Les membranes basales (MB) jouent un rôle majeur au cours des processus morphogénétiques. Elles et sont principalement composées de Collagène de type IV, de Perlecan et de Laminine. Les récepteurs d'adhésions/signalisations (Intégrines/Dystroglycans) localisés au pôle basal des cellules épithéliales, interagissent directement avec les MBs. De nombreuses études montrent l'importance de la composition des MBs dans le devenir cellulaire. Cependant, le rôle mécanique de la MB au cours du développement d'un organe multicouche n'est pas connu. Comme modèle de morphogenèse épithéliale, nous avons choisi d'étudier l'épithélium du follicule ovarien chez Drosophila melanogaster. La MB entoure chaque follicule ovarien qui est composé d'une monocouche de cellules épithéliales cuboïdes entourant un groupe interne formé de 16 cellules de la lignée germinale (15 cellules nourricières en postérieur et 1 ovocyte en antérieur). Au cours du développement folliculaire, les cellules épithéliales s'aplatissent suivant une vague antéro-postérieur. Cette transition cellulaire cuboïde – aplatie dépend du remodelage des jonctions d'adhérence et du cytosquelette. Mes travaux de thèse ont porté sur l'étude du rôle mécanique et moléculaire de la MB au cours de la morphogenèse épithéliale chez la Drosophile. J'ai ainsi pu montrer (i) que la rigidité de la MB augmente au fur et à mesure du développement du follicule, (ii) que l'aplatissement dépend de la structure de la MB et de la liaison a cette MB grâce aux intégrines (iii) que la MB s'assouplie lors de la transition cuboïde-squameux et que cette assouplissement dépend de ce processus. Ces résultats démontrent un dynamisme mécanique et moléculaire de la MB au cours de l'ovogenèse et de la morphogenèse, révélant le rôle central de la MB lors de ces processus. Parallèlement j'ai développé une approche par segmentation cellulaire afin d'extraire les valeurs métriques (hauteur, anisotropie, surface basale, volume) des cellules épithéliales et de mesurer les variations de ces paramètres au cours de la morphogenèse épithéliale (MARS-ALT) / Epithelial cell morphogenesis is an essential process for animal development. Epithelia are composed of polarized cells with a basal side interacting through Integrins, with a basement membrane (BM) and a lateral side containing cadherin-based junctional complexes. Integrins and Cadherins are, both, linked to actin filaments and are thus involved in cell shape regulation. While these links are well documented, it remains unclear how the components of the BM and the 3D organisation of this tissue influence epithelial cell morphogenesis. The model we are using to study this influence is the follicular epithelium in Drosophila melanogaster. It consists of a monolayer of 800 epithelial cells surrounding the egg chamber consisted of an internal cluster of 16 germline cells (15 nurse cells and one posteriorly-localized oocyte). An extracellular matrix (ECM), composed mainly of Collagen IV and Laminins, surrounds each follicle, directly secreted by follicular cells. During follicle development, the cuboidal epithelial cells become squamous around the nurse cells and columnar around the oocyte. The cuboidal-to-squamous transition depends on both Integrins (formed by the subunits aPS2/bPS) and Cadherin-based adherens junction remodelling. Here we designed an Atomic Force Microscopy (AFM) approach to investigate the elastic modulus of the ECM in a living ovarian follicle at different stages of development and particularly during epithelial cell morphogenesis. First, we found that the stiffness changes temporally during oogenesis with an increase of stiffness during Collagen IV deposition. Second, during cell morphogenesis, we observed a gradient of ECM stiffness. Third, by measuring the stiffness in mutants delaying or promoting cell flattening, we showed that the regional differences occurs in function of the cell ability to flatten. Fourth, to assess the involvement of Collagen IV or its structure for the ECM rigidity properties, we measured the stiffness of ECM produced by follicles mutant for Collagen IV or after collagenase treatment and concluded that collagen fibrils are the source of rigidity properties.Altogether, these results demonstrate the role of the regulation of the ECM stiffness for epithelial cell morphogenesis and highlight a new mechanical aspect in the comprehension of developmental processes

A two-step protocol has been devised as a rapid and selective alternative to conventional differential extraction techniques with an increased recovery of DNA. The protocol involves pressure cycling with the Barocycler® NEP 2320 from Pressure Biosciences. Inc. in alkaline conditions for epithelial cell lysis and removal. This step is followed by alkaline lysis at 95º C for extraction of sperm cell DNA. At 1:1 or 2:1 female to male cell ratios, high selectivity and complete separation can be achieved. But at higher ratios, male allelic dropout is observed. This protocol has been modified to generate a clean male profile at a 20:1 cell ratio through optimization of NaOH concentration and inclusion of an additional pressure cycling step. Validation studies have been performed to assess the efficiency of this method under various conditions. An additional immunomagnetic cell capture pretreatment allowed for nearly complete separation at cell ratios of up to 200:1.

Les peptides antimicrobiens (PAMs) sont des effecteurs de l'immunité innée et exercent leur activité microbicide sur un spectre de microorganismes. Au niveau de l'intestin, leur sécrétion est directement impliquée dans les processus homéostatiques existant entre un hôte et son microbiote. Ces dernières années, plusieurs études ont démontré une corrélation entre le niveau d'expression des PAMs et la susceptibilité des individus à différentes pathologies. Les PAMs peuvent être exprimés constitutivement ou de manière inductible. Dans les deux cas, les mécanismes de régulation (épi)génétique pour contrôler leur expression sont méconnus.Le but de ces travaux de thèse a été d'étudier la composante (épi)génétique des régulations contrôlant l'expression des gènes codant les PAMs. Utilisant un modèle de cellules épithéliales intestinales humaines exposées à des molécules inhibitrices de l'activité d'enzymes modifiant la chromatine, et stimulées par la bactérie Escherichia coli, nous avons identifié l'importance du processus d'acétylation dans l'expression des ces gènes. Nous avons montré que l'inhibition des enzymes de la famille des histones déacétylases augmente significativement le niveau d'induction des gènes antimicrobiens comme la béta-défensine-2, sans impacter celui des gènes pro-inflammatoires comme l'interleukine 8. Enfin, nous avons étudié le mécanisme moléculaire sous-jacent à cette observation, notamment le rôle du facteur de transcription NF- B et celui de l'histone acétyltransférase p300. Ces travaux démontrent l'existence d'un mécanisme (épi)génétique permettant de réguler différentiellement le niveau d'induction des gènes antimicrobiens et pro-inflammatoires. / Antimicrobial peptides (AMPs) are conserved molecules of the innate immune system and actively kill a wide variety of microorganisms. In the intestine AMPs secreted by the epithelium protect against pathogens and support homeostasis with the microbiota. Their importance becomes clear, as their expression has been correlated with susceptibility to infection and a multitude of severe pathologies. Some AMPs are expressed constitutively, while others are inducible. The regulation of inducible AMPs expression is widely undiscovered. The role of chromatin remodeling and histone modifications, as an additional regulatory level to transcription factor activation, in the expression of inducible genes is becoming more and more clear. The aim of this work was to investigate the (epi)genetic mechanisms, which are involved in the regulation of AMPs gene expression in the intestine. By the use of specific inhibitors of chromatin modifying enzymes, in an in vitro model of intestinal epithelial cells challenged with Escherichia coli strains, we discovered the importance of acetylation in the regulation of these genes. Inhibition of histone deacetylase enzymes significantly enhanced the bacteria-induced expression of the beta-defensin-2 and other AMPs, while the expression of the interleukin 8 and other inflammatory genes was not influenced. Furthermore, we detailed the molecular mechanism, especially involvement of the transcription factor NF-κB and the histone acetyltransferase p300 in this observation. This discovery presents a mechanism of (epi)genetic enhancement of AMPs expression, dissociated from the pro-inflammatory response.

O patótipo de Escherichia coli diarreiogênica denominado E. coli enteroagregativa (EAEC) é caracterizado pela expressão do padrão de adesão agregativo (AA) em células epiteliais cultivadas. Amostras de EAEC são classificadas como típicas ou atípicas, dependendo da presença ou não do gene aggR, respectivamente. O padrão AA no teste de adesão em células epiteliais é o diagnóstico padrão para a classificação de EAEC. Técnicas moleculares, como a PCR multiplex, são alternativas a esse teste. No presente estudo foi desenvolvida uma PCR multiplex para o diagnóstico molecular de EAEC típica e atípica, baseada na detecção dos genes aatA, aggR, aaiA e aaiG. A partir de colônias isoladas de E. coli de origem fecal o teste apresentou sensibilidade de 94,8%, especificidade de 94,7%, valor preditivo de teste positivo de 74,3% e valor preditivo de teste negativo de 99,1%. O teste desenvolvido mostrou-se uma alternativa diagnóstica sensível e específica para EAEC e permite um acréscimo significativo na detecção de EAEC atípica. / The diarrheagenic Escherichia coli pathotype known as enteroaggregative E. coli (EAEC) is characterized by the expression of the aggregative adherence pattern (AA) on cultured epithelial cells. EAEC strains are classified as typical or atypical, depending on the presence or absence of the aggR gene, respectively. The AA pattern in the adherence assay with epithelial cells is the gold standard diagnostic for EAEC. Molecular techniques, such as multiplex PCR, are alternatives to this test. In the present study we developed a multiplex PCR for the molecular diagnostic of typical and atypical EAEC, based on the detection of aatA, aggR, aaiA and aaiG genes. The test presented 96.5% of sensitivity, 94.8% of specificity, 74.3% of positive predictive value and 99.1% of negative predictive value, when tested in isolated colonies of E. coli from feces. The test developed is a sensitive and specific diagnostic alternative for EAEC and allows a significant increase in atypical EAEC detection.

Morphogenesis is the fundamental and remarkable biological process that produces elaborate and diverse tissues and organs from simple groups of cells, which can happen on timescales as short as minutes or as long as days. One of the biggest challenges in understanding morphogenesis is the gap between our knowledge of the molecular-scale activities of genes and proteins, and the large-scale behaviors of cells and tissues. To fill this gap, a complete understanding of both biochemical and mechanical factors involved in morphogenesis is needed. Morphogenesis is naturally a mechanical process in which tissues are physically sculpted by mechanical stress, strain, and movements of cells that are induced by these genetic and molecular programs. However, many of the mechanical factors involved in morphogenesis remain poorly understood partially due to the strong coupling of mechanical factors and biological factors, the active responses of living tissues to the environment, and the lack of experimental methods to study the mechanics of tissues in vivo. Epithelial tissues play crucial roles in shaping early embryos and are widely spread in mature animals to serve as boundaries and barriers. They are robust tissues that not only support the structure of embryos and organs, but also actively change shape and structure, displaying a fluid behavior during morphogenesis. Contractile tension and cell-cell adhesion are thought to be the main mechanical factors involved in epithelial tissue morphogenesis, but how the balance between these two determines epithelial tissue mechanics remains unclear. To build a fundamental understanding of the mechanical mechanisms underlying epithelial tissue morphogenesis, this dissertation studies the germband epithelial tissue in the early Drosophila melanogaster embryo and addresses two important open questions in the field of mechanics in morphogenesis: (1) what mechanical factors are involved in the morphogenesis of epithelial tissues; (2) how does a cell control these factors to tune tissue mechanical behaviors. In this dissertation, we developed a systematic, quantitative, in vivo experimental approach to explore mechanics of epithelial tissue morphogenesis in the Drosophila embryo by integrating molecular genetics approaches, live confocal fluorescence imaging, and quantitative image analysis. Combining our experimental studies in the Drosophila embryo with our collaborators’ theoretical modeling approaches, we showed that the shapes and alignment of cells within tissues can help us understand and predict epithelial tissue mechanical behaviors, such as tissue fluidity, during morphogenesis and how defects in these processes can result in abnormalities in embryo shape. We also observed that the Drosophila germband tissue transitions from more solid-like to more fluid-like behavior to help accommodate dramatic tissue flows during convergent extension, which indicates that the mechanical properties of developing tissues might be tuned during morphogenetic events. To elucidate molecular mechanisms underlying how tissue mechanical properties may be regulated during morphogenesis, this dissertation explores the role of cell-cell adhesion in controlling epithelial tissue mechanics. By systematically modulating cell-cell adhesion levels in the Drosophila germband tissue and combining live imaging and quantitative image analysis, we studied the effects of cell-cell adhesion levels on cellular and tissue behaviors. We found biphasic dependencies of cell rearrangements, cell shape, and tissue fluidity on cell-cell adhesion levels, which are surprisingly linked to each other by cell patterns in the tissue. In particular, tissues comprising cells with either lower or higher cell-cell adhesion levels tend to rearrange faster and show cell patterns indicating more fluid-like tissue behaviors. Further studies suggested that cell-cell adhesion works with cytoskeletal molecules to achieve these effects. The experimental approaches developed for exploring mechanics in 2-D in the Drosophila germband epithelial tissue are expanded upon in order to investigate germband tissue mechanics in 3-D. These approaches are also used to study mechanics in the inner ear round window membrane of the guinea pig for clinical application. This dissertation advances our understanding of mechanics of epithelial tissue morphogenesis in vivo and provides a practical, quantitative, and appealing platform for exploring mechanics in living tissues during morphogenesis. This helps fill the gap in our knowledge of molecular-scale activities and tissue-level behaviors, provides insight into building tissues with precise shapes and structures in the lab, and sheds light on human diseases associated with improper regulation of tissue mechanics such as birth defects, aberrant wound healing, and cancer metastasis.

Chlamydia trachomatis infections represent the predominant cause of bacterial sexually transmitted infections. As an obligate intracellular bacterium, C. trachomatis is dependent on the host cell for survival, propagation, and transmission. Thus, factors that affect the host cell, including nutrition, cell cycle, and environmental signals, have the potential to impact chlamydial development. Previous studies have demonstrated that activation of Wnt/β-catenin signaling benefits C. trachomatis infections in fallopian tube epithelia. In cervical epithelial cells chlamydiae sequester β-catenin within the inclusion. These data indicate that chlamydiae interact with the Wnt signaling pathway in both the upper and lower female genital tract (FGT). However, hormonal activation of canonical and non-canonical Wnt signaling pathways is an essential component of cyclic remodeling in another prominent area of the FGT, the endometrium. Given this information, we hypothesized that Wnt signaling would impact chlamydial infection in endometrial epithelial cells. To investigate this hypothesis, we analyzed the effect of Wnt inhibition on chlamydial inclusion development and elementary body (EB) production in two endometrial cell lines, Ishikawa (IK) and Hec-1B, in nonpolarized cell culture and in a polarized endometrial epithelial (IK)/stromal (SHT-290) cell co-culture model. Inhibition of Wnt by the small molecule inhibitor (IWP2) significantly decreased inclusion size in IK and IK/SHT-290 cultures (p < 0.005) and chlamydial infectivity (p ≤ 0.01) in both IK and Hec-1B cells. Confocal and electron microscopy analysis of chlamydial inclusions revealed that Wnt inhibition caused chlamydiae to become aberrant in morphology. EB formation was also impaired in IK, Hec-1B and IK/SHT-290 cultures regardless of whether Wnt inhibition occurred throughout, in the middle (24 hpi) or late (36 hpi) during the development cycle. Overall, these data lead us to conclude that Wnt signaling in the endometrium is a key host pathway for the proper development of C. trachomatis.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19592号 / 医博第4099号 / 新制||医||1014(附属図書館) / 32628 / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 伊達 洋至, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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