Orientador: Adriana Franco Paes Leme / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-21T23:53:23Z (GMT). No. of bitstreams: 1 Flores_IsadoraLuana_M.pdf: 6195465 bytes, checksum: 1730d0ae0783d2ec60f422de2cb20fa1 (MD5) Previous issue date: 2013 / Resumo: O carcinoma espinocelular oral (CEC oral) é a neoplasia maligna de cabeça e pescoço mais frequente e com grande morbidade. As melhorias nos protocolos terapêuticos não têm sido associadas com a melhora no prognóstico dos pacientes nas últimas décadas. Além disso, a taxa de descoberta de biomarcadores com utilização clínica de rotina ainda é um desafio para a grande maioria dos cânceres, inclusive para o CEC oral. Neste cenário, a associação da microdissecção a laser (ML) a espectrometria de massas (MS) tem sido considerada uma abordagem com alta robustez na descoberta de biomarcadores para o câncer. Diante disso, o objetivo deste estudo foi identificar potenciais biomarcadores para o CEC oral através da associação da ML a MS além de buscar possíveis mecanismos biológicos associados aos principais biomarcadores identificados com auxílio de ferramentas de bioinformática. Para isso, 10 pares de amostras teciduais frescas de CEC oral e mucosa oral normal foram obtidos para isolamento por ML para análise por cromatografia líquida acoplada a espectrometria de massas em tandem (LC-MS/MS) seguida por análises de bioinformática. Foram identificadas 2529 proteínas e entre essas, 107 proteínas apresentaram maior expressão nas amostras de CEC oral comparadas com a mucosa oral normal (teste t Student não-pareado, p<0,05 e razão >1,5). Os dados também foram submetidos às análises multivariadas pelos modelos Partial Least Squares Discriminant Analysis (PLS-DA) e Support Vector Machine (SVM) que indicaram, respectivamente, um painel de 40 e 70 potenciais biomarcadores. No geral, as análises pelo Ingenuity, KEGG (banco de dados Enciclopédia Kyoto para genes e genomas) e Gene Ontology mostraram que os processos mais significantes foram relacionados à sinalização célula-célula, a replicação do DNA, a recombinação e ao reparo, a adesão focal, a regulação do citoesqueleto de actina e a interação do receptor da matriz extracelular, entre outras. O fator eucariótico de elongação delta 1 (EEF1D) foi à proteína indicada para validação por ter apresentado reprodutibilidade de expressão entre as amostras de CEC oral, alta significância estatística segundo o teste t Student, expressão aumentada em CEC oral, por ter sido indicada como um marcador de classe na análise por PLS-DA e porque a sua função biológica ainda não foi investigada para esta neoplasia. Os resultados dos ensaios de validação confirmaram uma maior expressão de EEF1D por western blot utilizando-se células SCC-9 e tecido tumoral originado de modelo ortotópico. Além disso, uma intensidade de marcação maior para EEF1D foi observada em CEC oral pela validação por imuno-histoquímica (IHQ). Portanto, esse estudo combinou técnicas de ML, MS e bioinformática para a identificação de potenciais biomarcadores sendo que a proteína EEF1D foi considerada um potencial biomarcador para o CEC oral e validada por WB e IHQ podendo futuramente ser indicada também para validação em estudos translacionais / Abstract: The oral squamous cell carcinoma (OSCC) is a malignant head and neck with more frequency of occurrence and with greater morbidity. Improvements in treatment protocols have not been associated with improved prognosis of patients in recent decades. Furthermore, the rate of discovery of biomarkers with clinical routine use is still a challenge for the large majority of cancers, including OSCC. In this scenario, the combination of laser microdissection (LM) and mass spectrometry (MS) has been considered an approach with high robustness to biomarker discovery for cancer. Thus, the aim of this study was to identify potential biomarkers for OSCC through the association of the LM, MS and bioinformatics aiming at exploring biological mechanisms associated with major candidate biomarkers identified. For this, 10 pairs of fresh tissue samples of OSCC and normal oral mucosa were obtained for isolation by LM for analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis. 2529 proteins were identified and among these, 107 proteins have higher expression in oral SCC samples compared with normal oral mucosa (unpaired Student's t test, p <0.05 and ratio >1.5). The data were also submitted to multivariate models by Partial Least Squares Discriminant Analysis (PLS-DA) and Support Vector Machine (SVM) that, respectively, indicated a panel of 40 and 70 potential biomarkers. Overall, the Ingenuity analysis, KEGG (Kyoto Encyclopedia database for genes and genomes) and Gene Ontology showed that the most significant processes were related to cell-cell signaling, DNA replication, recombination and repair, adhesion focal, regulation of acting cytoskeleton and the interaction of the receptor extracellular matrix. The eukaryotic elongation factor 1 delta (EEF1D) was the protein indicated to validation because presented reproducibility of expression between OSCC samples, high statistical significance according to the Student t test, increased expression in OSCC, having been appointed as a class marker analysis for PLS-DA and because its biological function has not yet been investigated for this cancer. The higher expression of EEF1D was confirmed by western blot using proteins extracted from SCC-9 cells and tumor tissue originated from orthotropic model. Moreover, a higher marking intensity was observed in EEF1D for OSCC for validation by immunohistochemistry (IHC). Therefore, this study combined techniques ML, MS, and bioinformatics to identify potential biomarkers and EEF1D was the protein identified as a potential biomarker for OSCC and validated by WB and IHC and may also be indicated for validation in future translational studies / Mestrado / Estomatologia / Mestra em Estomatopatologia

Les maladies cardiovasculaires (MCV) sont la première cause de décès dans le monde. Une alimentation riche en précurseurs de méthyles pourrait diminuer le risque de survenue des MCV. Le statut en PDM a de très nombreuses conséquences car son altération va des risques de défaut de fermeture du tube neural au cours de l'embryogenèse au développement de maladies chroniques. Les PDM interviennent dans la voie de reméthylation de l'homocystéine en méthionine. Une diminution des niveaux de PDM entraîne une hyperhomocystéinémie considérée comme un facteur de risque de MCV via notamment son rôle dans le développement de l'athérosclérose. Relativement peu d'études se sont intéressées à l'effet d'une déficience en PDM sur le coeur. Afin de mieux comprendre les conséquences de la déficience en PDM sur cet organe, nous avons analysé les modifications du protéome myocardique chez des ratons de 21 jours nés de mères nourries avec un régime carencé en PDM comparativement à des ratons issus de mères normalement nourries. Les résultats de notre analyse protéomique ont montré que l'abondance de 22 protéines était augmentée entre 1,1 et 1,7 fois, alors que celle de 17 autres était diminuée entre 1,1 et 2,3 fois. Ces protéines interviennent surtout dans la production d'énergie cellulaire (21%), le métabolisme lipidique (18%) et le stress du réticulum endoplasmique (RE) (15%). Au moyen de l'outil bio-informatique "Ingenuity Pathway Analysis", nous avons montré que 34 d'entres-elles appartenaient à un réseau métabolique en relation avec "une perturbation du développement cardiaque et une altération cellulaire et du métabolisme lipidique". De plus, la carbonylation, principale modification posttraductionnelle oxydative des protéines, quantifiée par une technique de Dot-blot était augmentée de 1,9 fois dans le myocarde des ratons déficients en PDM comparativement aux contrôles. Afin d'étudier les mécanismes à l'origine de ces changements, nous avons développé un modèle in vitro de cellules cardiaques déficientes uniquement en folates ou en PDM. Des cellules de la lignée H9c2, dérivée de cardiomyoblastes embryonnaires de rat, ont été cultivées jusqu'à 4 jours dans un milieu carencé en folates (F) ou en PDM, comparativement à un milieu complet (C). Ceci nous a permis de disposer de deux modèles, constitués : (1) de cellules uniquement déficientes en folates et ne produisant pas davantage d'Hcy que les cellules contrôles, et (2) de cellules déficientes en au moins 2 PDM (folates et vitamine B12) et produisant des quantités accrues d'Hcy. Une analyse comparative des protéomes des cellules F et PDM vs C a permis d'identifier des différences et similitudes entre les 2 déficiences. Treize protéines présentant des abondances significativement différentes entre les cellules carencées pendant 4 jours en folates ou en PDM et leurs contrôles ont été mises en évidence : 7 avaient une abondance accrue et 6 une abondance diminuée. La comparaison des résultats obtenus a montré que des protéines mitochondriales ou intervenant dans la structure cellulaire n'ont été identifiées que dans le modèle de déficience en PDM. Nos résultats ont aussi démontré que les 2 types de carences affectaient des voies similaires à celles retrouvées dans notre étude in vivo : métabolisme énergétique et stress du RE. Nous avons alors confirmé que des protéines chaperones, a-crystalline B et prohibitine, avaient leur expression modifiée de la même manière dans les modèles de déficience in vivo (myocarde des ratons) et in vitro (cardiomyoblastes). Notre étude a également montré que l'induction rapide du stress du RE dans les cardiomyoblastes déficients en PDM est suivie par l'activation plus tardive du système d'ubiquitination des protéines et probablement de la voie de dégradation protéolytique dépendante du protéasome. En conclusion, cette thèse ouvre de nouvelles perspectives dans la compréhension des mécanismes des effets de la déficience en PDM ou en folates au niveau cardiaque. / No abstract available

As interações entre as plantas cultivadas e os outros organismos do agroecossistema podem afetar as características dos alimentos, trazendo implicações que abrangem desde as condições socioeconômicas dos produtores até a qualidade nutricional e sensorial dos produtos agrícolas. No caso de agroecossistemas equilibrados, a conservação da biodiversidade na propriedade contribui para diminuir a dependência de insumos externos, principalmente para o controle de pragas e doenças. Nesse sentido a produção de bananas no Vale do Ribeira constitui um mosaico de agroecossistemas, que pressionam os maiores e mais conservados remanescentes florestais de Mata Atlântica. A banana é um fruto climatérico típico, que mostrou grande potencial para o presente estudo, primeiro por ter boa parte de seus processos bioquímicos parcialmente elucidados, segundo, por apresentar seu genoma sequenciado e, terceiro, por ser um cultivo que permeia as áreas de Mata Atlântica do Vale do Ribeira. A perspectiva de análise do Proteoma label-free do fruto foi escolhida por sua ampla capacidade de compreensão de respostas biológicas, especialmente em delineamentos experimentais inéditos como esse, onde não é possível fazer grandes especulações acerca da resposta esperada. Dessa forma, inserido a um projeto de objetivo mais amplo, o objetivo deste trabalho foi avaliar a influência da biodiversidade atlântica no entorno do agroecossistema sobre a expressão de proteínas das bananas, considerando os aspectos físico-químicos, fisiológicos e bioquímicos, de modo a estimar as vias metabólicas influenciadas por esta condição ambiental. O projeto foi desenvolvido a partir da comparação de duas áreas comerciais de bananicultura que possuem idade e tratos culturais idênticos, sendo que a única diferença é que uma delas encontra-se cercada exclusivamente pelo monocultivo de bananeiras (parcela Controle) e a outra possui 60% do perímetro adjacente a um fragmento de Mata Atlântica (parcela Biodiversidade). Foi utilizada uma estratégia holística, contemplando diversos fatores do ambiente (fertilidade do solo e aspectos climáticos), da fisiologia da bananeira (diagnose foliar e infestação por doenças) e da banana (aspectos de qualidade, comportamento pós-colheita e proteoma da polpa). Os resultados mostram que as plantas da parcela biodiversidade apresentaram menor Índice de Severidade de Sigatoka Negra e produziram frutos com maior vida verde. Em relação ao Proteoma, as vias do Ciclo do ácido cítrico, do Metabolismo do piruvato e da Alanina, aspartato e glutamato foram as mais alteradas entre os frutos das duas parcelas, sinalizando uma maior tendência na síntese de ácidos graxos nos frutos da parcela Biodiversidade, que parece ter sido desviada para a síntese de aminoácidos nos frutos da parcela Controle. Algumas evidências reunidas sugerem que a presença da biodiversidade da Mata Atlântica no entorno do agroecossistema favorece o restabelecimento da homeostase vegetal, trazendo efeitos benéficos para o cultivo e para o fruto. / Food quality is affected by crop and other agroecosystem organism interaction. These are a broad and diverse field of study, with unclear central issues, implying since socioeconomic condition of the producer, up to food quality, in terms of nutritional and sensorial issues. In this sense, banana production on Vale do Ribeira represents an agroecosystem mosaic, among the hugest and most conserved remaining Atlantic forest. Banana is a climacteric fruit with great potential for this study, firstly because its biochemical processes has been partially clarified, secondly, because its genome is already sequenced and, finally, because its cultivation area is surrounded by Atlantic forest areas from Vale do Ribeira. Proteomic label-free has been chosen, because of its great capability to understand biological response, especially in unprecedented experimental approaches, in which expectations cannot be done. Thereby, inserted on a broader project, the aim of this work was to evaluate the influence of Atlantic forest biodiversity surrounding the agroecosystem on protein expression of banana fruit, considering physic-chemical, physiological and biochemical aspects, in order to highlight metabolic pathways influenced by this environmental condition. The development of this project is based on the comparison of two banana commercial plots, with similar age and cultural practices, being the only difference between plots the presence of an Atlantic forest remanant on 60% of the Biodiversity plot, while the Control plot is exclusively surrounded by banana crop. It has been adopted an holistic approach, including several environmental factors (soil fertility and climatic factors), crop physiology factors (foliar diagnosis and disease severity) and banana fruit (quality attributes, post-harvest behavior and pulp proteome). Results revealed a reduction on disease severity and a longer fruit greenlife, which represents the time available to transport and marketing, for plants of the Biodiversity plot. The Proteome has shown alterations on metabolic pathways, as Citric acid cycle, Piruvate metabolism and Alanine, aspartate and glutamate metabolism, suggesting a greater tendency on fatty acid biosynthesis on fruits from Biodiversity plots, whereas fruits from Control plot seems to enhance amino acid biosynthesis. Some evidence suggest that the Atlantic forest surrounding the agroecosystem can be helpful to plant homeostasis, with benefits to the crop and fruit.

Euglenophyta are monophyletic group of euglenids defined by presence of green, three membrane- bound plastid which has been aquired via secondary endosymbiosis with chlorophyte alga. Mechanism of transport of nuclear-encoded proteins into this plastid is not yet completely understood. It was observed that the proteins are transported to the outermost plastid membrane in vesicles passing through ER and Golgi, but the mechanism of their recognition and fusion with the target membrane remains unclear. Translocation system of inner two membranes is still completely unknown, regarding the situation in other plastids, it has been proposed that homologues of TOC and TIC complexes are present. In this work we analyzed sequence data from proteome of isolated plastid membranes of model organism Euglena gracilis and transcriptome of E. gracilis and its distant relative Eutreptiella gymnastica. We studied whether they contain proteins potentially involved in transport and homologues of proteins of transport systems known from plastids in other organisms (TOC/TIC, ERAD-like transport, SNARE). However, all our results are negative. It is hard to determine whether these findings indicate the possible absence of TOC and TIC complexes in euglenid plastid, or rather the insufficiency of our data. Powered by TCPDF (www.tcpdf.org)

Amastigotes are forms of Leishmania, naturally occurring in vertebrate hosts within phagocytic cells - especially the macrophages. The aim of this project was to compare three types of amastigotes of Leishmania that can be used for experiments under laboratory conditions - namely the axenic amastigotes, cultured extracellularly (without vertebrate phagocytic cells), amastigotes isolated from macrophages infected ex vivo, and "true" amatigotes isolated from lesions of the infected BALB/c mice. Amastigotes were compared with respect to the development in the natural vector and at the proteome level. L. mexicana, the causative agent of cutaneous leishmaniasis in the New World, was chosen for this comparison. In experiments comparing the development of Leishmania in the natural vector Lu. longipalpis we found significantly weaker infections in the sand flies infected with axenic amastigotes compared to other types of amastigotes. In addition to the intensity of infection, we compared the localization of promastigotes in the digestive tract of the phlebotomine sand flies. The following localizations were observed: the abdomen, the thorax, the cardia and the stomodeal valve, which is crucial for infectivity of the sand fly. There was no significant difference in localization in any of the groups of...

There is a very few papers to provide an overview of the characteristics of the estrous cycle, the relationship of the estrous cycle to physiological manifestations such as the pH of the vaginal environment, as well as the dynamics of the vaginal microbiota in wild mice. The aim of this thesis is to contribute to the understanding of the dynamic relationship between external influences and the physiology of the female reproductive system, to develop a reliable methodology for measuring the pH of the vaginal microenvironment in mice as well as to quantify the overall abundance of some bacterial taxons by comparing sequencing and qPCR methods. The results suggest that the physical presence of the male in the cage has the most significant effect on the prolongation of the estrus phase, in contrast to non-significant olfactory stimulation of the urine. Fluctuation in the pH of the vaginal environment have also been shown to be cyclic, and the qPCR method shows that the composition of the vaginal microbiota, during the estrus phase, differs significantly from other phases of the estrous cycle, as we confirmed by 16S rRNA sequencing. Thus, these results provide a comprehensive view of the variability of the estrous cycle with an emphasis on the variability of the vaginal microbiota and the change in the...

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19774号 / 農博第2170号 / 新制||農||1040(附属図書館) / 学位論文||H28||N4990(農学部図書室) / 32810 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 栗原 達夫, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of ArrF-encoding gene resulted in a 300old increase in the production of polyhydroxybutyrate (PHB), a polymer of industrial importance. This ∆arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. SDS-PAGE and MALDI-MS/MS revealed the overexpression of acetyl-CoA reductase, a phbBAC operon enzyme and the proteins that would alleviate the stress due to PHB accumulation in the ∆arrF mutant. Real-time RT-PCR revealed that phbR, phbB, phbA and phbC were upregulated in the mutant. Increased levels of activator PhbR in the mutant elevates the expression of phbB, phbA and phbC, resulting in the PHB overproduction. The proteins differentially expressed in the ∆arrF mutant were determined by gel-based proteomics and confirmed by real time RT-PCR. 6-phosphogluconolactonase that involve in the production of NADPH and acetyl-CoA, was upregulated, while the proteins involved in the TCA cycle that consumes acetyl-CoA were downregulated. Heat-shock proteins such as HSP20 and GroEL were overexpressed in the mutant. In addition, antioxidant proteins such as Fe-containing supeoxide dismutase (FeSOD), a putative oxidoreductase with unknown function, alkyl hydroperoxide reductase, flavorprotein WrbA and cysteine synthase were also upregulated, indicating that the PHB accumulation is highly stressful to the cells. Upregulated in the ∆arrF mutant were acetyl-CoA carboxylase, flagellin, and adenylate kinase. Among the genes upregulated in the ∆arrF mutant, sodB gene coding for Fe-superoxide dismutase and phbF gene encoding PHB synthesis regulator appears to be negatively regulated by small RNA ArrF in an antisense mechanism. However, all the TCA cycle genes were downregulated in the ∆arrF mutant. In addition to the TCA cycles enzyme, glutamate synthetase, elongation factor-Tu, iron ABC transporter, and major outer membrane porin OprF were downregulated in the ∆arrF mutant. Based on the results, it is concluded that several factors are responsible for the overproduction of PHB polymer in the ∆arrF mutant and one of which is the direct effect of small RNA ArrF on the expression of PhbF .

Cell dedifferentiation is a cell fate switching process in which a differentiated cell reverts to a status with competence for cell division and organ regeneration like an embryonic stem cell. Although the phenomenon of cell dedifferentiation has been known for over two and a half centuries in plants, little is known of the underlying mechanisms. Here, the proteome map of Arabidopsis cotyledons has been established and investigated the dynamic change of the cotyledon proteome in the time course of cell dedifferentiation. Among the 353 distinct genes, corresponding to 500 2-DE gel protein spots identified with high confidence, 12% have over twofold differential regulations within the first 48 h of induction of cell dedifferentiation. The distributions of these genes among different Gene Ontology categories and gene differential regulations within each of the categories have been examined. In addition, the cotyledon phosphoproteome has been investigated using Pro-Q Diamond Phosphoprotein in Gel Stain followed by mass spectrometry analyses. Among the 53 identified putative phosphoproteins, nine are differentially regulated during cell dedifferentiation. Arabidopsis cotyledon proteome at four different time points after the induction of cell dedifferentiation with MudPIT approach has been investigated and analyzed the protein quantity change using two labelree methods, the Spectral Count (SC) and SEQUEST Cross Correlation Coefficient (ÓXcorr) methods. Among the 662 MudPIT identified proteins, one hundred forty eight displayed differential regulation. The up-regulated proteins include transcription factors, calmodulins, translational regulators, and stress response proteins. The Spectral Count and the cross correlation coefficient quantification results are highly consistent in over 81% of the differentially regulated proteins. These studies have provided significant new insight into cell dedifferentiation process in Arabidopsis thaliana and also enhanced the Arabidopsis cotyledon proteome database established using gel based and non gel based methods. The results show that cell dedifferentiation involves extensive protein quantitative and qualitative changes in almost every cellular compartment and cellular process. Proteins like 14-3-3 proteins, Translational controlled tumor protein (TCTP) and its possible interaction protein-Translational elongation factor eEF1 alpha chain, GTP binding nuclear protein RAN2, GTP binding protein SAR1B and several other hypothetical and expressed proteins and nine other phosphoproteins showed significant differential expression during early dedifferentiation. Deciphering the molecular mechanisms regulating the cellular dedifferentiation certainly enhances the understandings and mechanisms of reprogramming all types of differentiated cells including animal cells.

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