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Chickens from lines that have been divergently selected for either low (LWS) or high (HWS) body weight at 56 days of age for more than 57 generations serve as unique models to study eating disorders. The LWS have different severities of anorexia while all HWS become obese. Over the past decade our groups has demonstrated that these lines have differential food intake threshold responses to a range of intracerebroventricular (ICV) injected neurotransmitters. The major brain region regulating homeostatic regulation of appetite is the hypothalamus, and hence this dissertation was focused on understanding how the hypothalamus is different between LWS and HWS lines. Experiments 1 and 2 were performed as follows: whole hypothalamus as well as individual hypothalamic nuclei, respectively, were collected from 5 day-old chicks that had been fasted for 180 min or had free access to food. The hypothalamic nuclei included those primarily associated with appetite including the lateral hypothalamus, paraventricular nucleus (PVN), ventromedial hypothalamus, dorsomedial nucleus, and arcuate nucleus (ARC). Total RNA was isolated, reverse transcribed, and real time PCR performed. Hypothalamic expression of anorexigenic factors was greater in LWS than HWS, those factors including calcitonin, corticotropin-releasing factor receptor 1, leptin receptor, neuropeptide S, melanocortin receptor 3 (MC3R), and mesotocin. The gene expression data from individual hypothalamic nuclei revealed that mesotocin from the PVN may play an important role in the inhibition of appetite in the LWS. Experiment 3 was then designed to evaluate the effects of stress on food intake: besides the differences in hypothalamic gene expression between the lines, they also have different feeding responses when stressed: ICV injection of neuropeptide Y (0.2 nmol, NPY) did not increase food intake in LWS on day 5 after stress exposure. Experiment 4 was thus designed to study the molecular mechanisms underlying conditional feeding responses to exogenous NPY after stress in the LWS. The melanocortin system (AgRP and MC3R) changed in the hypothalamus after stress in the LWS, and hence may be responsible for the loss of responsiveness to exogenous NPY in stressed LWS. Experiment 5 was designed to evaluate whether hypothalamic differences exist at the protein level: label-free liquid chromatography coupled to tandem-mass spectrometry was used to measure the abundance of proteins in the hypothalamus. Hypothalamus was obtained from fed and 180 minute-fasted 5 day-old male LWS and HWS chicks. Proteins involved in energy metabolism were different between the lines. Differences were also found in proteins involved in GABA synthesis and uptake as well as protein ubiquitination. In conclusion, these results suggest that different feeding behaviors of LWS and HWS may be due to differences in gene and protein expression in the hypothalamus. / Ph. D.

In this study, the soluble protein fraction of P. chrysosporium grown under standard conditions was analyzed by using 2D-PAGE approach and a 2-D reference map was constructed. 910 spots could be separated and detected on Coomassie-stained 2-D gels by the help of Delta2D image analysis software. 720 spots could be cut from the master gel and were subjected to MALDI-TOF MS analysis followed by MASCOT search. A total of 517 spots out of 720 were assigned to specific accession numbers from the P. chrysosporium genome database. Further analysis of the data revealed 314 different gene products (distinct ORFs). The theoretical pI and MW values were plotted against the experimental migration distances. Results indicated the existence of 124 protein spots whose horizontal migration differed significantly from the expected migration according to the calculated pI values and 52 spots with an apparent molecular weight that is significantly different from their theoretical molecular weight. While protein modification could be predicted by these analyses, the main support was the presence of multiple spots of the same gene product. As much as 118 ORFs yielded multiple spots on the master gel, corresponding to 37.5% of the all distinct ORFs identified in this work. The relative abundance of each of the 517 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be housekeeping ones. When the relative distribution of the proteins into four main functional categories was taken into consideration, &ldquo / Metabolism&rdquo / appeared the most important category with a share of 50.6% among identified proteins. However, among the functional classes, &ldquo / Posttranslational modifications, protein turnover, chaperones&rdquo / which is listed under the main category &ldquo / Cellular Processing and Signalling&rdquo / was represented by the highest number (104) of the identified proteins. Only 6 of the proteins listed in this study were assigned to hypothetical proteins. Out of the 314 identified gene products shown in P. chrysosporium, 29 were predicted to have a signal peptide sequence according to the SignalP algorithm. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, 147 of the proteins were predicted to be located in cytoplasm. The phosphorylated proteins of P. chrysosporium were detected by ProQ phosphoprotein staining of the 2-D gel. 380 out of 910 distinct protein spots (40%) were found to be phosphorylated in exponentially growing cells of P. chrysosporium. Of these spots, 96 could be matched to the identified proteins.

Made available in DSpace on 2014-12-17T14:05:20Z (GMT). No. of bitstreams: 1 VivianeKSM_TESE.pdf: 2717769 bytes, checksum: dc3d156a1c1b3c1791c93a0e2266556f (MD5) Previous issue date: 2011-12-13 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Chromobacterium violaceum is a free-living bacillus, Gram-negative commonly found in water and sand of tropical and subtropical regions. One of its main characteristic it's the ability to produce the purple pigment named violacein, that shows countless biological activities. In 2003, the genome of this organism was totally sequenced and revealed important informations about the physiology of this bacteria. However, few post-genomics studies had been accomplished. This work evaluated the protein profile of C. violaceum cultivated in LB medium at 28?C that allowed the identification and characterization of proteins related to a possible secretion system that wasn't identified and characterized yet in C. violaceum, to the quorum sensing system, to regulatory process of transcription and translation, stress adaptation and biotechnological potential. Moreover, the response of the bacteria to UVC radiation was evaluated. The comparison of the protein profile, analyzed through 2-D electrophoresis, of the control group versus the treatment group allowed the identification of 52 proteins that arose after stress induction. The obtained results enable the elaboration of a stress response pathway in C. violaceum generated by the UVC light. This pathway, that seems to be a general stress response, involves the expression of proteins related to cellular division, purine and pirimidine metabolism, heat chock or chaperones, energy supply, regulation of biofilm formation, transport, regulation of lytic cycle of bacteriophages, besides proteins that show undefined function. Despite the response present similarities with the classic SOS response of E. coli, we still cannot assert that C. violaceum shows a SOS-like response, mainly due to the absence of characterization of a LexA-like protein in this organism / Chromobacterium violaceum ? um bacilo de vida-livre, Gram-negativo comumente encontrado no solo e nas ?guas de regi?es tropicais e subtropicais. Uma das principais caracter?sticas deste organismo ? sua capacidade de produzir o pigmento violace?na, o qual apresenta in?meras atividades biol?gicas. Em 2003, o genoma deste organismo foi completamente sequenciado e revelou informa??es importantes sobre a fisiologia desta bact?ria. Por?m, poucos estudos p?s-gen?micos tem sido realizados. Este trabalho avaliou o perfil proteico de C. violaceum cultivada em meio LB a 28?C, o que permitiu a identifica??o de prote?nas relacionadas a um poss?vel sistema de secre??o ainda n?o identificado e caracterizado em C. violaceum, ao sistema quorum sensing, a processos regulat?rios da transcri??o e tradu??o, adapta??o ao estresse e ao potencial biotecnol?gico. Al?m disso, a resposta desta bact?ria ? radia??o UVC foi avaliada. A compara??o do perfil prot?ico, analisado por eletroforese 2-D, do controle versus tratado possibilitou a identifica??o de 52 prote?nas que surgiram ap?s a indu??o do estresse. Os resultados obtidos permitiram a elabora??o de uma via de resposta de C. violaceum ao estresse gerado pela luz UVC. Esta via, que parece ser de resposta geral ao estresse, envolve a express?o de prote?nas relacionada ? divis?o celular, metabolismo de purinas e pirimidinas, choque t?rmico ou chaperonas, fornecimento de energia, regula??o da forma??o de biofilme, transporte, regula??o do ciclo l?tico de bacteri?fagos, al?m de prote?nas que ainda n?o apresentam fun??o caracterizada. Apesar da reposta apresentar similaridades com a SOS cl?ssica de E. coli, ainda n?o podemos afirmar que C. violaceum apresenta uma resposta SOS-like, principalmente devido a aus?ncia da caracteriza??o de um prote?na LexA-like neste organismo

Les bactéries du genre Deinococcus sont extrêmement tolérantes à de fortes doses de radiations. Des études antérieures ont montré que IrrE est nécessaire à la radiotolérance et à l'induction des gènes de réparation de l'ADN après exposition des cellules à l'irradiation. Pendant des années il est resté inconnu comment IrrE active l'expression de ces gènes. L'objectif de ma thèse était la caractérisation de la voie de signalisation dépendent de IrrE chez Deinococcus deserti. Pour cela, des approches biochimiques et génétiques ont été utilisées. Les premiers résultats ont fortement suggéré que IrrE agit indirectement sur l'activation de l'expression des gènes. En utilisant des expériences in vitro et in vivo, nous avons montré que IrrE de Deinococcus deserti interagit avec DdrO, un régulateur potentiel qui est codé par un gène radio-induit et qui est, comme IrrE, conservé chez les Deinococcus. De plus, IrrE clive DdrO in vitro mais aussi in vivo lorsque les deux protéines sont co-exprimées chez Escherichia coli. Ce clivage est abolit en présence d'un agent chélateur de métaux, l'EDTA. Chez D. deserti, le clivage de DdrO dépendent de IrrE a été observé mais seulement après exposition à l'irradiation. En parallèle, nous avons montré que la répression du promoteur d'un gène radio-inductible est dépendante de DdrO. Nos résultats montrent donc que IrrE est une métalloprotéase et nous proposons que le répresseur DdrO soit désactivé après clivage par IrrE conduisant à l'induction de différents gènes indispensables pour la réparation de l'ADN et la survie des cellules après exposition de Deinococcus à l'irradiation. / Deinococcus bacteria are famous for their extreme tolerance to high doses of radiation. Earlier studies have shown that IrrE protein is required for radiation tolerance and for induction of DNA repair genes after exposure of cells to radiation. However, for years it has remained unknown how IrrE activates gene expression. The aim of my thesis was to characterize the IrrE-dependent regulation pathway in Deinococcus deserti. For this, biochemical and genetic approaches were used. The first results strongly suggested that IrrE activates gene expression in an indirect manner. Then, using other in vivo and in vitro experiments, IrrE from Deinococcus deserti was found to interact with DdrO, a predicted regulator encoded by a radiation-induced gene that is, like irrE, highly conserved in Deinococcus. Moreover, IrrE was found to cleave DdrO in vitro and also in vivo when the proteins were co-expressed in Escherichia coli. This cleavage was not observed in the presence of the metal chelator EDTA. In D. deserti, IrrE-dependent cleavage of DdrO was observed only after exposure to radiation. Furthermore, DdrO-dependent repression of the promoter of a radiation-induced gene was shown. Our results demonstrate that IrrE is a metalloprotease and we propose that IrrE-mediated cleavage inactivates repressor protein DdrO, leading to transcriptional induction of various genes required for DNA repair and cell survival after exposure of Deinococcus to radiation.

Animal models have been recognized for the valuable roles they serve in both animal and human medicine. Dogs share many of the same naturally occurring tumors as humans including osteosarcoma, lymphoma, and mammary tumors. In addition, dogs share the same environment as humans, have a shorter lifespan, and often have a quicker progression of disease, making them an attractive model of human disease. Platelets are small anucleate cell fragments that have essential roles in hemostasis, angiogenesis, and wound healing, and, more recently recognized, roles in development, survival, growth, and metastasis of various cancers. Their roles in angiogenesis has proven to be both directly and indirectly linked to tumor growth due to the angiogenic roles they play in the development of tumor blood supply. Being able to study the interactions and mechanisms between platelets and tumor cells at the protein level, through proteomics, would allow great insight into the effects of platelets on tumor cell behavior as well as potential biomarker identification and therapeutic development. The objective of this research is to integrate the roles of canine platelet proteins into a better understanding of the effects and interactions that platelets have with different tumor cells while utilizing the canine model of neoplasms commonly affecting their human counterparts. The first study in this research describes an efficient technique developed for the purification of canine platelets from clinically relevant volumes of whole blood with high platelet recovery and minimal contamination from other blood cells. The second study describes a non-electrophoretic detergent fractionation technique used for the digestion of canine platelet samples for proteomic analysis as well as description of the proteomic findings for the normal canine platelet. Lastly, the third study describes the proteomic analysis of proteins differentially expressed by canine osteosarcoma and mammary tumor cells following incubation with canine platelet lysate in vitro. Overall, findings of this research support the canine model of human cancers and provide comprehensive information regarding canine platelet proteomics as well as novel efficient techniques that aid the future of canine platelet-tumor cell interaction research

Résumé : INTRODUCTION : Les protéines doivent se retrouver dans le bon compartiment cellulaire afin de pouvoir exercer leurs fonctions. Une fois synthétisées dans le cytoplasme, les protéines se déplacent à travers les différents compartiments cellulaires, guidés par des signaux de ciblage protéique. Des modifications post-traductionnelles jouent un rôle dans la régulation et le contrôle dynamique de la localisation des protéines. Des exemples dans la littérature indiquent que cette régulation existe également au niveau pré-traductionnel. Ce projet cherche à investiguer cette régulation pré-traductionnelle sur les motifs d’import et d’export nucléaires choisi comme signaux modèles. A l’aide de la bio-informatique, nous caractérisons cette régulation pré-traductionnelle, pour ensuite analyser de façon quantitative le niveau du contrôle de l’inclusion de ces motifs dans les transcrits en considérant des données de RNA-seq. MÉTHODE : Des données du transcriptome humain ont été regroupées dans une base de données locale MySQL. Une curation extensive de bases de données publiques a permis d’identifier les motifs d’import et d’export nucléaires, validés expérimentalement. Des scripts dans les langages Python et PHP ont été créés afin d’interroger la base de données et d’implémenter les algorithmes. Par la suite des ensembles de données de RNA-seq ont été analysés pour quantifier le niveau d’inclusion de ces motifs. RÉSULTATS : La majorité de ces motifs varie pour les gènes dont seulement certaines isoformes contiennent ce motif. Nous avons pu déterminer la distribution de la position des motifs chez l’humain, et caractériser quatre différents types de régulation pré-traductionnelles pour ces motifs. Ces catégories sont : les sites d’initiation et de terminaison différentiels de transcription /traduction, l’épissage (d’introns/d’exons) et le décalage du cadre de lecture. L’index d’inclusion du motif (MII) varie pour les gènes à travers différents tissus d’un même ensemble de données et varie également dans des tissus cancéreux. Certains gènes produisent des isoformes dont le MII est tissu-spécifique. CONCLUSION : La régulation pré-traductionnelle de l’inclusion de motifs de ciblage d’import et d’export nucléaires joue un rôle important sur la localisation de la protéine résultante. Les données de RNA-seq ont abouti à une analyse quantitative sur ces motifs dans différents tissus normaux et cancéreux. / Abstract : INTRODUCTION: Proteins have to be in the right compartment in order to perform their functions. Once synthesized in the cytosol, proteins are guided by sorting signals as they move through different subcellular compartments. Post-translational modifications play a role in the regulation and dynamic control of protein localization. Some examples in the literature show that this regulation also exist at the pre-translational level. The purpose of this research is to investigate this regulation on nuclear import and export motifs, chosen as model signals. Using bioinformatics, we characterize this pre-translational regulation then using RNA-seq data, perform a quantitative analysis on the level of motif inclusion among transcripts. METHODS: Data of the human transcriptome was put in MySQL in house. For this study, we used experimental data. Extensive manual curation was performed on nuclear import and export motifs that were experimentally validated and obtained from public databases. In order to interrogate the database and implement the algorithms, scripts in Python and PHP were used. RNA-seq data were used and analyzed in order to quantify the level of inclusion of these motifs. RESULTS: The majority of these motifs are only present in a subset of the coding isoforms of the genes. The position distribution of these motifs in the human proteome was determined. We characterized four different types of pre-translational regulation for alternative motifs. The categories a re: differential initiation and termination sites of transcription/translation, splicing (of introns/exons) and frameshift mutation. Genes have a motif inclusion index (MII) that varies among different types of tissues within the same dataset and varies as well in cancer tissues. Some genes produce isoforms with MII that are tissue-specific. CONCLUSION: Pre-translational regulation plays an important role in the inclusion of nuclear import and export motifs and the localization of proteins containing them. Quantitative analyses showing the behaviour of the motifs in different types of normal and cancer tissues were performed with RNA-seq data.

In this dissertation, mesoporous silica thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway, with the aim of specifically harvesting the low molecular weight peptides and proteins from human serum, which has been regarded as a potential source of diagnostic biomarkers for the early detection of disease. The superior properties of mesoporous silica have been demonstrated in applications which include chemical sensing, filtration, catalysis, drug-delivery and selective biomolecular uptake. These properties depend on the architectural, physical and chemical properties of the materials, which in turn are determined by the processing parameters in evaporation-induced self-assembly (EISA). Using the different polymer templates and polymer concentration in the precursor solution, various pore size distributions, pore structures and surface hydrophilicities were obtained and applied for nanotexture-selective recovery of low mass proteins. With the assistance of mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin film and the specificity and efficacy of low mass proteome harvesting. In addition, to overcome the limitations of the pre-functionalization method in polymer selection, plasma ashing was used for the first time for the treatment of the mesoporous silica surface prior to chemical modification. Opposite surface charges due to the different functional groups used, resulted in a distinctive selectivity of the low molecular weight proteins from the serum sample. The mesoporous silica chips operate with extraordinary rapidity, high reproducibility, no sample pre-processing, and substantial independence from sample acquisition and storage temperature.In conclusion our study demonstrates that the ability to tune the physicochemical properties of mesoporous silica surfaces has the potential to promote the use of this material as a tool for the selective separation and concentration of the low molecular weight proteome from complex biological fluids. / text

Avec une consommation de 19600 milliers de tonnes équivalent carcasses en 2005, la viande de porc est la plus consommée en Europe. Elle montre, toutefois, une forte variabilité, en partie due à des variations dans le métabolisme énergétique musculaire post-mortem. Celui-ci est défini comme l'ensemble des réactions biochimiques et leurs interactions survenant dans le muscle au cours de la période allant de la mort de l'animal jusqu'à la préparation de la viande avant consommation. De nombreux facteurs, dépendant de l'animal (génétique, mode d'élevage, alimentation, réactivité au stress d'abattage...) ou de la technologie (mode d'étourdissement...), peuvent influencer le métabolisme post-mortem et par conséquent, les qualités de viande. Un plan expérimental 2x2x2 comparait le protéome et l'expression de certains gènes d'intérêt chez des porcs femelles ou mâles castrés, de 2 origines génétiques différentes (pères Large White ou Duroc, mères Large White x Landrace), élevés dans 2 conditions différentes (conventionnelle en intérieur ou alternative en extérieur). Les résultats obtenus mettent en évidence l'important effet des facteurs de variation sur les quantités de protéines ainsi que sur les voies biochimiques impliquées dans le déterminisme des qualités des viandes. Ils ont également permis l'identification des marqueurs protéiques caractéristiques de l'origine génétique ou environnementale de l'animal. Les facteurs de variations n'influence pas l'expression des gènes étudiés. Le déterminisme des qualités des viandes n'est probablement pas une conséquence des modifications dans le niveau d'expression des gènes, mais plutôt dans la régulation des processus traductionnels.

<p>Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.</p> / Dissertation