<p>  </p> <p>Congenital diseases of the kidney are the leading cause of chronic kidney disease in pediatric patients. Tissue engineering models used to investigate these diseases are limited by an immature phenotype. Models cultured in an extracellular matrix (ECM), a network of proteins and glycosaminoglycans surrounding cells and providing structural support that mimic the matrix found in development will be likely more mature. However, developing kidney ECM composition and structural dynamics are unknown. To address this gap, we studied ECM composition using mass spectrometry and organization by visualizing the ECM in 3D.</p> <p>In this work, we used mass spectrometry to resolve ECM basement membrane and interstitial matrix dynamics between embryonic, perinatal, and adult kidneys. Surprisingly, we observed a transient upregulation of interstitial matrix structures that corresponded to dynamic 3D structures in the cortex (vertical fibers) and at the corticomedullary junction (medullary ray sheath fibers). Notably, in a model of abnormal <em>Foxd1</em>+ stromal cells, the vertical fibers were disorganized, and medullary ray sheath fibers were no longer associated with blood vessels, suggesting the dynamic 3D structures depended on stromal cell modulation.</p> <p>One of the effects of abnormal kidney development is decreased amniotic fluid, which limits embryonic movement and subsequent limb development. In additional studies, we looked at the implications of the lost motility in the muscular dysgenesis (<em>mdg</em>) mouse on the development of the myotendinous junction (MTJ). The MTJ links contractile muscle with tendon. We found the ECM protein COL22A1 was specific to the developing MTJ as early as embryonic day (E)13.5. The development of the MTJ from a linear structure to a cap-like structure with invaginations in adolescent mice depended on muscle contraction. Furthermore, we used a model to decouple the muscle-tendon-bone complex at an ectopic lateral triceps insertion (<em>Prrx1Cretg/+; Tbx3fl/fl</em>). We observed disorganized tendon and MTJ markers at the termination of the ectopic lateral triceps muscle but negligible cartilage markers. Together, this indicated MTJ maturation depended on motility but not on the enthesis.</p> <p>The information gleaned from our studies on how stromal cells affect dynamic 3D interstitial ECM structures and composition change during kidney development can be used as a template for 3D kidney culture systems. Combined with forelimb MTJ development, our results indicate the importance of the interstitial matrix in tissue morphogenesis.</p>

La maladie de Wilson est une atteinte génétique rare associée à des mutations du gène codant pour l’ATP7B, protéine de transport et d’excrétion du cuivre dans l’organisme, entrainant une accumulation toxique de cuivre dans l’organisme au niveau du foie et du cerveau. La difficulté d’établir une corrélation génotype-phénotype et la grande hétérogénéité du tableau clinique conduisent à des difficultés de prise en charge, notamment concernant le diagnostic et le suivi biologique et thérapeutique des patients. Dans cette étude, nous avons orienté nos recherches vers la découverte et l’évaluation de candidats biomarqueurs de diagnostics et de pronostics précoce de l’évolution vers des formes neurologiques de la maladie. L’accessibilité au modèle préclinique murin Atp7b-/- nous a permis d’engager une étude préclinique permettant dans un premier temps d’optimiser la partie expérimentale pour l’identification la plus fiable de nouveaux candidats biomarqueurs plasmatiques. Cette étude a permis l’identification d’un panel de 7 candidats biomarqueurs. Ces résultats nous ont permis de susciter l’intérêt des équipes médicales du Centre National de Référence Wilson (CNR) à Lyon et à Paris et d’engager une étroite collaboration pour débuter l’étude clinique. L’obtention d’une première cohorte d’échantillons plasmatiques provenant de patients atteints de la maladie de Wilson a permis d’évaluer la valeur translationnelle et clinique des candidats biomarqueurs identifiés et de débuter l’étude clinique d’exploration du protéome plasmatique de patients atteints de la maladie de Wilson. En outre, la compréhension des mécanismes moléculaires associés au développement de la physiopathologie hépatique a été étudiée et a permis de mettre en évidence de nouvelles cibles pour, à terme, améliorer la prise en charge clinique des patients atteints de la maladie de Wilson. / Wilson’s disease is a rare genetic disorder triggered by mutations in the ATP7B gene, which encodes a transport protein involved in copper transport and excretion, triggering toxic copper overloads in the liver and the brain. The lack of genotype-phenotype correlation and phenotype variability lead to clinical care difficulties, especially for the diagnosis and biological follow up of patients. In this study, we initiated the discovery and evaluation of biomarker candidates for the diagnosis of Wilson’s disease and for early prognosis towards neurological manifestation. With the availability of the Atp7b-/- mice model, we engaged a preclinical study leading to the qualification of a panel of 7 biomarker candidates. These results allowed us to raise the interest of the National Reference Center for Wilson’s disease (CNR) medical teams in Lyon and Paris and to engage a close collaboration to initiate clinical study. Using a first plasma cohort from Wilson’s disease patients, we assessed the translational and clinical value of the 7 biomarker candidates and engage discovery study on patients’ plasma samples. Furthermore, we also studied the molecular mechanisms involved in liver pathophysiology using the Atp7b-/- mice model using discovery proteomics. These investigations led to the identification of a new potential therapeutic target.

The work here presents two main parts. In the first part, chapters 1 – 3 focus on dynamical systems modeling in plant immunity, whereas chapters 4 – 6 describe contributions to computational modeling and analysis of proteomics and genomics data. Chapter 1 investigates dynamical and biochemical patterns of reversibly oxidized cysteines (RevOxCys) during effector-triggered immunity (ETI) in Arabidopsis, examines the regulatory patterns associated with Arabidopsis thimet oligopeptidase 1 and 2’s (TOP1 and TOP2), roles in the RevOxCys events during ETI, and analyzes the redox phenotype of the top1top2 mutant. The second chapter investigates the peptidome dynamics during ETI in wild-type (WT) and top1top2 mutant, introduces a novel method to learn the cleavage motif for TOPs and predicts and validates bioactive peptides association with TOPs activity. The third chapter examines gene expression dynamics during Systemic Acquired Resistance (SAR). Time-series clustering identifies unique oscillatory patterns in gene transcription associated with the early onset of SAR. It then describes a mathematical model using ordinary differential equations to represent WT's transcriptional dynamics. The second part of this dissertation explores imputation and statistical modeling for proteomics data analysis and proposes a network inference methodology for polymorphic cysteines. The fourth chapter analyzes the performance of linear models (limma) and the effect of imputation in proteomics data. It shows the advantage of data imputation over filtering and the benefit of using limma over t-test for the statistical decision of differences in means between conditions for different peptides, PTMs, etc. The fifth chapter proposes a statistical model for proteomics data analysis using mean-variance (M-V) trend modeling. It describes a gamma regression to model the dependency of the variance on the mean of observations. Finally, a Bayesian decision model is proposed; the model shows an improvement over existing methods in statistical decision performance. The sixth and final chapter describes a network inference procedure that identifies genetic dependencies between polymorphic cysteines. It models the interactions between cysteines (nodes) as signed edges for positive or inhibitory relations. It utilizes local network structures for inferences about the relationship between the cysteines. The algorithm exhibits stability and efficiency, converging rapidly to inferred solutions.

Made available in DSpace on 2016-06-02T20:21:32Z (GMT). No. of bitstreams: 1 5298.pdf: 2436195 bytes, checksum: 47a2b6b8993059d60e028c4518c3ca75 (MD5) Previous issue date: 2013-05-24 / Universidade Federal de Sao Carlos / The citrus canker is an economically important disease for citrus crop. At the moment, there is no effective means of prevention or cure for this disease, which has contributed to citrus canker wide distribution around the world. The etiologic agents are bacteria of the genus Xanthomonas classified into two species, X. citri and X. fuscans. This study aimed to perform the differential proteomic analysis of cell surface proteins of Xanthomonas citri subsp. citri (XAC), the more virulent specie, between infectious (in vivo) and non-infectious (in vitro) conditions of growth. Additionally, the same analysis was performed by shotgun for XAC against the Xanthomonas fuscans subsp. aurantifolii type B (XauB), less virulent specie, both after growth in vivo. Initially, we performed growth curves of both bacteria on leaves of a common citrus host (Citrus aurantifolia) in order to investigate the dynamics of population growth in vivo and the efficiency of cell recovery by two different methods. For proteomic analysis, intact bacterial cells had their surface proteins labeled with fluorescence (DIGE CyDye Fluor minimal dyes), were then lysed and the total protein extract analyzed by differential gel electrophoresis (2D-DIGE), as standardized in this study. Protein profiles were analyzed by DeCyder 7.0 software and spots differentially expressed (ANOVA p <0.05) were isolated from gels, identified by mass spectrometry and search in protein databases of the annotated genome sequence of the bacteria. Seventy-nine spots from XAC were analyzed and thirty different proteins were identified, of which 10 correspond to known membrane or cell surface proteins: Ton-B dependent receptors and OmpA-related proteins exhibited lower expression in infectious condition, differently of Ferric enterobactin receptors, 60 kDa chaperonin (GroEL) and DnaK which showed higher expression after host interaction. XAC and XauB total extraction analysis by shotgun identified just two XAC proteins. Cell surface proteins with increased in vivo expression in virulent strain (XAC) could provide future targets of biotechnological interest for fighting citrus canker for being possibly related to phytopathogenicity and/or host spectrum. / O cancro cítrico é uma doença economicamente importante para a citricultura. Devido à inexistência de medidas eficazes de prevenção e combate, o cancro cítrico ainda é uma doença de ampla distribuição. Os agentes etiológicos são bactérias do gênero Xanthomonas, sendo classificadas em duas espécies, X. citri e X. fuscans, as quais diferem em virulência e espectro de hospedeiros cítricos. Este trabalho teve como objetivo a análise diferencial do subproteoma da superfície celular de Xanthomonas citri subsp. citri (XAC), espécie mais virulenta e causadora da cancrose A, entre duas condições de crescimento, infectante (in vivo) e não infectante (in vitro). Adicionalmente, a análise proteômica total por shotgun (LC-MS/MS) foi realizada para comparação de XAC com Xanthomonas fuscans subsp. aurantifolii tipo B (XauB), espécie menos virulenta, ambas após crescimento in vivo. Inicialmente, foram realizadas curvas de crescimento de ambas as bactérias em folhas de um hospedeiro cítrico comum (Citrus aurantifolia) a fim de se conhecer a dinâmica de crescimento populacional in vivo e a eficiência da recuperação bacteriana por dois diferentes métodos. Para as análises proteômicas, células bacterianas intactas tiveram suas proteínas de superfície marcadas com fluorescência (CyDye DIGE Fluor minimal dyes) e em seguida foram lisadas, sendo o extrato proteico total analisado por eletroforese diferencial em gel bidimensional (2D-DIGE), técnica padronizada neste trabalho. Os perfis proteicos de XAC foram analisados pelo software DeCyder 7.0 (GE Healthcare) e spots com expressão diferencial (ANOVA p<0,05) foram isolados dos géis e identificados por espectrometria de massas seguida de busca pela ferramenta Mascot em bancos de proteínas anotadas a partir da sequência genômica. Dos 79 spots de XAC analisados foram identificadas 30 diferentes proteínas, sendo que 10 correspondem a proteínas reconhecidamente de membrana e/ou superfície celular: receptores dependentes de Ton-B e proteínas relacionadas a OmpA foram encontradas com menor expressão na condição in vivo, enquanto que receptor de enterobactina, chaperonina 60 kDa (GroEL) e DnaK apresentaram maior expressão após interação com hospedeiro cítrico. Em relação à comparação do extrato total de XAC e XauB por shotgun foi possível identificar apenas duas proteínas de XAC. Proteínas da superfície celular com maior expressão na linhagem virulenta (XAC) na condição in vivo poderão ser futuros alvos de interesse biotecnológico para combate ao cancro cítrico por estarem possivelmente relacionadas com a fitopatogenicidade e/ou maior espectro de hospedeiros cítricos.