No description available.

Schistosomiasis is one of the most widespread parasite diseases in the world, whereas Schistosoma mansoni is a major schistosome species in Africa, America, and the Caribbean islets. Many antigenic vaccine candidates have been postulated, including sm22.6 and GST. Although the lower level of re-infection of human schistosomiasis is related to the higher level of IgE against rsm22.6, unfortunately, the observation of the experimental vaccination in mice finds some difficulties in further development of vaccine. In addition, the biochemical and biophysical properties of the antigen are virtually unknown, thus the present study intends to characterize sm22.6 from biochemistry and cell biology. To do this, sm22.6 was expressed in E. coli (BL21DE3) and purified to homogeneity. Analyses of the recombinant protein showed that the antigen was highly hydrophobic and formed polymers readily as judged by both native and denatured electrophoreses. Because various technologies including NMR and DNA binding which had been applied to the study of the antigen generated vague results, we decided to express the antigen in human breast cancer cell (MDA-MB-435s) to locate in the subcellular compartments where the antigen is situated. Results showed that the antigen, not like the recombinant expressed in E. coli, located in both cellular fluids and membrane, suggesting that the antigen might not be a skeleton protein as predicted by proteomics.

This dissertation consists of four different topics in the areas of proteomics, genomics, and cardiology. First, a data-based method was developed to assign the subcellular localization of proteins. We applied the method to data on the bacteria Rhodobacter sphaeroides 2.4.1 and compared the results to PSORTb v.3.0. We found that the method compares well to PSORTb and a simulation study revealed that the method is sound and produces accurate results. Next, we investigated genomic features involved in the lethality of the knockout mouse using the random forest technique. We achieved an accuracy rate of 0.725 and found that among other features, the evolutionary age of the gene was a good predictor of lethality. Third, we analyzed DNA breakpoints across eight different cancer types to determine if common hotspots or cancer-type specific hotspots can be well-predicted by various genomic features and investigated which of the genomic features best predict the number of breakpoints. Using the random forest technique, we found that cancer- type specific hotspots are poorly predicted by genomic features but common hotspots can be predicted using the relevant genomic features. Additionally, we found that among the genomic features analyzed, indel rate and substitution rate were consistently chosen as the top predictors of breakpoint frequency. Lastly, we developed a method to predict the hypothetical heart age of a subject based on the subject’s electrocardiogram (ECG). The heart age predictions are consistent with current ECG science and knowledge of cardiac health.

Subcellular localization is crucial for determining the functions of proteins. A system called prediction of protein subcellular localization (P2SL) that predicts the subcellular localization of proteins in eukaryotic organisms based on the amino acid content of primary sequences using amino acid order is designed. The approach for prediction is to nd the most frequent motifs for each protein in a given class based on clustering via self organizing maps and then to use these most frequent motifs as features for classication by the help of multi layer perceptrons. This approach allows a classication independent of the length of the sequence. In addition to these, the use of a new encoding scheme is described for the amino acids that conserves biological function based on point of accepted mutations (PAM) substitution matrix. The statistical test results of the system is presented on a four class problem. P2SL achieves slightly higher prediction accuracy than the similar studies.

国立情報学研究所で電子化したコンテンツを使用している。

Listeria monocytogenes est l'un des meilleurs organismes modèles pour l'étude des interactions bactérie-hôte. Ce pathogène intracellulaire facultatif peut infecter, survivre et se répliquer dans le cytoplasme des cellules eucaryotes, démontrant la co-évolution étroite de Listeria avec son hôte. Le style de vie intracellulaire de ce pathogène implique la manipulation de divers composants de la cellule hôte, dont l'un est la chromatine. En induisant des modifications de la chromatine au niveau des histones, Listeria peut influencer le programme transcriptionnel de l'hôte. Ce projet de thèse porte sur une modification spécifique des histones, la désacétylation de la lysine 18 de l'histone H3, induite par la désacétylase de l'hôte Sirtuin 2 (SIRT2) lors de sa relocalisation du cytoplasme vers le noyau pendant l'infection. Le détournement de SIRT2 par Listeria fournit un système idéal pour étudier les mécanismes de la localisation subcellulaire de SIRT2, qui est mal comprise, et c'est le but de cette thèse. En utilisant la spectrométrie de masse, nous avons identifié une nouvelle modification posttraductionnelle de SIRT2, la phosphorylation de la sérine 25 (S25), ciblée spécifiquement par l'infection, et essentielle pour l'association de SIRT2 à la chromatine. Nous avons caractérisé le complexe moléculaire impliqué dans la déphosphorylation de SIRT2-S25 et nous montrons que cette modification est essentielle pour contrôler la fonction de SIRT2 en tant que répresseur transcriptionnel, et est nécessaire pour une infection efficace. Notre approche protéomique a aussi permis la caractérisation d'un interactome de SIRT2. De nombreuses protéines ont été identifiées et quelques-unes ont été confirmées et étudiées pour leur rôle dans le transport nucléo-cytoplasmique de SIRT2. De plus, une collaboration au laboratoire a mis au jour un mécanisme de subversion de la réponse aux dommages de l'ADN de l'hôte par Listeria. Dans son ensemble, ce travail a contribué à la compréhension de mécanismes originaux de l’interaction entre les bactéries et la chromatine et a révélé un processus cellulaire contrôlant la localisation subcellulaire et la fonction de la protéine de l’hôte SIRT2. / One of the best model organisms for the study of bacterial-host interactions is Listeria monocytogenes. This facultative intracellular pathogen can infect, survive, and replicate in the cytoplasm of eukaryotic cells, demonstrating the close co-evolution of Listeria with itshost. The intracellular life style of this pathogen involves manipulation of various host cellcomponents, one of which is chromatin. By inducing chromatin modifications at the level of histones, Listeria can influence the transcriptional program of the host. This thesis focuses on one specific histone modification, deacetylation of histone H3 of lysine 18, which is induced by the host deacetylase Sirtuin 2 (SIRT2) upon its relocalization from the cytoplasmto the nucleus during infection. Hijacking of SIRT2 by Listeria provides an ideal system tostudy the mechanisms of SIRT2 subcellular localization, which is poorly understood, and is the purpose of this thesis. By using mass spectrometry we have identified a novel posttranslational modification of SIRT2, Serine 25 (S25) phosphorylation, specifically targeted byinfection, and essential for SIRT2 chromatin association. We have characterized themolecular complex involved in dephosphorylating SIRT2-S25 and we show that this modification is essential for controlling SIRT2 function as a transcriptional repressor andnecessary for productive infection. Our proteomic approach further allowed the characterization of a SIRT2 interactome. Many proteins were identified and a few wereconfirmed and studied for their role in nucleo-cytoplasmic shuttling of SIRT2. In addition, a laboratory collaboration uncovered a mechanism for subversion of the host DNA DamageResponse by Listeria. As a whole, this work has contributed to the understanding of original mechanisms of chromatin-bacteria cross talk, and has revealed a cellular process controlling subcellular localization and function of the host protein SIRT2.

Phototransduction is a well characterized system for study of G protein coupled receptor (GPCR) signaling. The GPCR rhodopsin couples to the heterotrimeric G protein transducin. Light-stimulated activation of transducin in turn activates phosphodiesterase (PDE), leading to closure to cGMP-gated channels and inhibition of glutamate release. Rod and cone photoreceptors are highly polarized neurons consisting of the outer segment (OS) where phototransduction biochemistry occurs, the inner segment containing mitochondria and other organelles, the nuclear layer, an axon, and a glutamatergic synapse. Upon illumination, activated G protein transducin redistributes from the rod OS (where it is localized in the dark) to the inner compartments of the cell. Interestingly, cone transducin does not translocate in light. Opposite to this, visual arrestin migrates from the inner compartments to the OS, where it binds to rhodopsin. Previous reports from other groups and our lab argue for either an active or passive mechanism for transducin and arrestin redistribution. Our lab has shown that arrestin migration occurs by diffusion which is restricted by molecular sinks (Nair et al, 2005b). The focus of my dissertation was to unravel the molecular mechanism of rod transducin translocation. Specifically, I found energy (ATP) was not required for transducin movement within photoreceptors. Also, I found that the disc membranes of the rod outer segments as well as protein-protein interactions with retinal guanylate cyclase serve to restrict transducin diffusion through the cell. In addition, I used the insights gained from these studies of transducin to re-examine the relationship of other G proteins' subcellular localization and signal transduction. Ultimately, I found that most G proteins do not undergo subunit dissociation under physiological activating conditions.

Correct localization of lipidated cytosolic proteins to the plasma membrane (PM) is mediated by interactions between lipid anchors of proteins and cell membranes. Previously, dietary fish oil and its major n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), have been shown to decrease Ras membrane association, concomitantly reducing rat colon tumor incidence and Ras signaling, compared with corn oil and linoleic acid (LA), a highly prevalent vegetable fat and dietary PUFA in the U.S. diet. In order to explore the potential regulatory role of the cellular lipid environment in PM targeting of lipidated proteins, young adult mouse colon (YAMC) cells were treated with 50 µM DHA, LA, or oleic acid (OA) 24 h prior to and 36-48 h after transfection with green fluorescent protein (GFP) fusion constructs of various lipidated cytosolic proteins. Relative expression of each GFP fusion protein at the PM and the Golgi in living cells was quantified using z-serial confocal microscopy and digital image processing. DHA differentially altered the subcellular localization of Ras isoforms and Src-related tyrosine kinases in a reversible manner. DHA significantly decreased the PM localization and increased the endomembrane association of H-Ras, N-Ras, and Lck, which are targeted to the PM via the exocytic pathway, regardless of their functional state. In contrast, the subcellular distribution of K-Ras and Fyn, of which transport is independent of the vesicular transport pathway, was unaffected by DHA. Moreover, DHA selectively inhibited lipidated cytosolic protein targeting since the PM delivery of transmembrane protein cargo was unaffected, indicating that DHA does not alter the bulk flow of secretory vesicular traffic. Overall, the present study presents compelling evidence that select dietary constituents with membrane lipid-modifying properties can differentially modulate subcellular localization of important lipidated signaling proteins depending on their intracellular trafficking route to the PM.

With rapid advances in sequencing technologies and precipitous decreases in cost, public sequence databases have increased in size apace. However, experimental characterization of novel genes and their products remains prohibitively expensive and time consuming. For these reasons, bioinformatics approaches have become increasingly necessary to generate hypotheses of biological function. Phylogenomic approaches use phylogenetic methods to place genes, chromosomes, or whole genomes within the context of their evolutionary history and can be used to predict the function of encoded proteins. In this thesis, two new phylogenomic methods and software implementations are presented that address the problems of subcellular localization prediction and functional divergence prediction within protein families respectively. Most of the widely used programs for subcellular localization prediction have been trained on model organisms and ignore phylogenetic information. As a result, their predictions are not always reliable when applied to phylogenetically divergent eukaryotes, such as unicellular protists. To address this problem, PhyloPred-HMM, a novel phylogenomic method was developed to predict sequences that are targeted to mitochondria or mitochondrion-related organelles (hydrogenosomes and mitosomes). This method was compared to existing prediction methods using an existing test dataset of mitochondrion-targeted sequences from well-studied groups, sequences from a variety of protists, and the whole proteomes of two protists: Tetrahymena thermophila and Trichomonas vaginalis. PhyloPred-HMM performed comparably to existing classifiers on mitochondrial sequences from well-studied groups such as animals, plants, and Fungi and better than existing classifiers on diverse protistan lineages. FunDi, a novel approach to the prediction of functional divergence was developed and tested on 11 biological datasets and two large simulated datasets. On the 11 biological datasets, FunDi appeared to perform comparably to existing programs, although performance measures were compromised by a lack of experimental information. On the simulated datasets, FunDi was clearly superior to existing methods. FunDi, and two other prediction programs, was then used to characterize the functional divergence in two groups of plastid-targeted glyceraldehyde-3-phosphate dehydrogenases (GAPDH) adapted to roles in the Calvin cycle. FunDi successfully identified functionally divergent residues supported by experimental data, and identified cases of potential convergent evolution between the two groups of GAPDH sequences.

Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-22Bitstream added on 2014-06-13T19:49:29Z : No. of bitstreams: 1 martins_pmm_me_rcla.pdf: 7124946 bytes, checksum: 0e5fc6b0551611dda7e75caf8cd99fad (MD5) / O cancro cítrico é uma doença causada pela bactéria Xanthomonas axonopodis pv. citri (Xac), e que afeta plantas de citros por todo o mundo. O genoma de Xac foi completamente seqüenciado, o que revelou grandes quantidades de ORFs (~30%) codificando para produtos com função desconhecida (proteínas hipotéticas). Baseando-se no princípio de que muitos eventos bioquímicos acontecem em sítios específicos no interior celular, a localização de proteínas em fusão com GFP tem sido amplamente utilizada para a obtenção de informações valiosas a respeito de suas funções. Para iniciarmos estudos de localização de proteínas hipotéticas em Xac, construímos um vetor integrativo capaz de expressá-las em fusão com o polipeptídio GFP, pPM2a. O vetor de expressão para Xac carrega um cassete promotor/repressor de xilose (xylR/pxyl), o gene gfp, um RBS sintético e um fragmento do gene de α-amilase de Xac, para direcionar a integração do sistema de expressão no lócus amy do cromossomo bacteriano. Mostramos aqui a integração estável do vetor no lócus amy de Xac. Além disso, mutantes de Xac expressando o polipeptídio GFP não apresentam nenhuma alteração em seu fenótipo de patogenicidade para o hospedeiro (laranja doce). Mutantes de Xac expressando versões marcadas com GFP para as proteínas ParB e ZapA, ambas codificadas por Xac, foram utilizadas para a padronização dos estudos de localização subcelular. GFP-ZapAXac apresentou um padrão de localização análogo ao de seu ortólogo presente em Bacillus subtilis: uma estrutura semelhante a uma barra, posicionada no meio do bacilo, onde o septo se desenvolve, orientado perpendicularmente com relação ao eixo longitudinal da célula. Este é o primeiro relato de um estudo de localização realizado em Xac. Ao contrário de GFP-ZapAXac, ParBXac-GFP não mostrou nenhum padrão de localização, apesar de a fusão... / Citrus canker is a disease caused by the bacterium Xanthomonas axonopodis pv. citri (Xac), which affects citrus plants worldwide. The genome of Xac was completely sequenced, which unveiled an expressive amount of ORFs (~30%) coding for products of unknown function (hypothetical proteins). Based on the principle that many biochemical events happen at specific sites within the cells, protein localization studies have been extensively used to gather valuable information about function. In order to start subcellular localization studies of hypothetical proteins encoded by Xac using fluorescent microscopy, we constructed an integrative expression vector for GFP-tagging of proteins in this bacterium, pPM2a. The expression vector for Xac carries a xylose repressor/promoter cassette (xylR/pxyl), the gfp gene, a synthetic Ribosome Binding Site (RBS), and a fragment of the α-amylase gene of Xac, to drive the integration of the whole expression system into the amy locus of the bacterial chromosome. We show here stable integration of the expression vector into the amy locus of Xac. Furthermore, Xac mutants expressing the polypeptide GFP do not exhibit any alteration in pathogenicity to the host plant sweet orange. Mutants of Xac expressing GFPtagged versions of ParB and ZapA proteins, both encoded by Xac, were used to standardize the subcellular localization studies. GFP-ZapAXac showed a localization pattern analogous to its ortholog encoded by Bacillus subtilis: a bar-like structure positioned in the middle of the rods, where the septum develops, oriented perpendicularly to the longitudinal axis of the cell. This is the first report of a protein localization study performed in Xac Unlike GFP-ZapAXac, ParBXac-GFP did not display any detectable localization pattern, despite the fact that we were able to detect the production of the fusion ParBXac-GFP in Western blot experiments... (Complete abstract click electronic access below)