O mamão papaia (Carica papaya L.) é uma fruta tropical de grande relevância comercial uma vez que o Brasil é o maior produtor mundial e terceiro maior exportador da fruta. Contudo, por ser uma fruta climatérica, tem uma vida pós-colheita limitada, devido ao rápido Amaciamento da polpa. Neste trabalho foi realizada uma investigação proteômica comparativa de polpa do mamão verde e maduro. Várias centenas de componentes protéicos (spots) foram resolvidos em géis 2-DE (faixa de pH 4-7) utilizando a eletroforese diferencial em gel (DIGE) e posteriormente as imagens geradas foram analisadas pelo programa PDQuest. Os spots diferencialmente expressos foram retirados do gel, digeridos e sequenciados (ESI-Q-TOF-MS/MS). Em geral, as proteínas diferencialmente expressas foram associadas ao metabolismo do etileno, resposta ao estresse, metabolismo de carbono e outros importantes processos fisiológicos. Os papéis de algumas das proteínas identificadas foram discutidos em relação à qualidade dos frutos do mamão. Este estudo fornece a primeira caracterização das mudanças no proteoma da polpa do mamão durante o amadurecimento. Deste modo a identificação de proteínas envolvidas na instalação ou desenvolvimento do amadurecimento pode contribuir para o entendimento deste processo e, também, gerar subsídios para avanços tecnológicos visando à qualidade e diminuição das perdas pós-colheita. / Papaya (Carica papaya L.) fruit is a relevant tropical crop since Brazil is the world\'s largest producer and third largest exporter of fruit. However being a climacteric fruit, has a limited green life due to the rapid pulp softening. We report here a comparative proteomic investigation of fruit pulp from unripe and ripe papayas. Several hundreds of protein components were resolved on 2-DE gels (pH range 4-7) using the differential gel electrophoresis (DIGE) approach, and images were analyzed by PDQuest. The variable components were excised, in-gel digested and were analyzed by ESI-Q-TOF-MS/MS. In general, the differentially expressed proteins were associated to ethylene metabolism, stress response, carbon metabolism and other important physiological processes. The roles of some of the identified proteins were discussed in relation to papaya fruit quality. To the best of our knowledge, this investigation provides the first characterization of the changes in papaya fruit pulp proteome during ripening. Thus the identification of proteins involved in the installation or development of the ripening may contribute to the understanding of this process and also provide subsidies for technological advances aimed at quality and reduction of post harvest losses.

Infestações com Rhipicephalus microplus, o carrapato dos bovinos, causam enormes prejuízos econômicos para a pecuária. Os carrapatos estão desenvolvendo resistência aos carrapaticidas que, além dessa desvantagem, deixam resíduos em carne e leite. Vacinas anticarrapato representam uma alternativa sustentável de controle de infestações, mas as atualmente disponíveis têm efeitos parciais e transitórios. Surge, assim, a necessidade de identificar novos antígenos vacinais. Para alcançar esse objetivo este trabalho explora o fato de que bovinos apresentam fenótipos contrastantes e herdáveis de infestações que são específicos de certas raças. Além disso, o nível de imunidade do hospedeiro afeta a transcrição de genes de glândulas salivares do carrapato, órgão que produz proteínas que medeiam o parasitismo. A hipótese de trabalho é a que os diferentes níveis da imunidade anticarrapato do hospedeiro afetam, também, a composição salivar do parasita. Assim, em carrapatos alimentando-se em hospedeiros resistentes as proteínas que são cruciais ao parasitismo poderão estar ausentes ou deficientes na sua saliva e por isso os carrapatos não terminam sua refeição de sangue. A neutralização dessas mesmas proteínas pela imunidade humoral pode ter o mesmo efeito e por isso, essas proteínas constituem bons antígenos vacinais. Assim, o objetivo do trabalho foi identificar novos antígenos vacinais em saliva de fêmeas e glândulas salivares de ninfas, machos e fêmeas de carrapatos alimentados em hospedeiros resistentes e suscetíveis, bem como em larvas não alimentadas oriundas de ovos de fêmeas alimentadas nestes mesmos hospedeiros. Para isso, foram empregadas abordagens de sequenciamento de nova geração \"RNA-Seq\" (454) e abordagens proteômicas, como análise diferencial em gel (DIGE) e Western Blots (imunoproteoma) seguido de sequenciamento de massa, além da tecnologia de identificação de proteínas multidimensionais (ou Multidimensional Protein Identification Technology, MudPIT) para descrever o proteoma das glândulas salivares e da saliva de fêmeas. A análise transcriptômica resultou no sequencimanto de 1.999.086 reads que permitiu identificar e classificar 11.676 sequências codificadoras (CDS), muitas das quais (3.600 CDS) contêm peptídeo sinal que é indicativo de secreção, portanto podendo estar presente na saliva e Resumo Gustavo Rocha Garcia apresentar função importante na hematofagia. Por meio de MudPIT, identificamos 321 proteínas salivares diferentes, além de 126 proteínas no DIGE e 266 proteínas nos imunoproteomas. Muitas dessas proteínas podem ser consideradas antígenos potenciais por estarem associadas com a hematofagia/parasitismo, tais como proteases, nucleases, inibidores de proteases, peptídeos antimicrobianos, proteínas de fixação, entre outros, inclusive proteínas ainda não caracterizadas. A maioria dos genes codificantes dessas proteínas está mais expressa em carrapatos alimentados em hospedeiros suscetíveis, principalmente em carrapatos machos. Além disso, muitas dessas proteínas não são reconhecidas por soros bovinos, inclusive soros de bovinos infestados, embora soros de bovinos infestados e resistentes ao carrapato apresente a maioria das reatividades. O conjunto dos resultados sugere que em nível de proteína a composição da saliva também é afetada pelos diferentes níveis de imunidade dos hospedeiros, além de variar com o ciclo de vida do carrapato. Desse modo, concluímos que as estratégias de investigação empregadas foram satisfatórias para identificar um conjunto de antígenos salivares do carrapato R. microplus que representam proteínas alvos para compor vacinas multicomponentes anticarrapato. / Infestation with Rhipicephalus microplus, the cattle tick, causes huge economic losses to livestock. Ticks are developing resistance to acaricides that, besides this disadvantage, leave residues in meat and milk. The anti tick vaccines represent a sustainable alternative of the infestations control, but the currently available has partial and transient effects. Thus arises the need to identify new vaccine antigens. To achieve this goal, this work explores the fact that cattle exhibit contrasting phenotypes and inheritable of infestations that are specific to certain breeds. Furthermore, the level of immunity of the host affects gene transcription tick salivary gland, organ that produces proteins that mediate the parasitism. The working hypothesis is that different levels of anti tick immunity of host affect also the salivary composition of the parasite. So in ticks feeding on resistant hosts the proteins that are crucial to parasitism may be absent or deficient in their saliva, and by this the ticks do not finish your meal blood. The neutralization of these same proteins by humoral immunity can have the same effect and by this, these proteins are good vaccine antigens. So, the aim of the study was to identify new vaccine antigens in saliva from females and salivary glands of nymphs, males and females of ticks fed on resistant and susceptible hosts as well as in unfed larvae originating from eggs of females fed on these same hosts. To this, were employed sequencing approaches of new generation \"RNA-Seq\" (454) and proteomic approaches, such as differential analysis in gel (DIGE) and Western Blots (immunoproteomics) followed by sequencing mass, besides the Multidimensional Protein Identification Technology (MudPIT) to describe the proteome of the salivary glands and saliva of females. The transcriptomics analysis identified 11,676 coding sequences (CDS), many of which (3,600 CDS) contain predicted signal peptide indicative of secretion, therefore may be present in saliva and provide an important function in blood feeding. Through MudPIT, we identify 321 different salivary proteins, besides 126 proteins in DIGE and 266 proteins in immunoproteomics. Many of these proteins may be considered as potential antigens to be associated with the blood meal/ parasitism, such as proteases, nucleases, protease inhibitors, antimicrobial peptides, proteins of attachment, among Abstract Gustavo Rocha Garcia others, including proteins not yet characterized. Most of the genes encoding of these proteins are more expressed in ticks fed on susceptible hosts, especially in male ticks. Moreover, many of these proteins are not recognized by bovine sera, including sera from infested hosts, although sera from infested and resistant host to tick present the most reactivities. The overall results suggest that in protein level, the composition of saliva is also affected by the different levels of immunity of the host, besides vary with the tick life cycle. Thus, we conclude that the research strategies employed were satisfactory to identify a set of tick salivary antigens from R. microplus that represent target proteins for composing anti tick multicomponent vaccines.

Infestações com Rhipicephalus microplus, o carrapato dos bovinos, causam enormes prejuízos econômicos para a pecuária. Os carrapatos estão desenvolvendo resistência aos carrapaticidas que, além dessa desvantagem, deixam resíduos em carne e leite. Vacinas anticarrapato representam uma alternativa sustentável de controle de infestações, mas as atualmente disponíveis têm efeitos parciais e transitórios. Surge, assim, a necessidade de identificar novos antígenos vacinais. Para alcançar esse objetivo este trabalho explora o fato de que bovinos apresentam fenótipos contrastantes e herdáveis de infestações que são específicos de certas raças. Além disso, o nível de imunidade do hospedeiro afeta a transcrição de genes de glândulas salivares do carrapato, órgão que produz proteínas que medeiam o parasitismo. A hipótese de trabalho é a que os diferentes níveis da imunidade anticarrapato do hospedeiro afetam, também, a composição salivar do parasita. Assim, em carrapatos alimentando-se em hospedeiros resistentes as proteínas que são cruciais ao parasitismo poderão estar ausentes ou deficientes na sua saliva e por isso os carrapatos não terminam sua refeição de sangue. A neutralização dessas mesmas proteínas pela imunidade humoral pode ter o mesmo efeito e por isso, essas proteínas constituem bons antígenos vacinais. Assim, o objetivo do trabalho foi identificar novos antígenos vacinais em saliva de fêmeas e glândulas salivares de ninfas, machos e fêmeas de carrapatos alimentados em hospedeiros resistentes e suscetíveis, bem como em larvas não alimentadas oriundas de ovos de fêmeas alimentadas nestes mesmos hospedeiros. Para isso, foram empregadas abordagens de sequenciamento de nova geração \"RNA-Seq\" (454) e abordagens proteômicas, como análise diferencial em gel (DIGE) e Western Blots (imunoproteoma) seguido de sequenciamento de massa, além da tecnologia de identificação de proteínas multidimensionais (ou Multidimensional Protein Identification Technology, MudPIT) para descrever o proteoma das glândulas salivares e da saliva de fêmeas. A análise transcriptômica resultou no sequencimanto de 1.999.086 reads que permitiu identificar e classificar 11.676 sequências codificadoras (CDS), muitas das quais (3.600 CDS) contêm peptídeo sinal que é indicativo de secreção, portanto podendo estar presente na saliva e Resumo Gustavo Rocha Garcia apresentar função importante na hematofagia. Por meio de MudPIT, identificamos 321 proteínas salivares diferentes, além de 126 proteínas no DIGE e 266 proteínas nos imunoproteomas. Muitas dessas proteínas podem ser consideradas antígenos potenciais por estarem associadas com a hematofagia/parasitismo, tais como proteases, nucleases, inibidores de proteases, peptídeos antimicrobianos, proteínas de fixação, entre outros, inclusive proteínas ainda não caracterizadas. A maioria dos genes codificantes dessas proteínas está mais expressa em carrapatos alimentados em hospedeiros suscetíveis, principalmente em carrapatos machos. Além disso, muitas dessas proteínas não são reconhecidas por soros bovinos, inclusive soros de bovinos infestados, embora soros de bovinos infestados e resistentes ao carrapato apresente a maioria das reatividades. O conjunto dos resultados sugere que em nível de proteína a composição da saliva também é afetada pelos diferentes níveis de imunidade dos hospedeiros, além de variar com o ciclo de vida do carrapato. Desse modo, concluímos que as estratégias de investigação empregadas foram satisfatórias para identificar um conjunto de antígenos salivares do carrapato R. microplus que representam proteínas alvos para compor vacinas multicomponentes anticarrapato. / Infestation with Rhipicephalus microplus, the cattle tick, causes huge economic losses to livestock. Ticks are developing resistance to acaricides that, besides this disadvantage, leave residues in meat and milk. The anti tick vaccines represent a sustainable alternative of the infestations control, but the currently available has partial and transient effects. Thus arises the need to identify new vaccine antigens. To achieve this goal, this work explores the fact that cattle exhibit contrasting phenotypes and inheritable of infestations that are specific to certain breeds. Furthermore, the level of immunity of the host affects gene transcription tick salivary gland, organ that produces proteins that mediate the parasitism. The working hypothesis is that different levels of anti tick immunity of host affect also the salivary composition of the parasite. So in ticks feeding on resistant hosts the proteins that are crucial to parasitism may be absent or deficient in their saliva, and by this the ticks do not finish your meal blood. The neutralization of these same proteins by humoral immunity can have the same effect and by this, these proteins are good vaccine antigens. So, the aim of the study was to identify new vaccine antigens in saliva from females and salivary glands of nymphs, males and females of ticks fed on resistant and susceptible hosts as well as in unfed larvae originating from eggs of females fed on these same hosts. To this, were employed sequencing approaches of new generation \"RNA-Seq\" (454) and proteomic approaches, such as differential analysis in gel (DIGE) and Western Blots (immunoproteomics) followed by sequencing mass, besides the Multidimensional Protein Identification Technology (MudPIT) to describe the proteome of the salivary glands and saliva of females. The transcriptomics analysis identified 11,676 coding sequences (CDS), many of which (3,600 CDS) contain predicted signal peptide indicative of secretion, therefore may be present in saliva and provide an important function in blood feeding. Through MudPIT, we identify 321 different salivary proteins, besides 126 proteins in DIGE and 266 proteins in immunoproteomics. Many of these proteins may be considered as potential antigens to be associated with the blood meal/ parasitism, such as proteases, nucleases, protease inhibitors, antimicrobial peptides, proteins of attachment, among Abstract Gustavo Rocha Garcia others, including proteins not yet characterized. Most of the genes encoding of these proteins are more expressed in ticks fed on susceptible hosts, especially in male ticks. Moreover, many of these proteins are not recognized by bovine sera, including sera from infested hosts, although sera from infested and resistant host to tick present the most reactivities. The overall results suggest that in protein level, the composition of saliva is also affected by the different levels of immunity of the host, besides vary with the tick life cycle. Thus, we conclude that the research strategies employed were satisfactory to identify a set of tick salivary antigens from R. microplus that represent target proteins for composing anti tick multicomponent vaccines.

No description available.

Besides playing its structural function in cellular membranes, ceramide has been recognized as a bioactive signalling molecule playing roles in the regulation of cell growth, differentiation, senescence and programmed cell death. Apoptosis can be induced in cancer cells by elevation of endogenous ceramide levels in response to a variety of apoptotic stimuli such as cytokines (TNF, IL-1), death receptor ligands (Fas ligand), heat stress, oxidative stress, chemotherapeutic agents, and ionizing or ultraviolet radiation. It was shown that use of exogenous cell-permeable short-chain ceramide can also promote apoptotic pathways in cancer cells. Several studies have attempted to further define the specific role of ceramide in cell death. However, the mechanisms by which ceramide mediates antiproliferative pathways or inhibits prosurvival effects are not yet well-defined. So, we investigated the signalling pathways triggered by exogenously-supplied natural long chain ceramide, especially C16-ceramide, to better understand how this messenger induces its biological effects in cancer cells. We first showed that C16-ceramide induced a decrease in viability of adenocarcinoma cells (HCT116), partly due to apoptosis. Using two-dimensional differential in-gel electrophoresis (2D-DIGE) proteomic approach, we identified new proteins involved notably in cell proliferation, apoptosis, protein transport and transcriptional regulation in response to exogenous C16-ceramide. Among them, the death promoting factor Btf (Bcl-2-associated transcription factor) was found to be involved in the ceramide-dependent pro-apoptotic signalling pathway. Indeed, Btf-depleted colon cancer cells were found to be more resistant to death triggered by C16-ceramide. Transfection of GFP-Btf expression plasmid up-regulated p53 and BAX protein levels whereas pBcl-2 and Mdm2 expression were down-regulated. Furthermore, we identified a new signalling pathway specifically induced by C16-ceramide, depending on Btf and leading to down-regulation of the Mdm2 protein expression and MDM2 promoter activity. Thus, we provided new information on molecular mechanisms involved in the ceramide-mediated cell death. Then, we investigated the regulation of Emerin expression and its post-translational modifications induced by ceramide. We found that cAMP-dependent protein kinase A (PKA) could be involved in the C16-ceramide induced-Emerin phosphorylation. However, we did not demonstrate the interaction between Btf and phosphorylated-Emerin upon ceramide treatment. Nevertheless, we showed that one of the pathway induced by ceramide implies Emerin and leads to down-regulation of the MDM2 promoter activity. We also hypothesized that GCL (germ-cell-less) could be an intermediate in the Emerin-Mdm2 pathway triggered by C16-ceramide. Furthermore, we showed that Emerin-depleted cells were not more sensitive to apoptosis induced by C16-ceramide. These results should allow us to further explore the potential functions of Emerin in a ceramide-dependent pathway.

Mollicutes (mycoplasmas) are pathogenic in a wide range of mammals (including humans), reptiles, fish, arthropods, and plants. Of the medically important mollicutes, Mycoplasma gallisepticum is of particular relevance to avian agriculture and veterinary science, causing chronic respiratory disease in poultry and turkey. Using two-dimensional electrophoresis based quantitative expression proteomics, the current study investigated the molecular mechanisms behind the phenotypic variability between a M. gallisepticum vaccine strain (6/85) and a competitive, virulent field strain (K5234), two strains which were indistinguishable using commonly accepted genetic methods of identification. Twenty-nine proteins showed a significant variation in abundance (fold change > 1.5, p-value < 0.01). Among others, the levels of putative virulence determinants were increased in the virulent K5234, while the levels of several proteins involved with pyruvate metabolism were decreased. It is hoped that the data generated will further the understanding of M. gallisepticum virulence determinants and mechanisms of infection, and that this may contribute to the optimization of diagnostic methodologies and control strategies.

El càncer d'endometri és la malaltia ginecològica més comuna i representa la quarta neoplàsia més freqüent en la dona en els països desenvolupats. Actualment és possible diagnosticar el 80% dels casos de EEC en estadiatge I FIGO, per tant, poder aplicar cirurgia i presentar un bon pronòstic. L'estadiatge I FIGO inclou tres subtipus: afecta l'endometri (IA), infiltra el miometri <50% (IB) i >50% (IC). El nostre grup es va centrar en la identificació de nous gens implicats en el EEC mitjançant l'estudi dels patrons d'expressió gènica diferencial entre teixit endometrial sa, hiperplàsic i tumoral, així com la cerca de nous gens associats al fenotip carcinomatós endometrial amb potencial valor diagnòstic i/o pronòstic. Es va trobar que els dos gens majorment sobreexpressats eren RUNX1/AML1 i ERM/ETV5 i alhora, es troben sobreexpressats en la fase en la qual el EEC esdevé invasiu. Partint d'aquí, vam plantejar la caracterització del mecanisme d'acció del factor de transcripció ERM/ETV5 en el EEC durant els esdeveniments inicials d'invasió i disseminació. La sobreexpressió d'ERM/ETV5 a la línia cel·lular de càncer d'endometri Hec-1A indueix dispersió cel·lular que correlaciona amb un augment de l'activitat gelatinasa de la MMP2. Tant els experiments de ChiP com amb iRNA o l'ús d'un inhibidor específic de MMP2 mostren un nexe funcional entre la sobreexpressió d'ERM/ETV5 i l'activació de MMP2. En el model animal ortotòpic de EEC es demostra que l'augment de l'activitat de MMP2 associat a la sobreexpressió d'ERM/ETV5 confereix major capacitat invasiva als tumors endometrials i aquests mostren un patró més agressiu i infiltrant. Es va confirmar que ERM/ETV5 tenia un paper en els passos inicials de la disseminació endometrial ja que es van localitzar ERM/ETV5 i MMP-2 en el front d'invasió de mostres de EEC humanes amb infiltració miometrial. Per tant podem proposar que en el EEC, ERM/ETV5 actua a través de l'activitat gelatinolítica de MMP-2 per a donar major capacitat invasiva, associat al punt d'inici de la infiltració miometrial. Tot seguit vam plantejar determinar les alteracions moleculars associades a la sobreexpressió del factor de transcripció ERM/ETV5 durant la infiltració miometrial en el EEC, identificant nous marcadors moleculars o dianes terapèutiques que podrien estar sota el control de l'activitat transcripcional d'ERM/ETV5. L'anàlisi comparatiu mitjançant 2D-DIGE i espectrometria de masses dels patrons d'expressió proteica diferencial de la línia cel·lular que sobreexpressa de forma estable GFP-ERM/ETV5 anvers la línia cel·lular sense transfectar i la que presenta el vector GFP buit, determina un llistat de proteïnes diferencialment expressades que es troben implicades en la regulació d'actina i la senyalització per TGF-beta i progesterona. Vam caracteritzar la sobreexpressió específica de la proteïna Hep27 que presenta localització mitocondrial depenent d'ERM/ETV5. Estudis funcionals van demostrar associació amb l'estrès oxidatiu. L'anàlisi dels resultats obtinguts reforcen el paper d'ERM/ETV5 com a factor regulador de la migració i invasió tumoral, i assenyala la seva implicació en la resposta a l'estrès oxidatiu associat a l'inici de la invasió en el carcinoma endometrial. Fins ara, la immunohistoquímica ha estat l'eina utilitzada per a la identificació de proteïnes implicades en la invasió del carcinoma endometrial, sense tenir en compte la percepció global del front d'invasió. En aquest últim treball hem dut a terme una aproximació proteòmica per a caracteritzar components específics del front d'invasió o l'estroma reactiu mitjançant la comparació de l'àrea invasiva d'un carcinoma endometrial anvers l'àrea tumoral superficial no invasiva i el teixit normal provinents de la mateixa pacient. Així hem pogut identificar proteïnes diferencialment expressades en el front d'invasió i que es troben implicades en la morfologia cel·lular, acoblament i moviment, així com els mecanismes moleculars relacionats amb la senyalització i interacció cèl·lula-cèl·lula i la resposta moduladora a l'estrès oxidatiu. / We have recently described the Ets family transcription factor, ERM/ETV5, specifically up-regulated in EEC, and associated with myometrial infiltration. Ets family members have been correlated to tumor progression by up-regulating the expression of matrix-degrading proteases. We investigated the possibility that in EEC, ERM/ETV5 may induce the expression of genes involved in extra-cellular matrix remodeling. The overexpression of ERM/ETV5 induced scattering in the EEC cell line Hec-1A, correlating to increased MMP-2 gelatinase activity. Both ChIP and iRNA experiments and specific MMP-2 inhibitor demonstrated a functional link between ERM/ETV5 overexpression and MMP-2 activation. Orthotopically implanted overexpressing ERM/ETV5 tumors presented a more aggressive and infiltrative pattern of myometrial invasion. The specific localization of ERM/ETV5 and MMP-2 at the invasive front of myometrial infiltrating human EEC reinforced the hypothesis of a role for ERM/ETV5 in the early steps of endometrial dissemination.To understand the role of ETV5 during myometrial infiltration, we analysed by 2D-DIGE technology those proteins whose expression was altered in endometrial cell lines stably over-expressing ERM/ETV5. Pathway analysis pointed to actin regulation and TGF-beta and progesterone signalling as processes regulated by ERM/ETV5. We characterized the specific up-regulation of the nuclear dehydrogenase/reductase Hep27, its ERM/ETV5-dependent mitochondrial localization, and functional studies demonstrated a link with oxidative stress.Overall, the ETV5-related proteomic approach performed in the Hec-1A cell line reinforces a role of this transcription factor in the regulation of the migratory and invasive tumour behaviour, and points to a modulated response to oxidative stress associated with the promotion of invasion in endometrial cancer. To date, the identification of proteins involved in endometrial carcinoma invasion has been essentially conducted by immunohistochemical methods, without a global perception on the invasive front. In this work we attempted a proteomic approach to characterise specific components of the invasive front or reactive stroma by comparing the invasive area of an endometrial carcinoma with the non¬invasive superficial area and normal tissue from the same patients. This led us to identify proteins involved in cellular morphology, assembly and movement, differentially expressed at the invasive front, as well as pathways like cell-to-cell signalling and interaction and a modulated response to oxidative stress as events related to endometrial carcinoma invasion. In conclusion, we describe a novel proteomic approach that specifically deals with endometrial carcinoma invasion front, allowing the identification of new players of myometrial infiltration.

Staphylococcus aureus is a remarkably successful pathogen, accounting for an estimated 95,000 invasive infections annually in the U.S. alone. The burden of MRSA infections on public healthcare continues to rise, particularly with the continued spread of antibiotic resistant strains and the hyper-virulent CA-MRSA strains. The pathogenic nature of S. aureus can be attributed to the cache of virulence factors encoded within the genome of this organism. Typically, these are secreted toxins which directly interact with the host during infection, and facilitate pathogenesis. A previous screen in our laboratory investigating proteases in S. aureus identified a mutant in aminopeptidase Z as being attenuated in disease causation. Classically aminopeptidases function in the bioactivation/inactivation of proteins; and/or the utilization of imported peptides for cellular nutrition. We therefore hypothesize that cells deficient in one of these two processes would have decreased fitness levels, resulting in reduced virulence. We therefore sought to explore the role of PepZ in S. aureus; either in the processing of exogenous oligopeptides for nutrition, or in protein bioactivation/inactivation, and protein stability. We determined that S. aureus strains deficient in PepZ are less viable when cultured under conditions of starvation or while in competition for nutrients with the parent strain, and does not appear to be peptide driven. Using protein analysis approaches we have identified PepZ externalization, suggesting a potential for the aminopeptidase beyond the confines of the cell membrane. Additionally, we have also identified a potential role for PepZ in protein stability in this organism. Lastly, we present the essential role for PepZ in S. aureus virulence.

Physical activity can prevent cardiovascular disease, obesity, type II diabetes and some types of cancer. With only 3.5% of adults meeting the recommended physical activity guidelines, research has focused on the regulatory factors that influence physical activity level. Genetic influence accounts for the majority of physical activity regulation. However, there is limited information on the mechanisms that affect physical activity, in part, because of a lack of reliable methods to silence genes in vivo. The purpose of this dissertation was to identify mechanisms in skeletal muscle that influence physical activity. The methods used to accomplish the purpose of this dissertation were the evaluation of Vivo-morpholinos as a gene silencing tool in skeletal muscle and brain, identification of proteins in skeletal muscle associated with increased physical activity level, and the use Vivo-morpholinos to transiently knockdown the identified skeletal muscle proteins as a means to elucidate mechanisms for the peripheral regulation of physical activity. Overall, this study showed that Vivo-morpholinos effectively silenced genes in skeletal muscle yet required the use of a pharmacological aid to achieve gene silencing in the brain. Additionally proteins associated with calcium regulation (Annexin A6 and Calsequestrin 1) and the Kreb’s (TCA) cycle were found to be over expressed in the high active animals. The knockdown of Annexin A6 and Calsequestrin 1 resulted in a significant decrease in physical activity, thus showing that calcium regulation could influence the physical activity response. While these results provide a potential mechanism for the peripheral regulation of physical activity, a side effect observed was that Vivo-morpholinos can hybridize resulting in increased mortality rates of the treatment animals. Therefore, we developed methods to alleviate the toxic effects of Vivo-morpholinos. Thus, this dissertation refined a technique for determining a gene’s effect in an in vivo model and identified two candidate proteins (Annexin A6 and Calsequestrin 1) that play a role in regulating daily physical activity.

A complete proteome analysis of the chloroplast stroma, using 2D-PAGE, from spinach and Arabidopsis was performed. To improve the identification of proteins a computer program named SPECLUST was used. In SPECLUST, peak masses that are similar in many spots cluster together because they originate from the same protein with different locations on the gel. Within this program peaks in a cluster can be investigated in detail by peaks-in-common, and the unidentified masses that differ between spots in a cluster could be caused by protein modifications, which was analysed further by MS/MS. The thylakoid is an internal membrane system in the chloroplast where protein complexes involved in photosynthesis are housed. Enclosed in the thylakoid membrane is the chloroplast lumen, with a proteome estimated to contain 80-200 different proteins. Because the chloroplast lumen is close to the photosynthesis machinery in the plant, one can expect that the lumen proteome will change depending on if the plant is dark or light adapted. DIGE analysis of lumen proteins found that 15 lumen proteins show increased relative abundance in light-adapted plants. In addition co-expression analysis of lumen protein genes suggests that the lumen protein genes are uniformly transcriptionally regulated, not only by light but in a general manner. Plastocyanin is one of the proteins involved in the electron transfer in photosynthesis. Two homologous plastocyanin isoforms are encoded by the genes PETE1 and PETE2 in the nuclear genome of Arabidopsis, where PETE2 is the more abundant isoform. Knockout mutants of each of the plastocyanin isoforms shows that a 90% reduction of plastocyanin levels affects rates of photosynthesis and growth only slightly. A corresponding over-expression of plastocyanin in each of the two knockout mutants results in essentially wild-type photosynthetic performance. Reduced plastocyanin levels make the plant sensitive to Cu stress and therefore plastocyanin plays a major role as a Cu sink. A by-product of photosynthesis is hydrogen peroxide, which may be harmful for the plant. The discovery that an abundant protein found in the chloroplast lumen, TL29, shared sequence homology to Ascorbate Peroxidase (APX) was therefore of interest. We have evidence that TL29 is not an APX protein; it lacks the heme-binding active site and shows no activity. TL29 is located in the grana region and is electrostaticaly attached to the thylakoid membrane. It has four isoforms, with different pIs, both in the native and denatured form. It has no interaction with ascorbate, when compared to raAPX1. TL29 has two cysteine residues and one of them seems to have redox-regulated function, proposing that it may interact with other proteins close to PSII.