RdoA, a serine/threonine kinase, is a member of the Cpx regulon, a stress response pathway, in Salmonella enterica serovar Typhimurium. Phenotypic characterization of rdoA null mutants suggested that RdoA kinase activity affects a wide range of cell functions, which could be the result of both direct and indirect phosphorylation of targets. In a search for RdoA’s target(s), the phosphoproteome profile of wild-type and rdoA null S. enterica was examined through phosphoprotein enrichment followed by 2-dimensional gel electrophoresis coupled with phospho-specific fluorescent stains and western blots using phospho-specific antibodies. Three different phosphoprotein enrichment protocols, all based on metal-ion affinity chromatography, were compared for yield and phosphoprotein specificity to determine which would be the most suitable for S. enterica. This study showed that the Phostag Enrich Phosphoprotein kit (PerkinElmer) gave the highest yield, the majority of which were phosphoproteins. These studies also showed that western blots using phospho-specific antibodies were more sensitive than phosphoprotein-specific fluorescent stain ProQ Diamond in detecting phosphoproteins. The phosphoproteome profile of S. typhimurium cells grown under Cpx activating conditions included phosphoproteins involved in the heat shock response, cellular metabolism and protein synthesis. This work also identified changes in the phosphoproteome that were dependent upon the presence or absence of RdoA. Phosphoproteins that showed a significant change in phosphorylation were identified by mass spectroscopy using peptide mass fingerprinting. Proteins identified included protein foldases (DnaK and GroEL), proteins involved in metabolism (glycerol kinase, enolase and E1 subunit of the pyruvate dehydrogenase complex), and in protein synthesis (elongation factor-Tu). These proteins may be phosphorylated in an RdoA-dependent manner to allow normal cell functioning under envelope stress. Several proteins unlikely to be phosphoproteins were also RdoA-dependent. SrgA, encoded on the virulence plasmid, is a disulfide oxidoreductase specific for the PEF fimbriae that was shown to be repressed by RdoA. This work also showed that integration host factor, previously suggested to be an RdoA target, was not affected in terms of expression or phosphorylation by RdoA. The several RdoA-dependent changes in protein expression levels and phosphorylation that were identified contribute to the elucidation of RdoA’s role in the envelope stress response and provided further insight in determining RdoA target(s). / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-11-06 10:51:01.253

Yes / Resistance exercise training (RET) can counteract negative features of muscle ageing but older age associates with reduced adaptive capacity to RET. Altered muscle protein networks likely contribute to ageing RET adaptation; therefore, associated proteome-wide responses warrant exploration. We employed quantitative sarcoplasmic proteomics to compare age-related proteome and phosphoproteome responses to RET. Thigh muscle biopsies were collected from eight young (25 ± 1.1 years) and eight older (67.5 ± 2.6 years) adults before and after 20 weeks supervised RET. Muscle sarcoplasmic fractions were pooled for each condition and analysed using Isobaric Tags for Relative and Absolute Quantification (iTRAQ) labelling, tandem mass spectrometry and network-based hub protein identification. Older adults displayed impaired RET-induced adaptations in whole-body lean mass, body fat percentage and thigh lean mass (P > 0.05). iTRAQ identified 73 differentially expressed proteins with age and/or RET. Despite possible proteomic stochasticity, RET improved ageing profiles for mitochondrial function and glucose metabolism (top hub; PYK (pyruvate kinase)) but failed to correct altered ageing expression of cytoskeletal proteins (top hub; YWHAZ (14-3-3 protein zeta/delta)). These ageing RET proteomic profiles were generally unchanged or oppositely regulated post-RET in younger muscle. Similarly, RET corrected expression of 10 phosphoproteins altered in ageing, but these responses were again different vs. younger adults. Older muscle is characterised by RET-induced metabolic protein profiles that, whilst not present in younger muscle, improve untrained age-related proteomic deficits. Combined with impaired cytoskeletal adhesion responses, these results provide a proteomic framework for understanding and optimising ageing muscle RET adaptation. / TE was supported by a postdoctoral fellowship from the Japan Society for the Promotion of Science and the Royal Society (JSPS/FF1/435). This work was supported by grants from the Medical Research Council (MR/T026014/1 and G0801271) and the Biotechnology and Biological Sciences Research Council (BB/X510697/1 and BB/C516779/1).

La doxorubicine (DXR) est l’un des médicaments les plus efficaces en chimiothérapie, mais sonapplication clinique est limitée par ses effets cardiotoxiques. Malgré des décennies de recherche, sesmécanismes pathogéniques ne sont toujours pas entièrement compris. Il s’ensuit qu’aucun traitementsatisfaisant, curatif ou préventif, n’existe. Dans cette étude, nous recherchons les mécanismes designalisation cellulaire impliqués. Deux modèles expérimentaux de toxicités, aigue d’une part (coeurisolé et perfusé de rat avec la DXR), et chronique d’autre part (rat traité à la DXR), ont permis deréaliser une étude ciblée (sur les voies de signalisation énergétiques) et deux études systémiques(phosphoprotéomique et transcriptomique). Les résultats combinés de ces travaux ont montré que laDXR modifiait le niveau de phosphorylation (activation) ou l’expression génique de protéinesimpliquées dans trois domaines fonctionnels distincts : métabolisme énergétique, réponses au stress,et structure/fonction du sarcomère. (i) Métabolisme énergétique : nous avons confirmé la surprenanteinhibition de l’AMPK, probablement provoquée par un contrôle négatif exercé par des partenaires designalisation (Akt et ERK), plutôt que par une modification des kinases activatrices en amont. Nousavons également montré l’augmentation du niveau de phosphorylation de la PDH, ce qui, en inhibantl’enzyme, ralentit le cycle de Krebs. Cependant, nous avons également observé un phénomènecompensatoire de surexpression de gènes codant pour des enzymes de la glycolyse et du cycle deKrebs ; (ii) Réponses au stress : dans nos modèles, la DXR génère des stress énergétique,génotoxique et oxydatif. Cependant, seuls quelques mécanismes compensatoires sont activés (lesvoies de signalisation de DNA-PK–Akt–GSK3, diverses chaperonnes). Les autres semblent êtreinhibées suggérant que l’amoindricement des réponses au stress serait un des mécanismes de lacardiotoxicité de la DXR; (iii) Structure/fonction du sarcomère: L’augmentation de la phosphorylationde la desmine ainsi que la réduction du nombre de transcrits codant pour des protéines essentiellesau développement cardiaque normal pourraient être la cause de la désorganisation du réseaumyofibrillaire. En conclusion, ces résultats révèlent potentiellement de nouveaux mécanismes de lacardiotoxicité induite par la DXR et permettent d’envisager de nouvelles cibles moléculaires pour ledéveloppement de stratégies protectrices. / Doxorubicin (DXR) is an efficient anticancer drug, the use of which is limited by seriouscardiotoxicity. Despite decades of research, its pathogenic mechanisms are not fully understood, andefficient preventive or curative strategies are not available. Here we address the question whethermechanisms in cardiac cell signaling contribute to the toxicity phenotype. Using experimental modelsfor acute (DXR-perfused, isolated rat hearts) or chronic toxicity (rats injected with DXR), we conducteda targeted study (focusing on energy signaling pathways) and two non-biased studies(phosphoproteomics and transcriptomics). The combined data reveal DXR-induced alterations inphosphorylation (activation) status or gene expression of proteins in mainly three functional domains:energy metabolism, stress responses, and sarcomere structure. (i) Energy metabolism: We confirm aparadox inhibition of AMPK signaling, that is rather due to inhibitory cross-talk with related signalingpartners (Akt, ERK) than impaired AMPK upstream signaling. We also show, among others, theincrease of inhibitory phosphorylation of pyruvate dehydrogenase, slowing down Krebs cycle, but alsoa compensating upregulation of glycolysis and Krebs cycle enzyme transcripts. (ii) Stress-responses:In our models, DXR generates energetic, oxidative and genotoxic stress, but only some compensatorystress responses are activated (DNA-PK–Akt–GSK3 pathway, chaperones). Many others seem to beinhibited, suggesting a blunted response to stress as component of DXR toxicity. (iii) Sarcomerestructure/function: We detect increased phosphorylation of desmin and reduced transcripts essentialfor e.g. normal heart development as potential causes for a disorganized myofibrillar network. Inconclusion, these results reveal some novel potential mechanisms of DXR-induced cardiotoxicity andsuggest new targets for protective strategies.

Genome of S. pneumoniae contains only one copy of the gene coding eukaryotic type protein kinase StkP and corresponding phosphatase PhpP. These two enzymes form a functional signaling pair regulating cell division, which could be used in the future for the design of new bacteriostatic compounds. Not only kinase and phosphatase are important components of the system, but also other members of this pathway - specific substrates of these enzymes. The identification of the Ser/Thr phosphoproteom with a focus on the membrane fraction provided information not only about already known substrates such as LocZ, Jag and DivIVA but also about an unknown protein P15 with a molecular weight about 15 kDa. In this thesis the protein was identified as rhodanase (spr0595) by MS MALDI TOF. However, its subsequent deletion did not confirm it as a StkP/PhpP substrate. Therefore we investigated another substrate, protein FtsA, which has already been identified as a substrate of this kinase in a previous study (Beilharz et al., 2012). FtsA is an essential cell division protein that anchors FtsZ filaments into the membrane. Phosphorylation of this protein was detected on the Thr residue at position 404. Using phosphoablative substitution we found out, that Thr404 is indeed phosphorylated by protein kinase StkP, however, FtsA...

Cell dedifferentiation is a cell fate switching process in which a differentiated cell reverts to a status with competence for cell division and organ regeneration like an embryonic stem cell. Although the phenomenon of cell dedifferentiation has been known for over two and a half centuries in plants, little is known of the underlying mechanisms. Here, the proteome map of Arabidopsis cotyledons has been established and investigated the dynamic change of the cotyledon proteome in the time course of cell dedifferentiation. Among the 353 distinct genes, corresponding to 500 2-DE gel protein spots identified with high confidence, 12% have over twofold differential regulations within the first 48 h of induction of cell dedifferentiation. The distributions of these genes among different Gene Ontology categories and gene differential regulations within each of the categories have been examined. In addition, the cotyledon phosphoproteome has been investigated using Pro-Q Diamond Phosphoprotein in Gel Stain followed by mass spectrometry analyses. Among the 53 identified putative phosphoproteins, nine are differentially regulated during cell dedifferentiation. Arabidopsis cotyledon proteome at four different time points after the induction of cell dedifferentiation with MudPIT approach has been investigated and analyzed the protein quantity change using two labelree methods, the Spectral Count (SC) and SEQUEST Cross Correlation Coefficient (ÓXcorr) methods. Among the 662 MudPIT identified proteins, one hundred forty eight displayed differential regulation. The up-regulated proteins include transcription factors, calmodulins, translational regulators, and stress response proteins. The Spectral Count and the cross correlation coefficient quantification results are highly consistent in over 81% of the differentially regulated proteins. These studies have provided significant new insight into cell dedifferentiation process in Arabidopsis thaliana and also enhanced the Arabidopsis cotyledon proteome database established using gel based and non gel based methods. The results show that cell dedifferentiation involves extensive protein quantitative and qualitative changes in almost every cellular compartment and cellular process. Proteins like 14-3-3 proteins, Translational controlled tumor protein (TCTP) and its possible interaction protein-Translational elongation factor eEF1 alpha chain, GTP binding nuclear protein RAN2, GTP binding protein SAR1B and several other hypothetical and expressed proteins and nine other phosphoproteins showed significant differential expression during early dedifferentiation. Deciphering the molecular mechanisms regulating the cellular dedifferentiation certainly enhances the understandings and mechanisms of reprogramming all types of differentiated cells including animal cells.

Esquistossomose é uma das doenças parasitárias com maior incidência mundial. Schistosoma mansoni, a única espécie encontrada no Brasil, tem um ciclo de vida complexo que inclui seis estágios de desenvolvimento distintos e dois hospedeiros, sendo um deles o homem. Nosso grupo identificou um gene que codifica um receptor que possui homologia com o receptor de TNF-&#945; humano em S. mansoni (SmTNFR) e descrevemos o efeito in vitro da citocina humana TNF-&#945; sobre a expressão gênica em larga escala no parasita. A via de sinalização através da qual o TNF-&#945; atua sobre o parasita permanece desconhecida. Em humanos, o TNF-&#945;, ao se ligar à isoforma do receptor que é mais semelhante ao SmTNFR, inicia uma cascata de fosforilação que conduz à ativação de diversas proteínas quinases que levam à ativação de fatores de transcrição. A nossa hipótese é a de que esta citocina humana atuaria no parasita de forma semelhante, resultando na alteração da expressão gênica do parasita observada em nosso trabalho anterior. No presente trabalho tivemos como objetivo verificar se há aumento de proteínas fosforiladas após o tratamento in vitro de vermes com o TNF-&#945; humano, e identificá-las, elucidando a via de sinalização induzida por esta citocina em S. mansoni. A fim de identificar as proteínas que são significativamente diferencialmente fosforiladas após exposição de S. mansoni à citocina humana, vermes machos adultos foram incubados em cultura por 15 min com TNF-&#945; (20 ng / ml), ou somente com o veículo, em controles, seguido da extração de proteínas totais. Utilizamos a abordagem fosfoproteômica por meio da realização de eletroforese bidimensional, seguida de coloração com corante específico para fosfoproteínas Pro-Q Diamond (Invitrogen). Para identificar quais proteínas tiveram fosforilação alterada pelo tratamento, fizemos uma análise quantitativa dos spots (volume dos spots) nas imagens dos géis corados com Pro-Q, utilizando o software 2D Platinum (GE). Em seguida a coloração de proteína total dos géis foi realizada utilizando Coomassie Blue Coloidal, para localização de todos os spots de proteínas de cada gel. Cortamos dos géis os spots de proteínas que apresentaram mudanças estatisticamente significativas na fosforilação (n = 3 réplicas; p <0,05, teste-t), com base na comparação de seus volumes relativos nas imagens dos géis das amostras tratadas comparadas com as controles, e em seguida as proteínas das amostras foram identificadas por espectrometria de massa. Analisamos três réplicas biológicas e observamos 45 spots que tiveram fosforilação significativamente alterada, sendo que 42 deles apresentaram fosforilação aumentada e 3 apresentaram fosforilação diminuída após o tratamento com a citocina. Entre os spots identificados por espectrometria de massa, verificou-se que proteínas relacionadas a processos metabólicos, sinalização celular, citoesqueleto e contração muscular estavam entre as que sofreram aumentos de fosforilação com o tratamento com TNF-&#945; humano. Embora a abordagem experimental adotada para este estudo não tenha sido sensível o suficiente para concluir qual via canônica de transdução de sinal é ativada por TNF-&#945; humano em S. mansoni, este estudo confirmou o aumento da fosforilação de proteínas-alvo induzido por TNF-&#945; humano, abrindo novos caminhos para uma investigação mais aprofundada que caracterize o papel das proteínas identificadas como alteradas por essa via de sinalização, e permita entender melhor a importância do TNF-&#945; humano na biologia do S. mansoni e na interação parasita-hospedeiro. / Schistosomiasis remains one of the parasitic diseases with the highest incidence worldwide. Schistosoma mansoni, the only species found in Brazil, has a complex life cycle which includes six life stages and two hosts, one of them being humans. Our group has identified a gene that encodes a TNF-alpha receptor-like in S. mansoni (SmTNFR) and described the in vitro effect of human TNF-alpha cytokine on large-scale gene expression of the parasite. The signaling pathways by which TNF-alpha acts upon the parasite remain unknown. In humans, when TNF-alpha binds to the receptor isoform that is most similar to SmTNFR it initiates a phosphorylation cascade that activates a signaling pathway leading to the activation of transcription factors. Our hypothesis is that this cytokine could act in the parasite through a phosphorylation cascade that activates transcription factors resulting in the change of gene expression observed in our previous work. The aim of this work was to verify if there were proteins that showed increased phosphorylation upon the in vitro treatment of worms with human TNF-alpha, identifying them, to try and elucidate the signaling pathway by which the cytokine acts in S. mansoni. In order to identify which proteins are significantly differentially phosphorylated upon exposure of S. mansoni to the human cytokine, we incubated male adult worms for 15 min in culture with human TNF-alpha (20 ng / ml), or with vehicle only, in controls, and we extracted total proteins from the parasites. We used the phosphoproteomics approach of bidimensional gel electrophoresis followed by phospho-specific staining of gels using Pro-Q Diamond dye (Invitrogen). To identify which proteins were phosphorylated or had their phosphorylation changed due to the treatment, we quantified the spots (determined the spots volumes) from Pro-Q stained gels using Image Master 2D Platinum software (GE). A total protein staining of the gels was performed using Coomassie Brilliant Blue (CBB) in order to localize all protein spots in the gel. We excised from CBB stained gels the protein spots that showed statistically significant changes in phosphorylation (n= 3 replicates; p-value<0.05, t-test), based on the relative volumes of their spot images with respect to the total volume of spots in the gel, with samples from treated compared to control parasites, and subsequently the proteins in these samples were identified by mass spectrometry. We have analyzed three biological replicates and observed 45 spots that were differentially phosphorylated; 42 of them had their phosphorylation increased and 3 had their phosphorylation decreased upon a 15-min-treatment of male adult worms with the cytokine. Among the spots identified by mass spectrometry, we found proteins related to metabolic process, cell signaling, and cytoskeleton and muscle contraction. Although the experimental approach adopted for this study was not sensitive enough to conclude which canonical signal transduction pathway is activated by human TNF-alpha in S. mansoni, this kind of study confirmed the increase in phosphorylation of target proteins induced by human TNF-alpha, opening new paths for further investigation in order to characterize the role of the proteins identified as altered by this specific signaling, which will lead to a better understanding of the importance of this cytokine in the biology of S. mansoni and in host-parasite interaction.

La doxorubicine (DXR) est l'un des médicaments les plus efficaces en chimiothérapie, mais sonapplication clinique est limitée par ses effets cardiotoxiques. Malgré des décennies de recherche, sesmécanismes pathogéniques ne sont toujours pas entièrement compris. Il s'ensuit qu'aucun traitementsatisfaisant, curatif ou préventif, n'existe. Dans cette étude, nous recherchons les mécanismes designalisation cellulaire impliqués. Deux modèles expérimentaux de toxicités, aigue d'une part (coeurisolé et perfusé de rat avec la DXR), et chronique d'autre part (rat traité à la DXR), ont permis deréaliser une étude ciblée (sur les voies de signalisation énergétiques) et deux études systémiques(phosphoprotéomique et transcriptomique). Les résultats combinés de ces travaux ont montré que laDXR modifiait le niveau de phosphorylation (activation) ou l'expression génique de protéinesimpliquées dans trois domaines fonctionnels distincts : métabolisme énergétique, réponses au stress,et structure/fonction du sarcomère. (i) Métabolisme énergétique : nous avons confirmé la surprenanteinhibition de l'AMPK, probablement provoquée par un contrôle négatif exercé par des partenaires designalisation (Akt et ERK), plutôt que par une modification des kinases activatrices en amont. Nousavons également montré l'augmentation du niveau de phosphorylation de la PDH, ce qui, en inhibantl'enzyme, ralentit le cycle de Krebs. Cependant, nous avons également observé un phénomènecompensatoire de surexpression de gènes codant pour des enzymes de la glycolyse et du cycle deKrebs ; (ii) Réponses au stress : dans nos modèles, la DXR génère des stress énergétique,génotoxique et oxydatif. Cependant, seuls quelques mécanismes compensatoires sont activés (lesvoies de signalisation de DNA-PK-Akt-GSK3, diverses chaperonnes). Les autres semblent êtreinhibées suggérant que l'amoindricement des réponses au stress serait un des mécanismes de lacardiotoxicité de la DXR; (iii) Structure/fonction du sarcomère: L'augmentation de la phosphorylationde la desmine ainsi que la réduction du nombre de transcrits codant pour des protéines essentiellesau développement cardiaque normal pourraient être la cause de la désorganisation du réseaumyofibrillaire. En conclusion, ces résultats révèlent potentiellement de nouveaux mécanismes de lacardiotoxicité induite par la DXR et permettent d'envisager de nouvelles cibles moléculaires pour ledéveloppement de stratégies protectrices.

Esquistossomose é uma das doenças parasitárias com maior incidência mundial. Schistosoma mansoni, a única espécie encontrada no Brasil, tem um ciclo de vida complexo que inclui seis estágios de desenvolvimento distintos e dois hospedeiros, sendo um deles o homem. Nosso grupo identificou um gene que codifica um receptor que possui homologia com o receptor de TNF-&#945; humano em S. mansoni (SmTNFR) e descrevemos o efeito in vitro da citocina humana TNF-&#945; sobre a expressão gênica em larga escala no parasita. A via de sinalização através da qual o TNF-&#945; atua sobre o parasita permanece desconhecida. Em humanos, o TNF-&#945;, ao se ligar à isoforma do receptor que é mais semelhante ao SmTNFR, inicia uma cascata de fosforilação que conduz à ativação de diversas proteínas quinases que levam à ativação de fatores de transcrição. A nossa hipótese é a de que esta citocina humana atuaria no parasita de forma semelhante, resultando na alteração da expressão gênica do parasita observada em nosso trabalho anterior. No presente trabalho tivemos como objetivo verificar se há aumento de proteínas fosforiladas após o tratamento in vitro de vermes com o TNF-&#945; humano, e identificá-las, elucidando a via de sinalização induzida por esta citocina em S. mansoni. A fim de identificar as proteínas que são significativamente diferencialmente fosforiladas após exposição de S. mansoni à citocina humana, vermes machos adultos foram incubados em cultura por 15 min com TNF-&#945; (20 ng / ml), ou somente com o veículo, em controles, seguido da extração de proteínas totais. Utilizamos a abordagem fosfoproteômica por meio da realização de eletroforese bidimensional, seguida de coloração com corante específico para fosfoproteínas Pro-Q Diamond (Invitrogen). Para identificar quais proteínas tiveram fosforilação alterada pelo tratamento, fizemos uma análise quantitativa dos spots (volume dos spots) nas imagens dos géis corados com Pro-Q, utilizando o software 2D Platinum (GE). Em seguida a coloração de proteína total dos géis foi realizada utilizando Coomassie Blue Coloidal, para localização de todos os spots de proteínas de cada gel. Cortamos dos géis os spots de proteínas que apresentaram mudanças estatisticamente significativas na fosforilação (n = 3 réplicas; p <0,05, teste-t), com base na comparação de seus volumes relativos nas imagens dos géis das amostras tratadas comparadas com as controles, e em seguida as proteínas das amostras foram identificadas por espectrometria de massa. Analisamos três réplicas biológicas e observamos 45 spots que tiveram fosforilação significativamente alterada, sendo que 42 deles apresentaram fosforilação aumentada e 3 apresentaram fosforilação diminuída após o tratamento com a citocina. Entre os spots identificados por espectrometria de massa, verificou-se que proteínas relacionadas a processos metabólicos, sinalização celular, citoesqueleto e contração muscular estavam entre as que sofreram aumentos de fosforilação com o tratamento com TNF-&#945; humano. Embora a abordagem experimental adotada para este estudo não tenha sido sensível o suficiente para concluir qual via canônica de transdução de sinal é ativada por TNF-&#945; humano em S. mansoni, este estudo confirmou o aumento da fosforilação de proteínas-alvo induzido por TNF-&#945; humano, abrindo novos caminhos para uma investigação mais aprofundada que caracterize o papel das proteínas identificadas como alteradas por essa via de sinalização, e permita entender melhor a importância do TNF-&#945; humano na biologia do S. mansoni e na interação parasita-hospedeiro. / Schistosomiasis remains one of the parasitic diseases with the highest incidence worldwide. Schistosoma mansoni, the only species found in Brazil, has a complex life cycle which includes six life stages and two hosts, one of them being humans. Our group has identified a gene that encodes a TNF-alpha receptor-like in S. mansoni (SmTNFR) and described the in vitro effect of human TNF-alpha cytokine on large-scale gene expression of the parasite. The signaling pathways by which TNF-alpha acts upon the parasite remain unknown. In humans, when TNF-alpha binds to the receptor isoform that is most similar to SmTNFR it initiates a phosphorylation cascade that activates a signaling pathway leading to the activation of transcription factors. Our hypothesis is that this cytokine could act in the parasite through a phosphorylation cascade that activates transcription factors resulting in the change of gene expression observed in our previous work. The aim of this work was to verify if there were proteins that showed increased phosphorylation upon the in vitro treatment of worms with human TNF-alpha, identifying them, to try and elucidate the signaling pathway by which the cytokine acts in S. mansoni. In order to identify which proteins are significantly differentially phosphorylated upon exposure of S. mansoni to the human cytokine, we incubated male adult worms for 15 min in culture with human TNF-alpha (20 ng / ml), or with vehicle only, in controls, and we extracted total proteins from the parasites. We used the phosphoproteomics approach of bidimensional gel electrophoresis followed by phospho-specific staining of gels using Pro-Q Diamond dye (Invitrogen). To identify which proteins were phosphorylated or had their phosphorylation changed due to the treatment, we quantified the spots (determined the spots volumes) from Pro-Q stained gels using Image Master 2D Platinum software (GE). A total protein staining of the gels was performed using Coomassie Brilliant Blue (CBB) in order to localize all protein spots in the gel. We excised from CBB stained gels the protein spots that showed statistically significant changes in phosphorylation (n= 3 replicates; p-value<0.05, t-test), based on the relative volumes of their spot images with respect to the total volume of spots in the gel, with samples from treated compared to control parasites, and subsequently the proteins in these samples were identified by mass spectrometry. We have analyzed three biological replicates and observed 45 spots that were differentially phosphorylated; 42 of them had their phosphorylation increased and 3 had their phosphorylation decreased upon a 15-min-treatment of male adult worms with the cytokine. Among the spots identified by mass spectrometry, we found proteins related to metabolic process, cell signaling, and cytoskeleton and muscle contraction. Although the experimental approach adopted for this study was not sensitive enough to conclude which canonical signal transduction pathway is activated by human TNF-alpha in S. mansoni, this kind of study confirmed the increase in phosphorylation of target proteins induced by human TNF-alpha, opening new paths for further investigation in order to characterize the role of the proteins identified as altered by this specific signaling, which will lead to a better understanding of the importance of this cytokine in the biology of S. mansoni and in host-parasite interaction.

No description available.

Characterization of phosphoproteins-including detection, identification of phosphoproteins and identification of phosphorylation sites-is mostly done with radiolabeling and proteomic techniques. Three main topics related to phosphoprotein characterization are included in this dissertation. First, large-scale characterization of the CHO (Chinese hamster ovary) cell phosphoproteome was done using two dimensional gel electrophoresis (2DE) separation, visualization of phosphoproteins by radiolabeling or a phosphoprotein specific dye, followed by MALDI-TOF identification. Because radiolabeling of phosphoproteins is very sensitive and straightforward to quantify, such analysis can give a clear picture of the relative phosphosphorylation of proteins present in a sample. But there are also limitations to this approach, such as the inability of 2DE to separate hydrophobic, acidic and large proteins and the poor detection limits of common protein stains such as Coomassie stain. Additionally, it is difficulty to excise the right spots for identification because of the low abundance of phosphoproteins which have been visualized by radiolabeling. Furthermore, there are problems associated with metabolic radiolabeling. A second topic of the dissertation is the development of a novel strong cation exchange monolithic column for MudPIT (multidimensional protein identification technology) and phosphopeptide isolation. This column, a poly(AMPS-co-PEGDA) monolith containing as high as 40% AMPS, has several favorable features, such as high binding capacity, extraordinarily high resolution, and high peak capacity, making it ideal for resolving complex peptide samples. Application of this novel column to isolate model phosphopeptides was shown. More general use of this column in MudPIT (strong cation exchange column followed by reverse-phased MS/MS) is probably somewhat limited, due to the hydrophobicity of the AMPS monomer. A better monolith could be obtained if a more hydrophilic monomer was used. In the third area of the dissertation, several individual protein phosphorylation sites were analyzed, employing different strategies. Phosphorylation sites of one multiply phosphorylated tryptic peptide from CK2-phosphorylated phosducin-like protein (PhLP) was well characterized using enrichment with a MonoTip® TiO Pipette Tip. Analysis of syntaxin 1a phosphorylation by AMPK (AMP-activated protein kinase) was done by peptide level mapping for potential phosphopeptides after its unsuccessful trial with enrichment using the MonoTip® TiO Pipette Tip. Several criteria such as existence of non-phosphorylated forms of potential phosphopeptides, controls and reasonable retention times were used to rule out false positives. Phosphorylation of syntaxin 1a by AMPK was narrowed down to tryptic peptide T32 with evidence from different sources. Three phosphorylation sites of syntaxin 4 by AMPK were identified within the same peptide (Q65QVTILATPLPEESMK80). Further pinpointing of phosphorylation site(s) for syntaxin 1a by AMPK and further confirmation of these phosphorylation sites in syntaxin 4 by AMPK are required in vivo. The role of phosphorylation in syntaxin 4 by AMPK is the next step toward elucidation of AMPK activation and regulation of the glucose uptake mechanism.