La taille du fruit et la morphologie des tissus du fruit sont des caractères clés définissant la qualité finale du fruit. Parmi la grande diversité de fruits observée dans la nature, la domestication et la sélection ont entrainé d’importantes modifications de la taille et de la morphologie des tissus du fruit. Jusqu'à présent, seuls quelques régulateurs génétiques ont été identifiés, et les mécanismes cellulaires et moléculaires par lesquels la morphologie des tissus du fruit est définie restent imprécis. Dans ce contexte, l'objectif de ma thèse est d'identifier et de caractériser de nouveaux régulateurs impliqués dans la morphologie des tissus du fruit. Pour cela, j'ai utilisé une collection de mutants EMS de tomate comme source de diversité génétique et phénotypique et j'ai sélectionné deux mutants présentant des tendances opposées et extrêmes d'épaisseur du péricarpe. Grace à une stratégie de cartographie par séquençage, j’ai identifié une région génétique du chromosome 10, associée au phénotype péricarpe épais. J'ai également étudié le rôle de la Guanylate Binding Protein (GBP) à l’origine du phénotype péricarpe fin chez la tomate. La GBP est une grosse GTP binding protein qui n’a jamais été caractérisée chez les plantes. Afin d'approfondir l’étude de cette protéine, j'ai étudié en parallèle son rôle dans les modèles tomate et Arabidopsis" thaliana. J'ai démontré que les deux protéines homologues sont localisées dans le noyau. La mutation de la GBP chez la tomate induit de fortes altérations de la division et de l'expansion cellulaire à l'intérieur du péricarpe ainsi qu'une altération de la croissance des racines latérales chez la tomate et Arabidopsis, une caractéristique classiquement retrouvée chez les mutants altérés dans la mitose. Cette étude suggère que le GBP joue un rôle dans le contrôle précis des divisions cellulaires dans le péricarpe de tomate. / Fruit size and morphology are key characters defining the final fruit quality. Among the large fruit diversity observed in the nature, human domestication and selection has induced changes in fruit size and tissue morphology. Only a few genetic regulators have been identified so far, thus cellular and molecular mechanisms by which fruit tissue morphology is defined remain incomplete. In this context, the aim of my thesis is to identify and characterize new regulators of fruit tissue morphology. For this purpose, I used a collection of tomato EMS mutants as a source of genetic and phenotypic diversity. I selected two mutants presenting opposite trends of pericarp thickness. Through a mapping-by-sequencing strategy, I identified a genetic region on chromosome 10, associated with an extreme thick pericarp phenotype. I also investigated the role of the Guanylate Binding Protein (GBP) at the origin of a thin pericarp phenotype. The GBP is a large GTP binding protein that was never characterized in plants so far. In order to go deeper into its functional characterization in plants, I studied in parallel the role of the protein in tomato and Arabidopsis "thaliana" models. I showed that both homolog proteins are localized at the nucleus. Mutation of GBP in tomato induced strong alterations in cell division and cell expansion inside the pericarp and altered lateral root growth in tomato and Arabidopsis, a classical feature for mutants impaired in mitosis. This study suggests a role for the GBP in the fine control of cell division in the tomato pericarp.

The aortic valve functions in a complex mechanical environment which leads to force dependent cellular and tissue responses. Characterization of these responses provides a fundamental understanding of valve pathogenesis. The aim of this work was to develop an ex vivo organ culture system capable of simulating physiological aortic pressures and flow rates, and study the biological characteristics of native porcine aortic valves cultured in the system. Collagen, sGAG and elastin content of the valve leaflets were measured and cusp morphology, cell phenotype, cell proliferation and apoptosis were examined. Presence of endothelial cells (ECs) on the leaflet surface was also evaluated. The differences in collagen, sGAG and elastin contents were not significant (p greater than0.05) between the cultured and fresh valve leaflets. The cultured valves maintained the structural integrity of the leaflets while preserving the native morphology and cell phenotype. Cell phenotype in leaflets incubated statically under atmospheric conditions decreased compared to fresh and cultured valve leaflets, indicating the importance of mechanical forces in maintaining the natural biology of the valve leaflets. ECs were retained on the surfaces of cultured leaflets with no remodeling of the leaflets. The number of apoptotic cells in the cultured leaflets was significantly (p less than 0.05) less than in the statically incubated leaflets and comparable to fresh leaflets. The sterile ex vivo organ culture system thus maintained the viability and native biological characteristics of the aortic valves that were cultured under dynamic conditions for a period of 48 hours.

<p>In 2011, an estimated 230,480 new cases of invasive breast cancer were diagnosed among women, as well as an estimated 57,650 additional cases of in situ breast cancer [1]. Breast conserving surgery (BCS) is a recommended surgical choice for women with early stage breast cancer (stages 0, I, II) and for those with Stage II-III disease who undergo successful neo-adjuvant treatment to reduce their tumor burden [2, 3]. Cancer within 2mm of a margin following BCS increases the risk of local recurrence and mortality [4-6]. Margin assessment presents an unmet clinical need. Breast tissue is markedly heterogeneous which makes identifying cancer foci within benign tissue challenging. Optical spectroscopy can provide surgeons with intra-operative diagnostic tools. Here, ex-vivo breast tissue is evaluated to determine which sources of optical contrast have the potential to detect malignancy at the margins in women of differing breast composition. Then, H&E images of ex-vivo breast tissue sites are quantified to further deconstruct the relationship between optical scattering and the underlying tissue morphology. </p><p>Diffuse reflectance spectra were measured from benign and malignant sites from the margins of lumpectomy specimens. Benign and malignant sites were compared and then stratified by tissue type and depth. The median and median absolute deviance (MAD) was calculated for each category. The frequencies of the benign tissue types were separated by menopausal status and compared to the corresponding optical properties. </p><p>H&E images were then taken of the malignant and benign sites and quantified to describe the % adipose, % collagen and % glands. Adipose sites, images at 10x, were predominantly fatty and quantified according to adipocyte morphology. H&E-stained adipose tissue sections were analyzed with an automated image processing algorithm to extract average cell area and cell density. Non-adipose sites were imaged with a 2.5x objective. Grids of 200µm boxes corresponding to the 3mm x 2mm area were overlaid on each non-adipose image. The non-adipose images were classified as the following: adipose and collagen (fibroadipose); collagen and glands (fibroglandular); adipose, collagen and glands (mixed); and malignant sites. Correlations between <&mus&#8242;> and % collagen in were determined in benign sites. Age, BMI, and MBD were then correlated to <&mus&#8242;> in the adipose and non-adipose sites. Variability in <&mus&#8242;> was determined to be related to collagen and not adipose content. In order to further investigate this relationship, the importance of age, BMI and MBD was analyzed after adjusting for the % collagen. Lastly, the relationship between % collagen and % glands was analyzed to determine the relative contributions of % collagen and % glands <&mus&#8242;>. Statistics were calculated using Wilcoxon rank-sum tests, Pearson correlation coefficients and linear fits in R. </p><p> The diagnostic ability of the optical parameters was linked to the distance of tumor from the margin as well as menopausal status. [THb] showed statistical differences from <&mus&#8242;> between malignant (<&mus&#8242;>: 8.96cm-1±2.24MAD, [THb]: 42.70&muM±29.31MAD) compared to benign sites (<&mus&#8242;>: 7.29cm-1±2.15MAD, [THb]: 32.09&muM±16.73MAD) (p<0.05). Fibroglandular (FG) sites exhibited increased <&mus&#8242;> while adipose sites showed increased [&beta-carotene] within benign tissues. Scattering differentiated between ductal carcinoma in situ (DCIS) (9.46cm-1±1.06MAD) and invasive ductal carcinoma (IDC) (8.00cm-1±1.81MAD), versus adipose sites (6.50cm-1±1.95MAD). [&beta-carotene] showed marginal differences between DCIS (19.00&muM±6.93MAD, and FG (15.30&muM±5.64MAD). [THb] exhibited statistical differences between positive sites (92.57&muM±18.46MAD) and FG (34.12&muM±22.77MAD), FA (28.63&muM±14.19MAD), and A (30.36&muM±14.86MAD). Due to decreased fibrous content and increased adipose content, benign sites in post-menopausal patients exhibited lower <&mus&#8242;>, but higher [&beta-carotene] than pre-menopausal patients.</p><p>Further deconstructing the relationship between optical scattering and tissue morphology resulted in a positive relationship between <&mus&#8242;> and % collagen (r=0.28, p=0.00034). Increased variability was observed in sites with a higher percentage of collagen. In adipose tissues MBD was negatively correlated with age (r=-0.19, p=0.006), BMI (r=-0.33, p=2.3e-6) and average cell area (r=-0.15, p=0.032) but positively related to the log of the average cell density (r=0.17, p=0.12). In addition, BMI was positively correlated to average cell area (r=0.31, p=1.2e-5) and negatively related to log of the cell density (r=-0.28, p=7.6e-5). In non-adipose sites, age was negatively correlated to <&mus&#8242;> in benign (r=-0.32, p=4.7e-5) and malignant (r=-0.32, p=1.4e-5) sites and this correlation varied significantly by the collagen level (r=-0.40 vs. -0.13). BMI was negatively correlated to <&mus&#8242;> in benign (r=-0.32, p=4e-5) and malignant (r=-0.31, p=2.8e-5) sites but this relationship did not vary by collagen level. MBD was positively correlated to <&mus&#8242;> in benign (r=0.22, p=0.01) and malignant (r=0.21, p=4.6e-3) sites. Optical scattering was shown to be tied to patient demographics. Lastly, the analysis of collagen vs. glands was narrowed to investigate sites with glands between 0-40% (the dynamic range of the data), the linear model reflected an equivalent relationship to scattering from % glands and the % collagen in benign sites (r=0.18 vs. r=0.17). In addition, the malignant sites showed a stronger positive relationship (r=0.64, p=0.005) to <&mus&#8242;> compared to the benign sites (r=0.52, p=0.03).</p><p>The data indicate that the ability of an optical parameter to differentiate benign from malignant breast tissues is dictated by patient demographics. Scattering differentiated between malignant and adipose sites and would be most effective in post-menopausal women. [&beta-carotene] or [THb] may be more applicable in pre-menopausal women to differentiate malignant from fibrous sites. Patient demographics are therefore an important component to incorporate into optical characterization of breast specimens. Through the subsequent stepwise analysis of tissue morphology, <&mus&#8242;> was positively correlated to collagen and negatively correlated to age and BMI. Increased variability of <&mus&#8242;> with collagen level was not dependent on the adipose contribution. A stronger correlation between age and <&mus&#8242;> was seen in high collagen sites compared to low collagen sites. Contributions from collagen and glands to <&mus&#8242;> were independent and equivalent in benign sites; glands showed a stronger correlation to <&mus&#8242;> in malignant sites than collagen. This information will help develop improved scattering models and additional technologies from separating fibroglandular sites from malignant sites and ultimately improve margin assessment.</p> / Dissertation

Correct nuclear position is crucial for cellular function. The cytoskeletal mechanisms of nuclear positioning have been studied intensely in cultured cells. However, it is less clear if and how tissue morphology can influence nuclear positioning in developing tissues. To address this question, this thesis compares nuclear migration in straight and curved neuroepithelia of the developing zebrafish. Neuroepithelial nuclei occupy different apicobasal positions in interphase but migrate to the apical surface before mitosis, a process essential for epithelial development. While apical migration in the straight hindbrain and the curved retina depends on actomyosin, it is unclear how the necessary forces are generated and if tissue morphology influences the force generation mechanisms. Remarkably, this study demonstrates that in neuroepithelia of different shape nuclei move with distinct kinetics and undergo distinct deformations. Such differences are explained by the action of disparate forces that propel hindbrain and retinal nuclei. In agreement with this conclusion, hindbrain and retinal cells display distinct actomyosin distribution and regulation during nuclear migration. Apical movement is shown to depend on Rho-ROCK activity in the hindbrain and formin activity in the retina. Therefore, hindbrain and retinal cells employ distinct actin-dependent mechanisms of nuclear positioning. Comparison of nuclear movements in another pair of straight and curved neuroepithelia shows that in tissues with similar morphology nuclei have conserved modes of apical migration. The different mechanisms of apical migration used in tissues of different shape argue that tissue morphology can indeed influence the mechanism of nuclear positioning. The findings in this thesis suggest that different mechanisms arise due to differences in actin arrangements during development of tissues with distinct curvature. Furthermore, they emphasize the importance of developmental context, tissue and cell morphology for the execution of intracellular processes.:1. INTRODUCTION 1.1. The cytoskeleton is a versatile tool to perform a variety of cellular functions 1.1.1. Introduction to microtubules and actomyosin 1.1.2. Functions of the cytoskeleton 1.1.3. The cytoskeleton and intracellular transport 1.2. The cytoskeleton in nuclear positioning 1.2.1. The nucleus can be coupled to the cytoskeleton 1.2.2. Mechanisms of nuclear positioning by microtubules and actin 1.2.3. Nuclear positioning in the pseudostratified epithelium 1.3. Objective of the study 2. MATERIALS AND METHODS 2.1. Zebrafish methods 2.1.1. Zebrafish husbandry 2.1.2. RNA and DNA injections 2.1.3. Cloning strategies 2.1.4. List of constructs 2.1.5. Heat shock of embryos 2.1.6. Drug treatments 2.1.7. Immunofluorescence 2.2. Image acquisition 2.2.1. Confocal scans 2.2.2. Time-lapse imaging using spinning disk confocal microscope (SDCM) 2.2.3. Time-lapse imaging using light-sheet fluorescent microscope (LSFM) 2.3. Laser ablations 2.3.1. PSE laser ablations 2.3.2. Nuclear laser ablations 2.4. Image analysis 2.4.1. Sample drift correction 2.4.2. Actin, myosin, and nuclear intensity distribution 2.4.3. Nuclear segmentation, shape measurements, and tracking in 3D 2.4.4. Analysis of the kinetics of apical nuclear migration 2.4.5. Tissue and cell shape measurements 3. RESULTS 3.1. Characterization of apical nuclear migration in zebrafish neuroepithelia 3.1.1. The overall duration of G2 and apical migration differ in hindbrain and retina 3.1.2. Hindbrain and retinal nuclei move with distinct kinetics during apical migration 3.2. Nuclear deformations can be used to study the forces experienced by the organelle 3.2.1. The absence of lamin A/C is likely to enable nuclear deformations 3.2.2. Neuroepithelial nuclei can respond to applied forces by deformation 3.2.3. Retinal nuclei deform more strongly during apical migration 3.2.4. Deformation of ablated regions in the nucleus suggests that retinal nuclei are pushed to the apical side 3.3. Apical nuclear migration depends on actomyosin with distinct distribution in hindbrain and retina 3.3.1. Apical nuclear migration in the hindbrain depends on actin and not on microtubules 3.3.2. Actin and myosin are locally enriched basally of the nucleus in retinal cells but not in hindbrain G2 cells 3.4. Apical nuclear migration is regulated differently in hindbrain and retinal cells 3.4.1. Initial screening for possible actomyosin regulators of apical nuclear migration 3.4.2. Apical nuclear migration is controlled by different actomyosin regulators in hindbrain and retinal cells 3.5. Cells of neuroepithelia with distinct curvature use different mechanisms of apical migration 3.5.1. Tissue-wide contractile actomyosin that is enriched basally in the retina, is absent in the hindbrain 3.5.2. Tissue curvature and cell shape differ between hindbrain and retina 3.5.3. Characterization of the straight and the curved regions of the MHB 3.5.4. Nuclei in straight and curved neuroepithelia move with distinct kinetics 3.5.5. Nuclear shape changes during apical migration are stronger in curved compared to straight neuroepithelia 3.5.6. Basal cytoplasmic actomyosin follows the nucleus in cells of curved neuroepithelia 4. DISCUSSION 4.1. The deformability of neuroepithelial nuclei as a prerequisite for migration in the crowded PSE 4.2. Possible mechanisms of apical nuclear migration 4.2.1. Cortical flow-dependent mechanism in the hindbrain 4.2.2. Basal pushing mechanism in the retina 4.2.2.a. Pushing of the nucleus by a cytoplasmic flow 4.2.2.b. Pushing of the nucleus by an expanding actin network 4.2.2.c. Possible roles of myosin in a basal pushing mechanism 4.3. The adaptability of the cytoskeleton ensures robust apical nuclear migration 4.3.1. Cytoskeleton adaptability ensures the robustness of apical nuclear migration 4.3.2. Adaptation of the actomyosin cytoskeleton to different tissue curvature 5. OUTLOOK LIST OF ABBREVIATIONS LIST OF TABLES LIST OF FIGURES MOVIE LEGENDS REFERENCES APPENDIX