Avec plus de 600 000 nouveaux cas par an, les cancers des voies aéro-digestives supérieures (VADS) se classent au sixième rang mondial. Ces cancers, traditionnellement causés par la consommation chronique de tabac et d’alcool, connaissent depuis une trentaine d’années de profonds changements épidémiologiques. Alors que l’incidence des cancers développés dans la cavité orale, le larynx et l’hypopharynx tend à se stabiliser voire à régresser en raison de la diminution du tabagisme, ceux développés dans l’oropharynx sont en nette augmentation. Cette augmentation est attribuée aux papillomavirus oncogènes et notamment au génotype 16 (HPV16). Les cancers de l’oropharynx (COP) HPV-induits représentent une pathologie distincte des autres cancers des VADS tant au niveau épidémiologique, clinique, histologique que biologique. Ils affectent des sujets plus jeunes, sont extrêmement lymphophiles et leur pronostic est significativement meilleur. L’émergence de ces cancers impose dès aujourd’hui de réfléchir à des stratégies diagnostiques, thérapeutiques et de surveillance spécifiques. Néanmoins, ces objectifs ne pourront être pleinement atteints qu’à condition de mieux comprendre leur histoire naturelle ainsi que leurs mécanismes oncogéniques propres. L’objectif de ce travail est de contribuer à une meilleure compréhension des mécanismes biologiques distinguant les COP HPV-induits de leurs homologues HPV-négatifs sur la base de l’analyse de leur profil d’expression génomique. A partir d’une cohorte de 38 COP, sélectionnés sur des critères stricts, nous avons identifié un set d’ARNm et de miRNA dont l’expression est exclusivement corrélée au statut HPV. L’analyse fonctionnelle de ces sets confirme que les bases biologiques des COP varient en fonction de leur statut HPV et confortent au niveau moléculaire des données cliniques et pathologiques déjà connues ou fortement suspectées (différenciation tumorale, infiltration lymphocytaire…). Cette étude souligne également le rôle potentiel de plusieurs voies de signalisation dont les dérégulations contribueraient au développement de ces tumeurs. L’exploration plus approfondie de ces voies pourrait à terme permettre de mieux comprendre ces tumeurs et avoir d’éventuelles retombées thérapeutiques. / Head and neck squamous cell carcinomas (HNSCCs) represent the sixth most common form of cancer with an annual incidence of approximately 600,000 new cases worldwide. Tobacco and alcohol abuse are the traditional risk factors. Whilst the incidence of oral cavity, larynx and hypopharynx cancers is stabilizing or falling, because of a drop in tobacco consumption, those arising in the oropharynx are on the increase. This epidemiologic change has been attributed to high-risk human papillomavirus and particularly to type 16 (HPV16), which is now recognized as a causative agent in a growing subset of oropharyngeal squamous cell carcinomas (OPSCCs).HPV-induced OPSCCs represent a distinct subgroup, separate from other HNSCCs, with unique epidemiologic, clinical, pathological and molecular characteristics. They affect young patients, are highly lymphophilic and have markedly improved survival outcomes compared to those with HPV-negative HNSCC. The emergence of these cancers demands special attention, as in the coming years diagnosis, treatment and follow up in HNSCC may vary according to HPV status. However, these objectives will not be fully achieved without a better understanding of their natural history and specific oncogenic mechanisms. The goal of this work is to contribute to a better understanding of the biological basis that differentiates HPV-induced OPSCCs from their HPV-negative counterparts. To this end, we have investigated global changes in gene expression in a cohort of 38 strictly selected OPSCCs. We have identified a set of mRNA and miRNA that discriminated between OPSCCs solely according to HPV16 status. The functional analysis of these 2 sets confirms that the biological basis of OPSCCs varies according to their HPV status and consolidates at the molecular level known or suspected clinical and pathological data (e.g tumoral differentiation, lymphoid infiltrations…). This study highlights the potential role of several pathways that, once deregulated, could contribute to the development of HPV-induced OPSCC. Further investigation is required for a more comprehensive understanding of the biological properties of HPV related OPSCCs. These properties may be exploited to develop novel therapeutic agents.

Human activities have being responsible for the global environmental changes, resulting in an increase number of incident of vector- and rodent-borne diseases worldwide. Rodents and arthropods-borne viruses are important globally emerging and re-emerging viruses and most of them are RNA viruses. Efficient and early diagnosis of these infections are very important to prevent their spread, to improve clinical management of the patients, as wells to protect livestock and domestic animals. Currently, available diagnostic methods such as immunoassays, polymerase chain reaction and virus isolation can detect only one or few viruses in a single assay. The DNA microarray platform has emerged as diagnostic tool suitable for high throughput screening of pathogenic agents. The aim of this study was to develop a DNA microarray platform (RoboArboVirusChip) for detecting rodent- and arthropod-borne viruses, which belong to seven families: Bunyaviridae (genera Orthobunyavirus, Nairovirus and Phlebovirus), Flaviviridae (genus Flavivirus), Togaviridae (genus Alphavirus), Reoviridae (genera Orbivirus, Seadornavirus and Coltvivirus), Rhabdoviridae (genera Vesiculovirus and Ephemerovirus), and Asfarviridae (genus Asfarvirus). Specific oligonucleotide probes of 60-mer (n=4209) targeting 412 virus species and generic probes of 25-35-mer (n=87) targeting viruses at the genus level were designed. A total of 17 reference viruses belonging to the Bunyaviridae, Flaviviridae, Rhabdoviridae and Togaviridae families were used to standardize RoboArboVirusChip. All reference viruses were specifically detected without any cross hybridization; however, the generic probes were not able to identify the viruses at the genus level. The RoboArboVirusChip was able to specifically identify four viruses contained in three different mixtures: i) virus of different families, ii) virus of the Flavivirus genus, and iii) the Dengue virus (DENV) serotypes. The four DENV serotypes were use to evaluate the sensitivity of the RoboArboVirusChip, which was able to detect a minimum of 25 RNA copies/mL of the viruses, confirming its high sensitivity. The applicability of the RoboArboVirusChip to detect viruses in clinical samples was tested with serum samples obtained from dengue suspected cases (four positive cases and 40 negative cases). DENV was detected in the four positive serum samples, while in the 40 negative serum samples, it was not detected any virus. The results obtained in this study suggest that the RoboArboVirusChip platform could be a useful tool for early diagnosis of robovirus and arbovirus infections during epidemic outbreaks, helping in the rapid implementation of disease containment strategies / As atividades humanas têm sido responsável por mudanças ambientais globais, resultando num aumento do número de casos de doenças transmitidas por vetores e roedores em todo o mundo. Os vírus transmitidos por roedores e artrópodes são vírus emergentes e re-emergentes de importância global, sendo que a maioria deles são vírus de RNA. O diagnóstico eficiente e precoce dessas infecções são muito importantes para evitar a sua propagação, para melhorar o manejo clínico dos pacientes e também, para proteger o gado e os animais domésticos. Atualmente, os métodos de diagnóstico disponíveis, tais como os imunoensaios, a reação em cadeia da polimerase e o isolamento viral podem detectar apenas um ou poucos vírus em um único ensaio. A plataforma de microarranjo de DNA tem surgido como uma ferramenta de diagnóstico apropriada para o monitoramento em larga escala de agentes patogênicos. O objetivo deste estudo foi desenvolver uma plataforma de microarranjo de DNA (RoboArboVirusChip) para a detecção de vírus transmitidos por roedores e artrópodes, os quais pertencem a sete famílias: Bunyaviridae (gêneros Orthobunyavirus, Nairovirus e Phlebovírus), Flaviviridae (gênero Flavivirus), Togaviridae (gênero Alphavirus), Reoviridae (gênero Orbivirus, Seadornavirus e Coltvivirus), Rhabdoviridae (géneros Vesiculovirus e Ephemerovirus), e Asfarviridae (gênero Asfarvirus). Sondas oligonucleotídicas de 60-mer (n=4209) específicas contra 412 espécies virais, e sondas genéricas de 25-35-mer (n=87) para detecção de vírus a nível do gênero foram desenhados. Um total de 17 vírus de referência, pertencentes às famílias Bunyaviridae, Flaviviridae, Rhabdoviridae e Togaviridae foram utilizados para padronizar o RoboArboVirusChip. Todos os vírus de referência foram detectados especificamente sem apresentação de hibridação cruzada, porem as sondas genéricas não foram capazes de detectar os vírus a nível do gênero. O RoboArboVirusChip foi capaz de identificar especificamente quatro vírus contidos em diferentes misturas: i) vírus de diferentes famílias, ii) vírus pertencentes ao gênero a Flavivirus, e iii) os sorotipos do vírus da dengue (DENV). Os quatro sorotipos do DENV foram utilizados para determinar a sensibilidade do RoboArboVirusChip, o qual foi capaz de detectar um mínimo de 25 copias de RNA/mL. A aplicabilidade do RoboArboVirusChip para detectar vírus em amostras clínicas foi avaliada testando amostras de soro de pacientes com suspeita de dengue (quatro casos positivos e 40 casos negativos). Os resultados obtidos neste estudo sugerem que o RoboArboVirusChip poderá ser uma ferramenta útil para o diagnóstico precoce da infecção causada por robovírus e arbovírus, auxiliando na rápida implementação de estratégias de contenção das doenças causadas por esses vírus

Point-of-care testing brings diagnosis and treatment monitoring to the site of the patient. It heavily relies on biosensors, which leverage the interactions between a target biomarker and a bioreceptor, to deliver fast and accurate results. However, non-specific binding of molecules and microorganisms on the biointerface can interfere with biomarker-bioreceptor interactions and diminish a biosensor’s sensitivity, specificity, and stability. In turn, this can lead to false diagnoses and ineffective treatments. Omniphobic-lubricant infused (OLI) coatings exhibit slippery, self-cleaning characteristics that repel untargeted molecules and microorganisms to augment the biosensor’s performance. In this work, we investigate the proficiency of OLI coatings in two specific applications: dissolved oxygen sensing and DNA biosensing. First, in water quality monitoring, an OLI coating is applied to the selectively permeable membranes of a dissolved oxygen sensor. Over a three-week incubation period in an environment with accelerated bacterial growth, the coated membranes exhibit a 160% higher reproducibility (10% deviation in sensitivity) and lower biofilm formation (96° static contact angle) in comparison to unmodified membranes (26%, 32°). The second application is in DNA biosensing, where a novel OLI coating uses carbon dioxide plasma activation to embed oligonucleotide probes. It demonstrates an optimized balance of slippery repellency (76° static contact angle, 10° sliding angle) and biosensing functionality, 19% longer clotting times than conventional blocking conditions, and equal sensitivity to PLL-PEG when capturing target DNA in whole blood. Going forward, our research will continue to expand the use of OLI coatings in biosensing applications, particularly exploiting its antibiofouling and anticoagulative capabilities. / Thesis / Master of Applied Science (MASc) / Biosensors are an integral tool in delivering quick and accurate point-of-care diagnosis and treatment monitoring. However, their performance can be impeded by the non-specific binding of undesirable molecules and microorganisms on the sensing surface. Omniphobic lubricant-infused (OLI) coatings have been shown to suppress biofouling and blood clotting on surfaces through exceptional repellency. This thesis focuses on the implementation of OLI coatings in biosensing applications. It investigates the antibiofouling capacity of an OLI coating on a membrane for dissolved oxygen detection. Then, it discusses a novel coating with integrated DNA biosensing functionality for working directly with blood samples. The enclosed work demonstrates that the OLI coating empowers biosensors to deliver more effective point-of-care testing.

The purpose of the current study was to test the hypothesis that chronic hypoxia associated with sleep-disordered breathing relates to abnormal Nitric Oxide (NO) production and vascular endothelial growth factor (VEGF) expression patterns that contribute to aberrancy of specific determinates of cardiovascular and cardiopulmonary function before, during, and after graded exercise. These patterns may further reflect pathologic alteration of signaling within the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt-1) transduction network. To this end, 7 medically diagnosed OSA patients (3 male, 4 female), mean age 48 years and 7 apparently healthy control subjects (3 male, 4 female), mean age 42 years, underwent baseline venous blood draws and maximal bicycle ergometry. Mononuclear cells isolated from peripheral blood were utilized as reporter cells for measurement of VEGF, Akt-1, hypoxia inducible factor-1 alpha (HIF-1 alpha), and vascular endothelial growth factor receptor-2 (VEGFR2) gene expression by redundant oligonucleotide DNA microarray and real-time PCR technologies. Circulating angiogenic progenitor cells expressing VEGFR2 were profiled by flow cytometry. Plasma and serum concentrations of VEGF, nitrates/nitrites, catecholamines, and dopamine were measured by enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC). Arterial blood pressure, cardiac output, oxygen consumption and total peripheral resistance were determined at Baseline, 100W, and peak ergometric stress by standard techniques. There were no apparent differences (p < .05) observed in biochemical markers relating to vascular function and adaptation including, serum nitrates/nitrites, norepinephrine, dopamine, and plasma VEGF. No differences were found relative to cardiac output, stroke volume, cardiopulmonary or myocardial oxygen consumption, expired ventilation, heart rate, arteriovenous oxygen difference, total peripheral resistance, and mean arterial pressure. Due to methodological issues related to the redundant oligonucleotide DNA microarray and real-time PCR gene expression analyses, results of these experiments were uninterpretable. Thus, the research hypothesis was rejected. Conversely, significant (p < .05) differences were observed in waist: hip ratios, recovery: peak systolic blood pressure ratio at 1 minute post-exercise and %VEGFR2 expression. OSA was associated with elevations in both waist: hip ratios and recovery: peak systolic blood pressure ratio at 1 minute post-exercise as well as significant depression of %VEGFR2 profiles. Moreover, significant negative correlations were found regarding waist: hip ratios and %VEGFR2 expression (r = -.69;p =.005) and recovery: peak systolic blood pressure ratio at 1 minute post-exercise and %VEGFR2 expression (r = -.65;p =.01). These findings did not provide evidence that NO-dependent vasoactive mechanisms are suppressed nor did they support the supposition that angiogenic mechanisms are pathologically activated in sleep-disordered breathing. / Ph. D.

We report the assembly of three-dimensional (3D) liver sinusoidal mimics comprised of primary rat hepatocytes, human or rat liver sinusoidal endothelial cells denoted as hLSECs and rLSECs respectively, and an intermediate chitosan-hyaluronic acid (HA) polyelectrolyte multilayer (PEM). The height of the PEMs ranged from 30-55nm and exhibited a shear modulus of ~ 100kPa. Primary rat hepatocytes coated with 5 and 15 PE layers exhibited stable urea and albumin production over a seven day period and these values were either comparable or superior to that in a collagen sandwich (CS). Hepatocyte-PEM-hLSEC liver mimics exhibited stable urea production and increasing albumin secretion over the culture period in comparison to hepatocyte-LSEC samples. In the 3D liver mimics, hLSEC phenotype was maintained and verified by the uptake of acetylated low-density lipoprotein (AcLDL). A sixteen-fold increase in CYP1A1/2 activity was observed for hepatocyte-PEM-10,000 hLSEC samples, thereby, suggesting that interactions between hepatocytes and hLSECs play a key role in enhancing hepatic phenotypes in in vitro cultures. As the first step towards elucidating key signaling pathways involved in cell-cell communications, global genome-wide transcriptional profiles of primary hepatocytes cultured in CS and hepatocyte monolayers (HMs) were performed over an eight-day period using DNA microarray measurements and Gene Set Enrichment Analysis (GSEA) in order to derive biologically meaningful information at the level of gene sets. The gene expression in CS cultures steadily diverged from that in HMs. Gene sets up-regulated in CS are those linked to liver metabolic and synthetic functions, such as lipid, fatty acid, alcohol and carbohydrate metabolism, urea production, and synthesis of bile acids. Monooxygenases such as CYP enzymes were significantly up-regulated starting on day 3 in CS cultures. These results serve as a baseline for further investigation into the systems biology of engineered liver tissues. 3D hepatic constructs were also assembled with primary rat hepatocytes and rLSECs, and a chitosan-HA PEM. In these hepatic models, the phenotype of hepatocytes and rLSECs were maintained. rLSEC phenotype was verified over a twelve-day period through immunostaining with the sinusoidal endothelial-1 (SE-1) antibody. In contrast, rLSECs cultured as monolayers lost their phenotype within 3 days. A two-fold increase in albumin production was observed only in the 3D liver models. rLSEC-PEM-hepatocyte cultures exhibited three- to six-fold increased CYP1A1/2 and CYP3A enzymatic activity. Well-defined bile canaliculi were observed in only 3D hepatic constructs. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte constructs can be used as liver models for future studies. / Ph. D.

No description available.

Die mittlere Lebenserwartung des Menschen ist über die letzten 200 Jahre kontinuierlich gestiegen. Da Langlebigkeit ohne Gesundheit wenig Wert besitzt, ist es ein zentrales Anliegen, das Auftreten altersbedingter Krankheiten zu mindern. Besonders pflanzliche Phytochemikalien, im speziellen Polyphenole (PPs), sollen erheblich an der Gesundheitsförderung mitwirken. Die exakten Mechanismen jedoch, welche die Wirkvielfalt erklären könnten, sind nicht im Detail bekannt. Diese Fragen können nur durch in vivo Studien an Modelorganismen beantwortet werden, die sowohl die Lebensdauer, sowie physiologische und genetische Parameter einschließen. In dieser Studie wurden drei PPs mit lebensverlängernden Eigenschaften in C. elegans identifiziert: Quercetin (Q), Kaffeesäure (CA) und Rosmarinsäure (RA). Für alle drei PPs wurden hormetische Konzentration-Wirkungs-Kurven gefunden, dennoch war die Hormetin-typische Aktivierung einer Stressantwort (gemessen als Geneexpressions-Level von Hitzeschock-Proteinen) auf Q und RA beschränkt. Eine Umverteilung von Ressourcen nach dem Prinzip der „Disposable Soma Theorie“ konnte anhand von Abweichungen in der Größe, verändertem Lipid-Metabolismus und verzögerter Reproduktion (bei gleichbleibender Anzahl der Nachkommen), für alle drei PPs gezeigt werden. Während direkte CR-Effekte ausgeschlossen wurden, ist dies nicht möglich für durch CA und RA ausgelöste indirekte CR-Effekte, da beide die Lebensspanne von sir-2.1 Mutanten nicht verlängern konnten. Alle drei PPs verlängerten die Lebensspanne von mev-1 Mutanten, jedoch wurde eine erhöhte TAC in vivo und eine reduzierte oxidative Schädigung, nur durch Q- und CA- Gabe erreicht. Die genetischen Wirkwege der PPs wurden durch Lebensdauer- und Thermotoleranztests mit in alters-relevanten Genen mutierten Nematoden definiert. Die gesundheitsfördernden Eigenschaften von CA und RA konnten so osr-1, sek-1, sir-2.1 and unc-43, sowie daf-16 im Falle von CA, zugeschrieben werden. Die Mechanismen von Q wurden in größerem Umfang, durch die Integration von durchgeführten Lebensdauertests und Microarray-Studien einerseits und einer umfassenden Meta-Analyse von veröffentlichten, alters-relevanten Genexpressions-Profilen andererseits, analysiert. Q wirkt vermutlich durch ein komplexes Zusammenspiel von konservierten genetischen Signalwegen, im Speziellen dem Insulin-ähnlichen (ILS), TGF-beta, p38 MAPK, CAMKII und möglicherweise auch über eine von der Keimbahn und somatischen Gonade ausgehenden Signalwirkung. Zusammenfassend lässt sich sagen, dass sowohl in vivo antioxidative und prooxidative Eigenschaften, die Modulation auf Genebene, sowie eine Umverteilung von Ressourcen zu gewissen Teilen (abhängig vom PP) zur Lebensverlängerung beitragen. / The mean life expectancy of humans has increased continuously over the last 200 years. Since longevity is of little value in the absence of health, it is a central request to prevent the increasing burden of age-related diseases. It is suggested that phytochemicals in plants, specifically the polyphenols (PPs), are important factors to support the overall well-being. However, the precise mechanisms that can explain, in full, the magnitude of impact remains elusive. This knowledge gap can only be plugged by in vivo model organism approaches that integrate lifespan assays with physiological, and genetic parameters following the ingestion of PPs. In this study, three PPs with life-extending properties in C. elegans were identified: Quercetin (Q), Caffeic acid (CA) and Rosmarinic acid (RA). The underlying mechanisms were systematically studied by a broad spectrum of functional and genetic investigations. For all three compounds, life extension was characterized by hormetic concentration-response curves, but stress-response induction, a hallmark of hormetin action, was restricted to Q and RA, at least when assessed at the level of gene expression of heat shock proteins. A reallocation of resources in a disposable soma-like pattern could be shown for all three PPs, because the exposure to Q, CA and RA resulted in variations in body size, altered lipid-metabolism and a tendency towards a delay in reproductive timing. However, the total number of offspring was unaltered. While direct CR effects arising from reduced food uptake could be rejected, an indirect CR effect cannot be excluded for CA and RA, as these PPs failed to provoke longevity in sir-2.1 mutants. Furthermore, the in vitro versus in vivo antioxidative properties were evaluated. While all three PPs could prolong mev-1 lifespan, only Q and CA were shown to increase the TAC in vivo and reduce oxidative damage in the nematodes. To define the genetic pathways of PP action, lifespan and thermotolerance assays were performed in mutant animals devoid of aging-relevant genetic players. These experiments revealed that the health gaining properties of CA and RA both rely on osr-1, sek-1, sir-2.1 and unc-43, plus daf-16 in the case of CA. The mechanisms of Q action are partly distinct and were analyzed in more detail by integrating own mutant lifespan assays and microarray studies with an extensive meta-analysis of published gene expression profiles obtained under aging-relevant conditions. Quercetin is proposed to act through a complex interplay of conserved genetic pathways, for example Insulin-like signaling (ILS), TGF-beta signaling, p38 MAPK, CaMKII, and possibly also due to germline and somatic gonad signaling. Taken together, hormesis, in vivo antioxidative/prooxidative properties, modulation of genetic players, as well as the re-allocation of resources all contribute (to some extent and dependent on the polyphenol) to life extension. Summary 1

Devido à grande diversidade de microrganismos desconhecidos no meio ambiente, 99% deles não podem ser cultivados nos meios de cultura tradicionais dos laboratórios. Para isso, projetos metagenômicos são propostos para estudar comunidades microbianas presentes no meio ambiente, a partir de técnicas moleculares, em especial o seqüenciamento. Dessa forma, para os próximos anos é esperado um acúmulo de seqüências produzidas por esses projetos. As seqüências produzidas pelos projetos genomas e metagenomas apresentam vários desafios para o tratamento, armazenamento e análise, como exemplo: a busca de clones contendo genes de interesse. Este trabalho apresenta uma abordagem algébrica que define e gerencia de forma dinâmica as regras para a seleção de clones em bibliotecas genômicas e metagenômicas, que se baseiam em álgebra de processos. Além disso, uma interface web foi desenvolvida para permitir que os pesquisadores criem e executem facilmente suas próprias regras de seleção de clones em bancos de dados de seqüências genômicas e metagenômicas. Este software foi testado em bibliotecas genômicas e metagenômicas e foi capaz de selecionar clones contendo genes de interesse. / Due to the wide diversity of unknown organisms in the environment, 99% of them cannot be grown in traditional culture medium in laboratories. Therefore, metagenomics projects are proposed to study microbial communities present in the environment, from molecular techniques, especially the sequencing. Thereby, for the coming years it is expected an accumulation of sequences produced by these projects. Thus, the sequences produced by genomics and metagenomics projects present several challenges for the treatment, storing and analysis such as: the search for clones containing genes of interest. This work presents an algebraic approach that defines it dynamically and manages the rules of the selection of clones in genomic and metagenomic libraries, which are based on process algebra. Furthermore, a web interface was developed to allow researchers to easily create and execute their own rules to select clones in genomic and metagenomic sequence database. This software was tested in genomics and metagenomics libraries and it was able to select clones containing genes of interest.

A análise da expressão gênica através de dados gerados em experimentos de microarrays de DNA vem possibilitando uma melhor compreensão da dinâmica e dos mecanismos envolvidos nos processos celulares ao nível molecular. O aprimoramento desta análise é crucial para o avanço do conhecimento sobre as bases moleculares das neoplasias e para a identificação de marcadores moleculares para uso em diagnóstico, desenho de novos medicamentos em terapias anti-tumorais. Este trabalho tem como objetivos o desenvolvimento de modelos de análise desses dados, propondo uma nova forma de normalização de dados provenientes de microarrays e dois modelos para a construção de redes regulatórias de expressão gênica, sendo uma baseada na conectividade dinâmica entre diversos genes ao longo do ciclo celular e a outra que resolve o problema da dimensionalidade, em que o número de experimentos de microarrays é menor que o número de genes. Apresenta-se, ainda, um pacote de ferramentas com uma interface gráfica de fácil uso contendo diversas técnicas de análise de dados já conhecidas como também as abordagens propostas neste trabalho. / The analyses of DNA microarrays gene expression data are allowing a better comprehension of the dynamics and mechanisms involved in cellular processes at the molecular level. In the cancer field, the improvement of gene expression interpretation is crucial to better understand the molecular basis of the neoplasias and to identify molecular markers to be used in diagnosis and in the design of new anti-tumoral drugs. The main goals of this work were to develop a new method to normalize DNA microarray data and two models to construct gene expression regulatory networks. One method analyses the dynamic connectivity between genes through the cell cycle and the other solves the dimensionality problem in regulatory networks, meaning that the number of experiments is lower than the number of genes. We also developed a toolbox with a user-friendly interface, displaying several established statistical methods implemented to analyze gene expression data as well as the new approaches presented in this work.

L’émergence et l’augmentation continue de la résistance aux médicaments chez Mycobacterium tuberculosis (MTB) constituent un défi majeur pour la lutte antituberculeuse. Pour résoudre le problème de temps (2 à 8 semaines) posé par les tests classiques de sensibilité aux médicaments (DST), des tests moléculaires ont été développés pour la détection précoce des mutations associées à la résistance. Jusqu'à présent, à l'exception du séquençage complet (incompatible avec un diagnostic de routine dans les pays en développement), aucun test ne détecte simultanément les différents types de résistance majeurs en une seule réaction. Sur la base de la littérature et de travaux antérieurs menés au Vietnam, au Laos et au Cambodge, une puce à ADN capable de détecter 184 mutations liées à la résistance aux médicaments de première et de deuxième lignes a été mise au point. En comparaison avec les données de DST, la puce a montré une sensibilité (84,3%-100%) et une spécificité élevées (89,2%-100%). Par rapport au séquençage, la puce a donné des résultats comparables, avec une sensibilité entre 90% et 100% et une spécificité entre 98,2% et 100%. Elle offre, de plus, une meilleure couverture que les tests moléculaires approuvés par l'OMS, car elle permet la détection des résistances aux médicaments de première et de deuxième lignes dans un seul test. Le temps de traitement des isolats issus de la culture est d’environ 6 à 7 heures, ce qui réduit considérablement le temps de diagnostic par rapport au DST. En conclusion, la puce à ADN a été développée avec succès. Certains aspects techniques doivent maintenant être améliorés pour en faire un outil de diagnostic abordable et facile à utiliser. / The emergence and continuous increase of drug resistance in Mycobacterium tuberculosis (MTB) is a major challenge for tuberculosis (TB) control. To overcome the time-consuming problem of conventional drug susceptibility testing (DST), many molecular-based tests have been recently developed for early detection of drug resistance-associated mutations. Up to now, except whole genome sequencing (not ready for routine diagnostic in low and middle-income countries), no test has the capacity to simultaneously detect the different types of drug resistance to first- and second-line drugs in one reaction. Based on the literature and previous works carried out in Vietnam, Laos and Cambodia, in this study, a DNA chip was developed able to detect 184 main mutations conferring resistance to both first- and second-line drugs. Compared to DST, the DNA chip showed high sensitivity (between 84.3% and 100%) and high specificity (between 89.2% and 100%). Compared to sequencing, the DNA chip showed comparable accuracy, with sensitivity between 90% and 100% and specificity between 98.2% and 100%. The DNA chip showed a better coverage than the WHO endorsed molecular tests since it enables the detection of both first- and second-line drug resistances in one test. The turn-around time is about 6-7h from cultured isolates reducing considerably the diagnostic time compared to cultured-based DST. Finally, the DNA chip has been successfully developed, even if certain technical aspects need to be improved to make an affordable and easy-to-use diagnostic tool.