Le cancer est un véritable problème de santé publique et représentera en 2010 la première cause de mortalité dans le monde. La lutte contre cette maladie est donc, plus que jamais, un enjeu capital. Les processus tumoraux associés à la carcinogénèse, tels que la métastase, l'invasion ou l'angiogenèse, dépendent étroitement de la composition du milieu extracellulaire, lui-même affecté par la sécrétion de protéines par les cellules tumorales. La mutation ou la variation d'expression de facteurs pro- ou anti-oncogénique joue un rôle important dans le développement et la progression de la tumeur. Durant ce travail de thèse, nous avons voulu apporter des éléments de réponse à la problématique suivante : l'étude par protéomique des protéines sécrétées par la tumeur apporte t'elle des informations contribuant à la compréhension des mécanismes de la tumorogenèse et peut elle permettre de mettre en valeur de nouvelles cibles d'intérêt clinique ? Nous avons ciblé notre étude sur le cancer du poumon, le plus fréquent au niveau mondial tant en terme d'incidence que de mortalité. Ce cancer présente le plus fort taux de mutation du gène p53, un anti-oncogène clé de la cellule. Cette perte de fonction module la sécrétion de nombreuses protéines dont l'investigation est primordiale, bien que paradoxalement, peu étudiée. Notre travail s'est ainsi focalisé sur l'étude de l'influence de p53 sur la modulation de la sécrétion de protéines par les tumeurs du poumon. Nous avons ainsi développé un procédé d'analyse protéomique adapté, basé notamment sur un marquage iTRAQ, une séparation par isoélectrofocalisation de type OFFGEL et une analyse LC-MALDI-MS/MS. Ce procédé a été appliqué à l'étude du rôle joué par l'expression conditionnelle de p53 sur la modulation du secretome d'une lignée cellulaire d'adénocarcinome du cancer du poumon non à petites cellules. Plusieurs protéines d'intérêt ont ainsi été caractérisées et confirmées in vivo chez la souris au niveau tumoral et plasmatique. Ces résultats apportent une meilleure compréhension du rôle primordial joué par l'altération de p53 dans la modulation de l'environnement tumoral et font des secretomes cellulaires un modèle de choix pour l'identification de marqueurs tumoraux.

This thesis examines the influence of nutritional phosphate (Pi) deprivation on extracellular proteins secreted by the model plant Arabidopsis thaliana. Initial studies compared the secretome of Pi-sufficient (+Pi) versus Pi-deficient (-Pi) Arabidopsis cell cultures by 2-dimensional gel electrophoresis. Mass spectrometry identified 18 different secreted proteins that were upregulated by at least 2-fold by –Pi Arabidopsis. They were predicted to function in Pi scavenging, cell wall and ROS metabolism, proteolysis, and pathogen responses. The relationship between mRNA levels and relative amounts of selected secretome proteins was assessed. The results indicate that transcriptional control is but one of many factors contributing to Arabidopsis Pi starvation responses and highlight the importance of parallel biochemical and proteomic studies of –Pi plants. Three purple acid phosphatase (APase) isoforms were fully purified from the culture media of –Pi Arabidopsis cells and identified as AtPAP12 (At2g27190) and two AtPAP26 (At5g34850) glycoforms. As each purple APase exhibited broad substrate specificities and pH-activity profiles, it is hypothesized that their combined activities facilitate Pi scavenging from soil-localized organophosphates during nutritional Pi deprivation. AtPAP26 is dual-targeted during Pi stress since an earlier report demonstrated that it is also the principal intracellular (vacuolar) APase upregulated by -Pi Arabidopsis. The results indicate that differential glycosylation influences AtPAP26’s substrate specificity and subcellular targeting. An atpap26 T-DNA insertional mutant lacking AtPAP26 transcripts and immunoreactive AtPAP26 polypeptides exhibited: (i) 9- and 5-fold lower shoot and root APase activity, respectively, which did not change in response to Pi starvation, (ii) a 40% reduction in secreted APase activity during Pi deprivation, (iii) 35 and 50% reductions in free and total Pi concentration, respectively, in shoots of –Pi plants, and (iv) impaired shoot and root development when subjected to Pi deficiency. By contrast, no deleterious influence of AtPAP26 loss of function was apparent in +Pi plants. The results establish a firm role for AtPAP26 in the acclimation of Arabidopsis to Pi deficiency. The identification and functional characterization of secreted proteins upregulated by –Pi Arabidopsis is relevant to applied efforts to engineer Pi-efficient transgenic plants, needed to minimize the input of expensive, unsustainable, and polluting Pi fertilizers in crop production. / Thesis (Ph.D, Biology) -- Queen's University, 2010-04-28 17:20:46.892

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-02-16T17:00:17Z No. of bitstreams: 2 Dissertação - Edilânia Gomes Araújo Chaves - 2013.pdf: 3289607 bytes, checksum: 174bfb3cce0a9109022321f4adf3368d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2017-02-17T16:53:18Z (GMT) No. of bitstreams: 2 Dissertação - Edilânia Gomes Araújo Chaves - 2013.pdf: 3289607 bytes, checksum: 174bfb3cce0a9109022321f4adf3368d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-02-17T16:53:18Z (GMT). No. of bitstreams: 2 Dissertação - Edilânia Gomes Araújo Chaves - 2013.pdf: 3289607 bytes, checksum: 174bfb3cce0a9109022321f4adf3368d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-03-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccicoidioides is the etiological agent of paracoccidioidomycosis (PCM), a disease considered one of the main causes of mortality among systemic mycoses in Brazil. The success in establishing of the infection is related with the ability of fungus to adhere and degrade components of the extracellular matrix (ECM). Human plasminogen (hPlg) is a protein of blood plasma of the host that presents fibrinolytic activity when activated into plasmin. Many pathogens are able to subvert the plasminogen/plasmin system using linker molecules and promote the degradation of tissue barriers. In this work, we identified through Far Western and proteomic analysis, a total of 15 proteins secreted of Paracoccidioides that are plasminogen binding proteins. Those proteins are probable targets of the interaction of the fungus with the host and could contribute to the invasiveness of the fungus. The fructose 1,6-biphosphate aldolase was described in other organisms such as plasminogen binder and presentes participation in the adherence of Paracoccidioides to host cells. This protein selected for validation tests and their presence was observed on the surface and secretory vesicles of the fungus. FBA confirm the ability to convert plasminogen to plasmin in the presence of tissue plasminogen activator (tPA) and this interaction promoted the degradation of fibrin. In infection assays, the addition of antibodies blocking the FBA binding site reduced the interaction of the fungus with macrophages and the interaction of FBA with Plg increased the rate of cell invasion. These data suggest that the FBA can contribute to adhesion, invasion and spread process of the fungus. / Paracoccicoidioides é o agente etiológico da paracoccidioidomicose (PCM), uma doença considerada como primeira causa de mortalidade entre as micoses sistêmicas no Brasil. O sucesso no estabelecimento da infeção está relacionado com a capacidade do fungo de aderir e degradar componentes da matriz extracelular (MEC). O plasminogênio humano (hPlg) é uma proteína presente no plasma, a qual possui atividade fibrinolítica quando é ativado em plasmina. Muitos micro-organismos patogênicos são capazes de subverter o sistema plasminogênio/plasmina através de moléculas ligantes e promovem a degradação de barreiras teciduais. Neste trabalho foram identificadas, através de Far-Western e análise proteômica, um total de 15 proteínas secretadas por Paracoccidioides com habilidade de ligação ao plasminogênio. Essas proteínas são prováveis alvos da interação do patógeno com o hospedeiro, que contribui para o potencial invasivo do fungo. A frutose 1,6-bifosfato aldolase (FBA) foi descrita em outros organismos como ligante de plasminogênio e possui participação na adesão de Paracoccidioides às células do hospedeiro. Selecionamos esta proteína para ensaios de validação e foi observada sua presença na superfície e em vesículas secretoras do fungo. Confirmamos a capacidade da FBA de converter plasminogênio em plasmina na presença do ativador tecidual de plasminogênio (tPA) e essa interação promoveu a degradação de fibrina. Em ensaios de infecção, a adição de anticorpos que bloqueiam o sítio de ligação da FBA reduziu a interação do fungo com macrófagos e a interação de FBA com Plg aumentou o índice de invasão celular. Esses dados sugerem que a FBA pode contribuir no processo de adesão, invasão e disseminação do fungo.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted glycoprotein that promotes degradation of low-density lipoprotein receptors. Gain- and loss-of-function variants of PCSK9 cause hypercholesterolemia and hypocholesterolemia, respectively. Although it has been a decade since the discovery of PCSK9, its effect in terms of global protein changes and interactions still require further understanding. This study provided a global outlook at the protein changes caused by PCSK9 and its variants in human hepatic HUH7 cell line. First, a proteomics-based method for protein subcellular distribution analysis has been developed. Second, through secretome analyses, six apolipoproteins and six proteins involved in the coagulation pathway were found with >2-fold changes between wild type PCSK9 and its variants. Third, through secreted interactome analyses, a list of 159 PCSK9 interactor candidates was identified. Two interacting proteins, FASN and PSMD2, were validated and demonstrated with dynamic interacting patterns between PCSK9 and its variants.

No description available.

No / Skeletal muscle has long been established as a highly multifunctional organ, playing a vital role in locomotion, whole-body metabolic and energy homeostasis, and thermoregulation. More recently, emergent evidence has highlighted a potent secretory role for muscle, producing and releasing “myokine” molecules that act in autocrine, paracrine, or endocrine fashion to govern muscle physiology and regulate whole-body homeostasis via multi-tissue cross talk mechanisms. Myokines represent promising therapeutic targets in health and disease, with their discovery, measurement, and functional importance being a hotbed of research across numerous physiological contexts. Here, we provide an overview of myokines and summarize current understanding of their biological role(s). We also outline primary approaches for myokine analysis, including detailed methodology for performing omics-driven myokine prediction, while further appraising both method-specific and general technical considerations to provide an evidence-based approach for designing and conducting myokine experiments.

Prostate cancer (PCa) is the second most common cancer in men with more than 1 million new cases worldwide each year. While some of the genomic, genetic and molecular events characterizing PCa have been functionally associated with tumor onset, development and resistance to therapy, the meaning of many other molecular alterations remains poorly understood. Recent development of organoids technology and prostate organoid cultures has established an innovative and valuable model for the study of adult tissue homeostasis, physiology and disease. In this project we combined prostate organoids technology with genetic engineering and CLICK-chemistry coupled Mass Spectrometry approaches in order to better characterize molecular features of wild type and genetically engineered mouse prostate organoids modeling early steps of human prostate tumorigenesis. In details, by manipulating mPrOs to proxy ETS-related gene (ERG) precursor PIN/HGPIN lesions of human prostate, we identified possible novel pro-tumorigenic roles of ERG which unleashes cells proliferation from the tight control of growth stimuli, and, even more interesting, corrupts immune system components to escape immune surveillance. In conclusion, this project shows that coupling innovative biological systems and technological approaches can lead to significant improvements in the analysis and understanding of disease mechanisms.

Reproductive diseases, mainly those resulting in the infertility affect the chances of human being to reproduce. On the contrary, the heart disease, cancer and degenerative diseases currently account for majority of deaths in the world. Usually, these lifestyle diseases need longer lifespan to become the cause of death. The proteins secreted by cells carry important information about the cell's well-being, as well as about the condition of the tissues formed by these cells. Once secreted, these proteins may also be transferred throughout the body by means of body fluids, many of which are easily accessible for further 'in-depth' studies. Cellular and secreted proteins are often a focus of studies using proteomic means and the revelation of protein alterations can lead us to new ideas about the molecular mechanisms of diseases as well as possible identification of proteins that may be used as new targets for pharmaceutical intervention or molecules that could be used for diagnostic or prognostic purposes. Taking into consideration the above aspects, this research was undertaken to find proteins that could: (a) characterise the human follicular fluid as microenvironment of the maturing oocyte, to increase understanding of reproductive processes to improve the techniques of assisted repro- duction;...

L'établissement de toute symbiose repose sur un dialogue moléculaire hautement régulé entre les deux partenaires. Une approche par génétique inverse a été utilisée pour identifier des gènes fongiques jouant un rôle clé dans le dialogue moléculaire entre les partenaires de la symbiose ectomycorhizienne. La caractérisation phénotypique et moléculaire d'un mutant non-mycorhizien du champignon Hebeloma cylindrosporum a montré qu'il présente une insertion de l'ADN-T mutagène dans le promoteur de la subtilase HcSbt1. Le fait que ce mutant soit incapable de coloniser les racines de la plante hôte Pinus pinaster, indique que HcSbt1 joue un rôle crucial dans le dialogue moléculaire précoce entre les partenaires de la symbiose ectomycorhizienne. Lors de la formation des mycorhizes, HcSbt1 est réprimé dès le contact avec les racines, avant même la différenciation de toute structure symbiotique. La répression se maintient tant que dure la symbiose. Ceci suggère que HcSbt1 pourrait inhiber le processus symbiotique. Sa répression serait un prérequis à la formation des mycorhizes. L'analyse par Western Blot et séquençage du sécrétome du champignon a montré que HcSbt1 est exocellulaire. Elle pourrait inhiber l'établissement de la symbiose en dégradant/activant/inactivant des protéines exocellulaires de la plante ou/et du champignon. La comparaison des secrétomes de la souche sauvage et de la souche mutante a montré que HcSbt1 peut dégrader de petits peptides exocellulaires. Notre hypothèse est que cette subtilase inhibe l'établissement de la symbiose en dégradant des petits peptides exocellulaires qui seraient des effecteurs / The establishment of any symbiosis relies on a tightly regulated molecular dialogue between symbiotes. A reverse genetic approach was used to identify fungal genes playing a key role in the molecular dialogue between the partners of the ectomycorrhizal symbiosis. A non-mycorrhizal mutant of the fungus Hebeloma cylindrosporum has a single insertion of mutagenic T-DNA in the promoter of the subtilase HcSbt1. This mutant was unable to colonize Pinus pinaster roots, indicating that HcSbt1 plays a crucial role in the early molecular cross-talk between partners of the ectomycorrhizal symbiosis. During symbiotic interaction, HcSbt1 was repressed upon contact with the roots, even before the differentiation of any symbiotic structure. This repression was stable throughout the whole symbiotic process, suggesting that HcSbt1 could inhibit symbiotic structure differentiation. Subsequently, HcSbt1 repression would be a prerequisite for mycorrhiza differentiation. Western Blot analysis together with fungal secretome sequencing showed that HcSbt1 is extracellular. It could inhibit the symbiosis establishment by degrading / activating / inactivating extracellular proteins from plant and/or fungal origin. The comparison of wild-type and mutant secretomes showed that HcSbt1 could degrade small extracellular peptides. Based on this, we hypothesized that this subtilase could inhibit symbiosis establishment by degrading small extracellular peptides that would be effectors

Biotransformação é uma estratégia promissora para a descoberta de novas moléculas de interesse farmacêutico e industrial. Fungos têm demonstrado habilidade para biotransformar muitas moléculas naturais e sintéticas. Um produto natural muito estudado e de potencial para a indústria farmacêutica é o Lapachol, uma naftoquinona que apresenta uma ampla diversidade de atividades biológicas. Existem na literatura muitos estudos de biotransformação utilizando fungos, mas pouco se sabe sobre como esses microrganismos transformam essas moléculas, e quais proteínas e vias metabólicas estão envolvidas nesses processos. A proteômica tornou-se uma das principais abordagens para analisar e compreender os sistemas biológicos. Dessa forma, o objetivo desse estudo foi compreender o processo de biotransformação do lapachol pelo fungo Phanerochaete chrysosporium, identificando os biotransformados, suas atividades biológicas e investigando o proteoma do fungo durante o bioprocesso a fim de identificar as proteínas e as vias metabólicas envolvidas. O fungo foi capaz de biotransformar o lapachol e gerou dois análogos, um já teve sua estrutura elucidada e é inédita na literatura. O proteoma intracelular foi analisado por eletroforese bidimensional e, após análises quanti e qualitativas, as proteínas selecionadas foram submetidas à espectrometria de massas por MALDI-TOF/TOF. Após análises, com a ferramenta de bioinformática BLAST2GO, determinou-se que as enzimas identificadas hiperabundante e ou exclusivas da condição lapachol estavam principalmente envolvidas com processos de detoxicação e resposta ao estresse oxidativo. Além disso, foram detectadas diversas enzimas com atividade de oxirredução. Além do mais, a técnica de Shotgun LC-MS/MS foi realizada. A identificação e quantificação (label-free) das proteínas foram feitas através da análise de dados MS/MS brutos pelo software MaxQuant/Andromeda. As diferenças nas expressões das proteínas identificadas entre os grupos foram computadas usando o software Perseus. Este estudo foi capaz de identificar um total de 221 proteínas intracelulares e 28 extracelulares, sendo que 54 proteínas intracelulares e 4 proteínas extracelulares foram estatisticamente diferentes ou únicas para uma condição (lapachol ou controle). Manganês-peroxidase e ?-glicosidase, ambas enzimas com interesse biotecnológico, foram identificadas exclusivas e aumentada, respectivamente na condição lapachol, desssa forma, o lapachol pode ser um bom agente pró-oxidante, pois estimulou a produção dessas enzimas oxidativas. Époxido desidrogenase e álcool desidrogenase parecem estar envolvidas na rota de biotransformação do lapachol, a primeira catalisando a abertura do epóxido e a segunda, reduzindo regioseletivamente carbonilas. O derivado PC01 mostrou-se mais tóxico para células de adenocarcinoma mamário que o lapachol e 3 e 5 vezes menos tóxico para células epiteliais normais que cisplatina e lapachol, respectivamente, mostrando-se um composto promissor. Esta é a primeira vez que a proteômica foi utilizada para investigar a biotransformação do lapachol. / Biotransformation is a promising strategy to discovery of new molecules with pharmaceutical and industrial interest. Fungi have demonstrated the ability to biotransform many natural and synthetic molecules. A very studied natural product of potential for the pharmaceutical industry is Lapachol, a naphthoquinone with a great diversity of biological activities. There are many biotransformation studies in the literature using fungi but little is known about how these microorganisms transform these molecules, which proteins and metabolic pathways are involved in these processes. Proteomics has become one of the main approaches for analyzing and understanding biological systems. The objective of the present study was to understand the biotransformation process of lapachol by Phanerochaete chrysosporium, identifying the biotransformed and its biological activities and investigating the fungus proteome during the bioprocess to identify the proteins and the metabolic pathways involved. After 24 h of bioprocessing, the fungus was able to biotransform lapachol and generated two analogues, one already had its structure elucidated and is unpublished in the literature. The intracellular proteome was analyzed by two-dimensional electrophoresis and, after quantitative and qualitative analyzes, the selected proteins were submitted to MALDI-TOF/TOF mass spectrometry. After analysis, with the BLAST2GO bioinformatics tool, it was determined that the enzymes identified as hyperabundant and exclusive to the lapachol condition were mainly involved with detoxification and oxidative stress response processes. Moreover, several enzymes with oxreduction activity were detected. In addition, the Shotgun LC-MS/MS technique was performed on intracellular and extracellular proteins. Identification and quantification (label-free) of the proteins were performed by the analysis of MS/MS raw data using MaxQuant/Andromeda software. Differences in the expressions of the proteins identified between the groups were computed using Perseus software. This study was able to identify a total of 221 intracellular and 28 extracellular proteins. 54 intracellular proteins and 4 extracellular proteins were different or unique statistics for a condition (lapachol or control). Manganese-peroxidase and ?-glycosidase, both enzymes of biotechnological interest, were identified exclusively and increased respectively in the lapachol condition, thus, lapachol may be a good pro-oxidant agent, since it stimulated the production of these oxidative enzymes. Epoxide dehydrogenase and alcohol dehydrogenase appear to be involved in the lapachol biotransformation route, the first catalyzing the epoxide opening and the second, regioselectively reducing carbonyls. The PC01 derivative was shown to be more toxic to mammary adenocarcinoma cells than lapachol and approximately 3 to 5 times less toxic to normal epithelial cells than cisplatin and lapachol, showing a promising compaund. To our best knowledge, this is the first time that proteomics has been used to investigate the biotransformation of lapachol.