Oxidative stress can result in changes to many biomolecules and also affect their activities. We are interested in protein carbonylation, a type of unnatural oxidation which has been associated with numerous degenerative disease states and is also a consequence of the natural aging process. Protein carbonyls are stable species, but countless analytical barriers exist in terms of their identification. Thus, the main goal of this work was to develop and optimize analytical methods that could be used to help us better understand which, where, and how proteins are being carbonylated. Initial studies involved method validation for carbonylating, tagging, and enriching the model protein human serum albumin (HSA). We have developed a reproducible method of producing carbonylated protein in vitro in which HSA is treated with acrolein to carbonylate cysteines, histidines, and lysines. Protein carbonyls are compatible with various affinity labels and enrichment techniques. We strived to learn more about the efficiencies of various biotin affinity labels and avidin enrichment techniques using quantitative assays and mass spectrometry. Results showed a preference for different affinity labels based on their chemical properties and suggested that monomeric columns are selective for particular peptides. Most recently, method development and validation work was done involving a cleavable biotin tag that enables both enrichment and identification of protein carbonylation modification sites. This affinity tag offered the highest labeling efficiency of all tags tested in the past and greater coverage of modification sites than biotin hydrazide reagents. We applied our analytical methods to two sets of human blood samples. The first sample set was plasma taken from chronic kidney disease (CKD) patients. No carbonylation patterns were elucidated, but this project marked the beginning of blood analyses in which existing protocols were adapted to blood samples. The second sample set was serum/plasma taken from patients with traumatic injuries. We effectively applied our analytical methods to these sample sets and were able to visualize and quantitate temporal protein carbonylation patterns via Western blotting and iTRAQ-based mass spectrometry experiments. ProteoMiner experiments proved successful in that we were able to identify a larger and more diverse amount of carbonylated proteins via mass spectrometry.

The current environment for drug discovery and disease treatment relies heavily on genomic analysis, structural biology and chemical biology techniques. With the enormous advances in genomic analysis and structural biology, the use of and desire for targeted therapies has increased. However, as more genomic data for cancer disease state pathology becomes available we must ask increasingly difficult questions and even produce new technologies, such as activity-based probes, to answer these questions. In particular, targeted kinase inhibitors for the treatment of cancer has become a mainstay for drug development for both industry and academia, but it is evident that the genomic data is not always indicative of protein expression. Additionally, protein expression alone does not completely characterize functional activity. Therefore, in order to more accurately validate drug targets and predict drug efficacy, we must not only identify possible targets but also determine their activity in vivo. The goal of this work was to develop a probe for Protein Kinase A that would act by alkylating a conserved cysteine in the substrate-binding pocket of the enzyme. We hypothesized that by targeting the substrate-binding pocket we could effectively utilize the natural substrate selectivity filters as well as take into account multiple endogenous regulatory mechanisms. We produced probes utilizing portions of the pseudosubstrate inhibitor PKI that demonstrate the ability to label the catalytic subunit of Protein Kinase A in an activity-dependent manner, thus making it an important first step in a new class of activity-based probes for the kinome.

In the presented study, we identified that SRSF3 controls the alternative splicing of CPEB2 and consequently promotes a metastatic phenotype in triple negative breast cancer (TNBC). TNBC causes thousands of deaths annually, frequently due to a lack of effective treatments and a high rate of metastasis in patients. Alternative splicing has been found to be dysregulated in numerous cancers, while splicing factors such as SRSF3 are variably expressed. In this study we performed a siRNA panel to screen potential splicing factors, then used specific siRNA to study the effect of its knockdown on cellular function. These results showed that SRSF3 encourages the production of the pro-metastatic isoform of CPEB2, which contributes the aggressive phenotype of the tumor. We utilized numerous methods to measure the metastatic function of cultured TNBC cells to determine if SRSF3 strongly promoted the metastatic function. These data showed that siRNA reduction of SRSF3 was able to reduce the metastatic potential of cancer cells. These findings suggest that SRSF3 has great potential as a therapeutic measure to reduce and minimize the aggressiveness of TNBC tumors.

The emergence of genomics as a discrete field of biology has changed humanity’s understanding of our relationship with bacteria. Sequencing the genome of each newly-discovered bacterial species can reveal novel gene sequences, though the genome may contain genes coding for hundreds or thousands of proteins of unknown function (PUFs). In some cases, these coding sequences appear to be conserved across nearly all bacteria. Exploring the functional roles of these cases ideally requires an integrative, cross-species approach involving not only gene sequences but knowledge of interactions among their products. Protein interactions, studied at genome scale, extend genomics into the field of interactomics. I have employed novel computational methods to provide context for bacterial PUFs and to leverage the rich genomic, proteomic, and interactomic data available for hundreds of bacterial species. The methods employed in this study began with sets of protein complexes. I initially hypothesized that, if protein interactions reveal protein functions and interactions are frequently conserved through protein complexes, then conserved protein functions should be revealed through the extent of conservation of protein complexes and their components. The subsequent analyses revealed how partial protein complex conservation may, unexpectedly, be the rule rather than the exception. Next, I expanded the analysis by combining sets of thousands of experimental protein-protein interactions. Progressing beyond the scope of protein complexes into interactions across full proteomes revealed novel evolutionary consistencies across bacteria but also exposed deficiencies among interactomics-based approaches. I have concluded this study with an expansion beyond bacterial protein interactions and into those involving bacteriophage-encoded proteins. This work concerns emergent evolutionary properties among bacterial proteins. It is primarily intended to serve as a resource for microbiologists but is relevant to any research into evolutionary biology. As microbiomes and their occupants become increasingly critical to human health, similar approaches may become increasingly necessary.

Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did post translational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress.

Positional proteomics is emerging as an attractive technique to characterize protein termini, which play important biological roles in cells. Even with the advances in past decades, there still are areas for improvement. This thesis focuses on improving data quality and assignment confidence in positional proteomics. A novel workflow was designed for the large-scale identification of protein N-terminal sequences. 4-sulfophenyl isothiocyanate (SPITC) is used for N-termini sulfonation; Upon higher energy collisional dissociation (HCD), SPITC peptides in electrospray ionization ESI generate predominately y-type ion series; such simplification of spectra enables the identification of N-termini with high fidelity. The presence of b1 + SPITC product ions upon HCD furthers the confidence for N-terminal identifications. Secondly, sulfonated N-terminal peptides possess one negative charge site at low pH, which was exploited to enrich the SPITC modified N-terminal peptides by electrostatic repulsion hydrophilic interaction (ERLIC) chromatography. Such enrichment process allows both N-termini enriched and N-termini deficient fractions to be collected and analyzed by LC-MS/MS. This method was applied to an E. coli cell lysate, identifying approximately 350 N-terminal peptides (85% represented neo-N-termini from protein degradation and 15% from leading methionine excision). These N-terminal peptides represented 274 distinct E.coli proteins, 224 of which were also identified in the analysis of flow-through fractions from internal peptides. Another approach we took to boost the identification confidence is by exploiting iTRAQ (isobaric tag for relative and absolute quantitation) in the positional proteomics workflow. This approach allows for multiplexed comparison between different samples, and thus is well-suited for degradadomics analyses where degraded samples are compared to control samples. Both control and protease treated sample are labeled by different tags which allows direct comparison of protein N-termini with neo-N-termini. In addition, samples are analyzed duplicate by labeling with two tags, aiming for quick validation of peptides by internal replicates. In this study, Asp-N digested E.coli cell lysate is taken as a model system. A total of 500 N-terminal peptides, corresponding to 370 proteins, were identified with high confidence in one experiment, with 87% of those proteolytic products matching the expected protease digestion specificity, validating the assignment accuracy of this approach.

Orientador: Fabiana Ferreira de souza / Resumo: A obesidade e o diabetes têm alto índice mundial, ambas ligadas a infertilidade em diferentes espécies e em humanos. Pretende-se investigar os efeitos dessas doenças sobre a capacidade reprodutiva e o perfil proteico de células espermáticas. Foram utilizados 30 ratos Rattus novergicus (Wistar) com ~50 dias de idade, separados em 3 grupos: controle (n=10), diabéticos (n=10) e (obesos (n=10). O grupo obeso recebeu dieta de cafeteria e água ad libitum com 5% de sacarose, por 38 semanas. Os animais foram pesados uma vez por semana, para avaliação da evolução do peso para cálculo do índice de Lee. Foi induzida diabetes, com aplicação de 35 mg/kg de estreptozotocina, dose única, e foram considerados diabéticos animais com perda de peso corporal e níveis de glicemia maiores ou iguais a 120 mg/dL. Houve aumento e diminuição do peso corporal nos grupos obesos e diabéticos respectivamente. Os animais diabéticos apresentam aumento significativo no índice glicêmico. Quanto às alterações sistêmicas houve aumento da leptina no grupo obeso e redução da adiponectina nos diabéticos. Na citologia testicular houve aumento na concentração de das linhagens finais da espermatogênese, aumento das células de Sertoli, e na morfologia houve redução de células normais nos grupos diabéticos e obesos. As váriaveis foram normalizadas e analisadas pelo ANOVA one way, e comparações múltiplas pelo teste Newman-Keuls, os resultados foram apresentados como médias ± erro padrão (SEM), com P ≤ 0,05. Na análise p... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Obesity and diabetes have a high worldwide index, both linked to infertility in different species and in humans. It is intended to investigate the effects of these diseases on the reproductive capacity and protein profile of sperm cells. A total of 30 Rattus novergicus (Wistar) rats at ~50 days of age were divided into three groups: control (n = 10), diabetics (n = 10) and obese (n = 10) and water ad libitum with 5% sucrose for 38 weeks. The animals were weighed once a week to evaluate the evolution of the weight for the Lee's index calculation. Diabetes was induced with application of 35 mg / kg streptozotocin, and diabetic animals with a loss of body weight and glycemia levels greater than or equal to 120 mg / dL were observed to be diabetic, with increased and decreased body weight in the obese and diabetic groups, respectively. As for the systemic alterations, there was an increase in leptin in the obese group and reduction of adiponectin in diabetics. In the testicular cytology there was an increase in the concentration of the final spermatogenesis lines, increase of Sertol cells i, and in the morphology there was reduction of normal cells in the diabetic and obese groups. The variances were normalized and analyzed by ANOVA one way, and multiple comparisons by the Newman-Keuls test, the results were presented as means ± standard error (SEM), with P ≤ 0.05. In the proteomic analysis, 15 proteins were identified as important in the separation of the groups, 14 were less ex... (Complete abstract click electronic access below) / Mestre

A banana tem grande importância econômica, é a fruta mais produzida no mundo, e o Brasil é o segundo maior produtor. É uma fruta altamente perecível, de rápida maturação, sensível a choques mecânicos, suscetível a descoloração, ao amolecimento excessivo e a patógenos na fase pós-colheita. As mudanças ocorridas durante o amadurecimento levam a uma vida de prateleira muito reduzida e são dependentes da expressão de diversas proteínas. Portanto, a identificação de proteínas associadas com essas modificações pode contribuir para a melhor compreensão da regulação do amadurecimento e auxiliar no aprimoramento das estratégias de conservação pós-colheita e melhoria da qualidade dessa fruta. Através da análise proteômica diferencial podem ser identificadas proteínas com variação de abundância durante o amadurecimento e que estejam envolvidas nesse processo. O presente trabalho teve por objetivo comparar os mapas protéicos de polpa de banana (Musa acuminata cv. nanicão) nas fases pré-climatérica e climatérica, e a identificar spots de proteínas que diferem em abundância nesses dois estádios, através da eletroforese bidimensional acoplada à espectrometria de massas. Neste trabalho foram utilizadas três amostragens distintas de frutas, de modo a minimizar o efeito da variabilidade biológica, não relacionada com o amadurecimento. Para a obtenção dos perfis protéicos foi utilizada a metodologia 2D-DIGE. Os géis obtidos foram analisados com o software PDQuest e para a análise estatística foi utilizado o teste T. Chegou-se a uma lista de 50 spots que foram recortados dos géis, sendo as proteínas digeridas e seqüenciadas por espectrometria de massas. Destas proteínas, 26 tiveram a provável identidade apontada pela comparação com o banco de dados MSDB, empregando o software Mascot. A maioria das proteínas identificadas apresenta provável função durante o amadurecimento da banana e podem estar relacionadas com processos bioquímicos relacionados com a qualidade da fruta. / Banana has great economic importance, is the most widely produced fruit in the world, and Brazil is the second largest producer. It is a highly perishable fruit, ripening fast, sensitive to mechanical shock, susceptible to discoloration, excessive softening and pathogens in the post-harvest. The changes during ripening leads to a very limited shelf life and are dependent on the expression of several proteins. Therefore, the identification of proteins associated with these modifications may contribute to better understanding the regulation of maturation and help to improve strategies for post-harvest preservation and improvement of this fruit. Through differential proteomics analysis could be identified proteins with a range of abundance during ripening and that could be involved in this process. This study aimed to compare the protein maps of banana pulp (Musa acuminata cv. nanicão) in pre-climacteric and climacteric phases, and identify protein spots that differ in abundance in these two stages by two-dimensional electrophoresis coupled to mass spectrometry. In this study we used three different fruits samples, to minimize the effect of biological variability, non-ripening related. To obtain the protein profiles was used 2D-DIGE methodology. The gels were analyzed with the PDQuest software and Student´s t-Test was used to statistical analysis. A list of 50 spots differential acumulated were detected and then were extracted of gels, protein digested and sequenced by mass spectrometry. Of these proteins, 26 had the probable identity indicated by comparison with the MSDB database, using Mascot software. Most of the identified proteins has probable function during banana ripening and may be related to biochemical processes related to fruit quality.

Orientador: Pedro Luis da Costa Aguiar Alves / Coorientador: Martin Vila-Aiub / Banca: Ricardo Victória Filho / Banca: Caio Antonio Carbonari / Banca: Rubem Silverio de Oliveira Junior / Banca: Leonardo Bianco de Carvalho / Resumo: As culturas agrícolas estão sujeitas a conviver com plantas daninhas que podem, em determinadas situações, reduzir seu potencial genético de produção, causando prejuízos. Na maioria das vezes, devido à praticidade e ao custo, essas plantas são controladas pela aplicação de herbicidas, o que se denomina de controle químico. Dentre os produtos utilizados, está o glyphosate, que nos últimos anos vem sendo usado de maneira repetitiva devido à presença quase que exclusiva de culturas tolerantes a esse herbicida, como a soja, o algodão e o milho. Com isso, a utilização desse herbicida vem selecionando, nos últimos anos, plantas que apresentam adaptações para resistir a sua ação, dentre elas o azevém e o capim-pé-de-galinha. A resistência pode ser causada por diferentes mecanismos, envolvendo ou não a enzima-alvo de atuação do herbicida. Para o glyphosate, essa enzima é a 5-enolpiruvilshiquimato-3-fosfato, e essa pode apresentar mutações simples ou duplas. Essas mutações, além de afetar a tolerância da planta ao herbicida, podem modificar a fisiologia e o metabolismo da espécie, tornando-a mais ou menos adaptada ecologicamente, o que é denominado de fitness. Este trabalho teve por objetivo estudar os impactos da resistência ao glyphosate nas duas espécies supracitadas. Em um primeiro trabalho, plantas de azevém resistentes ao glyphosate foram comparadas a plantas suscetíveis quanto a seu perfil metabólico e proteico antes e após a aplicação do herbicida. As plantas suscetíveis apres... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Crops are subject to live with spontaneous plants that may in certain situations reduce their genetic potential of production, causing losses. Most of the time, due to the practicality and cost, these plants are controlled by the application of herbicides, what is called chemical control. Among the products for this control, there is glyphosate, which in recent years has been used repetitively due to the almost exclusive presence of crops tolerant to this herbicide, such as soybean, cotton and corn. The use of this herbicide has been selecting, therefore in the last years plants that present adaptations to resist its application, among them Italian ryegrass and goosegrass. The resistance can be caused by different mechanisms, involving or not the target enzyme of action of the herbicide. For glyphosate, this enzyme is 5-enolpyruvyl-silicon-3-phosphate and it may present single or double mutations. These mutations, in addition to affecting the tolerance of the plant to the herbicide, can modify the physiology and metabolism of the species, making it more or less ecologically adapted, which is called fitness. The objective of this work was to study the impacts of glyphosate resistance on the two species mentioned above. In a first work, glyphosate resistant Italian ryegrass plants were compared to susceptible plants for their metabolic and protein profile before and after herbicide application. Susceptible plants showed higher levels of amino acids produced from the shikimic acid route and lower levels of glyphosate in their leaves 72 hours after the application of the herbicide. It was observed that the susceptible plants presented greater development, proteins linked to the greater ryegrass physiology expressed and differential expression of proteins bound to vegetal defense against stresses, absent in resistant plants. After th... (Complete abstract click electronic access below) / Doutor

O câncer de bexiga é uma neoplasia urogenital que acomete homens e mulheres, sendo que somente no Brasil 8.600 novos casos ao ano são diagnosticados. Cistoscopia transuretral é a conduta padrão no diagnóstico e acompanhamento do câncer de bexiga. Entretanto, tal procedimento é extremamente invasivo e doloroso além de ter elevado custo e não garantir todos os resultados. Assim, busca-se por marcadores moleculares que possam auxiliar no diagnóstico e progressão do câncer de bexiga, bem como diminuir a necessidade de exames invasivos no acompanhamento de pacientes tratados. Nesse sentido, a urina tem papel de destaque como fonte de biomarcadores devido principalmente ao seu caráter não invasivo. <br /> Nesse trabalho foram utilizadas duas abordagens \'ômicas\': proteômica e metabolômica, para a busca de biomarcadores em urina para o diagnóstico e prognóstico do câncer de bexiga, respectivamente. Com a abordagem proteômica buscou-se apenas por biomarcadores para o diagnóstico da doença e, utilizando as técnicas de eletroforese 2-DE, OFFGEL e MS, juntamente com análise estatística multivariada, foi possível identificar 32 proteínas que apresentam-se como potenciais marcadores para o câncer de bexiga. A abordagem metabolômica foi empregada para a busca de biomarcadores para reincidência e progressão da doença. As técnicas analíticas utilizadas nessa abordagem, LC-MS e CE-MS, mostraram-se complementares e, dos resultados obtidos com ambas e avaliados com análise estatística multivariada foi possível indicar 19 metabólitos para reincidência e 23 metabólitos para progressão do câncer de bexiga. <br /> Assim, neste trabalho explorou-se como as ciências \'ômicas\', a qual abrange técnicas analíticas de high-throughput, estatística multivariada e ferramentas de bioinformática auxiliando na descoberta de potenciais biomarcadores não invasivos para o diagnóstico e prognóstico do câncer de bexiga. Portanto, o presente estudo foi de grande importância e relevância à medida que ilustrou como tais técnicas podem potencialmente auxiliar no diagnóstico e prognóstico de doenças e contribuir para tratamentos personalizados no futuro, indicando a potencialidade de estudos dessa natureza. / Bladder cancer is an urogenital cancer affecting men and women, and just in Brazil 8,600 new cases are diagnosed annually. Transurethral cystoscopy is a standard conduct in the diagnosis and monitoring of bladder cancer. However, this procedure is extremely invasive, painful, expensive and does not guarantee the best results. Thus, the searching for molecular markers may assist in the diagnosis and monitoring of bladder cancer, as well as decreasing the need for invasive tests in the monitoring of patients treatment. In this way, urine shows an important role as a source of biomarkers, mainly due to its non-invasive nature. <br /> In this work we used two \'omics\' approaches: proteomics and metabolomics, to search for biomarkers in urine for the diagnosis and progression of bladder cancer, respectively. The proteomics approach was explored for biomarkers for diagnosing disease. Using 2-DE, OFFGEL electrophoresis, and MS techniques, as well multivariate statistical analysis, they were identified 32 proteins that may be pointed as potential markers for bladder cancer. The metabolomics approach was used to search for biomarkers for progression and recurrence of the disease. The analytical techniques used for this approach, LC-MS and CE-MS, were complementary to each other and the results evaluated with multivariate statistical analysis indicated that 19 metabolites for recurrence and 23 metabolites for progression of bladder cancer could possibly be used for validation studies. <br /> Thus, we demonstrated how the \'omics\' sciences, which include high- throughput analytical techniques, multivariate statistical analysis, and bioinformatics tools, aid in the discovery of potential biomarkers for noninvasive diagnosis, evaluate recurrence and monitor progression of bladder cancer. Therefore, this study was of high relevance to demonstrate the potential of such techniques to contribute to studies of personalized medicine.