only provide new and critical information on neuronal migration and circuit formation in the BG, but also a new conceptual framework to investigate the formation of nuclear structures of the anterior brain.

Fish can have complex life histories and use multiple habitats and resources throughout their life span. Consequently, their life histories are often poorly understood. The Atlantic Tarpon, Megalops atlanticus, is a large, typically migratory, elopomorph fish that is both ecologically and economically important. Atlantic Tarpon are under threat due to regional exploitation, loss of natal and juvenile habitat, poor water management, and offshore impacts. In addition, little is known about its lifelong habitat and resource use. In Chapter 1, I used stable isotope analysis of eye lens δ13C and δ15N values to explore patterns in trophic history and habitat use of 16 Atlantic Tarpon from West-Central Florida and Louisiana. The stable isotope chronologies showed 100% use of backcountry habitats during the early life history and an ontogenetic habitat shift to coastal waters at approximately 10 years of age and 140 cm total length. During the coastal phase Atlantic Tarpon displayed among-individual variability and within-individual consistency in basal resource use. In Chapter 2, mark-recapture data from a multi-year genetic tagging program were used to investigate survival and growth rates, ontogenetic habitat use, and migration of juvenile Atlantic Tarpon in Florida. The study found that juvenile Atlantic Tarpon take approximately 10 years to reach the length associated with maturity, and appear to have a high survival rate (~80%), possibly due to effective use of habitats with reduced competition and predation. Atlantic Tarpon underwent several ontogenetic habitat shifts throughout the juvenile phase. In addition, juvenile Atlantic Tarpon did not migrate long distances but instead showed fidelity to systems wherein only short movements were needed to shift habitat types. This work serves to fill critical gaps in our knowledge of Atlantic Tarpon life history and may aid in better management and conservation of the species.

Slug est un membre de la famille des protéines Snail impliquées au cours du développement dans le contrôle de la forme et la différenciation cellulaire. Nous avons localisé Slug au cours du développement des glandes mammaires de souris dans les cellules qui participent aux mécanismes de croissance. Seule une sous-population de cellules épithéliales mammaires située dans le compartiment basal de la glande exprime Slug. Cette expression est maintenue pendant les différentes étapes du développement de la glande mammaire, de la puberté jusqu'au début de la gestation. Nous avons observé une perte d'expression dans lobules se différenciant en alvéoles sécrétoires, ensuite une re-expression au stade d' involution. La population exprimant Slug est positive pour la cytokératine 5, décrit comme un marqueur des cellules basales et myoépithéliales, également considéré comme un marqueur de cellules souches ou progénitrices, et elle est incluse dans la population CD24 positive et surexprimant le CD49, connue pour contenir les cellules souches de l'épithélium mammaire. Nous avons constaté que canaux epithéliaux des glandes mammaires de souris Slug knock-out envahissent moins le coussinet adipeux mammaire. En outre, Ils montrent un phénotype de branchements latéraux, ce qui suggère une différenciation précoce. Ce phénotype ressemble le phénotype des glandes mammaires de souris Knock-out pour la P-cadhérine. Nous avons également constaté une diminution de l'expression de la P-cadhérine in vivo dans les glandes mammaires des souris SlugLaZ, et in vitro, dans les cellules épithéliales mammaires transfectées avec un siARN ciblant Slug. Ces cellules montrent un retard de migration cellulaire. Ces observations valident à notre hypothèse que le facteur de transcription Slug contrôle la différenciation des cellules épithéliales au cours de la croissance physiologique de la glande mammaire murine. / Slug is a member of the Snail protein family involved during development in the control of cell shape and differentiation. We located Slug during mammary gland development in mouse in cells participating to the growth mechanisms. Only a distinct sub-population of mammary epithelial cells located in the basal compartment was found to express Slug. This expression is maintained during the various stages of mammary gland development, from puberty until the beginning of gestation. We observed a loss of expression in lobules differentiating into secreting alveoli, followed by re-expression at involution stage. The Slug expressing population was positive for Cytokeratin 5, described as a basal and myoepithelial cell marker, also considered as a stem/progenitor marker, and was included into the (CD24+ CD49++) population, known to contain the mammary epithelial progenitor cells. We found that mammary gland from Slug-deprived mice were slower to invade the mammary fat pad. In addition, they displayed increased lateral branching, suggesting precocious differentiation. This phenotype resembles the phenotype of mammary glands of P-cadherin Knock-out mice. We also found that P-cadherin is down regulated in vivo in SlugLaZ mice mammary gland, and in vitro, in mammary epithelial cells transfected with an siRNA targeting Slug. These cells show a delay in migration. These observations lead to our hypothesis that Slug controls an early epithelial cell differentiation stage during mammary gland physiological growth.

O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX) and O-isobutyl-S-2-diethylaminoethyl methylphosphonothioate (R-VX), are considered chemical warfare agents due to their strong acetylcholinesterase-inhibiting properties. Subsequent to terrorist use of these V-type nerve agents in both Japan and the United States of America (the September 11, 2001 attacks) and the limited capability of anti-terrorist groups to detect such weapons, there has been an increased obligation by the Chemical Weapons Convection for specific detection and identification methods for VX and R-VX. Chemical and/or enzymatic hydrolysis yields sulfhydryl mimic products, diethylaminoethanethiol (DEAET) and dimethylaminoethanethiol (DMAET). This thesis investigates the electrocatalytic parameters of DEAET and DMAET using basal plane pyrolytic graphite electrodes (BPPGEs) modified with: (a) single-wall carbon nanotube (BPPGE-SWCNT); (b) SWCNT functionalised with cobalt (II) tetra-aminophthalocyanine by (i) physical (BPPGE-SWCNT-CoTAPc(mix)), (ii) chemical (BPPGE-SWCNT-CoTAPc(cov)) and (iii) electrochemical adsorption (BPPGE-SWCNT-CoTAPc(ads)) processes; (c) nickel powder (BPPGE-Ni); (d) BPPGE-Ni decorated with SWCNT (BPPGE-Ni-SWCNT), and (e) SWCNT functionalised with nickel (II) tetra-aminophthalocyanine (BPPGE-SWCNT-poly-NiTAPc). Electrochemical studies (performed by voltammetric and electrochemical impedance spectroscopic techniques) revealed that the SWCNT and SWCNT-CoTAPc(mix) films showed comparable electrocatalytic responses towards the detection of DEAET and DMAET whereas competitive electrochemical behaviour was seen between SWCNT and SWCNT-NiTAPc modified BPPGEs. Using the BPPGE-SWCNT-CoTAPc(mix), the estimated catalytic rate constants (k) and diffusion coefficients (D) were higher for DEAET than for the DMAET. Also, the detection limits of approximately 8.0 and 3.0µM for DMAET and DEAET were obtained with sensitivities of 5.0×10−2 and 6.0×10−2 AM−1 for DMAET and DEAET, respectively. Unlike BPPGE-SWCNT-CoTAPc(mix) that detected the two sulfhydryls at slightly different potentials, BPPGE-SWCNT did not. The BPPGE-Ni gave enhanced Faradaic response for the redox probe ([Fe(CN)6]3−/4−) and also displayed enhanced electrocatalytic behaviour towards the detection of DMAET and DEAET with high sensitivity (~23x10−3 AM−1) and low detection limits (4.0 – 9.0 µM range). In comparison to other electrodes reported in the literature, BPPGE-Ni exhibits more promising features required for a simple, highly sensitive, fast and less expensive electrode for the detection of the hydrolysis products of V-type nerve agents in aqueous solution. The efficient response of the BPPGE-Ni is attributed to the high microscopic surface area of the nickel powder. The poor response of the BPPGE-Ni-SWCNT suggests that the nickel impurity in SWCNT did not show any detectable impact on the heterogeneous electron transfer kinetics of SWCNT. Unlike the nickel powder, SWCNT and CoTAPc-SWCNT, the NiTAPc-SWCNT hybrid did not show significant electrocatalysis towards the detection of the sulfhydryls. It is interesting, however, to observe for the first time that SWCNT induced crystallinity on the electropolymer of NiTAPc, and that such electropolymer exhibit charge-storage /-transfer properties that greatly enhance the electrochemical response of nitric oxide. / Dissertation (MSc (Chemistry))--University of Pretoria, 2008. / Chemistry / unrestricted

Epithelial tissues are composed of a sheet of adherent cells and are present in all metazoans. Their broad function is to compartmentalize tissues and enable the regulated exchange of nutrients and waste between the internal and external environments. To accomplish this function, cells require a specific organization: an apico-basal polarity that provides directionality and intercellular adhesion mediated by adherens junctions that hold cells together. How the epithelia architecture is initiated and maintained remains to be fully elucidated. Adherens junctions and the polarity proteins are functionally linked, as a loss of the main component of AJs: E-cadherin leads to a loss of apico-basal polarity, while disturbing apico-basal polarity results in a re-localization of E-Cadherin. Therefore is challenging to study either pathway in isolation.During my thesis I explored the role of Crumbs, a polarity protein, in the regulation of E-Cadherin in both AJ maturation and maintenance. During maturation of AJs in Drosophila embryo, I demonstrated for the first time by using quantitative high-resolution microscopy PALM that Crumbs regulates E-Cadherin clusters size and their homogenous distribution along the junction. In conclusion, my thesis work provides the first dissection of polarity proteins in E-Cadherin regulation apart from polarity pathways. / Epithelial tissues are composed of a sheet of adherent cells and are present in all metazoans. Their broad function is to compartmentalize tissues and enable the regulated exchange of nutrients and waste between the internal and external environments. To accomplish this function, cells require a specific organization: an apico-basal polarity that provides directionality and intercellular adhesion mediated by adherens junctions that hold cells together. How the epithelia architecture is initiated and maintained remains to be fully elucidated. Adherens junctions and the polarity proteins are functionally linked, as a loss of the main component of AJs: E-cadherin leads to a loss of apico-basal polarity, while disturbing apico-basal polarity results in a re-localization of E-Cadherin. Therefore is challenging to study either pathway in isolation.During my thesis I explored the role of Crumbs, a polarity protein, in the regulation of E-Cadherin in both AJ maturation and maintenance. During maturation of AJs in Drosophila embryo, I demonstrated for the first time by using quantitative high-resolution microscopy PALM that Crumbs regulates E-Cadherin clusters size and their homogenous distribution along the junction. In conclusion, my thesis work provides the first dissection of polarity proteins in E-Cadherin regulation apart from polarity pathways.

Literature-based reading instruction -- Writing centers -- Library corners.

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