Normally present in water, soil and waste water, Acinetobacter baumannii has become an important nosocomial pathogen, as causal agent of pneumonias, septicemias and urinary tract infections, among other complications in compromised patients from hospital’s intensive care units. One of its last acquired abilities is the resistance to colistin (polymixin E), the last therapeutic option for its infections. In this thesis, descriptive and quantitative differential expression proteomics is used in the study of acquired colistin resistance. As result of this research, 1,097 proteins belonging to the Acinetobacter genus have been identified by combined application of bidimensional gel electrophoresis (2DE), differential gel electrophoresis (DIGE), and peptide labeling with stable isobaric isotopes tags (iTRAQ). Analyses have been performed on the global expressed proteome of a reference, colistin-sensible strain (A. baumannii ATCC 19606) and, for comparative purposes, on a derived strain on which colistin resistance has been induced in vitro. The resistant phenotype shows reduced fitness, with significant differences in expression found in outer membrane proteins, membrane active transporters, diverse metabolic enzymes (fatty acids, citrate, phenylacetate, piruvate and nitrogen), proteins involved in stress response and biofilm formation, as well as in protein synthesis and folding pathways. The work has allowed to assess the strengths and weaknesses of the different techniques currently used in this type of proteomic analysis. / Acinetobacter baumannii, normalmente aislado en suelos y aguas (corrientes o residuales), se ha convertido en importante patógeno nosocomial, siendo agente causal de, entre otras complicaciones, neumonías, septicemias e infecciones del tracto urinario de pacientes comprometidos en unidades hospitalarias de cuidados intensivos. La más reciente de sus capacidades adquiridas es la resistencia a colistina (polimixina E), antibiótico peptídico considerado la última opción terapéutica en contextos clínicos. Esta tesis doctoral emplea la proteómica descriptiva y de expresión diferencial cuantitativa para investigar la resistencia adquirida por A. baumannii a dicho antibiótico. Los resultados han supuesto la identificación de 1.097 proteínas de Acinetobacter mediante el empleo combinado de electroforesis bidimensional convencional (2DE), 2DE diferencial (DIGE) y marcaje peptídico mediante isótopos isobáricos estables (iTRAQ). Los análisis se han realizado en el proteoma expresado por una cepa de referencia sensible a colistina (A. baumannii ATCC 19606), así como en una cepa derivada de ésta en la que se ha inducido, a efectos comparativos, resistencia a colistina in vitro. El fenotipo resistente manifestó reducida adaptabilidad biológica, encontrándose las principales diferencias en la estructura de la membrana externa, en la expresión de transportadores activos de membrana, en diversos enzimas metabólicos (ácidos grasos, citrato, fenilacetato, piruvato, nitrógeno) y de respuesta a condiciones de estrés, así como en la expresión de proteínas participantes en la formación de biopelículas y en el proceso de síntesis y plegamiento de proteínas. Además, el trabajo ha permitido evaluar los puntos fuertes y débiles de las técnicas empleadas actualmente en este tipo de análisis proteómicos.

Xanthomonas oryzae pv. oryzae (Xoo) est une bactérie phytopathogène responsable de la bactériose vasculaire du riz, maladie pouvant engendrer de fortes pertes de rendement à travers le monde. La course à l'armement entre la bactérie et sa plante hôte correspond d'une part à la mise en place de la virulence par le microorganisme et d'autre part en la résistance du végétal face à l'agression. Comprendre les mécanismes par lesquels Xoo accompli son cycle infectieux est d'une importance cruciale pour le développement futur de nouvelle méthode de luttes. Plusieurs approches complémentaires ont été mises en uvre afin de caractériser des éléments associés au pouvoir pathogène de Xoo.Dans un premier temps nous avons effectué une analyse protéomique comparative. Cette approche a permis l'identification chez une souche Africaine de Xoo d'un jeu de protéines induites par HrpX et susceptibles de jouer un rôle dans la virulence. Dans un second temps, l'implication de deux peptides dans la virulence Xoo a été étudiée. Le premier de ces peptides, supposé être le facteur d'avirulenceAvrXa21, a fait l'objet d'une caractérisation fonctionnelle et phylogénique. Le second peptide est synthétisé par un cluster NRPS, similaire à l'un de ceux présent chez Xanthomonas albilineans. Afin d'élucider l'importance de la molécule synthétisée par cette voie pour Xoo, une étude préliminaire impliquant la mutation d'un élément régulateur des NRPS a été effectuée. En dernier lieu, des informations nouvelles ont été apportées sur la topologie de la protéine membranaire HrcR qui est une composante essentielle du système de sécrétion de type III chez la plupart des bactéries appartenant au genre Xanthomonas. / Xanthomonas oryzae pv. oryzae (Xoo) is the agent of bacterial leaf blight BLB in rice, a disease which causes considerable yield losses throughout the world. In the arms race underlying the interactions between the microorganism and the host, the presence of virulence factors in the former parallels that of resistance factors in the latter. Understanding the mechanisms of Xoo's infectious cycle is of paramount importance for the elaboration of new fighting strategies to combat BLB. To achieve this, several complementary approaches to characterize components of Xoo's pathogenicity have been employed.First, we performed comparative proteomics that allowed us to identify novel HrpX-induced candidate pathogenicity factors of an African Xoo strain. Second, the involvement of two peptides in Xoo's pathogenicity has been investigated. One was speculated to be the avirulence factor AvrXa21 and has been characterized both functionally and phylogenetically. The other one was found to be synthesized by a Non-Ribosomal Peptide Synthetase (NRPS), reminescent to NRPS genes found in Xanthomonas albilineans. In order to determine the role of NRPS-mediated synthesis in Xoo virulence, we studied a strain carrying a mutated regulatory gene of the NRPS pathway. Finally, we provide new information on the topology of the HrcR membrane protein which is a conserved component of the type III secretion system of most Xanthomonas.

Depuis les révolutions industrielles des années 1700 et 1800, et pendant l'industrialisation des siècles les suivant, une accumulation de composés polluants a eu lieu dans l'atmosphère, principalement due à la combustion de matières fossiles comme le charbon et le pétrole. Outre les polluants directement émis comme les oxydes de souffre et d'azote, des polluants secondaires comme l'ozone se sont accumulés. Aujourd'hui, l'ozone est le troisième gaz responsable du réchauffement climatique, mais est également dangereux pour la santé humaine et responsable de dommages sur la végétation. Depuis les années cinquante, les effets de l'ozone sur les plantes et les réponses de ces dernières ont été étudiés de façon ciblée. Aujourd'hui, avec l'apparition de techniques permettant l'étude globale du transcriptome ou du protéome, il est possible d'aborder le problème d'une façon non biaisée, permettant des études plus complètes du stress. Dans le cadre de ce travail, une étude protéomique des effets de l'ozone sur les processus foliaires du peuplier a été proposée. Cette technique, complétée par des approches biochimiques, physiologiques, et des observations morphologiques, a permis de confirmer certains résultats d'études ciblées, mais également d'émettre des nouvelles hypothèses sur les mécanismes d'action de l'ozone sur le métabolisme du peuplier. En parallèle, l'étude du stress a aussi permis d'éclaircir les changements du métabolisme lors de la croissance de feuilles en conditions de stress et en conditions normales. Dans les pages de ce document, les procédures, résultats et conclusions de ce travail seront présentés en détail / After the industrial revolution of the 1700s and 1800s, and the subsequent industrialization, many pollutants have accumulated in the atmosphere, mainly due to the use of coal and fossil fuels. Besides the primary pollutants such as nitrogen oxides and sulfur oxides, secondary oxides such as ozone started to accumulate. Nowadays, ozone is the third gas involved in global climate change, but is also a major health risk for humans, and induces considerable damage to vegetation. Starting in the 50s, ozone research was based on targeted studies. Nowadays, with the advent of global techniques such as transcriptomics and proteomics, new results can be produced in an unbiased way. In the thesis presented here, a proteomic study of the effects of ozone on poplar leaf processes was carried out. With the help of this technique, complemented with biochemical and physiological approaches and with morphological observations, it was possible to confirm previous results, but also to elaborate new hypotheses concerning the effects of ozone on poplar leaf metabolism. In parallel, studying the stress also allowed to clarify some of the changes that occur in metabolism during leaf development, under stress conditions and under control conditions. In this document, the procedures, results and conclusions obtained during this study are presented in detail

<p>Developmental toxicology is an important part of preclinical drug toxicology as well as environmental toxicology. Assessing reproductive and developmental toxicity is especially expensive and time demanding, since at least two generations of animals are needed in the tests. In light of this there is a great need for alternative test methods in many areas of developmental toxicity testing.</p><p>The complete set of RNA transcripts in any given organism is called the transcriptome. Proteomics refers to the study of the proteins in a given organism or cell population. The work of this thesis has focused on the use of high throughput screening methods in transcriptomics and proteomics to search for molecular markers of developmental toxicity.</p><p>We have studied the global gene expression effects of the developmentally toxic substance valproic acid (VPA) using microarray technology. Several genes were found that display the same gene expression pattern <i>in vivo</i> using mouse embryos as the pattern seen <i>in vitro</i> using the embryocarcinoma cell line P19. Based on these observations, the gene Gja1 was suggested as one potential molecular marker of VPA induced developmental toxicity and potential marker of histone deacetylase (HDAC) inhibition <i>in vitro</i>. </p><p>Using 2D-DIGE technology, which measures relative protein abundances, the effect of neonatal exposure to the flame retardant PBDE-99 was studied in mouse brain (cortex, hippocampus and striatum) 24 hr after exposure. Differentially expressed proteins in the cortex and the striatum indicate that PBDE-99 may alter neurite outgrowth.</p><p>Finally, we have suggested several improvements in the use of the 2D-DIGE technology. Novel methods for normalizing data were presented, with several advantages compared to existing methods. We have presented a method named DEPPS that makes use of all identified proteins in a dataset to make comprehensive remarks about biological processes affected.</p>

Developmental toxicology is an important part of preclinical drug toxicology as well as environmental toxicology. Assessing reproductive and developmental toxicity is especially expensive and time demanding, since at least two generations of animals are needed in the tests. In light of this there is a great need for alternative test methods in many areas of developmental toxicity testing. The complete set of RNA transcripts in any given organism is called the transcriptome. Proteomics refers to the study of the proteins in a given organism or cell population. The work of this thesis has focused on the use of high throughput screening methods in transcriptomics and proteomics to search for molecular markers of developmental toxicity. We have studied the global gene expression effects of the developmentally toxic substance valproic acid (VPA) using microarray technology. Several genes were found that display the same gene expression pattern in vivo using mouse embryos as the pattern seen in vitro using the embryocarcinoma cell line P19. Based on these observations, the gene Gja1 was suggested as one potential molecular marker of VPA induced developmental toxicity and potential marker of histone deacetylase (HDAC) inhibition in vitro. Using 2D-DIGE technology, which measures relative protein abundances, the effect of neonatal exposure to the flame retardant PBDE-99 was studied in mouse brain (cortex, hippocampus and striatum) 24 hr after exposure. Differentially expressed proteins in the cortex and the striatum indicate that PBDE-99 may alter neurite outgrowth. Finally, we have suggested several improvements in the use of the 2D-DIGE technology. Novel methods for normalizing data were presented, with several advantages compared to existing methods. We have presented a method named DEPPS that makes use of all identified proteins in a dataset to make comprehensive remarks about biological processes affected.

Biological processes in living organisms consist of a vast number of different molecular networks and interactions, which are complex and often hidden from our understanding. This work is focused on recovery of such details for two quite distant examples: acclimation to extreme freezing tolerance in Siberian spruce (Picea obovata) and detection of proteins associated with prostate cancer. The first biological system in the study, upon P. obovata, is interesting by this species ability to adapt and sustain extremely low temperatures, such as -60⁰C or below. Despite decades of investigations, the essential features and mechanisms of the amazing ability of this species still remains unclear. To enhance knowledge about extreme freezing tolerance, the metabolome and proteome of P. obovata’s needles were collected during the tree’s acclimation period, ranging from mid August to January, and have been analyzed. The second system within this study is the plasma proteome analysis of high risk prostate cancer (PCa) patients, with and without bone metastases. PCa is one of the most common cancers among Swedish men, which can abruptly develop into an aggressive, lethal disease. The diagnostic tools, including PSA-tests, are insufficient in predicting the disease’s aggressiveness and novel prognostic markers are urgently required. Both biological systems have been analyzed following similar steps: by two-dimensional difference gel electrophoresis (2D-DIGE) techniques, followed by protein identification using mass spectrometry (MS) analysis and multivariate methods. Data processing has been utilized for searching for proteins that serve as unique indicators for characterizing the status of the systems. In addition, the gas chromatography-mass spectrometry (GC-MS) study of the metabolic content of P.obovata’s needles, from the extended observation period, has been performed. The studies of both systems, combined with thorough statistical analysis of experimental outcomes, have resulted in novel insights and features for both P. obovata and prostate cancer. In particular, it has been shown that dehydrins, Hsp70s, AAA+ ATPases, lipocalin and several proteins involved in cellular metabolism etc., can be uniquely associated with acclimation to extreme freezing in conifers. Metabolomic analysis of P. obovata needles has revealed systematic metabolic changes in carbohydrate and lipid metabolism. Substantial increase of raffinose, accumulation of desaturated fatty acids, sugar acids, sugar alcohols, amino acids and polyamines that may act as compatible solutes or cryoprotectants have all been observed during the acclimation process. Relevant proteins for prostate cancer progression and aggressiveness have been identified in the plasma proteome study, for patients with and without bone metastasis. Proteins associated with lipid transport, coagulation, inflammation and immune response have been found among them.

The developing brain goes through a number of developmental periods during which it displays an increased sensitivity to exogenous disturbances. On such period is the so called “Brain growth spurt” (BGS) which in humans takes place starting from the third trimester of pregnancy and throughout the first few years of life. The corresponding period in rats and mice is the first postnatal weeks. Exposure to relatively modest concentrations of the brominated flame retardant PBDE-99 during the second week of life in mice causes a more or less permanent impairment in the ability of the animals to adjust properly to environmental changes at adulthood. This “late response on early exposure” reflects the long-term consequences of disrupting the developing brain during a sensitive time period. The cellular mechanisms underlying the behavioral effects are far from clear. To address the initial damage occurring around the time of exposure, the approach used in this thesis is to use proteomics to analyze the effects of PBDE-99 on protein expression soon (24 hours) after exposure of the neonatal mouse on postnatal day (PND) 10.The thesis comprises the effects on the proteome in three distinct brain parts: cerebral cortex, striatum and the hippocampus. In addition, an in vitro model was developed and used to evaluate the PBDE-99 effects on cultured cerebral cortex cells from embryonic rat brains. Gel-based proteomics (2D-DIGE) coupled to MALDI- or ESI-MS has been used throughout for the proteomics experiments, but other techniques aimed at analyzing both proteins and mRNA have also been used to better characterize the effects. Even if the protein complements expressed by the different brain parts and separated with 2D-DIGE are seemingly similar, the effects are apparently specific for the different brain regions. In hippocampus, PBDE induces effects on proteins involved in metabolism and energy production, while the effects in striatum point towards effects on neuroplasticity. PBDE-99 changes the expression of cytoskeletal proteins in the cerebral cortex 24 hours after exposure. Interestingly, in vitro exposure of cerebral cortex cells to a PBDE-99 concentration in the same order of magnitude as in the in vivo neonatal brain also induces cytoskeletal effects, in the absence of cytotoxicity. This may suggest effects on regulatory aspects of cytoskeletal dynamics such as those involved in neurite sprouting. This thesis also addresses the problems involved in presenting proteomics data. Many of the available methods and approaches for presenting transcriptomics data are not suitable for isoform rich protein data. Modifications of existing methods and the development of a new approach (DEPPS) is also presented. Most importantly, the thesis presents the application and usefulness of proteomics as hypothesis generating techniques in neurotoxicology.

The catfish industry suffers losses primarily due to enteric septicemia of catfish and columnaris disease caused by Edwardsiella ictaluri and Flavobacterium columnare, respectively. Understanding the host-pathogen interactions is vital for prevention and eradication of these diseases. Hence, the overall objective of this study was to analyze whole cell proteomes of these two bacteria, and to determine the changes in E. ictaluri protein expression against in vitro iron-restriction and host serum treatment. High-throughput proteomic analysis of these bacteria was conducted using two-dimensional liquid chromatography followed by electrospray ionization tandem mass spectrometry (2-D LC ESI MS/MS) and two-dimentional gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-oflight mass spectrometry (2-DE MALDI TOF/TOF). Identified proteins were clustered into functional groups using clusters of orthologous groups, and subcellular locations as well as possible functional relationships were determined. A total of 788 unique E. ictaluri and 621 unique F. columnare proteins were identified, which represented 12 and 28 pathways, respectively. Vertebrate hosts tend to chelate free iron of their body and make the environment hostile for bacteria. Hence, reduced availability of iron may cause significant stress for pathogens and is considered a signal that leads to alteration in virulent gene expression. Similarly, E. ictaluri might use the catfish blood stream effectively for quick systemic invasion. Hence, exposure to catfish serum components might reveal the ability of E. ictaluri to protect against host defense mechanisms. Using two-dimensional difference gel electrophoresis, responses of E. ictaluri due to in vitro iron-restriction and host serum treatment were determined. A total of 50 and 19 proteins were identified to be differentially expressed due to in vitro iron-restriction and catfish serum treatment, respectively. Among the differentially expressed proteins, several putative virulent determinants, immunogenic proteins, chaperones, and housekeeping genes were noted. To initiate functional studies, four differentially expressed E. ictaluri genes (lamB, glyS, malE, and sdhA) were mutated by inrame deletion. Results from this study provided experimental evidence for many predicted proteins. In addition, identification of differentially expressed proteins provided targets for further functional analysis, which could help elucidate pathogenic mechanisms of E. ictaluri.

Today, assessment of the health of coastal waters is recognized as being important for both the conservation of nature and well-being of humans. Anthropogenic pollution has been the focus of extensive research for some time and a variety of programs for the monitoring and assessment of environmental pollution have been developed. Determination of the levels of pollution in sensitive ‘sentinels’ such as mussels, allows monitoring of these levels in a given area over a prolonged period of time. Furthermore, the biological effects of pollution are reflected in a series of biomarkers, none of which provides a general picture of the sentinel’s state of health and all of which are individually specific for certain pollutants and influenced by both biotic and abiotic factors. In an attempt to improve biomonitoring of marine pollution, we have developed two proteomic approaches here. In the first portion of the thesis, a proteomic analysis was performed on peroxisomes isolated from mussels exposed either to one of three model anthropogenic pollutants, or two different types of crude oil, or from mussels exposed to the Prestige oil spill. Application of two-dimensional electrophoresis (2-DE) provided protein expression signatures (PES) for exposure to these different pollutants.Furthermore, several individual protein components of these PES could be putatively identified. In the second portion of this work, such analysis of subproteomes was developed further in order to improve the applicability of this approach to biomonitoring. A simple fractionation procedure in combination with liquid chromatography and 2-DE provided samples from mussels residing in different regions of a pollution gradient around the harbor of Gothenburg, as well as from mussels exposed to two types of fuel oil similar to that of the Prestige that were suitable for environmental proteomics. In addition, we constructed a model for this approach that can be cross-validated in the future and applied to assess sources of fuel oil pollution in connection with biomonitoring programs.

Made available in DSpace on 2016-06-02T20:21:32Z (GMT). No. of bitstreams: 1 5298.pdf: 2436195 bytes, checksum: 47a2b6b8993059d60e028c4518c3ca75 (MD5) Previous issue date: 2013-05-24 / Universidade Federal de Sao Carlos / The citrus canker is an economically important disease for citrus crop. At the moment, there is no effective means of prevention or cure for this disease, which has contributed to citrus canker wide distribution around the world. The etiologic agents are bacteria of the genus Xanthomonas classified into two species, X. citri and X. fuscans. This study aimed to perform the differential proteomic analysis of cell surface proteins of Xanthomonas citri subsp. citri (XAC), the more virulent specie, between infectious (in vivo) and non-infectious (in vitro) conditions of growth. Additionally, the same analysis was performed by shotgun for XAC against the Xanthomonas fuscans subsp. aurantifolii type B (XauB), less virulent specie, both after growth in vivo. Initially, we performed growth curves of both bacteria on leaves of a common citrus host (Citrus aurantifolia) in order to investigate the dynamics of population growth in vivo and the efficiency of cell recovery by two different methods. For proteomic analysis, intact bacterial cells had their surface proteins labeled with fluorescence (DIGE CyDye Fluor minimal dyes), were then lysed and the total protein extract analyzed by differential gel electrophoresis (2D-DIGE), as standardized in this study. Protein profiles were analyzed by DeCyder 7.0 software and spots differentially expressed (ANOVA p <0.05) were isolated from gels, identified by mass spectrometry and search in protein databases of the annotated genome sequence of the bacteria. Seventy-nine spots from XAC were analyzed and thirty different proteins were identified, of which 10 correspond to known membrane or cell surface proteins: Ton-B dependent receptors and OmpA-related proteins exhibited lower expression in infectious condition, differently of Ferric enterobactin receptors, 60 kDa chaperonin (GroEL) and DnaK which showed higher expression after host interaction. XAC and XauB total extraction analysis by shotgun identified just two XAC proteins. Cell surface proteins with increased in vivo expression in virulent strain (XAC) could provide future targets of biotechnological interest for fighting citrus canker for being possibly related to phytopathogenicity and/or host spectrum. / O cancro cítrico é uma doença economicamente importante para a citricultura. Devido à inexistência de medidas eficazes de prevenção e combate, o cancro cítrico ainda é uma doença de ampla distribuição. Os agentes etiológicos são bactérias do gênero Xanthomonas, sendo classificadas em duas espécies, X. citri e X. fuscans, as quais diferem em virulência e espectro de hospedeiros cítricos. Este trabalho teve como objetivo a análise diferencial do subproteoma da superfície celular de Xanthomonas citri subsp. citri (XAC), espécie mais virulenta e causadora da cancrose A, entre duas condições de crescimento, infectante (in vivo) e não infectante (in vitro). Adicionalmente, a análise proteômica total por shotgun (LC-MS/MS) foi realizada para comparação de XAC com Xanthomonas fuscans subsp. aurantifolii tipo B (XauB), espécie menos virulenta, ambas após crescimento in vivo. Inicialmente, foram realizadas curvas de crescimento de ambas as bactérias em folhas de um hospedeiro cítrico comum (Citrus aurantifolia) a fim de se conhecer a dinâmica de crescimento populacional in vivo e a eficiência da recuperação bacteriana por dois diferentes métodos. Para as análises proteômicas, células bacterianas intactas tiveram suas proteínas de superfície marcadas com fluorescência (CyDye DIGE Fluor minimal dyes) e em seguida foram lisadas, sendo o extrato proteico total analisado por eletroforese diferencial em gel bidimensional (2D-DIGE), técnica padronizada neste trabalho. Os perfis proteicos de XAC foram analisados pelo software DeCyder 7.0 (GE Healthcare) e spots com expressão diferencial (ANOVA p<0,05) foram isolados dos géis e identificados por espectrometria de massas seguida de busca pela ferramenta Mascot em bancos de proteínas anotadas a partir da sequência genômica. Dos 79 spots de XAC analisados foram identificadas 30 diferentes proteínas, sendo que 10 correspondem a proteínas reconhecidamente de membrana e/ou superfície celular: receptores dependentes de Ton-B e proteínas relacionadas a OmpA foram encontradas com menor expressão na condição in vivo, enquanto que receptor de enterobactina, chaperonina 60 kDa (GroEL) e DnaK apresentaram maior expressão após interação com hospedeiro cítrico. Em relação à comparação do extrato total de XAC e XauB por shotgun foi possível identificar apenas duas proteínas de XAC. Proteínas da superfície celular com maior expressão na linhagem virulenta (XAC) na condição in vivo poderão ser futuros alvos de interesse biotecnológico para combate ao cancro cítrico por estarem possivelmente relacionadas com a fitopatogenicidade e/ou maior espectro de hospedeiros cítricos.