A cana-de-açúcar, uma das maiores culturas agrícolas no mundo, é uma antiga fonte de energia para os seres humanos e mais recentemente, tem sido utilizada como fonte para a produção de etanol. Em estudos moleculares sobre a cana-de-açúcar, sua complexidade genética e o fato de seu genoma não estar completamente sequenciado, dificultam a análise dos proteomas de seus diferentes tecidos. Recentemente, em um estudo de um grupo brasileiro sobre os transcriptomas de vários tecidos de cana-de-açúcar, 17.563 transcritos completos foram sequenciados, os quais correspondem a aproximadamente 53% do genoma predito desta planta. Entretanto, sabe-se que o conteúdo de mRNA nem sempre se correlaciona diretamente com os níveis de expressão de proteínas. Dessa forma, a caracterização do proteoma é importante para identificar as proteínas que são de fato expressas, em complementaridade aos estudos sobre o genoma, e fornece informação preditiva sobre a composição do proteoma de diferentes cultivares. Entretanto, estudos sobre folhas de cana-de-açúcar são raros. Neste estudo, com o objetivo de caracterizar o proteoma de folhas +1 de cana-de-açúcar, foram selecionadas duas espécies ancestrais (Saccharum officinarum [S. off] e S. spontaneum [S. sp]) e uma cultivar híbrida (SP80-3280 [SP]), as quais diferem-se na capacidade de produção de sacarose e na resistência a patógenos. A análise de parâmetros fisiológicos (taxa de fotossíntese, condutância estomática, taxa de transpiração e eficiência no uso de água), em folhas de 12 meses revelaram que em S. off e SP existe uma relação melhor entre o número de moléculas de água perdidas na transpiração e o número de moléculas de CO2 assimiladas na fotossíntese do que em S. sp. Os mesmos parâmetros foram avaliados em folhas +1 de SP com 4, 8, 11 e 13 meses de crescimento e revelaram o papel dos estômatos na manutenção do potencial hídrico das folhas, atuando na modulação da taxa de respiração em resposta às mudanças ambientais, entretanto sem induzir alterações drásticas na taxa de fotossíntese, no período avaliado neste estudo. Com o objetivo de obter proteínas em condições ótimas para análise proteômica, foram testados diferentes métodos de extração de proteínas, que foram avaliados quanto ao rendimento e perfil eletroforético das proteínas extraídas, e selecionado um protocolo que utiliza uma combinação de fenol e dodecil sulfato de sódio para este estudo. Para caracterizar e comparar os proteomas de folhas +1 de S. off, S. sp e SP, foram utilizadas as abordagens proteômicas Shotgun/bottom-up, GeLC-MS/MS e Multidimensional Protein Identification Technology (MudPIT) (off-line e \"on-line\"), e as plataformas MaxQuant e Perseus, como ferramentas de bioinformática para análise dos dados. Os dados obtidos por meio destas abordagens foram combinados e resultaram na identificação de 5.825 grupos de proteínas em folhas +1 de S. off, S. sp e SP, e a análise de enriquecimento de termos pelo Gene Ontology destacou a natureza fotossintética desses proteomas, além da representatividade de organelas envolvidas em processos de fotossíntese e metabolismo de carbono. A análise quantitativa comparativa dos 1.060 grupos de proteínas identificados por meio da abordagem Shotgun/bottom-up revelou que 120 proteínas encontravam-se diferencialmente expressas em folhas de S. off, S. sp e SP, e destas, aquelas que mostraram diferença em abundância de pelo menos quatro vezes, são relacionadas com processos biológicos envolvidos com os estresses biótico e abiótico (resposta a outros organismos, resposta ao íon cádmio, defesa contra fungos e defesa ao estresse oxidativo). Para a análise da variação ontogenética no proteoma de folhas +1 de SP (de 4, 8, 11 e 13 meses de crescimento) foi aplicada a abordagem Shotgun/bottom-up, que resultou na identificação de 5.780 peptídeos, correspondendo a 1.615 grupos de proteínas únicos. Sessenta e cinco proteínas foram identificadas com abundância diferencial entre as amostras, e a análise de agrupamento hierárquico revelou que as folhas de 13 meses mostraram um perfil mais distinto, enquanto que as outras formaram um grupo separado. A caracterização dos fosfoproteomas de folhas +1 de S. off, S. sp e SP utilizando a abordagem Immobilized Metal Affinity Chromatography (IMAC) como método de enriquecimento de fosfopeptídeos, e identificação por LC-MS/MS, resultou na identificação de 633 fosfopeptídeos únicos em 515 proteínas. A caracterização dos fosfoproteomas de folhas +1 de SP (de 4, 8, 11 e 13 meses de crescimento) utilizando a abordagens IMAC e Hydroxy Acid-Modified Metal Oxide Chromatography (HAMMOC) como métodos de enriquecimento de fosfopetídeos, e identificação por LC-MS/MS, resultou na identificação de 558 fosfopeptídeos únicos em 413 proteínas. Em ambas análises, foram identificadas várias proteínas quinases fosforiladas, e de forma geral, a análise de enriquecimento de termos pelo Gene Ontology revelou o núcleo e o citoplasma, entre os componentes celulares, e a regulação da transcrição, a resposta ao estresse abiótico e a glicólise, entre os processos biológicos, como os termos mais enriquecidos. Este é o primeiro estudo objetivando a caracterização dos proteomas totais de folhas de cultivares e genótipos de cana-de-açúcar no Brasil, e seus resultados representam um significante esforço para o uso de diferentes abordagens de proteômica para aprofundar o conhecimento sobre os complexos processos fisiológicos que modulam o crescimento e a performance em cana-de-açúcar / Sugarcane, one of the major crops worldwide, is an old energy source for humans and, more recently, has been used as a source for ethanol production. In sugarcane molecular studies, its complex genetic background and the lack of a complete genome sequencing hinder the analysis of proteomes of different tissues. Recently, in a Brazilian research study focused on the transcriptomes of different sugarcane tissues, 17,563 full-length transcripts were sequenced and covered approximately 53% of the predicted sugarcane genome. However, it is known that mRNA levels are not always directly correlated with protein expression levels. Therefore, the study of the sugarcane proteome is important to identify proteins that are actually expressed, complementing the genome studies, and provides predictive information on the proteome composition of different cultivars. However, studies on sugarcane leaves are rare. In this study, with the aim of characterizing the proteome of sugarcane +1 leaves, we selected two ancestral species (Saccharum officinarum [S. off] and S. spontaneum[S. sp]), and a hybrid cultivar (SP80-3280 [SP]), which differ in sucrose production and pathogen resistance. The analysis of physiological parameters (photosynthesis rate, stomatal conductance, transpiration and efficiency in water use) of 12 month-old +1 leaves revealed that in S. off and SP there is a better relationship between the number of H2O molecules lost by transpiration and the number of CO2 molecules assimilated upon photosynthesis than in S. sp. The same parameters were evaluated in +1 leaves of SP upon 4, 8, 11 and 13 months of growth and revealed the role of stomata in the maintenance of leaf water potential, acting in the modulation of the transpiration rate in response to environmental changes, however without inducing drastic changes in the photosynthesis rate, during the period evaluated in this study. In order to obtain proteins in optimal conditions for proteomic analysis, we carried out different protein extraction methods and evaluated the yield and electrophoretic profiles of extracted proteins, and selected a protocol using a combination of phenol and sodium dodecyl sulfate to carry out this study. To characterize and compare the +1 leaf proteomes of S. off, S. sp and SP we used Shotgun/bottom-up, GeLC-MS/MS and Multidimensional Protein Identification Technology (MudPIT) (off-line and \"on-line\") as proteomic approaches, and the software MaxQuant and Perseus as bioinformatic tools for data analysis. The combined results of these approaches allowed for the identification of 5,825 protein groups in +1 leaves of S. off, S. sp and SP, and the Gene Ontology enrichment analysis highlighted the photosynthetic nature of these proteomes, along with high representativeness of organeles involved in the processes of photosynthesis and carbon metabolism. The comparative quantitative analysis of 1,060 protein groups identified using the Shotgun/bottom-up approach revealed that 120 proteins were differentially expressed in +1 leaves of S. off, S. sp and SP, and of these, those that showed at least a 4-fold change are related to biological processes involved in both abiotic and biotic stress (response to other organism, response to cadmium ion, defense response to fungus, and defense to oxidative stress). For the analysis of the ontogenetic variation in the proteome of +1 leaves of SP (of 4, 8, 11 and 13 months old plants) the Shotgun/bottom-up proteomic approach was employed and resulted in the identification of a total of 5,780 peptides corresponding to 1,615 unique protein groups. Sixty five proteins were identified as differentially abundant between the samples and a hierarchical cluster analysis revealed that the 13 months-old leaves showed the most distinct profile, while the others clustered together. The characterization of the phosphoproteomes of +1 leaves of S. off, S. sp and SP using Immobilized Metal Affinity Chromatography (IMAC) as an enrichment method for phosphopeptides, and LC-MS/MS analysis, resulted in the identification of 633 unique phosphopeptides in 515 proteins. The characterization of the phosphoproteome of +1 leaves of SP (of 4, 8, 11 and 13 months old plants) using IMAC and Hydroxy Acid-Modified Metal Oxide Chromatography (HAMMOC) as enrichment methods for phosphopeptides, and LC-MS/MS analysis, resulted in the identification of 558 unique phosphopeptides in 413 proteins. In both analyses various phosphorylated kinases were identified, and the overall Gene Ontology enrichment analysis showed as most enriched terms nucleus and cytoplasm, as cellular components, and regulation of transcription, response to abiotic stress and glycolysis in biological process. This is the first study to characterize the whole proteome of leaves from sugarcane cultivars and genotypes in Brazil, and its results constitute a significant effort to use different proteomic approaches to advance the knowledge on the complex physiological processes modulating sugarcane growth and performance

No description available.

Introduction: Chronic myeloid leukaemia is a blood cancer which progresses from a chronic phase to an acute blast crisis if untreated. Disease progression and treatment resistance may be precipitated by the mutator action of BCR/ABL protein tyrosine kinase (PTK), but only few protein phosphosites involved in the DNA damage response have been investigated with respect to BCR/ABL action. Aim: The aim of this PhD project was to demonstrate that BCR/ABL PTK expression can affect the response to genotoxic stress signalling at the protein phosphorylation level. Methodology: Etoposide-induced DNA damage response has been studied in control and BCR/ABL PTK-expressing Ba/F3 cells using apoptosis and γH2AX assays. Quantitative phosphoproteomics was performed with iTRAQ peptide labelling to discover putative modulated phosphorylation sites. Absolute quantification (AQUA ) performed with selected reaction monitoring was used to validate discovery phosphoproteomics. The effect of genotoxic stress on the THO complex protein Thoc5/Fmip was studied using western blots. Results: The expression of BCR/ABL PTK induced γH2AX phosphorylation after etoposide exposure. This was associated with the modulation of H2AX tyrosine 142 phosphorylation, MDC1 (serines 595 and 1053) and Hemogen serine 380 phosphorylation among proteins regulated by both BCR/ABL PTK and etoposide. We identified that leukaemogenic PTKs mediate Thoc5/Fmip phosphorylation on tyrosine 225 via Src proto-oncogene and oxidative stress, while ATM and MEK1/2 may control its phosphorylation. Human CD34+ CD38- leukaemic stem cells showed pronounced level of THOC5/FMIP tyrosine phosphorylation. Expression of phosphomutant Thoc5/Fmip Y225F might reduce apoptosis mediated by etoposide and H2O2. Conclusion: BCR/ABL PTK can sustain, create, block and change the intensity of protein phosphorylation related to genotoxic stress. Modulation of H2AX, MDC1, Hemogen and Thoc5/Fmip post-translational modifications by BCR/ABL PTK might promote unfaithful DNA repair, genomic instability, anti-apoptotic signalling or abnormal cell differentiation, resulting in leukaemia progression.

As fraturas e perdas ósseas representam altos riscos para o Sistema público de Saúde (SUS), além de afetar a qualidade de vida do paciente, portanto é necessário o entendimento das bases moleculares que envolvem os mecanismos de reparo ósseo. Citocinas secretadas por células do sistema imune presentes no local da inflamação, como as IL-6, IL-10 e TNFα atuam como fatores quimiotáticos para células mesenquimais, que proliferam e se diferenciam em osteoblastos pela ação autócrina e parácrina de Proteínas Morfogenéticas Ósseas (BMPs), principalmente a BMP2. Embora seja conhecido que a ação de BMP2 ocorra através de sua ligação nos receptores ActRI/BMPR, que ativam proteínas SMADS 1/5/8 efetoras, pouco se sabe sobre os mecanismos intracelulares que participam do processo de diferenciação osteoblástico. Neste estudo propôs-se analisar as diferenças no conteúdo de proteínas totais e de proteínas fosforiladas em células mesenquimais de pele induzidas à osteogênese pelo tratamento com BMP2 por diferentes períodos de tempo, utilizando-se de Isótopos Estáveis de Dimetila acoplado ao LC/MS. A partir de 150µg de material inicial, foi possível identificar 2.264 proteínas, as quais foram quantificadas nos diferentes pontos de indução, sendo que 235 são fosforiladas. Análise de motivos de quinases mostrou que diversos substratos possuem sítios fosforilados correspondentes àqueles dos motivos de fosforilação das quinases Casein Kinase, p38, CDK e JNK. A análise da ontologia gênica mostrou um aumento de processos biológicos relacionados com sinalização e diferenciação após a primeira hora de indução com rhBMP2. Além disso, proteínas envolvidas com o rearranjo do citoesqueleto e com vias de sinalização Wnt e Ras foram encontradas como tendo fosforilação diferencial durante todos os períodos estudados. Os dados revelaram novos substratos intracelulares que são fosforilados nos primeiros momentos do comprometimento com a diferenciação osteoblástica mediada pelo tratamento com rhBMP2 em células mesenquimais derivadas da pele. Além disso, clones celulares que superexpressam as proteínas recombinantes humanas BMP2 e BMP4 foram gerados, e sua atividade verificada in vitro. Paralelamente, a rhBMP7, obtida anteriormente, foi purificada por cromatografia de afinidade utilizando-se uma coluna de Heparina-Sepharose, que foi posteriormente utilizada para ensaios in vitro e in vivo, nos quais se mostrou capaz de gerar osteoblastos e tecido ósseo, respectivamente, o que abre novas possibilidades para o uso destas proteínas como biofármacos no Brasil. / Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated human skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. From 150 µg of starting material, 2,264 proteins containing two or more peptides were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during commitment to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. Cell clones overexpressing the human BMP 2 and 4 recombinant proteins were also generated, and their biological activity was confirmed in vitro. In parallel, chromatography-affinity purified rhBMP7, obtained using heparin-Sepharose columns, was used for in vivo and in vitro assays to evaluate the ability of this purified protein to generate osteoblasts and bone tissue, respectively, opening new avenues for the use of these proteins as biopharmaceuticals in Brazil.

Células tronco embrionárias (CTE) são capazes de proliferar indefinidamente mantendo a sua pluripotência, isto é, a capacidade de se diferenciar em diversos tipos celulares perante estímulos adequados. Esse potencial tem sido intensamente estudado, de modo a permitir a utilização dessas células em terapias de reposição celular. Trabalhos anteriores demonstraram que as proteínas kinases C (PKC) são importantes moduladores moleculares de cascatas de sinalização que levam ao processo de proliferação e auto-renovação das CTE. Porém o papel exato das diferentes isoenzimas das PKCs ainda não foi elucidado. Isso ocorre porque a família das PKCs é composta por pelo menos dez isoenzimas e apenas, recentemente, desenvolveram-se moduladores específicos para as diferentes isoenzimas, o que permitirá estudar o papel específico dessas quinases. No presente trabalho verificamos que a ativação da PKCδ induziu a proliferação de CTE indiferenciadas sem induzir a diferenciação das mesmas. Para tentar elucidar as vias de sinalização mediadas pela PKC&#948 que levam à proliferação das CTE indiferenciadas realizamos estudos de fosfoproteômica o que possibilitou a identificação de potenciais alvos diretos e indiretos da PKC&#948. Dentre os alvos identificados foram encontradas diversas proteínas relacionadas com proliferação, transcrição, tradução e resposta ao stress (chaperonas), contribuindo para a hipótese de que a ativação da PKCδ leva à proliferação das CTE indiferenciadas. Em diversos sistemas, a ativação da PKCδ leva à ativação da MAPK, em particular das ERK1/ 2, sendo essa via capaz de induzir a proliferação de diversas linhagens celulares. Identificamos diversas proteínas alvos da PKC&#948, que interagem também com componentes da via das MAPKs. Desta forma, verificamos a influência da ativação da PKC&#948 na via das MAPKs. De fato, a ativação da PKC&#948 na linhagem de CTE murinas indiferenciadas, E14TG2a, ativou a MEK, ERK1/ 2 e o fator de transcrição ELK-1. Como estudos anteriores demonstraram que a inibição da ERK1/ 2 mantém CTE indiferenciadas e que a ativação desta via poderia levar à diferenciação de CTE, investigamos a cinética de ativação da ERK pela PKC&#948. Demonstramos que a ativação da ERK pela PKC&#948 se da de modo transiente e que apesar da PKC&#948 não translocar para o núcleo, sua ativação induz a fosforilação e translocação nuclear da ERK, que atuará na fosforilação do fator de transcrição ELK-1. Desta forma, concluímos que a PKC&#948 induz a proliferação das CTE murinas indiferenciadas ativando transitoriamente a via das ERK1/ 2, que translocam para o núcleo fosforilando fatores de transcrição como a ELK1 e levando possivelmente ao aumento de proliferação dessas células. A ativação transiente das ERK1/ 2 pela PKC&#948 é importante para a auto-renovação das CTE. / Embryonic stem cells (ESC) are able of proliferating indefinitely maintaining their pluripotency, which is the capability to differentiate in different cell types upon appropriate stimuli. Pluripotency has been intensely investigated in order to allow the use of these cells in cellular replacement therapies. Previous work has demonstrated that the serine/ threonine kinases, such as, Protein kinases C (PKC) are important modulators of signaling cascades that lead to the process of proliferation and self-renewal of ESC. However, the exact role of the different PKC isoenzymes still remains to be elucidated. Due to the fact that the PKC family is composed of at least ten different isoenzymes and only recently isoenzyme specific modulators have been developed, which now allows the elucidation of these kinases roles. In the present work we verified that activation of PKC&#948 induced undifferentiated ESC have their proliferation rate increased. Trying to elucidate the signaling pathways mediated by PKC&#948 that lead to the proliferation increase we performed phosphoproteomic studies to identify potential PKC&#948 targets. Between the targets identified we found several proteins related with proliferation, protein transcription, translation and stress response (chaperones). These targets contributed to the hypothesis that PKC&#948 activation leads to undifferentiated ESC proliferation. In different cell lines, PKC&#948 activation leads to MAPK activation, through ERK1/ 2 activation, which are frequently involved with cellular proliferation. We also identified several targets of PKC&#948 that Interact with several components of MAPK`s signaling cascade. PKC&#948 activation in murine undifferentiated ESC line, E14TG2a, led to MEK, ERK1/ 2 and the transcription factor Elk-1 activation. Some articles demonstrate that the inhibition of ERK1/2 are responsible to maintains ESC undifferentiated and that it`s activation could lead to ESC differentiation. Analysing the kinetics of ERK activation in the ESC by PKC&#948, we show that ERK activation was transient and despite the fact that PKC&#948 does not translocated to the nucleus upon activation, but induces ERK activation and it`s nuclear translocation, where ERK could phosphorylate the transcription factor Elk-1. In conclusion PKC&#948 induces undifferentiated murine ESC proliferation increase by a transient ERK activation and it`s nuclear translocation.

As fraturas e perdas ósseas representam altos riscos para o Sistema público de Saúde (SUS), além de afetar a qualidade de vida do paciente, portanto é necessário o entendimento das bases moleculares que envolvem os mecanismos de reparo ósseo. Citocinas secretadas por células do sistema imune presentes no local da inflamação, como as IL-6, IL-10 e TNFα atuam como fatores quimiotáticos para células mesenquimais, que proliferam e se diferenciam em osteoblastos pela ação autócrina e parácrina de Proteínas Morfogenéticas Ósseas (BMPs), principalmente a BMP2. Embora seja conhecido que a ação de BMP2 ocorra através de sua ligação nos receptores ActRI/BMPR, que ativam proteínas SMADS 1/5/8 efetoras, pouco se sabe sobre os mecanismos intracelulares que participam do processo de diferenciação osteoblástico. Neste estudo propôs-se analisar as diferenças no conteúdo de proteínas totais e de proteínas fosforiladas em células mesenquimais de pele induzidas à osteogênese pelo tratamento com BMP2 por diferentes períodos de tempo, utilizando-se de Isótopos Estáveis de Dimetila acoplado ao LC/MS. A partir de 150µg de material inicial, foi possível identificar 2.264 proteínas, as quais foram quantificadas nos diferentes pontos de indução, sendo que 235 são fosforiladas. Análise de motivos de quinases mostrou que diversos substratos possuem sítios fosforilados correspondentes àqueles dos motivos de fosforilação das quinases Casein Kinase, p38, CDK e JNK. A análise da ontologia gênica mostrou um aumento de processos biológicos relacionados com sinalização e diferenciação após a primeira hora de indução com rhBMP2. Além disso, proteínas envolvidas com o rearranjo do citoesqueleto e com vias de sinalização Wnt e Ras foram encontradas como tendo fosforilação diferencial durante todos os períodos estudados. Os dados revelaram novos substratos intracelulares que são fosforilados nos primeiros momentos do comprometimento com a diferenciação osteoblástica mediada pelo tratamento com rhBMP2 em células mesenquimais derivadas da pele. Além disso, clones celulares que superexpressam as proteínas recombinantes humanas BMP2 e BMP4 foram gerados, e sua atividade verificada in vitro. Paralelamente, a rhBMP7, obtida anteriormente, foi purificada por cromatografia de afinidade utilizando-se uma coluna de Heparina-Sepharose, que foi posteriormente utilizada para ensaios in vitro e in vivo, nos quais se mostrou capaz de gerar osteoblastos e tecido ósseo, respectivamente, o que abre novas possibilidades para o uso destas proteínas como biofármacos no Brasil. / Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated human skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. From 150 µg of starting material, 2,264 proteins containing two or more peptides were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during commitment to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. Cell clones overexpressing the human BMP 2 and 4 recombinant proteins were also generated, and their biological activity was confirmed in vitro. In parallel, chromatography-affinity purified rhBMP7, obtained using heparin-Sepharose columns, was used for in vivo and in vitro assays to evaluate the ability of this purified protein to generate osteoblasts and bone tissue, respectively, opening new avenues for the use of these proteins as biopharmaceuticals in Brazil.

Le récepteur de la sérotonine (5-hydroxytryptamine, 5-HT) 2A a été identifié comme la cible principale des hallucinogènes psychédéliques comme le diéthylamide de l'acide lysergique (LSD). Ces agonistes sont connus pour reproduire quelques uns des principaux symptômes de la schizophrénie. Un paradoxe non résolu est que seuls certains agonistes des récepteurs 5-HT2A présentent une activité hallucinogène, alors que des composés de structure apparentée avec une affinité comparable au niveau du récepteur n'ont pas des propriétés psychoactives. En utilisant une approche quantitative phosphoproteomic combinant un marquage isotopique stable en acides aminés dans la culture cellulaire (SILAC), un double enrichissement en phosphopeptides par chromatographie d'interaction hydrophile (HILIC) / chromatographie d'affinité (IMAC) et la spectrométrie de masse haute résolution, nous avons comparé les phosphoprotéome dans des cellules HEK-293 cellules exprimant de manière transitoire le récepteur 5-HT2A sous trois conditions: cellules non stimulées, cellules exposées aux hallucinogènes [2,5-diméthoxy-4-iodophényl]-2-aminopropane (DOI) et LSD les cellules exposées aux agonistes non- hallucinogènes Lisuride et Ergotamine. Parmi les 5996 phosphopeptides identifiés, 454 sont spécifiquement régulés par les hallucinogènes. Il s'agit notamment d'un résidu sérine du récepteur 5-HT2A éventuellement impliqués dans la régulation de la désensibilisation des récepteurs qui a été spécifiquement phosphorylée lors de l'exposition aux deux hallucinogènes. La phosphorylation différentielle des récepteurs 5-HT2A dans les cellules exposées aux agonistes hallucinogènes (DOI et le LSD) vs non hallucinogènes (lisuride et l'ergotamine) a été confirmé par l'analyse par spectrométrie de masse du récepteur purifié. Parallèlement, l'exposition des cellules aux agonistes hallucinogènes induit une internalisation et une désensibilisation des récepteurs moins prononcée qu'après exposition à la non-agonistes hallucinogènes. En conclusion, les résultats de cette thèse révèlent que la stimulation du récepteur 5-HT2A par les hallucinogènes et les agonistes non hallucinogènes induit deux modèles différents de phosphorylation qui pourraient être impliqués directement dans leurs réponses comportementales distinctes. ils fournissent également l'une des premières manifestations de la phosphorylation différentielle d'un récepteur couplé aux protéines G lors de la stimulation du récepteur par des agonistes biaisée. / The serotonin (5-hydroxytryptamine, 5-HT)2A receptor has been identified as the primary target of psychedelic hallucinogens such as lysergic acid diethylamide (LSD), which reproduce some of the core symptoms of schizophrenia. A non-resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related compounds with comparable affinity and agonist activity lack psychoactive properties. Using a quantitative phosphoproteomic approach combining stable isotope labelling by amino acids in cell culture (SILAC), phosphopeptide enrichment by hydrophilic interaction chromatography (HILIC) / immobilized metal affinity chromatography (IMAC) and high resolution mass spectrometry, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor under three conditions: non-stimulated cells, cells exposed to the phenethylamine hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) and cells exposed to the non-hallucinogenic 5-HT2A agonist lisuride. Among the 5,996 identified phosphopeptides, 454 were specifically regulated by DOI but not by lisuride. These include a serine residue of 5-HT2A receptor possibly involved in regulation of receptor desensitization which was specifically phosphorylated upon DOI exposure. Differential phosphorylation of 5-HT2A receptor in cells exposed to hallucinogenic (DOI and LSD) vs. non-hallucinogenic (lisuride and ergotamine) agonists was further confirmed by mass spectrometry analysis of purified receptor. Correspondingly, cell exposure to hallucinogenic agonists induced a less pronounced receptor desensitization and internalization than exposure to non-hallucinogenic agonists. In conclusion, our phosphoproteomic analysis revealed that 5-HT2A receptor stimulation by hallucinogenic and non hallucinogenic agonists induces different phosphorylation patterns that might underlie their distinct behavioural responses. It also provides one of the first demonstrations of differential phosphorylation of a G protein-coupled receptor upon stimulation by biased agonists.

A cana-de-açúcar, uma das maiores culturas agrícolas no mundo, é uma antiga fonte de energia para os seres humanos e mais recentemente, tem sido utilizada como fonte para a produção de etanol. Em estudos moleculares sobre a cana-de-açúcar, sua complexidade genética e o fato de seu genoma não estar completamente sequenciado, dificultam a análise dos proteomas de seus diferentes tecidos. Recentemente, em um estudo de um grupo brasileiro sobre os transcriptomas de vários tecidos de cana-de-açúcar, 17.563 transcritos completos foram sequenciados, os quais correspondem a aproximadamente 53% do genoma predito desta planta. Entretanto, sabe-se que o conteúdo de mRNA nem sempre se correlaciona diretamente com os níveis de expressão de proteínas. Dessa forma, a caracterização do proteoma é importante para identificar as proteínas que são de fato expressas, em complementaridade aos estudos sobre o genoma, e fornece informação preditiva sobre a composição do proteoma de diferentes cultivares. Entretanto, estudos sobre folhas de cana-de-açúcar são raros. Neste estudo, com o objetivo de caracterizar o proteoma de folhas +1 de cana-de-açúcar, foram selecionadas duas espécies ancestrais (Saccharum officinarum [S. off] e S. spontaneum [S. sp]) e uma cultivar híbrida (SP80-3280 [SP]), as quais diferem-se na capacidade de produção de sacarose e na resistência a patógenos. A análise de parâmetros fisiológicos (taxa de fotossíntese, condutância estomática, taxa de transpiração e eficiência no uso de água), em folhas de 12 meses revelaram que em S. off e SP existe uma relação melhor entre o número de moléculas de água perdidas na transpiração e o número de moléculas de CO2 assimiladas na fotossíntese do que em S. sp. Os mesmos parâmetros foram avaliados em folhas +1 de SP com 4, 8, 11 e 13 meses de crescimento e revelaram o papel dos estômatos na manutenção do potencial hídrico das folhas, atuando na modulação da taxa de respiração em resposta às mudanças ambientais, entretanto sem induzir alterações drásticas na taxa de fotossíntese, no período avaliado neste estudo. Com o objetivo de obter proteínas em condições ótimas para análise proteômica, foram testados diferentes métodos de extração de proteínas, que foram avaliados quanto ao rendimento e perfil eletroforético das proteínas extraídas, e selecionado um protocolo que utiliza uma combinação de fenol e dodecil sulfato de sódio para este estudo. Para caracterizar e comparar os proteomas de folhas +1 de S. off, S. sp e SP, foram utilizadas as abordagens proteômicas Shotgun/bottom-up, GeLC-MS/MS e Multidimensional Protein Identification Technology (MudPIT) (off-line e \"on-line\"), e as plataformas MaxQuant e Perseus, como ferramentas de bioinformática para análise dos dados. Os dados obtidos por meio destas abordagens foram combinados e resultaram na identificação de 5.825 grupos de proteínas em folhas +1 de S. off, S. sp e SP, e a análise de enriquecimento de termos pelo Gene Ontology destacou a natureza fotossintética desses proteomas, além da representatividade de organelas envolvidas em processos de fotossíntese e metabolismo de carbono. A análise quantitativa comparativa dos 1.060 grupos de proteínas identificados por meio da abordagem Shotgun/bottom-up revelou que 120 proteínas encontravam-se diferencialmente expressas em folhas de S. off, S. sp e SP, e destas, aquelas que mostraram diferença em abundância de pelo menos quatro vezes, são relacionadas com processos biológicos envolvidos com os estresses biótico e abiótico (resposta a outros organismos, resposta ao íon cádmio, defesa contra fungos e defesa ao estresse oxidativo). Para a análise da variação ontogenética no proteoma de folhas +1 de SP (de 4, 8, 11 e 13 meses de crescimento) foi aplicada a abordagem Shotgun/bottom-up, que resultou na identificação de 5.780 peptídeos, correspondendo a 1.615 grupos de proteínas únicos. Sessenta e cinco proteínas foram identificadas com abundância diferencial entre as amostras, e a análise de agrupamento hierárquico revelou que as folhas de 13 meses mostraram um perfil mais distinto, enquanto que as outras formaram um grupo separado. A caracterização dos fosfoproteomas de folhas +1 de S. off, S. sp e SP utilizando a abordagem Immobilized Metal Affinity Chromatography (IMAC) como método de enriquecimento de fosfopeptídeos, e identificação por LC-MS/MS, resultou na identificação de 633 fosfopeptídeos únicos em 515 proteínas. A caracterização dos fosfoproteomas de folhas +1 de SP (de 4, 8, 11 e 13 meses de crescimento) utilizando a abordagens IMAC e Hydroxy Acid-Modified Metal Oxide Chromatography (HAMMOC) como métodos de enriquecimento de fosfopetídeos, e identificação por LC-MS/MS, resultou na identificação de 558 fosfopeptídeos únicos em 413 proteínas. Em ambas análises, foram identificadas várias proteínas quinases fosforiladas, e de forma geral, a análise de enriquecimento de termos pelo Gene Ontology revelou o núcleo e o citoplasma, entre os componentes celulares, e a regulação da transcrição, a resposta ao estresse abiótico e a glicólise, entre os processos biológicos, como os termos mais enriquecidos. Este é o primeiro estudo objetivando a caracterização dos proteomas totais de folhas de cultivares e genótipos de cana-de-açúcar no Brasil, e seus resultados representam um significante esforço para o uso de diferentes abordagens de proteômica para aprofundar o conhecimento sobre os complexos processos fisiológicos que modulam o crescimento e a performance em cana-de-açúcar / Sugarcane, one of the major crops worldwide, is an old energy source for humans and, more recently, has been used as a source for ethanol production. In sugarcane molecular studies, its complex genetic background and the lack of a complete genome sequencing hinder the analysis of proteomes of different tissues. Recently, in a Brazilian research study focused on the transcriptomes of different sugarcane tissues, 17,563 full-length transcripts were sequenced and covered approximately 53% of the predicted sugarcane genome. However, it is known that mRNA levels are not always directly correlated with protein expression levels. Therefore, the study of the sugarcane proteome is important to identify proteins that are actually expressed, complementing the genome studies, and provides predictive information on the proteome composition of different cultivars. However, studies on sugarcane leaves are rare. In this study, with the aim of characterizing the proteome of sugarcane +1 leaves, we selected two ancestral species (Saccharum officinarum [S. off] and S. spontaneum[S. sp]), and a hybrid cultivar (SP80-3280 [SP]), which differ in sucrose production and pathogen resistance. The analysis of physiological parameters (photosynthesis rate, stomatal conductance, transpiration and efficiency in water use) of 12 month-old +1 leaves revealed that in S. off and SP there is a better relationship between the number of H2O molecules lost by transpiration and the number of CO2 molecules assimilated upon photosynthesis than in S. sp. The same parameters were evaluated in +1 leaves of SP upon 4, 8, 11 and 13 months of growth and revealed the role of stomata in the maintenance of leaf water potential, acting in the modulation of the transpiration rate in response to environmental changes, however without inducing drastic changes in the photosynthesis rate, during the period evaluated in this study. In order to obtain proteins in optimal conditions for proteomic analysis, we carried out different protein extraction methods and evaluated the yield and electrophoretic profiles of extracted proteins, and selected a protocol using a combination of phenol and sodium dodecyl sulfate to carry out this study. To characterize and compare the +1 leaf proteomes of S. off, S. sp and SP we used Shotgun/bottom-up, GeLC-MS/MS and Multidimensional Protein Identification Technology (MudPIT) (off-line and \"on-line\") as proteomic approaches, and the software MaxQuant and Perseus as bioinformatic tools for data analysis. The combined results of these approaches allowed for the identification of 5,825 protein groups in +1 leaves of S. off, S. sp and SP, and the Gene Ontology enrichment analysis highlighted the photosynthetic nature of these proteomes, along with high representativeness of organeles involved in the processes of photosynthesis and carbon metabolism. The comparative quantitative analysis of 1,060 protein groups identified using the Shotgun/bottom-up approach revealed that 120 proteins were differentially expressed in +1 leaves of S. off, S. sp and SP, and of these, those that showed at least a 4-fold change are related to biological processes involved in both abiotic and biotic stress (response to other organism, response to cadmium ion, defense response to fungus, and defense to oxidative stress). For the analysis of the ontogenetic variation in the proteome of +1 leaves of SP (of 4, 8, 11 and 13 months old plants) the Shotgun/bottom-up proteomic approach was employed and resulted in the identification of a total of 5,780 peptides corresponding to 1,615 unique protein groups. Sixty five proteins were identified as differentially abundant between the samples and a hierarchical cluster analysis revealed that the 13 months-old leaves showed the most distinct profile, while the others clustered together. The characterization of the phosphoproteomes of +1 leaves of S. off, S. sp and SP using Immobilized Metal Affinity Chromatography (IMAC) as an enrichment method for phosphopeptides, and LC-MS/MS analysis, resulted in the identification of 633 unique phosphopeptides in 515 proteins. The characterization of the phosphoproteome of +1 leaves of SP (of 4, 8, 11 and 13 months old plants) using IMAC and Hydroxy Acid-Modified Metal Oxide Chromatography (HAMMOC) as enrichment methods for phosphopeptides, and LC-MS/MS analysis, resulted in the identification of 558 unique phosphopeptides in 413 proteins. In both analyses various phosphorylated kinases were identified, and the overall Gene Ontology enrichment analysis showed as most enriched terms nucleus and cytoplasm, as cellular components, and regulation of transcription, response to abiotic stress and glycolysis in biological process. This is the first study to characterize the whole proteome of leaves from sugarcane cultivars and genotypes in Brazil, and its results constitute a significant effort to use different proteomic approaches to advance the knowledge on the complex physiological processes modulating sugarcane growth and performance

Indiana University-Purdue University Indianapolis (IUPUI) / This dissertation studies the effects of fatty acids on prostate cancer. Prostate cancer is one of the most common malignant diseases in males in the U.S. Because of the slow progression of this disease, early intervention methods, especially, dietary fatty acid interventions are considered very important to control the disease in early stages. This study describes how the depletion of the enzyme for endogenous fatty acid synthesis, fatty acid synthase, influences the expression of enzymes that metabolize dietary fatty acids and show how dietary fatty acids affect prostate cancer protein expression and function. Fatty acid synthase is an oncoprotein overexpressed in prostate cancer and its expression is suppressed with omega-3 fatty acid treatment. This study finds that the depletion of fatty acid synthase by siRNA knockdown induces suppression of cyclooxygenase-2 and fatty acid desaturase-1. Our results also show that fish oil (omega-3 fatty acid), but not oleic acid (omega-9 fatty acid), suppresses prostate cancer cell viability. Assessment of fatty acid synthesis activity indicates that oleic acid is a more potent inhibitor than fish oil of de novo fatty acid biosynthesis. In addition, the inhibition of its activity occurs over several days while its effects on cell viability occur within 24 hours. To better understand this relationship, label free LC-MS/MS based mass spectrometry was carried out to determine global proteomic and phosphoproteomic profiles of the prostate cell line PC3, with longitudinal treatment with fish oil or oleic acid. With short-term fish oil treatment, sequestosome-1was elevated. Prolonged treatment induced downregulation of microseminoprotein, a proinflammation factor, as well as proteins in the glycolysis pathway. In the phosphoproteomics study, we confidently identified 828 phosphopeptides from 361 phosphoproteins. Quantitative comparison between fish oil or oleic acid treated groups and the untreated group suggests that the fish oil induces changes in phosphorylation of proteins involved in the pathways associated with cell viability and metabolic processes, with fish oil inducing significant decreases in the levels of phospho-PDHA1Ser232 and phospho-PDHA1Ser300 and they were accompanied by an increase in PDH activity, suggesting a role for n-3 polyunsaturated fatty acids in controlling the balance between lipid and glucose oxidation.

Post-translationale Modifikationen (PTMs) sind wesentlich für die Regulierung von zellulären Mechanismen. Um diese Prozesse besser zu verstehen, ist es essentiell Methoden für deren Erforschung zu entwickeln. In dieser Arbeit wurden zwei chemoproteomische Ansätze entwickelt, um die PTMs, Proteinphosphorylierung und Proteinpyrophosphorylierung zu untersuchen. Die Proteom-weite Erforschung von Proteinphosphorylierungen beruht gewöhnlich auf der LC-MS/MS-Analyse von enzymatisch verdauten Proteomen und da die Phosphorylierung von niedriger Abundanz ist, wird ein Phosphopeptid-Anreicherungsschritt benötigt. Die Identifizierung von bestimmten Phosphopeptiden ist allerdings abhängig von der gewählten Anreicherungsmethode. Die Entwicklung von neuen Prozeduren ist daher bedeutsam, um neue Phosphorylierungsstellen zu identifizieren. Im ersten Projekt wurde eine milde und selektive Phosphopeptid-Anreicherungsmethode entwickelt und optimiert. Die Methode zeigte die Fähigkeit Phosphopeptide anzureichern und somit das Potential, das Repertoire der vorherigen Methoden zu erweitern, um neue Phosphorylierungsstellen zu identifizieren. Proteinpyrophosphorylierung ist eine unlängst identifizierte PTM, die nicht-enzymatisch an Proteine angefügt wird und es ist nur wenig ist über ihre Funktion bekannt. Vorherige Studien wiesen darauf hin, dass diese Modifikation enzymatisch entfernt wird, allerdings sind die verantwortlichen Enzyme („Proteinpyrophosphatasen“) nicht bekannt. Hier wurde eine Peptidaffinitätsmethode entwickelt, um potentielle Pyrophosphatasen und weitere interagierende Proteine aus humanen Zellen zu identifizieren. Damit wurden 6 Phosphatasen als potentielle Pyrophosphatase-Kandidaten identifiziert und weitere interagierende Proteine gaben Aufschlüsse über die Funktion der Proteinpyrophosphorylierung. Dadurch wurde das Potential der Methode aufgezeigt, interagierende Proteine der Proteinpyrophosphorylierung zu identifizieren, um die zelluläre Rolle zu verstehen. / Post-translational modifications (PTMs) are crucial for the regulation of cellular mechanisms. To better understand these processes, the development of chemical tools to investigate them is of high importance. In this thesis, two chemoproteomics approaches were established to investigate the PTMs protein phosphorylation and protein pyrophosphorylation. The proteome-wide study of protein phosphorylation usually relies on LC-MS/MS analysis of enzymatically digested proteomes, requiring a phosphopeptide enrichment step, due to the low abundance of phosphorylation. However, the identification of certain sets of phosphopeptides is dependend on the choice of enrichment method. Therefore, the development of new workflows is important to identify new phosphorylation sites. In the first project, a mild and selective phosphopeptide enrichment method was developed and optimized. The method was able to enrich phosphopeptides and therefore, showed the potential to complement the repertoire of current methods to identify new phosphorylation sites. Protein pyrophosphorylation is a recently discovered PTM, which is non-enzymatically attached to proteins and there is only sparse knowledge about the function. Previous studies have indicated the enzymatic removal of this modification, but the responsible enzymes (‘protein pyrophosphatases’) are unknown. Here, a peptide affinity capture method was developed to identify potential pyrophosphatases and further interacting proteins from human cells. Therewith, 6 phosphatases were identified as potential pyrophosphatase candidates and further interacting proteins gave insights into the function and mechanisms of protein pyrophosphorylation. Thereby, the potential of this method was demonstrated to identify interacting proteins of protein pyrophosphorylation to understand the cellular role.