Thesis (doctoral)--Université de Clermont-Ferrand II, 1983. / Errata slip inserted. Includes bibliographical references (p. 241-263).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 14, 2010). Includes bibliographical references.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 59 pages. Includes Vita. Includes bibliographical references.

Intellectual disability (ID) is a common phenotype of brain-development disorders and is heterogeneous in etiology with numerous genetic causes. PAK3 is one gene with multiple mutations causing ID. Affected individuals have microcephaly, and other brain-structure defects have been reported. Additionally, PAK3 is in a genetic network with eighteen other genes whose mutations cause ID, suggesting the molecular mechanisms by which PAK3 regulates of cognitive function may be shared by other genetic ID disorders. Studies in rodent models have shown that the orthologs of PAK3 are important for regulating dendrite spine morphology and postnatal brain size. In Drosophila melanogaster, the morphological processes of oogenesis, dorsal closure during embryogenesis, and salivary gland-lumen formation require Pak, the Drosophila ortholog of PAK3. Additionally, Pak is important for development of the subsynaptic reticulum of the neuromuscular junction, sensory axon pathfinding and terminal arborization in the Drosophila central nervous system (CNS). However, the role of Pak in mushroom body (MB) structure and intrinsic neurite arbor morphogenesis, as well as details of the underlying cellular and molecular mechanisms are unknown. To address this gap, I used Drosophila models of PAK3 gene mutations, Pak, and a combination of immunostaining, primary cell culture, and genetic interaction studies to elucidate these mechanisms. I performed a detailed characterization of the previously reported adult Pak phenotypes of decreased survival as well as leg and wing morphology. I found that decreased survival is a low-penetrance phenotype that is enhanced by chromosomes from the same mutagenesis. Defects of the adult wing include folding and misalignment between the layers, blisters, and missing or partial cross veins. The Pak-mutant legs are short and often misdirected in the pupal case with morphological defects in the shape of the leg segments themselves. The mushroom bodies are important insect learning and memory brain structures whose lobes are composed of axon bundles with individual axons bifurcating to form the α and β lobes. Mutations in Pak cause defects in the length, thickness, and direction of the MB α and β lobes. These defects increase in severity during metamorphosis, when neurogenesis and differentiation of these structures occur, suggesting that Pak stabilizes the branches of the α/β mushroom body neurons. Pak-mutant cultured neurons have reduced neurite arbor size with defects in neurite caliber. Initial outgrowth was normal, followed by a decrease in neurite branch number, again supporting the role of Pak in neurite-branch stability. There are defects in the cytoskeleton in growth cones at six hours post-plating as well as in neurons after three days in vitro. The Pak-mutant phenotype severity depends on the phosphorylation status of myosin regulatory light chain, supporting the mechanistic hypothesis that Pak regulates neurite-branch stability by inhibiting myosin light chain kinase. The neuronal phenotype of decreased branch stability suggests a mechanism of excessive retraction as the cellular pathogenesis underlying PAK3 mutation-associated brain disorders. I used western blotting to characterize the protein products of four nonsense mutations in Drosophila Pak to interpret genotype-phenotype relationships. Each allele has molecularly unique consequences: Pak¹¹, stop-codon read through and truncated protein; Pak¹⁶, no read through, but truncated protein; Pak⁶, read through with no truncated protein; Pak ¹⁴, neither readthrough nor truncated protein. Truncated proteins produced by Pak¹¹ and Pak¹⁶ alleles retained partial function for survival, wing blistering, leg morphology, and neurite length. Conversely, truncated protein increased the severity of the mushroom body defects. Truncated proteins have no effect on neuron branch number, wing folding, or vein defects. Together, these results demonstrate a role of Pak in regulating epithelial morphology, brain structure, and neurite arbor size and complexity. These closely resemble features of the human disorder, providing evidence that this is a good genetic model for this cause of ID.

No description available.

The monoclonal antibody Sp12 recognizes an epitope shared by a diverse group of developmentally regulated cell surface and extracellular matrix antigens which are expressed during Strongylocentrotus purpuratus development. Immunofluorescent localization reveals antigen in the cortical granules of eggs and in the hyaline layer from fertilization through to gastrula. Primary (skeletogenic) mesenchyme and two secondary mesenchyme derivatives (blastocoelar cells and pigment cells) express antigen after their release into the blastocoel and maintain it throughout the remainder of larval development. The antibody allows detailed descriptions of blastocoelar cells, a prominent yet poorly described fibroblast-like mesenchyme lineage. In adults antigen is localized to the organic matrix which invests the calcified stereom of the test and spines. Immunogold electron microscopy shows antigen in the cortical granules of eggs, and on cell surfaces, within membrane bound vesicles, and within the Golgi apparatus of mesenchyme cells. On western blots a confluent smear of antigen (Mr primarily >$180K) is present in eggs, but is resolved into seven antigen bands (Mr from 35K to >$200K) after fertilization. A prominent antigen at 140K is newly expressed coincident with mesenchyme immunoreactivity. Antigens at 140K and 120K fade as prisms develop into plutei, while an antigen at 105K appears in older larvae. In adult test six antigens are shared with larvae, and there are two novel antigens at 75K and 110K. Immunoreactivity is eliminated by digestion of samples with endoglycosidase F, is reduced by periodate oxidation, but is unaffected by boiling. Calcium-magnesium-free-seawater or 1M glycine extract a subset of antigens from dissociating embryos, leaving a complementary subset of antigens associated with cell membranes. Membrane antigens are not extracted, at 4 C, with high or low ionic strength, organic solvents, or non-ionic detergents but are solubilized by ionic detergents. Whole antibody has no effect on development of embryos cultured in it from fertilization on, nor is there a specific effect from injection of antibody into the blastocoel of developing embryos. The shape of dissociated cells cultured in Sp12 whole antibody is markedly constrained compared to that of controls, however, this difference is not seen in cells incubated in Sp12 Fab fragments. Sp12 appears to recognize a carbohydrate moiety shared by ten glycoproteins of widely variable Mr and cell membrane affinity which are differentially expressed on mesenchyme throughout S. purpuratus development. The antibody does not disrupt development in vivo, but does affect cell shape in vitro, probably by crosslinking cell surface antigens. / Graduate

The cardiac conduction system is responsible for maintaining and orchestrating the rhythmic contractions of the heart. Results from lineage tracing studies indicate that precursor cells in the ventricles give rise to both cardiac muscle and conduction cells. Using chick embryonic hearts, we have found that Notch signaling plays an important role in the differentiation of cardiac muscle and conduction cell lineages in the ventricles. Notch1 expression coincides with a conduction marker at early stages of conduction system development. Mis-expression of constitutively active Notch1 (NIC) in early heart tubes exhibited multiple effects on cardiac cell differentiation. Cells expressing NIC had a significant decrease in the expression of cardiac muscle markers, but an increase in the expression of conduction cell markers. Loss-of-function studies further support that Notch1 signaling is important for the differentiation of these cardiac cell types. Functional electrophysiology studies show that the expression of constitutively active Notch1 resulted in abnormalities in ventricular conduction pathway patterns. During cardiogenesis, groups of myocardial cells become separated from each other, and migrate to form the trabeculae. These finger-like projections found within the ventricular chamber coalesce to generate the muscular portions of the interventricular septum, the thickened myocardium, and future sites of the conduction system. We have found that Notch signaling regulates the migration of cardiac cells during cardiogenesis. Over-expression of constitutively active Notch causes cells to localize more centrally within the heart, while loss-of-Notch function results in cells distributed within the periphery of the heart. Staining of heart sections shows that Notch signaling regulates the expression of N-cadherin, the predominant adhesion molecule in cardiomyocytes. We find that the effects of Notch on cell migration are two-fold: delamination and cell motility. Time-lapse studies demonstrate that Notch signaling increases cell motility, but does not affect speed or directionality of migration. Furthermore, we find that the effects of Notch on cell migration is independent of its effects on differentiation.

L’élongation épithéliale joue un rôle prépondérant au cours du développement. Ce processus morphogénétique peut être décrit schématiquement par trois paramètres : le signal permettant d’orienter l’élongation, la force capable de générer le mouvement et le comportement cellulaire associé. Le follicule ovarien de drosophile, initialement rond s’allonge au cours de son développement résultant en un œuf 2.5 fois plus long que large. J’ai mis en évidence que les follicules traversent au moins trois phases d’élongation successives et je me suis focalisé sur la phase précoce dont les paramètres étaient inconnus. J’ai déterminé que le signal venait d’un groupe de cellules situées aux pôles par la sécrétion du ligand de la voie Jak-Stat puis de l’activation consécutive de cette voie selon un gradient depuis les pôles. La force est apportée par un gradient de pulsations apicales des cellules dépendantes de la voie Jak-Stat générant des forces de traction aux extrémités des follicules. L’élongation est stabilisée par des intercalations cellulaires orientées selon l’axe d’élongation et par un gradient de constriction apicale des cellules. Ces données permettent de proposer un mécanisme où un gradient d’activité d’un facteur de transcription induit une élongation épithéliale indépendamment d’une polarité planaire. / Tissue elongation plays a key role during development. Schematically, this morphogenetic process can be described by three main parameters: the cue orienting the elongation, the movement generating force and the associated cellular behavior. The Drosophila ovarian follicle, initially spherical, elongates as it develops, ending as a 2.5 time longer than wide mature egg. I found that follicles elongate through at least three consecutive phases and I aimed to determine the parameters of the early phase. The signal comes from a cluster of cells located at each pole of the follicle secreting the Jak-Stat pathway ligand and the subsequent activation of the pathway as a gradient from the poles. A pulling force is generated by the Jak-Stat dependent apical pulsations of the cells following the same gradient. The elongation is stabilized by oriented cell intercalations along the elongation axis and a gradient of apical constriction. Our data allow proposing a mechanism where a gradient of transcription factor activity can lead to epithelial elongation without any planar polarity requirement.

La régénération de la nageoire caudale et de la peau chez le poisson zèbre impliquent la formation d’un tissu de remplacement, structurellement et fonctionnellement identique à celui précédant la lésion. Ces mécanismes nécessitent la mise en place et la prolifération de cellules progénitrices, capables de reformer lors d’une phase de « patterning » les tissus lésés.Au cours de ma thèse j’ai étudié les évènements prolifératifs qui permettent cette néo-formation des tissus. Dans le cas de la nageoire, mes travaux ont conduit à la mise en évidence d’un mécanisme commun entre régénération et développement de croissance saltatoire des segments osseux. Ils expliquent en partie le retour à la forme bilobée observée lors de la repousse. De la même façon, dans le cas de la peau, l’avènement de la prolifération dans le derme et dans l’épiderme précède la mise en place des signaux, communs au développement, requis pour la distribution et la formation des écailles.De plus, j’ai effectué une caractérisation précise de l’ensemble du processus cicatriciel chez le poisson zèbre, conduisant à la formation d’une peau intégralement régénérée. Au cours de la cicatrisation, la fermeture de la blessure est complétée rapidement en quelques heures par migration épidermique. Une fois la zone lésée fermée, un mécanisme de morphogénèse de la peau est réactivé chez l’adulte. La prolifération cellulaire est présente simultanément dans le derme et l’épiderme. Elle n’est déclenchée qu’après la mise en place de l’assise basale de l’épiderme. Dans celui-ci, elle affecte d’une part des cellules éparses au sein de la couche basale et d’autre part la majorité des cellules de la couche intermédiaire. Ces derniers travaux suggèrent que chez les téléostéens, les cellules souches épidermiques sont localisées dans la couche basale, alors que la prolifération des cellules transientes a lieu dans la couche intermédiaire. D’autre part, ils mettent en évidence un processus commun de cicatrisation rapide en milieu liquide impliquant une fermeture de la blessure par néo-épithélialisation, semblable au cas de la cornée et de la muqueuse orale chez les mammifères. / Caudal fin and skin regeneration in zebrafish both require new tissues formation, structurally and functionally identical to the former ones. They imply the formation and especially the proliferation of progenitor cells, and then during a patterning phase, they differentiate into a well-organized structure.During my PhD, I have studied in zebrafish model the proliferative events that conduct to the neoformation of caudal fin in one hand and the proliferative events implicated in the cutaneous wound healing in the other hand.The first part of this work supports the evidences of a common saltatory growth mechanism in both regeneration and development of caudal fin bony rays, and that the bi-lobed shape restoration of the fin could be a consequence of this bony segment salutary growth.During skin wound healing, proliferation is necessary in order to allow dermis and epidermis neo-morphogenesis and these events are over before the scale formation is initiated. In the second part of this study I characterized the entire skin wound healing process in the zebrafish model, from the wound closure to a fully regenerated skin including appendages. According to my results, the wound closure is a very fast event completed only in a few hours and it occurs only by epidermal cells migration. Cellular proliferation was detected after complete wound closure and once the epidermal basal layer differentiation is achieved. Cell proliferation appears to be restricted to a few basal cells whereas the major proliferation is detected in the intermediates layers of the epidermis.Taken together, these results suggest that in teleosteans, the epidermal stem cells and transient cells might be respectively located in the basal and intermediate layers. Moreover, there might be a common process due to aquatic environment, leading to a fast wound closure by re-epithelialization, between teleost skin and mammal’s cornea as well as oral mucosa.

Orientador: Hernandes Faustino de Carvalho / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T10:03:30Z (GMT). No. of bitstreams: 1 Cardoso_AlexandreBruni_D.pdf: 5087353 bytes, checksum: c88d322680c216b0a0f0c99aef894042 (MD5) Previous issue date: 2010 / Resumo: Importante glândula acessória do trato reprodutor de mamíferos, a próstata é um órgão alvo de várias doenças benignas e malignas, que ocorrem principalmente com o envelhecimento. Tanto o desenvolvimento prostático pós-natal como a regressão da glândula após a ablação hormonal são caracterizados por intensa modificação no comportamento das células e remodelação da matriz extracelular (MEC). As metaloproteinases de matriz (MMP) constituem uma família de enzimas que degradam principalmente componentes de MEC. Portanto, pareceu-nos plausível que as MMPs tenham papel crucial na remodelação tecidual que ocorre em decorrência dos eventos morfogenéticos da próstata ventral (PV) e na progressiva regressão prostática pós-castração. Assim, o objetivo desse trabalho foi investigar o papel da MMP-2 no desenvolvimento prostático pós-natal em roedores e das MMP-2, -7 e - 9 na regressão da PV de ratos pós-castração. Para isso, foram empregadas técnicas moleculares, bioquímicas e análises morfológicas. A aplicação do siRNA específico para MMP-2 comprometeu o crescimento, a ramificação, a formação de lúmen e a proliferação de células epiteliais da PV de ratos in vitro, além de provocar um acúmulo de fibras colagênicas no compartimento estromal. A PV do camundongo MMP-2-/- adultos apresentou peso relativo reduzido e um menor volume epitelial, que resultaram de menor proliferação epitelial, menor ramificação ductal e maior estabilização da matriz colagênica durante a primeira semana de desenvolvimento pós-natal. Na regressão da próstata ventral de ratos após a castração encontraram-se múltiplas ondas de morte celular e uma relação direta entre a expressão e atividade das MMP-2, -7 e -9 e o pico de apoptose que ocorre 11 dias após a castração. Conclui-se através dos resultados apresentados neste trabalho, que tanto o desenvolvimento prostático pós-natal, como a regressão prostática pós-castração são dependentes da expressão e atividade das MMPs. / Abstract: The prostate is an important gland of the reproductive tract of mammals, which is a target of several benign and malign diseases affecting the elder. Both postnatal prostate development and prostate regression after androgenic ablation are characterized by intense modification in cell behavior and remodeling of extracellular matrix (ECM). MMPs constitute a family of endopeptidases which are able to cleave preferentially ECM components. Thus, it seems reasonable that these enzymes play a crucial role in tissue remodeling that happens during the ventral prostate (VP) morphogenesis and in the prostate regression after castration. In this study, we aimed to define the involvment of MMP-2 in the postnatal prostate development of rodent and the involvement of MMP-2, -7 and -9 rat ventral prostate regression after castration. For this aim, we have used molecular, biochemical and morphological approaches. siRNA specific for MMP-2 compromised the rat VP growth, branching, lumen formation and epithelial cell proliferation, besides leading an accumulation of collagen fibers in the stroma. MMP-2-/- VP showed a reduced relative weight and epithelial volume, besides displaying a decreased epithelial proliferation and branching and a stabilization of collagen matrix at the end of the first postnatal week. In the prostate regression after castration, we found multiple waves of cell death and a direct association between activity and expression of MMP-2, -7 and -9 and an apoptotic peak that occurs at the 11th Day after castration. In conclusion, the results presented here showed that both postnatal prostate development and prostate regression after castration are dependent on the expression and activity of MMPs. / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural