Übergewicht und Adipositas führen zu Insulinresistenz und erhöhen deutlich das Risiko für die Entwicklung von Typ-2-Diabetes und kardiovaskulären Erkrankungen. Sowohl Adipositas als auch die Suszeptibilität gegenüber Diabetes sind zu einem erheblichen Teil genetisch determiniert. Die relevanten Risikogene, deren Interaktion mit der Umwelt, insbesondere mit Bestandteilen der Nahrung, und die Pathomechanismen, die zur Insulinresistenz und Diabetes führen, sind nicht vollständig aufgeklärt. In der vorliegenden Arbeit sollte durch Genexpressionsanalysen des weißen Fettgewebes (WAT) und der Langerhansschen Inseln die Entstehung und Progression von Adipositas und Typ-2-Diabetes untersucht werden, um relevante Pathomechanismen und neue Kandidatengene zu identifizieren. Zu diesem Zweck wurden Diät-Interventionsstudien mit NZO- und verwandten NZL-Mäusen, zwei polygenen Mausmodellen für das humane metabolische Syndrom, durchgeführt. Eine kohlenhydrathaltige Hochfett-Diät (HF: 14,6 % Fettanteil) führte in beiden Mausmodellen zu früher Adipositas, Insulinresistenz und Typ 2 Diabetes. Eine fettreduzierte Standarddiät (SD: 3,3 % Fettanteil), welche die Entstehung von Adipositas und Diabetes stark verzögert, sowie eine diabetesprotektive kohlenhydratfreie Hochfett-Diät (CHF: 30,2 % Fettanteil) dienten als Kontrolldiäten. Mit Hilfe der Microarray-Technologie wurden genomweite Expressionsprofile des WAT erstellt. Pankreatische Inseln wurden durch laserbasierte Mikropräparation (Laser Capture Microdissection; LCM) isoliert und ebenfalls hinsichtlich ihres Expressionsprofils analysiert. Differenziell exprimierte Gene wurden durch Real-Time-PCR validiert. Im WAT der NZO-Maus bewirkte die HF-Diät eine reduzierte Expression nukleärer Gene der oxidativen Phosphorylierung und von lipogenen Enzymen. Dies deutet auf eine inadäquate Fettspeicherung und -verwertung in diesen Tieren hin. Die Reduktion in der Fettspeicherung und -oxidation ist spezifisch für das adipöse NZO-Modell und konnte bei der schlanken SJL Maus nicht beobachtet werden, was auf eine mögliche Beteiligung an der Entstehung der Insulinresistenz hinweist. Zusätzlich wurde bestätigt, dass die Expansion des Fettgewebes bei der adipösen NZO-Maus eine zeitlich verzögerte Infiltration von Makrophagen in das WAT und dort eine lokale Immunantwort auslöst. Darüber hinaus wurde die Methode der LCM etabliert und zur Gewinnung hochangereicherter RNA aus den Langerhansschen Inseln eingesetzt. In erstmalig durchgeführten genomweiten Expressionsanalysen wurde zu einem frühen Zeitpunkt in der Diabetesentwicklung der Einfluss einer diabetogenen HF-Diät und einer diabetesprotektiven CHF-Diät auf das Expressionsprofil von pankreatischen Inselzellen verglichen. Im Gegensatz zum WAT bewirkt die diabetogene HF-Diät in Inselzellen einerseits, eine erhöhte Expression von nukleären Genen für die oxidative Phosphorylierung und andererseits von Genen, die mit Zellproliferation assoziiert sind. Zudem wurden 37 bereits annotierte Gene identifiziert, deren differenzielle Expression mit der Diabetesentwicklung korreliert. Das Peptidhormon Cholecystokinin (Cck, 11,8-fach erhöht durch die HF) stellt eines der am stärksten herauf regulierten Gene dar. Die hohe Anreicherung der Cck-mRNA in Inselzellen deutet auf eine bisher unbekannte Funktion des Hormons in der Regulation der Inselzellproliferation hin. Der Transkriptionsfaktor Mlxipl (ChREBP; 3,8-fach erniedrigt durch die HF) stellt in Langerhansschen Inseln eines der am stärksten herunter regulierten Gene dar. Ferner wurde ChREBP, dessen Funktion als glucoseregulierter Transkriptionsfaktor für lipogene Enzyme bislang in der Leber, aber nicht in Inselzellen nachgewiesen werden konnte, erstmals immunhistochemisch in Inselzellen detektiert. Dies deutet auf eine neue, bisher unbekannte regulatorische Funktion von ChREBP im Glucosesensor-Mechanismus der Inselzellen hin. Eine durchgeführte Korrelation der mit der Diabetesentwicklung assoziierten, differenziell exprimierten Inselzellgene mit Genvarianten aus humanen genomweiten Assoziationsstudien für Typ-2-Diabetes (WTCCC, Broad-DGI-T2D-Studie) ermöglichte die Identifizierung von 24 neuartigen Diabetes-Kandidatengenen. Die Ergebnisse der erstmals am polygenen NZO-Mausmodell durchgeführten genomweiten Expressionsuntersuchungen bestätigen bisherige Befunde aus Mausmodellen für Adipositas und Diabetes (z.B. ob/ob- und db/db-Mäuse), zeigen in einigen Fällen aber auch Unterschiede auf. Insbesondere in der oxidativen Phosphorylierung könnten die Ergebnisse relevant sein für das Verständnis der Pathogenese des polygen-bedingten humanen metabolischen Syndroms. / Overweight and obesity cause insulin resistance and increase the risk of developing type 2 diabetes and cardiovascular diseases. Both, obesity and susceptibility to diabetes, are to a major part genetically predisposed. The relevant genes, their interaction with the environment – especially with food components – and the pathomechanisms causing insulin resistance and diabetes are not fully known yet. In the present study the development and progression of obesity and type 2 diabetes should be investigated by the means of gene expression analyses of the white adipose tissue (WAT) and the islets of Langerhans to identify underlying pathomechanisms and new causative candidate genes. For this purpose diet intervention studies on NZO- and related NZL-mice – two polygenic mouse models for the human metabolic syndrome – were performed. A carbohydrate containing high fat-diet (HF: 14.6 % fat) caused early obesity, insulin resistance and type 2 diabetes in both mouse models. A fat reduced standard chow (SD: 3.3 % fat) which strongly delayed the onset of obesity and diabetes, and a diabetes protective carbohydrate free high fat-diet (CHF: 30.2 % fat) served as control diets. Using microarray technology genome wide expression profiles of the WAT were generated. Pancreatic islets were isolated by the means of laser capture microdissection (LCM) and expression profiles of them were created, too. Differentially expressed genes were validated by quantitative real time PCR. The HF-diet reduced the expression of nuclear genes of the oxidative phosphorylation and lipogenic enzymes in the WAT of the NZO-mouse. This suggests an inadequate storage and utilization of fat in these animals. This is specific for the obese NZO-model and wasn’t observed for the lean SJL-mouse, indicating a role in the development of insulin resistance. Additionally, there was proof that the enlargement of the WAT triggers a retarded infiltration of macrophages into the WAT and there a local immune response. Moreover, the LCM technique was established and used for the isolation of highly enriched RNA from islets of Langerhans. For the first time the influence of carbohydrates in a high fat-diet on the expression profile of pancreatic islets was investigated by the use of genome wide expression analyses at an early time point at the onset of diabetes. Contrary to the WAT the diabetogenic HF-diet in islets cells increased the expression of both nuclear genes coding for the oxidative phosphorylation and genes associated with cell proliferation. Furthermore 37 already annotated genes correlated with diabetes progression were identified. The peptide hormone cholecystokinin (Cck: 11.8-fold enriched by the HF-diet) is one of the most up-regulated genes. The strong enrichment of Cck-mRNA in islets suggests a previously unknown function of the hormone in the regulation of the islet cell proliferation. The transcription factor ChREBP (Mlxipl: 3.8-fold reduced by the HF-diet) is one of the most down-regulated genes in the islets of Langerhans. Moreover, ChREBP, which has been already identified as a glucose regulated transcription factor for lipogenic enzymes in the liver but not in islets of Langerhans, was detected for the first time in islet cells, using immunohistochemistry. This points to an until now unknown regulatory function of ChREBP in the glucosesensor mechanism of the islet cells. Correlation of the differentially expressed genes associated with diabetes progression with gene variants from human genome wide association studies for type 2 diabetes (WTCCC, Broad-DGI-T2D-study) made the identification of 24 new diabetes candidate genes possible. The results of the genome wide expression analyses, which were done for the first time on a polygenic mouse-model, corroborated previous results for monogenic mouse-models for obesity and diabetes (e.g. ob/ob- and db/db-mice), however also demonstrated differences in some instances. Especially the results concerning the oxidative phosphorylation could be relevant for the comprehension of the pathogenesis of the polygenic human metabolic syndrome.

Primary aldosteronism is the most common secondary cause of hypertension with a prevalence of about 10%. About half of PA cases are caused by aldosterone-producing adenomas (APA). Two APA subtypes, ZG-like and ZF-like APAs, have been described, according to the histological resemblance to normal zona glomerulosa (ZG) and zona fasciculata (ZF), underlying somatic mutations (KCNJ5 commonly found in ZF-like, CACN1AD, ATP1A1, ATP2B3, CTNNB1 in ZG-like APAs), and transcriptome profile. It is unknown if the process of tumorigenesis differs between ZG- and ZF-like APAs. In order to define ZG specific genes, we have compared the transcriptome of APAs and their adjacent adrenal glands by microarray assay. RNA was isolated by laser capture microdissection (LCM) from adjacent ZG, ZF and APAs from 14 patients with Conn’s and 7 patients with phaeocromocytoma. Two top hit genes from the comparison of ZG vs ZF were functionally studied, ANO4 and NEFM. NEFM, encoding neurofilament medium, was the fourth most up-regulated gene in ZG vs ZF, showing 14.8-fold-fold higher expression levels (p=9.16-12) in ZG than ZF. NEFM was also one of the most down-regulated genes in ZF-like vs ZG-like APAs. Immunohistochemistry (IHC) confirmed selective high expression of NEFM in ZG and ZG-like APAs. Silencing NEFM in H295R cells increased aldosterone secretion and cell proliferation. In addition, it increased stimulation and inhibition, respectively, of aldosterone secretion from H295R cells by the dopamine receptor D1R agonist fenoldopam and antagonist SCH23390. IHC showed predominantly intracellular staining for D1R in NEFM-rich ZG-like APAs, but membranous staining in NEFM-poor ZF-like APAs. Aldosterone secretion in response to fenoldopam in primary cells from ZG-like APAs was lower than in cells from ZF-like APAs. NEFM expression levels directly correlate with KCNJ5 phenotype: KCNJ5 mutations down-regulate NEFM mRNA and protein levels in H295R cells and in primary cells from ZG-like APAs. ANO4,encoding a Ca2+-activated chloride channel family member, was the third most upregulated gene, showing 19.9-fold higher expression levels (p=6.6x10-24) in ZG than ZF. IHC confirmed ZG selectivity of ANO4 protein in the adrenal cortex. The staining was mainly cytoplasmic. Unlike NEFM, there was no difference in expression of ANO4 between ZG- and ZF-like APAs, the levels being mid-way between those of ZF and ZG. Overexpression of ANO4 in H295R cells caused an increase in CYP11B2 and NR4A2 gene expression levels but basal aldosterone secretion was unchanged. In the presence of calcium agonists, ANO4 reduced aldosterone secretion. ANO4 subcellular localisation was confirmed as cytoplasmic by immunofluorescence microscopy of transfected cells. When exposed to calcium ionophores, ANO4 generated small chloride currents as detected by YFP assay. In summary, the comparison of transcriptome of ZG with paired ZF found unexpected up-regulated genes. Most of the highly up regulated genes in human ZG, including NEFM and ANO4, inhibit either basal or stimulated aldosterone secretion, and this may reflect an adaptive response to high salt intake. No clear-cut correspondence was found between transcriptome of APAs and their resembling zone of adrenal cortex. The down-regulation of NEFM following transfection of mutant KCNJ5 suggests that ZF-like properties may be a consequence of mutation, rather than tissue of origin.

Orientadores: Adriana Franco Paes Leme, Ricardo Della Coletta / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-28T02:02:25Z (GMT). No. of bitstreams: 1 Macedo_CarolinaCarneiroSoares_M.pdf: 3754068 bytes, checksum: 36ddb6796cb6690a37ce3e4e9447009c (MD5) Previous issue date: 2015 / Resumo: O carcinoma espinocelular oral (CEC) é o tipo de neoplasia maligna mais comum em cabeça e pescoço, com alta prevalência e morbidade. O tratamento é baseado em sistemas de classificação pouco precisos e o prognóstico é ruim em muitos casos. Diferentes padrões histológicos já foram descritos na tentativa de melhor compreender o curso da doença, predizer prognóstico e auxiliar no tratamento. As diferentes áreas do tumor apresentam características morfológicas e moleculares distintas resultando em comportamentos clínicos específicos, e estudos recentes apontam a região de invasão tumoral (do inglês invasive front tumor) como área de interesse para análises de perfil molecular e identificação de possíveis marcadores de prognóstico. O padrão de invasão neoplásico tem relação com a agressividade tumoral e a presença de ilhas no fronte invasivo já foi descrita como pior padrão. Assim, o objetivo desta dissertação foi comparar a composição diferencial de proteínas totais de ilhas neoplásicas grandes e pequenas do fronte e do interior do tumor, e correlacionar essas proteínas com o prognóstico. A proteômica foi associada à microdissecção a laser (ML), consideradas juntas como ferramentas de alta robustez para identificação de proteínas em tecidos neoplásicos em regiões específicas. Vinte peças cirúrgicas de CEC oral de língua fixadas em parafina foram submetidas a ML para obtenção das amostras compostas pelas seguintes regiões do tecido: 1) ilhas neoplásicas grandes da região frontal, 2) ilhas neoplásicas pequenas da região frontal, 3) ilhas neoplásicas grandes do interior do tumor e 4) ilhas neoplásicas pequenas do interior do tumor, seguida pela extração de proteínas e análise por cromatografia líquida acoplada à espectrometria de massas (LC-MS/MS). A anotação funcional das proteínas e a correlação aos dados clínico-patológicos dos pacientes foram realizadas. Foram identificadas 1906 proteínas totais, sendo 1480 proteínas comuns entre as quatros regiões estudadas. Duas proteínas foram exclusivas nas ilhas grandes do fronte e sete nas ilhas grandes do interior. Oitenta e cinco proteínas foram diferencialmente expressas entre a região do fronte e o interior tumoral, e destas, 57 foram relacionadas a dados clínico-patológicos importantes para o prognóstico. Os processos biológicos, como desenvolvimento da epiderme, adesão celular, apoptose, ciclo celular, degradação da matriz extracelular e expressão gênica, evidenciados entre as proteínas diferencialmente expressas confirmam as mudanças moleculares associadas à progressão neoplásica. A combinação de ML, MS e bioinformática foi capaz de identificar um painel de proteínas que podem auxiliar a desvendar o curso do CEC oral, predizendo agressividade e prognóstico. Em acréscimo, essa abordagem pode ajudar ainda na compreensão das diferenças e dos mecanismos de sinalização entre diferentes áreas do tecido / Abstract: Oral squamous cell carcinoma (SCC) is the most common type of malignant tumor in head and neck, with high prevalence and morbidity. Treatment is based on inaccurate classification systems and the prognosis is poor in many cases. Different histological patterns have been described in an attempt to better understand the course of the disease, predict prognosis and assist in treatment. Different areas of the tumor have different morphological and molecular characteristics resulting in specific clinical behaviors, and recent studies point to the region of tumor invasion as an area of interest for molecular profile analysis and identification of possible prognostic markers. The pattern of neoplastic invasion is related to tumor aggressiveness and the presence of islands in the invasive front has been described as worst invasion pattern. The objective of this work was to compare the protein differential expression of large and small islands neoplastic from the front and the inner tumor, and to correlate these proteins with prognosis. Proteomics was associated with laser microdissection (ML), and together they are considered robustness tools to identify proteins in neoplastic tissues in specific regions of interest. Twenty surgical specimens of oral tongue SCC fixed in paraffin were subjected to ML to obtain sample composed by the following regions of tissue: 1) large neoplastic frontal islands, 2) small neoplastic islands of the frontal region, 3) large neoplastic islands inside the tumor and 4) small islands within the neoplastic tumor, followed by extraction and analysis of proteins by liquid chromatography coupled to mass spectrometry (LC-MS/MS). The functional annotation of proteins and correlation with clinicopathological data from patients were performed. A total of 1906 proteins were identified, with 1480 common proteins between the four regions studied. Two proteins were exclusives in the large islands of the forehead and seven in large islands in the interior. Eighty-five proteins were differentially expressed between the front region and inner tumor, and of these, 57 were related to clinical and pathological data. The biological processes such as development of the epidermis, cell adhesion, apoptosis, cell cycle, disassembly of the extracellular matrix and gene expression, evidenced among the differentially expressed proteins confirmed the molecular changes associated with neoplastic progression. The combination of ML, MS, and bioinformatics was able to identify a panel of proteins that may help to unravel the course of oral SCC, predicting aggressiveness and prognosis. In addition, this approach may also help in understanding the differences and signaling mechanisms between different areas of the tumor tissue / Mestrado / Patologia / Mestra em Estomatopatologia

ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.

ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.

ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.

L’apport de fer est essentiel pour la plupart des organismes vivants, incluant la majorité des bactéries pathogènes. Cependant, le fer libre est toxique : il est lié à des protéines de stockage et de transport (e.g. ferritine, hémoprotéines…) et voit son homéostasie finement régulée. Afin d’extraire le fer de ces protéines, les bactéries utilisent divers systèmes tels que des protéines de surface ou encore des sidérophores. Bacillus cereus est une bactérie Gram-positive sporulante, pathogène opportuniste chez l’homme, 2ème cause en France de toxi-infection alimentaire collective. Chez B. cereus, la protéine de surface IlsA et le sidérophore bacillibactine (BB) sont impliqués dans l’acquisition du fer de la ferritine exogène et elles sont importantes pour l’infection de l’insecte modèle Galleria mellonella. Mes travaux présentaient deux parties : tout d’abord, l’étude de l’import du complexe BB-Fe3+ dans la cellule par FeuA, protéine de liaison de ce complexe à la surface de la bactérie, souligne le rôle central du couple BB-FeuA. La délétion des gènes codants pour ces deux molécules limite l’acquisition par B. cereus du fer de la ferritine, de l’hème, de l’hémoglobine et du fer inorganique in vitro. En revanche, elle présente un phénotype de virulence in vivo comparable à la souche de référence dans le cas d’injection intra-hémocœlique de larves de G. mellonella. Ce résultat surprenant suggère un probable rétrocontrôle sur l’expression de facteurs de virulence lorsque B. cereus ne produit ni BB ni FeuA, et se trouve par conséquent fortement carencé en fer. Le second volet de mes travaux s’intéresse à l’expression des gènes liés à l’homéostasie du fer in vivo, au cours de l’infection de l’intestin de larves de G. mellonella axéniques. Nous avons choisi une approche de type microgénomique, en prélevant les échantillons par microdissection laser, sur de façon à prélever de petits échantillons dans une zone définie, puis en analysant l’expression de quelques gènes ciblés par RT-qPCR et ddPCR à 3h et 16h post ingestion. Nos résultats montrent que : i) la colonisation intestinale de G. mellonella est impactée lorsque B. cereus est dépourvu du couple BB-FeuA ; ii) ilsA est exprimé lors de l’infection intestinale ; iii) les gènes ciblés impliqués dans l’homéostasie du fer sont activés dès le début de l’infection, suggérant un rôle dans l’adaptation et la pathogénicité ; iv) une faible modulation de l’expression est observée entre les deux temps. Ces travaux ouvrent de nouvelles connaissances fondamentales sur l’homéostasie du fer et des perspectives quant à l’utilisation de nouvelles techniques pour l’étude in situ des interactions hôte-pathogène. / Iron acquisition is essential for most living organisms, including many pathogenic bacteria. However, free iron is toxic: it is bound into storage or transport proteins (e.g. ferritin, hemoproteins…) and iron homeostasis is tightly regulated. To scavenge iron from these sources, bacteria possess several systems to acquire the bound iron, by surface proteins or siderophores. Bacillus cereus is a sporeforming Gram-positive bacterium, opportunistic human pathogen, 2nd cause of food-borne disease in France. It has been demonstrated that the B. cereus surface protein IlsA and the siderophore bacillibactin (BB) are involved in iron acquisition from ferritin and that these two molecules are important for infection of the insect model G. mellonella. My thesis project focused on two parts: first the study of the BB-Fe3+ complex import into the cell by the siderophore binding protein FeuA highlights the central role of both BB and FeuA. The deletion of the genes encoding for these two molecules limits iron acquisition by B. cereus from ferritin, heme, hemoglobin and inorganic iron in vitro. On the other hand, the virulence phenotype during intra-haemocelic infection of G. mellonella is similar to the Wild-type strain. These results suggest a possible feedback on the expression of virulence factor genes when B. cereus is unable to synthetize both BB and FeuA, and therefore are under high stress. The second part of my work focused on the expression of genes involved in iron homeostasis in vivo, during gut infection of germ-free larvae of G. mellonella. We chose to perform a microgenomic approach, using laser-capture microdissection to get small samples in targeted areas, and then analysing the expression of chosen genes by RT-qPCR and ddPCR at two time points post ingestion The results show that : i) the colonisation of G. mellonella gut is impacted when B. cereus is deprived of both BB and FeuA ; ii) ilsA is expressed during gut infection ; iii) iron homeostasis is involved in adaptation and pathogenicity from the early step of infection of the insect gut ; iv) only weak gene expression modulation occured between the two timepoints This work gives new fundamental knowledge about B. cereus iron homeostasis, and highlights the use of new techniques regarding the in situ study of host-pathogen interactions.

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Obwohl B-Zellen in der Pathogenese der Rheumatoide Arthritis (RA) eine wichtige Rolle spielen, ist über ihre Aktivierung und Differenzierung im Synovialgewebe (SG) nicht viel bekannt. Ein Merkmal von RA ist das Auftreten von Autoantikörpern (auto-AK). Trotz dessen sind bisher kaum Daten über den Einfluss des SG auf die auto-AK-Produktion und die Frequenz autoreaktiver synovialer B-Zellen bekannt. Diese Arbeit beschäftigt sich mit der Charakterisierung der synovialen B-Zell-Antwort und der Spezifität synovialer B-Lymphozyten. Dazu wurden B- und Plasmazellen (PC) aus dem Synovialgewebe von RA-Patienten mittels Mikrodissektion und Durchflusszytometrie isoliert und ihr Immunglobulin(Ig)-Repertoire bestimmt. Die Analyse der VH-Gene zeigte, dass sowohl naive als auch Gedächtnis-B-Zellen in das SG einwandern können. Ein Vergleich der VDJ-Rearrangements aus B-Zellen und PC belegte, dass hauptsächlich Gedächtnis-B-Zellen Antigen-abhängig aktiviert werden, klonal expandieren und zu PC differenzieren. Dabei können aktivierte B-Zellen ihre Ig-Klasse wechseln. Im Gegensatz dazu wurden nur rudimentäre Anzeichen somatischer Hypermutation nachgewiesen. Um die Spezifität synovialer B-Lymphozyten zu ermitteln, wurden rekombinante AK aus synovialen B-Zellen und PC generiert. Der Polyreaktivitätstest zeigte, dass naive B-Zellen aus dem SG einen hohen Anteil polyreaktiver Zellen besitzen. Im Gegensatz dazu ist die Frequenz von autoreaktiven Gedächtnis-B-Zellen und PC gegenüber naiven B-Zellen erhöht. Daneben konnten auch spezifische AK gegen bakterielle Antigene (insbesondere gegen Parodontitis-auslösende Bakterien) und gegen das Auto-Ag MCV identifiziert werden. Eine Affinitätsmessung des MCV-spezifischen Auto-AK zeigte, dass im SG sezernierte Auto-AK eine sehr hohe Affinität erreichen können. Die hier gewonnenen Daten verdeutlichen, dass B-Lymphozyten entscheidend an der Aufrechterhaltung oder gar Entstehung von RA beteiligt sind / Although B cells have an important impact on the pathogenesis of Rheumatoid arthritis (RA) still surprisingly little is known about their activation and differentiation within the inflamed synovial tissue (ST). A hallmark of RA is the presence of auto-antibodies (auto-Ab). However, still little is known about the frequency of self reactive synovial lymphocytes and it is unclear to which extent the inflamed ST contributes to auto-Ab production. These thesis deals with the characterization of the synovial B cell response and the specificity of synovial B lymphocytes. B and plasma cells (PC) from RA patients were isolated either by Laser Capture Microdissection or by FACS and their immunoglobulin(Ig)-repertoire was determined. The analysis of the VH-genes revealed that both naïve and memory B cells can immigrate the ST. A comparison of VDJ-rearrangements of B cells and PC showed that in ST without ectopic germinal centres mainly memory B cells become activated, expand clonally and differentiate into PC. During this process B cells can switch their Ig-class but do only hypermutate slightly. To determine the specificity of synovial B lymphocytes, recombinant Ab from synovial B cells and PC were generated. The polyreactivity assay showed that particularly naïve B cells were polyreactive. In contrast, the frequency of autoreactive memory B cells and PC was much higher than that of naïve B cells. In addition, Ab specific for bacterial antigens (especially for periodontal bacterias) and for the autoantigen MCV were identified. The affinity measurement of the MCV-specific autoantibody revealed that auto-Ab secreted in the ST can exhibit very high affinities. The data presented here show that B cells seem to play an important role in the maintenance and possibly the development of RA.

The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. Subplate plays a fundamental role in cortical development. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of corticofugal and thalamic axons. At later stages, these cells are involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. In my thesis, I aimed to further characterize the embryonic murine subplate by establishing a gene expression profile of this population at embryonic day 15.5 (E15.5) using laser capture microdissection combined with microarrays. I found over 250 transcripts with presumed higher expression in the subplate at E15.5. Using quantitative RT-PCR, in situ hybridization and immunohistochemistry, I have confirmed specific expression in the E15.5 subplate for 13 selected genes which have not been previously associated with this compartment. In the reeler mutant, the expression pattern of a majority of these genes was shifted in accordance with the altered position of subplate cells. These genes belong to several functional groups and likely contribute to the maturation and electrophysiological properties of subplate cells and to axonal growth and guidance. The roles of two selected genes - cadherin 10 (Cdh10) and Unc5 homologue c (Unc5c) - were explored in more detail. Preliminary results suggest an involvement of Cdh10 in subplate layer organization while Unc5c could mediate the waiting period of subplate corticothalamic axons in the internal capsule. Finally, I compared the expression of a selection of subplate-specific genes (subplate markers) between mouse and rat and found some surprising species differences. Confirmed subplate markers were used to monitor subplate injury in a rat model of preterm hypoxiaischemia and it appeared that deep cortical layers including subplate showed an increased vulnerability over upper layers. Further characterization of subplate-specific genes will allow us to broaden our understanding of molecular mechanisms underlying subplate properties and functions in normal and pathological development.