Transcription factors (TFs) are biologically important proteins that interact with transcription machinery and bind DNA regulatory sequences to regulate gene expression by modulating the synthesis of the messenger RNA. The regulatory sequences comprise of short conserved regions of a specific length called motifs . TFs have very diverse roles in different cells and play a very significant role in development. TFs have been associated with carcinogenesis in various tissue types, as well as developmental and hormone response disorders. They may be responsible for the regulation of oncogenes and can be oncogenic. Consequently, understanding TF binding and knowing the motifs to which they bind is worthy of attention and research focus. Various projects have made the study of TF binding their main focus; nevertheless, much about TF binding remains confounding. Chromatin immunoprecipitation in conjunction with deep sequencing (ChIP-seq) techniques are a popular method used to investigate DNA-TF interactions in vivo. This procedure is followed by motif discovery and motif enrichment analysis using relevant tools. Protein Binding Microarrays (PBMs) are an in vitro method for investigating DNA-TF interactions. We use a motif enrichment analysis tools (CentriMo and AME) and an empirical quality assessment tool (Area under the ROC curve) to investigate which method yields motifs that are a true representation of in vivo binding. Motif enrichment analysis: On average, ChIP-seq derived motifs from the JASPAR Core database outperformed PBM derived ones from the UniPROBE mouse database. However, the performance of motifs derived using these two methods is not much different from each other when using CentriMo and AME. The E-values from Motif enrichment analysis were not too different from each other or 0. CentriMo showed that in 35 cases JASPAR Core ChIP-seq derived motifs outperformed UniPROBE mouse PBM derived motifs, while it was only in 11 cases that PBM derived motifs outperformed ChIP-seq derived motifs. AME showed that in 18 cases JASPAR Core ChIP-seq derived motifs did better, while only it was only in 3 cases that UniPROBE motifs outperformed ChIP-seq derived motifs. We could not distinguish the performance in 25 cases. Empirical quality assessment: Area under the ROC curve values computations followed by a two-sided t-test showed that there is no significant difference in the average performances of the motifs from the two databases (with 95% confidence, mean of differences=0.0088125 p-value= 0.4874, DF=47) .

L'hyperplasie de la prostate (HBP) est l'une des affections les plus fréquentes de l'homme âgé. Les cliniciens utilisent le terme d'HBP pour décrire un syndrome clinique à trois composantes : l'hypertrophie prostatique bénigne, les symptômes du bas appareil urinaire, et l'obstruction sous vésicale. L'HBP peut être asymptomatique ou responsable d'une gêne urinaire, d'une rétention urinaire voire d'une insuffisance rénale obstructive. Histologiquement l'HBP est définie par une hyperplasie épithéliale et stromale de la zone de transition et de la zone périurétrale de la prostate. Le rôle des androgènes et de l'âge est connu dans le développement de l'HBP, mais les mécanismes moléculaires et cellulaires sous-sous-jacents restent largement méconnus. L'objectif de ce travail était de comprendre ces mécanismes pouvant conduire à la survenue de l'HBP, dans le but de développer éventuellement de nouvelles thérapeutiques. La première partie de ce travail a consisté à rechercher des modifications sur l'ADN extrait du tissu d'HBP en utilisant des SNP (Single Nucleotide Polymorphism) arrays. Cette étude a conclu à l'absence de larges zones de perte d'hétérozygoties, d'amplification ou de délétion sur l'ADN de l'HBP. La seconde partie de ce travail a consisté à analyser les profils d'expression génique de l'HBP sur des micropuces à ARN et à les comparer aux caractéristiques clinico-pathologiques des patients. Il a ainsi été observé un profil d'expression génique différent entre les prostates de volume inférieur à 60ml et celle supérieures à 60ml. Des voies de signalisation spécifiques telles que l'apoptose, les facteurs de croissance, les gène du cycle cellulaire et de l'inflammation, étaient significativement modifiées entre les deux groupes. Un des gènes identifiés, TGFBR2, était significativement sur-exprimé dans les grosses prostates. Une validation de ce résultat a été réalisée au niveau protéique sur tissu microarrays. Le niveau d'expression stromale de TGFBR2 était significativement supérieur dans les prostates de plus de 60ml que de moins de 60ml. En outre, le marquage épithélial de TGFBR2 était significativement associé à l'inflammation histologique de la prostate. Le rôle de l'inflammation dans l'HBP a été étudié également sur tissu microarrays par analyse cytologique de l'inflammation et marquage immunohistochimique. L'infiltrat inflammatoire tissulaire a été ainsi caractérisé avec précision. Il a été observé que les prostates ayant un score immunohistochimique d'inflammation élevé provenaient de patients ayant des prostates plus volumineuses et un score symptomatique plus élevé. Des travaux de recherche clinique ont aussi été effectués par le candidat. Ils ont apporté des éléments nouveaux concernant le rôle de l'HBP dans la prise en charge du cancer de prostate. Il a aussi été montré l'enjeu que constitue la prise en charge chirurgicale de l'HBP chez les patients toujours plus nombreux sous anti-thrombiques. Ce travail a conduit à une meilleure connaissance des mécanismes moléculaires et tissulaires de l'HBP : il a été clairement identifié que les profils d'expression génique de l'HBP variaient avec le volume prostatique, le rôle de TGFBR2 a été précisé et l'inflammation tissulaire a été caractérisée. L'HBP en pratique clinique a aussi été explorée dans ce travail notamment en ce qui concerne la prise en charge des patients sous antithrombiques. De nouvelles questions ont été soulevées appelant une poursuite des recherches : quel serait l'effet sur la croissance prostatique d'un blocage de TGFBR2 ? Quels sont les liens exacts entre TGFBR2 et l'inflammation ? Quels sont les niveaux d'expression protéique des nombreux autres gènes identifiés sur les puces à ARN ? / Benign prostate hyperplasia (BPH) is one of the most common proliferate disease affecting aging male. Clinicians use the term BPH to describe three components: benign prostatic enlargement, lower urinary tract symptoms, and bladder outlet obstruction. BPH can cause urinary bothersome, urinary retention, and even renal failure. BPH is histologicaly defined by hyperplesia of both epithelial and stromal component of the transition zone and the periurethral area of the prostate. Androgens and age are known to play a role in the development of BPH, but underlying molecular and cellular mechanisms are mostly unknown. The objective of this work was to understand mechanisms leading to BPH, with the aim of finding new therapeutic targets. The first part of thiswork aimed to find DNA changes on BPH using single nucleotide polymorphism arrays. We did not observe large loss of heterozygosity areas, neither amplification nor deletion on BPH DNA. The second part consisted in analyzing BPH gene expression profiles using RNA microaarays, and to compare them to patients' phenotypes. A different gene expression profile was observed between prostate larger than 60 ml and smaller ones. Several specific pathways including growth factors genes, cell cycle genes, apoptose genes, and inflammation genes, displayed major differences between small and large prostate glands. One of the genes we analized, TGFBR2, was significantly overexpressed in large prostate glands. A validation of what we analyzed at the RNA level was performed at the protein level using tissue microarrays. TGFBR2 stromal strainig was foung significantly associated with prostate volume. TGFBR2 epithelial staining was found significantly associated with inflammation. The role of inflammation was also analyzed using tissue microarrays. Inflammation in BPH was characterized. Patients whom BPH displayed high inflammation score tented to have higher prostate volume and higher symptom score. Clinical research works were also performed by the candidate. New elements were found concerning the role of BPH in the management of prostate cancer; some works focused on the surgical management of BPH patients on oral anticoagulation. This work allowed a better understanding of clinical management of BPH, as well as molecular and cellular mechanisms of BPH: it was clearly observed that BPH gene expression profile was related to prostate volume, the role of TGFBR2 was detailed, and inflammation infiltrate within BPH was characterized. More research is needed to find answers to other questions: what would be the effect of TGFBR2 blockage on prostate development? What are the connections between TGFBR2 and inflammation? What are protein expression levels of other genes we identified on RNA microarrays?

Microarrays on gold have been used to develop new methodologies for biocatalysis and proteomic applications. The technology applies the logic of solid phase supported chemistry using self-assembled monolayers (SAMs) on a gold chip. The advantages of this technology are: i) an easy to handle platform, ii) parallel screening (64 reactions at once), iii) microliter scale reactions (1µL per sample), iv the use of mild conditions (buffers and t=37 °C), v) the absence of purification steps (only chip washing is required), vi) quick and accurate analysis by MALDI ToF MS.The metallopeptidase thermolysin was studied in peptide-coupling reactions to profile reactivity and specificity (Chapter 2). Reactivity was further investigated in transpeptidation reactions. Comparing the serine peptidase chymotrypsin with the zinc dependent thermolysin, it was found that transpeptidations proceed via N-transacylation reactions independent of a specific enzymatic catalytic mechanism (Chapter 3). These transacylations may be exploited for modifications of biocompatible and selective surfaces in ‘bottom-up’ bionanofabrication technologies. Selected peptidases with different catalytic mechanism were also arrayed to investigate polymerisation ability of dipeptides (Chapter 4). It was shown that oligomerisation can be obtained under mild conditions and a set of peptides was synthesised. Chapter 5 describes a new chemical methodology by which crude tryptic peptide digests can be trapped on chip and analysed by MALDI ToF MS without further purification steps. This dramatically improves time and cost efficiency.Finally, a new stepwise native chemical ligation methodology is proposed for amino acids, and peptides containing N-terminal cysteine residues (Chapter 6).

A cutícula dos insetos é composta principalmente por uma variedade de proteínas que interagem com filamentos de quitina, um polímero de N-acetilglicosamina, para formar um envoltório rígido que protege e dá forma ao organismo. O crescimento dos insetos depende da renovação periódica da cutícula, que se desprende durante a apólise e é digerida enquanto a epiderme sintetiza uma nova cutícula substituta. Tal renovação caracteriza a muda e metamorfose e é coordenada por hormônios, com destaque para os ecdisteróides. O atual trabalho objetivou caracterizar a expressão diferencial de genes do tegumento (cutícula e epiderme subjacente), além de elucidar aspectos de regulação e função no contexto da muda e metamorfose, com foco nos genes codificadores de proteínas estruturais e enzimas cuticulares. Para este fim, utilizamos o tegumento de fases específicas da muda pupal-adulta, isto é, de pupas (Pw), de pupas em apólise (Pp) e de adultas faratas (Pbl) para análises de microarrays de cDNA. As análises dos microarrays mostraram 761 e 1173 genes diferencialmente expressos nos tegumentos de adultas faratas (Pbl) em comparação com pupas (Pw) ou pupas em apólise (Pp), respectivamente. A categorização destes genes, segundo os critérios do Gene Ontology, distinguiu totalmente o tegumento de adultas faratas (Pbl) dos tegumentos de pupas (Pw) ou pupas em apólise (Pp) tanto em relação ao critério Processo Biológico quanto em relação à Função molecular, evidenciando grande mudança na expressão gênica durante a construção do exoesqueleto definitivo nas adultas faratas (Pbl). Os microarrays mostraram aumento estatisticamente significante da expressão de 24 genes cuticulares no tegumento de adultas faratas. Este resultado foi validado por RT-PCR em tempo real (qRT-PCR) para 23 destes genes (AmelCPR3, AmelCPR4, AmelCPR6, AmelCPR14, AmelCPR15, AmelCPR17, AmelCPR23, AmelCPR24, AmelCPR25, AmelCPR28, AmelCPR29, AmelCPR30, apd-1, apd-2, apd-3, CPLCP1, Am-C, Am-D, AmelTwdl1, AmelTwdl2, GB12449, GB12811 e GB11550), e por RT-PCR semiquantitativa para o gene Amlac2. Além disto, a maior expressão de outros 2 genes cuticulares (AmelCPR1 e AmelCPR2) em adultas faratas foi demonstrada por qRT-PCR. Estes genes cuticulares positivamente regulados no tegumento de adultas faratas (Pbl) devem estar envolvidos com a formação e diferenciação do exoesqueleto definitivo. O aumento da expressão gênica neste período da muda (Pbl) é regulado pela variação do título de ecdisteróides e ocorre enquanto o título deste hormônio decai, após ter atingido o pico indutor da apólise na fase de desenvolvimento precedente (Pp). Ao contrário, as análises por qRT-PCR mostraram que 2 outros genes cuticulares (AmelCPF1 e AmelCPR1) são negativamente regulados no tegumento de adultas faratas em comparação com pupas, sugerindo que são específicos de cutícula pupal. Estes genes foram inibidos pelo aumento dos níveis de ecdisteróides, que induz a apólise. Vinte e um entre os 24 genes cuticulares diferencialmente expressos nos microarrays codificam proteínas pertencentes às famílias CPF, CPR, Apidermina, CPLCP, Análoga a peritrofina e Tweedle. Os outros 3 genes diferencialmente expressos (GB12449, GB12811, GB11550) não tinham sido ainda caracterizados como genes cuticulares. Dois deles, GB12449 e GB12811, foram sequenciados para validação da predição e para a caracterização das respectivas estruturas genômicas. Experimentos de hibridação in situ com sonda fluorescente (FISH) nos permitiram localizar altos níveis de transcritos destes genes no citoplasma de células da epiderme de adultas faratas, sugerindo fortemente sua natureza cuticular e envolvimento na construção do exoesqueleto definitivo. O presente estudo consiste na primeira análise global de expressão de genes do tegumento de uma espécie de himenóptero social. Os resultados apresentados levaram à identificação de genes com expressão associada à muda pupal-adulta e formação do exoesqueleto definitivo. Este trabalho contribui com novos dados moleculares para o aprofundamento do conhecimento da metamorfose de A. mellifera. / The insect cuticle is mainly composed of proteins that interact with chitin filaments to form a rigid structure that protects and shapes the organism. Insects grow through the periodic renewal of the cuticle, which is shed at each apolysis episode, and subsequently digested while the epidermis synthesizes the cuticle of the next stage. These molting events are coordinated by hormones, mainly ecdysteroids. The current work aimed to characterize differential gene expression in the integument (cuticle and underlying epidermis) during the ecdysteroid-regulated pupal-to-adult molt. Special attention was given to the structure and expression of genes encoding proteins and enzymes involved in cuticle formation and differentiation. To achieve these goals, we used thoracic integument of newly-ecdysed pupae (Pw), pupae in apolysis (Pp) and pharate adults (Pbl) in cDNA microarray analyses. The microarray analysis showed 761 and 1173 differentially expressed genes in the pharate adult integument (Pbl) in comparison to pupae (Pw) or pupae in apolysis (Pp), respectively. Gene Ontology terms for Biological Process and Molecular Function completely distinguished the integument of pharate adults (Pbl) from the integument of pupae (Pw) or pupae in apolysis (Pp). The microarray analysis discriminated 24 cuticular genes with a significant expression increase in the pharate adult integument. This was validated by real time RT-PCR analysis (qRT-PCR) for 23 of these genes (AmelCPR3, AmelCPR4, AmelCPR6, AmelCPR14, AmelCPR15, AmelCPR17, AmelCPR23, AmelCPR24, AmelCPR25, AmelCPR28, AmelCPR29, AmelCPR30, apd-1, apd-2, apd-3, CPLCP1, Am-C, Am-D, AmelTwdl1, AmelTwdl2, GB12449, GB12811 and GB11550), and by semiquantitative RT-PCR for Amlac2. In addition, the increased expression of other two cuticular genes (AmelCPR1 and AmelCPR2) was confirmed by qRT-PCR. These up-regulated cuticular genes in pharate adult integument apparently are involved in adult cuticle formation and differentiation, which occurs while the ecdysteroids titers decay, after reaching the peak that induces apolysis in the preceding phase (Pp). In contrast, two cuticular genes (AmelCPF1 e AmelCPR1) were confirmed by qRT-PCR analysis as negatively regulated in the integument of pharate adults compared to pupae, suggesting that they are specific to pupal cuticle. Therefore, these genes were inhibited by the increasing ecdysteroid levels that induce apolysis. Twenty one of the 24 cuticular genes differentially expressed in the microarrays encode proteins belonging to the CPF, CPR, Apidermin, CPLCP, Analogous to peritrofins and Tweedle families. The other three differentially expressed genes (GB12449, GB12811, GB11550) had not yet been assigned as cuticular genes. Two of them (GB12449 and GB12811) were sequenced, thus allowing prediction validation and gene structure characterization. In situ hybridization experiments using fluorescent probe (FISH) localized high expression of these genes in the pharate adult epidermis, strongly suggesting their involvement in the construction of the adult exoskeleton. This study is the first global gene expression analysis of the integument from a social hymenopteran species. The expression of genes in the integument was associated to the molting process and to the adult exoskeleton formation. This work contributes with new molecular data for a deeper understanding of A. mellifera metamorphosis.

Neste trabalho realizamos um programa de seqüenciamento em larga escala de cDNAs obtidos de bibliotecas construídas a partir de mRNA de células de B. emersonii submetidas ao choque térmico e ao estresse por cádmio. Obtivemos 6350 seqüências expressas (ESTs) de alta qualidade, que representam 2326 seqüências únicas putativas (unigenes) do fungo. Destes unigenes putativos, 1282 genes foram classificados em pelo menos uma das categorias do Consórcio Gene Ontology (GO). A análise do transcriptoma parcial de B. emersonii determinado até o momento permitiu a identificação de 78 unigenes codificando chaperones moleculares de todas as famílias conhecidas. Para avaliarmos a expressão global dos genes em resposta a estresses ambientais, como o choque térmico e o cádmio, realizamos ensaios de microarranjos de DNA nestas condições de estresse. Observamos que em resposta ao choque térmico, B. emersonii induz a expressão de genes que codificam proteínas relacionadas com o enovelamento de proteínas e com a proteólise, o que seria esperado em condições de temperaturas elevadas, assim como genes que codificam proteínas com propriedades antioxidantes, além de proteínas envolvidas no metabolismo de nucleotídeos e no metabolismo de carboidratos. Em resposta ao estresse por cádmio, verificou-se a indução de genes que codificam principalmente proteínas com propriedades antioxidantes, proteínas envolvidas no metabolismo de aminoácidos, proteínas relacionadas com o transporte celular e proteínas envolvidas no enovelamento de proteínas e proteólise. Uma das conseqüências do estresse por cádmio é o aumento do estresse oxidativo e proteínas antioxidantes têm um papel fundamental na resposta a este tipo de estresse. Dentre os genes observados durante o seqüenciamento das ESTs de B. emersonii, observamos dez genes codificando proteínas distintas da família Hsp70. Nove genes hsp70 são expressos em pelo menos um dos estágios do desenvolvimento do fungo e sete apresentam uma indução significativa após o choque térmico. Estes dados sugerem que estes genes desempenham um papel importante durante o desenvolvimento e em resposta ao estresse térmico em B. emersonii. Outro dado interessante obtido neste trabalho foi o enriquecimento de ESTs que continham íntrons em sua seqüência nas bibliotecas de estresse. Portanto, o choque térmico e o estresse por cádmio em B. emersonii diminuem a eficiência de processamento dos íntrons permitindo sua caracterização. O cDNA da proteína Hsp17 foi o que apresentou o maior número de ESTs seqüenciadas nas bibliotecas de estresse. Experimentos de Northern blot indicaram que o gene hsp17 possui um nível de expressão muito baixo durante o ciclo de vida de B. emersonii, no entanto, como esperado sua expressão aumenta drasticamente quando as células de esporulação ou germinação são submetidas a choque térmico. Os níveis da proteína Hsp17 acompanham os níveis do seu mRNA, indicando que o controle da expressão do gene hsp17 deve ocorrer em nível de transcrição. / In this work we realized a large scale, sequencing program of cDNAs libraries obtained from mRNA of B. emersonii cells submitted to heat shock and cadmium stress. A total of 6350 high quality expressed sequence tags (ESTs) were obtained, representing 2326 unique putative genes (unigenes) of this fungus. From these putative unigenes, 1282 genes were classified at least in one of the three Gene Ontology Consortium (GO) categories. The analysis of the partial transcriptome of B. emersonii, determined until now, allowed the identification of 78 unigenes encoding molecular chaperones of all known protein families. To evaluate the global expression of the genes in response to environmental stresses, such as heat shock and cadmium, DNA microarray assays were performed. We observed that in response to heat shock B. emersonii induces the expreession of genes encoding proteins related to protein folding and proteolysis, as expected under high temperature conditions, as well as genes encoding proteins with antioxidant properties and proteins involved in nucleotide and carbohydrate metabolism. In response to cadmium stress, we mainly verified the induction of genes for proteins with antioxidant properties, proteins involved in amino acid metabolism, proteins related to cellular transport and proteins related to protein folding and proteolysis. One of the consequences of the exposure to cadmium is the increase of oxidative stress, and antioxidant proteins have a fundamental role in the response to this kind of injury. Amongst the genes observed during the B. emersonii EST sequencing program, ten genes encoding distinct proteins from the Hsp70 family were observed. Nine of them are expressed at least in one stage of the fungus development and seven genes presented a significant induction during heat shock treatment. These data suggest that the hsp70 genes perform an important role during development and in response to heat stress in B. emersonii. Another interesting result from this work was the enrichment of ESTs containing introns in the stress libraries. Thus, heat shock and cadmium stress decrease the efficiency of intron processing in B. emersonii, allowing for intron characterization. The cDNA for the Hsp17 protein presented the highest number of ESTs sequenced from the stress libraries. Northern blot experiments indicated that the hsp17 gene is expressed at very low levels throughout the life cycle of B. emersonii, however, as expected its expression increases drastically when sporulation or germination cells are submitted to heat shock. Hsp17 protein levels accompany its mRNA levels, indicating that the control of expression of the hsp17 gene occurs at a transcriptional level.

O carcinoma de células renais (CCR) é o tumor mais agressivo que afeta o rim de pessoas adultas. O CCR é uma doença heterogênea, com diferentes alterações moleculares e variados patrões histológicos e clínicos que apresentam evolução diferente. Atualmente apenas variáveis anatomopatológicas clássicas são utilizadas para determinar o prognóstico dos pacientes. Utilizando uma plataforma de microarranjos de DNA, nosso grupo identificou em um trabalho anterior um conjunto de genes que se encontram diferencialmente expressos em tumores de rim. Neste estudo, nove candidatos foram selecionados para avaliação como marcadores de prognóstico no CCR. Foi confirmada a alteração na expressão dos genes ARNTL, ACTN4 e EPAS1 (p < 0,05) em amostras tumorais de CCR através de PCR em tempo real. Adicionalmente, foi observada a alteração da expressão dos genes ARNTL, EPAS1 e CASP7 em linhagens celulares imortalizadas derivadas de tumores renais, recapitulando por tanto, as alterações observadas nos tumores obtidos de pacientes. Posteriormente investigamos o padrão de expressão proteica destes candidatos por imunohistoquímica utilizando microarranjos de tecidos. Foi detectada a diminuição significativa (p < 0,05) da expressão das proteínas ACTN4, ARNTL, CASP7 e EPAS1 em tumores de pacientes com CCR relativamente ao tecido renal não tumoral. Além disso, foi possível determinar valores de imunomarcação capazes de estratificar pacientes com CCR em diferentes grupos de risco quanto à sobrevida câncer-específica, que adicionalmente apresentaram associação significativa com parâmetros anatomopatológicos utilizados na clínica. As imunomarcações de ACTN4, ARNTL, e EPAS1 se mostraram parâmetros independentes de prognóstico de sobrevida dos pacientes. A imunomarcação de CASP7 foi capaz de identificar subgrupos de pacientes com pior prognóstico dentro de um conjunto de pacientes de baixo risco em função do estadio clinico, além de identificar pacientes com menor risco de morte pelo câncer entre aqueles apresentaram recorrência em até 5 após a cirurgia. O conjunto de resultados obtidos aponta para um novo conjunto de biomarcadores moleculares com potencial relevância para auxiliar no prognóstico de pacientes com carcinoma de células renais. / The renal cell carcinoma (RCC) is the most aggressive tumor that affects the kidney in adult people. The RCC is a heterogeneous disease, with many different molecular alterations and varied histological and clinical patterns with different outcome. Currently, only classic anatomopathological variables are used to determine patients\' prognosis. Using a DNA microarray platform, our group identified in a previous work a set of genes differentially expressed in renal tumors. In this study, nine candidates were selected for evaluation as prognostic biomarkers in RCC. Alteration of the gene expression in RCC tumor samples was confirmed for ARNTL, ACTN4 and EPAS1 (p < 0.05) by real time PCR. Additionally, gene expression changes of ARNTL, EPAS1 and CASP7 were also observed in immortalized cell lines derived from renal tumors, recapitulating the expression changes detected in the patients\' tumors. Next, we used tissue microarrays to investigate the protein expression of the selected candidates by immunohistochemistry. Expression of the proteins ACTN4, ARNTL, CASP7 and EPAS1 was detected as significantly downregulated (p < 0.05) in patients´ tumors relative to non-tumor renal tissue. Furthermore, immunostaining patterns of the selected candidates were able to stratify patients with RCC in different risk groups according to cancer-specific survival, which also showed significant associations with anatomopathological parameters used in the clinics. ACTN4, ARNTL and EPAS1 immunostaining resulted as independent prognostic parameters of patient survival. CASP7 immunostaining was able to identify subgroups of patients with worse prognosis in a set of low risk patients as determined by their clinical stage, and also identified patients with lower risk of death from cancer amongst patients that relapsed within 5 years after surgery. Overall, these results point to a new set of molecular biomarkers with potential relevance to help in the prognosis of patients with renal cell carcinoma.

The PhD thesis is focused on a transcriptomics-based comparison of barley cultivars differing with respect to their low temperature acclimation capacity, with a particular focus on genes transcribed in the leaf and crown. The crown was of interest because of its importance for the winter survival of the plant. To involve both the first and the second phase of hardening, the test plants were exposed first to +3řC for 21 days, followed by - 3řC for one day. Freezing damage was assessed by measuring electrolyte leakage (Papers 2 and 3), using a modified version of a protocol developed by Prášil and Zámečník (1998). The same protocol was adapted to evaluate crown regrowth (Paper 2); for this purpose, the plants were cooled, then replanted and cut above the crown, and their survival rate calculated over the following week. Each RNA sample was queried by hybridization to an Affymetrix 22 K Barley1 GeneChip Genome Array (Close et al. 2004). The data were statistically analysed with the help of the software packages R, MAS 5.0 (Ihaka & Gentleman 1996) (Papers 2 and 3), Gene Spring GX 7.3 (Agilent Technologies, Santa Clara CA) and MapMan (Thimm et al. 2004; Usadel et al. 2005) (Paper 2), the "Self-Organizing Maps" algorithm (Kohonen et al. 1996) (Paper 3) and MIPS FunCat (Ruepp et al. 2004) (Paper 2). Paper...

In the field of protein research in general and the pharmaceutical industry in particular, it is now necessary to perform measurements of the secondary structure of proteins on many samples simultaneously, for instance to screen for molecules that stabilize proteins or to evaluate the action of multiple environmental conditions. In this context, we have proposed a new approach to evaluate the secondary structure of proteins on a very large scale (approximately 2000 to 4000 samples / cm2), by combining infrared imaging and 2D printing of protein microarrays. In view of the large amount of data, in a first step, methods for automating the extraction of spectra of interest from microarray infrared images and for automating the processing of the spectra were developed. Since the estimation of the secondary structure from infrared spectra is based on the construction of prediction models by chemometric methods, a relevant set of proteins for calibration was mandatory. A protein bank consisting of 92 commercially available proteins, the structure of which was well characterized by X-ray crystallography, was established for this purpose. After the development of predictive models for secondary structure determination and the validation of the protein microarray approach, we tried to optimize the models to improve the secondary structure prediction by different approaches as secondary structure definition, partial deuteration or subtraction of side chain contribution to the spectra. On the other hand, dealing with non-native structures not present in the reference protein library was a challenge. We took the opportunity to analyze the structural modifications of a subset of our protein library subjected to moderate denaturation conditions. Multivariate curve resolution-alternating least squares (MCR-ALS) was used to model a new spectral component appearing in the protein set subjected to denaturing conditions, which could represent a potential spectroscopic marker of aggregation and could allow a semi-quantitative evaluation of the aggregation. While the assessment of secondary structure was well established in the first part of this work, tertiary structure and stability are also critical. Hydrogen / deuterium exchange (HDX) is a potential approach for studying the structure and dynamics of proteins. In the last part of this work, we built a device which allowed to follow the HDX exchange kinetics simultaneously on the entire microarray. In conclusion, protein microarray FTIR imaging opens the door to high throughput analysis of protein secondary structure without any labelling and would allow better understanding of three-dimensional structure and dynamics of proteins through recording HDX curves. / Dans le domaine de la recherche sur les protéines et de l'industrie pharmaceutique, il s’avère désormais nécessaire d'effectuer des mesures de la structure secondaire des protéines sur de nombreux échantillons simultanément, de cribler des molécules qui stabilisent les protéines, ou d'évaluer l'action de multiples conditions environnementales. Dans ce contexte, nous avons proposé une nouvelle approche pour évaluer la structure secondaire des protéines à très grande échelle (environ 2 000 à 4 000 échantillons / cm2), en associant l'imagerie infrarouge et l'impression 2D de damiers de protéines. Dans un premier temps, des méthodes d'automatisation de l'extraction des spectres d'intérêt à partir des images infrarouges des damiers et d'automatisation des spectres ont été développées. L'estimation de la structure secondaire à partir des spectres infrarouges étant basée sur la construction de modèles de prédiction à partir de méthodes chimiométriques, un ensemble pertinent de protéines pour l'étape de calibration était obligatoire. Une banque de protéines constituée de 92 protéines disponibles dans le commerce, dont la structure était bien caractérisée par cristallographie aux rayons X, a été constituée dans ce but. Après élaboration des modèles prédictifs de la structure secondaire et la validation de l'approche des damiers de protéines, nous avons tenté d'optimiser les modèles pour améliorer les prédictions de structure secondaire par différentes approches. D'autre part, traiter des protéines présentant une structure jamais rencontrée dans les structures natives de notre bibliothèque de protéines de référence constituait un défi. Nous avons saisi l'opportunité d'analyser les modifications structurales d'un sous-ensemble de notre bibliothèque de protéines, caractérisé par un contenu structurel secondaire très différent en le soumettant à des conditions de dénaturation modérées La méthode de résolution de courbes multivariées des moindres carrés alternés (MCR-ALS) a été utilisée pour modéliser une nouvelle composante spectrale apparaissant dans l'ensemble protéique soumis à des conditions dénaturantes, et a permis de révéler un marqueur spectroscopique potentiel d'agrégation protéique permettant une évaluation semi-quantitative de son contenu. Alors que l’évaluation de la structure secondaire a été bien établie dans la première partie de ce travail, la structure tertiaire et la stabilité sont également critiques. L'échange hydrogène / deutérium (HDX) est une approche potentielle pour l’étude de la structure et de la dynamique des protéines. Dans la dernière partie de ce travail, nous avons construit un dispositif qui a permis de suivre la cinétique d’échange HDX simultanément sur l'ensemble d’un damier. En conclusion, l'imagerie FTIR de damiers de protéines ouvre la porte à une analyse à haut débit de la structure secondaire des protéines et permettrait de mieux comprendre la structure et la dynamique tridimensionnelles grâce à l'enregistrement des courbes HDX. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

While development in flies is well understood, pattem formation and the evolution thereof in arachnids have yet to be clarified. Flies and other metazoans primarily use two families of genes called Hox genes and Pax genes to regulate embryogenesis. Because of the high evolutionary conservation of Hox and Pax proteins, I hypothesize that arachnids also use this system to organize their body pattern. To enable studies of the Westem black widow spider, Latrodectus hesperus, an embryonic eDNA library and a fixation protocol were developed for L. hesperus embryos. The generation of these tools will allow comprehensive analysis of black widow spider development and give insight into whether, and how, spiders use Hox and Pax genes to organize their bodies. Finally, it will provide a more thorough understanding of how different developmental mechanisms have evolved and ultimately how changes in gene expression can lead to a change in overall body plan.

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