Among the factors important for maintaining productivity of recombinant proteins in mammalian cells, culture lifetime, cell density and cell viability are three simple but essential aspects that can greatly influence the final yield. During cell culturing, the degradation of environmental conditions such as nutrient depletion and accumulation of toxic waste products, often lead to premature apoptotic cell death in cultures and suboptimal protein yield. Although apoptosis has been extensively researched, the changes in the whole cell proteome during prolonged cultivation, where apoptosis is a major mode of cell death, have not been examined. The work presented in this thesis is the first whole cell proteome analysis of non-induced apoptosis in mammalian cells. Flow cytometry analyses of the level of activated executioner caspases, caspase 3 and 7, demonstrated the onset of apoptosis in CHO batch cultures after the exponential phase. The monitoring of apoptotic cell death assesses the culture conditions at different time points and allows the association of changes in the protein abundances to the degradation of culture conditions, in particular the degree of apoptosis in the whole cultures. The detection of activated caspase 8 and caspase 9, which trigger the extrinsic and intrinsic pathway respectively, has shown that the onset of apoptosis in CHO cells during prolonged cultivation predominantly is via the intrinsic pathway. To examine the proteomic changes in a monoclonal antibody-producing CHO cell culture at various phases of a batch culturing process, a differential in gel electrophoresis proteomic approach was employed in this study. CHO protein samples at four time points during cultivation were compared. A total of 40 differentially expressed protein spots were successfully identified by mass spectrometry sequencing, resulting in 28 unique protein identifications. These proteins include four structural proteins of the cytoskeleton, ten endoplasmic reticulum and cytosolic chaperones and folding proteins, seven metabolic enzymes and seven other proteins of varied function. The cleavage of cytoskeletal proteins is a known consequence of apoptosis and the activation of executioner caspases. On the other hand, the induction of seven ER chaperone and foldases is a solid indication for the onset of unfolded protein response, which is triggered by cellular and ER stresses, many of which are present during prolonged batch cultures. In addition, the upregulation of six glycolytic enzymes and another metabolic protein emphasizes a change in the energy metabolism likely occurred when culture conditions degraded and apoptosis advanced. Interestingly, although a significant portion of proteins identified are well known housekeeping proteins, recent studies have shown that many of them exhibit a wide variety of other roles, including apoptosis regulation and execution. Overall, this study shows that the most drastic changes in aging CHO cultures, where apoptosis is known to be part of, involve the onset of UPR and upregulation of proteins catalyzing glucose metabolism.

Among the factors important for maintaining productivity of recombinant proteins in mammalian cells, culture lifetime, cell density and cell viability are three simple but essential aspects that can greatly influence the final yield. During cell culturing, the degradation of environmental conditions such as nutrient depletion and accumulation of toxic waste products, often lead to premature apoptotic cell death in cultures and suboptimal protein yield. Although apoptosis has been extensively researched, the changes in the whole cell proteome during prolonged cultivation, where apoptosis is a major mode of cell death, have not been examined. The work presented in this thesis is the first whole cell proteome analysis of non-induced apoptosis in mammalian cells. Flow cytometry analyses of the level of activated executioner caspases, caspase 3 and 7, demonstrated the onset of apoptosis in CHO batch cultures after the exponential phase. The monitoring of apoptotic cell death assesses the culture conditions at different time points and allows the association of changes in the protein abundances to the degradation of culture conditions, in particular the degree of apoptosis in the whole cultures. The detection of activated caspase 8 and caspase 9, which trigger the extrinsic and intrinsic pathway respectively, has shown that the onset of apoptosis in CHO cells during prolonged cultivation predominantly is via the intrinsic pathway. To examine the proteomic changes in a monoclonal antibody-producing CHO cell culture at various phases of a batch culturing process, a differential in gel electrophoresis proteomic approach was employed in this study. CHO protein samples at four time points during cultivation were compared. A total of 40 differentially expressed protein spots were successfully identified by mass spectrometry sequencing, resulting in 28 unique protein identifications. These proteins include four structural proteins of the cytoskeleton, ten endoplasmic reticulum and cytosolic chaperones and folding proteins, seven metabolic enzymes and seven other proteins of varied function. The cleavage of cytoskeletal proteins is a known consequence of apoptosis and the activation of executioner caspases. On the other hand, the induction of seven ER chaperone and foldases is a solid indication for the onset of unfolded protein response, which is triggered by cellular and ER stresses, many of which are present during prolonged batch cultures. In addition, the upregulation of six glycolytic enzymes and another metabolic protein emphasizes a change in the energy metabolism likely occurred when culture conditions degraded and apoptosis advanced. Interestingly, although a significant portion of proteins identified are well known housekeeping proteins, recent studies have shown that many of them exhibit a wide variety of other roles, including apoptosis regulation and execution. Overall, this study shows that the most drastic changes in aging CHO cultures, where apoptosis is known to be part of, involve the onset of UPR and upregulation of proteins catalyzing glucose metabolism.

A manga (Mangifera indica L.) é cultivada em áreas tropicais e subtropicais, principalmente em países em desenvolvimento. Os maiores produtores são a Índia, China, México, Indonésia, Tailândia, Paquistão e Brasil, mas, por ser uma fruta altamente perecível, suas exportações têm sido limitadas. Durante o amadurecimento, as frutas adquirem características que as tornam adequadas para o consumo como conseqüência de alterações metabólicas dependentes, em larga medida, da expressão de genes específicos. Uma vez que as proteínas são os elementos efetores da expressão gênica, a análise de proteomas pode auxiliar na identificação de pontos de controle do metabolismo determinantes para a qualidade desses alimentos. Assim, o objetivo do trabalho é identificar spots de proteínas diferentemente abundantes durante o amadurecimento a partir dos mapas 2D-DIGE das polpas de mangas (Mangifera indica L.) da cultivar Keitt nos estádios pré-climatérico e climatérico. Após extração das proteínas e separação por 2D-DIGE, as imagens dos géis obtidas foram analizadas com o software PDQuest, utilizando o teste T de Student para a análise estatística. Dentre os spots protéicos bem resolvidos e considerados na análise, os 47 que apresentaram-se diferentemente abundantes entre os estádios estudados foram removidos dos géis, suas proteínas digeridas e, enfim, sequenciadas por espectrometria de massas. Foram obtidas as identidades prováveis de 58 proteínas diferentes a partir da comparação das sequencias obtidas com banco de dados NCBI2010, utilizando o software Mascot. / Mango fruit (Mangifera indica L.) is cultivated in tropical and subtropical areas, mainly in developing countries. India, China, Mexico, Indonesia, Thailand, Pakistan and Brazil are the major producers, but its trade has been limited due the highly perishable nature of the fruit. During ripening, the fruits acquire characteristics that make them appropriate for consumption as a consequence of metabolic changes dependent on the expression of specific genes. As proteins are the effector elements of gene expression, proteome analysis can help the identification of metabolism keypoints that could influence the fruit quality. Thus, the aim of this study was to compare the protein maps of mango pulp (Mangifera indica L. cv. Keitt) in pre-climacteric and climacteric stages, in order to identify protein spots that differ in abundance in these two stages. After protein extraction and separation by 2D-DIGE technique, the gel images obtained were analyzed with PDQuest software, using the Student´s T-test for statistical analysis. We obtained 47 spots differently abundant between the stages studied, that were excised from the gels and its proteins digested with trypsin and sequenced by mass spectrometry. We obtained the identities of 58 distinct proteins from the search of the peptide sequences against NCBI2010 database using the software Mascot.

P. falciparum est le principal responsable des formes graves du paludisme. Le parasiteévolue entre deux hôtes (homme et moustique) qui lui imposent différents environnements; ettout particulièrement, des modifications des pressions partielles d’O2 nécessitant des capacitésd’adaptation surprenantes pour un parasite microaérophile. Chez l’hôte vertébré, lesphénomènes de cytoadhésion, ralentissant la progression du parasite notamment au niveau despoumons, augmentent la durée d’exposition aux conditions hyperoxiques.La dynamique de la réponse parasitaire à l’hyperoxie a été étudiée par une approchecombinée de transcriptomique et de protéomique. Certains mécanismes de défense contre lesespèces réactives d’oxygène ont été appréciés, dont une éventuelle fonction oxydasealternative.L’exposition du parasite à 21% d’O2 induit un retard de croissance au niveau de laschizogonie. Le stress oxydatif induit par l’hyperoxie entraîne des perturbations métaboliquescomme une inhibition de la glycolyse en faveur de la respiration et un ralentissement dumétabolisme de la vacuole digestive. Cette action combinée sur le métabolisme mitochondrialet vacuolaire permet au parasite de s’adapter à un environnement hyperoxydant, en régulant laproduction d’espèces réactives d’oxygène. Nos travaux ont montré qu’un inhibiteur de lafonction oxydase alternative, l’acide salicylhydroxamique ou SHAM, avec un effet mineur surla croissance parasitaire en microaérophilie, avait un effet létal sur les parasites en hyperoxie.Une meilleure compréhension de la biologie parasitaire pourrait contribuer audéveloppement de nouveaux traitements antipaludiques associés à une thérapie hyperbarique. / P. falciparum is the main species responsible for severe case of malaria. The parasiteevolves between two hosts (human and mosquito), imposing to it different environments;especially changes in the O2 pressure, demanding astonishing adaptation skills for amicroaerophilic parasite. In the vertebrate host, the phenomena of cytoadhesion, which slowdown the spread of the parasite among others in the lungs, increase the timing of exposure tohyperoxic conditions.The parasitic response dynamic to hyperoxia has been analysed by a combinedtranscriptomic and proteomic approach. Some of the defense mechanisms against reactiveoxygen species have been evaluated, among which a potential alternative oxidase function.The exposure of the parasite to 21%O2 atmosphere leads to a growth delay atschizogony level. The oxidative stress resulting from the hyperoxia conducts to metabolicalterations, as an inhibition of the glycolysis in favour of respiration and as a slowdown of themetabolism of the digestive vacuole. This combined action on the mitochondrial and vacuolarmetabolisms allows the parasite to adapt itself to hyperoxic environment, by regulatingreactive oxygen species. Our works have shown that an inhibitor of the alternative oxidasefunction, the salicylhydroxamic acid or SHAM, with a minor effect on the parasite growth inmicroaerophily, had letal effect on parasites in hyperoxia.A better understanding of the parasitic biology could contribute to the development ofnew antimalarial treatments, associated with a hyperbaric oxygen therapy.

A manga (Mangifera indica L.) é cultivada em áreas tropicais e subtropicais, principalmente em países em desenvolvimento. Os maiores produtores são a Índia, China, México, Indonésia, Tailândia, Paquistão e Brasil, mas, por ser uma fruta altamente perecível, suas exportações têm sido limitadas. Durante o amadurecimento, as frutas adquirem características que as tornam adequadas para o consumo como conseqüência de alterações metabólicas dependentes, em larga medida, da expressão de genes específicos. Uma vez que as proteínas são os elementos efetores da expressão gênica, a análise de proteomas pode auxiliar na identificação de pontos de controle do metabolismo determinantes para a qualidade desses alimentos. Assim, o objetivo do trabalho é identificar spots de proteínas diferentemente abundantes durante o amadurecimento a partir dos mapas 2D-DIGE das polpas de mangas (Mangifera indica L.) da cultivar Keitt nos estádios pré-climatérico e climatérico. Após extração das proteínas e separação por 2D-DIGE, as imagens dos géis obtidas foram analizadas com o software PDQuest, utilizando o teste T de Student para a análise estatística. Dentre os spots protéicos bem resolvidos e considerados na análise, os 47 que apresentaram-se diferentemente abundantes entre os estádios estudados foram removidos dos géis, suas proteínas digeridas e, enfim, sequenciadas por espectrometria de massas. Foram obtidas as identidades prováveis de 58 proteínas diferentes a partir da comparação das sequencias obtidas com banco de dados NCBI2010, utilizando o software Mascot. / Mango fruit (Mangifera indica L.) is cultivated in tropical and subtropical areas, mainly in developing countries. India, China, Mexico, Indonesia, Thailand, Pakistan and Brazil are the major producers, but its trade has been limited due the highly perishable nature of the fruit. During ripening, the fruits acquire characteristics that make them appropriate for consumption as a consequence of metabolic changes dependent on the expression of specific genes. As proteins are the effector elements of gene expression, proteome analysis can help the identification of metabolism keypoints that could influence the fruit quality. Thus, the aim of this study was to compare the protein maps of mango pulp (Mangifera indica L. cv. Keitt) in pre-climacteric and climacteric stages, in order to identify protein spots that differ in abundance in these two stages. After protein extraction and separation by 2D-DIGE technique, the gel images obtained were analyzed with PDQuest software, using the Student´s T-test for statistical analysis. We obtained 47 spots differently abundant between the stages studied, that were excised from the gels and its proteins digested with trypsin and sequenced by mass spectrometry. We obtained the identities of 58 distinct proteins from the search of the peptide sequences against NCBI2010 database using the software Mascot.

One of the most concerning characteristics of microbial biofilms is that of increased resistance to antimicrobial agents such as the commonly used biocide chlorhexidine (CHX). This can have huge impact on clinical, household and environmental settings. This is particularly alarming when it involves opportunistic pathogenic environmental organisms such as Delftia acidovorans as routine mitigation practices may fail to be effective. This thesis examines tolerance mechanisms of D. acidovorans biofilms exposed to CHX at inhibitory and sub-inhibitory concentrations. To achieve the study goals and objectives, a CHX-tolerant D. acidovorans strain (WT15), (Minimum Inhibitory Concentration; MIC-15 μg ml-1) was compared to a CHX-sensitive strain (MT51, MIC-1 μg ml-1) that was obtained by mutating the wild type strain using transposon mutagenesis. Specific morphological, structural and chemical compositional differences between the CHX-treated and untreated biofilms of wild type and mutant strains were documented using microscopic techniques including confocal laser scanning microscopy (CLSM), scanning transmission x-ray microscopy (STXM), transmission electron microscopy (TEM) and infrared (IR) spectroscopy. Molecular level changes between biofilms formed by these two strains due to CHX treatment were compared using whole-cell proteomic analysis (determined using differential in-gel electrophoresis, or DIGE) along with fatty acid methyl ester (FAME) analysis. The gene disrupted by transposon insertion that led to increased susceptibility to CHX in the mutant strain was identified as tolQ. CLSM revealed differences in biofilm architecture and thickness between the biofilms formed by strains WT15 and MT51. STXM analyses showed that WT15 biofilms contained two morpho-chemical cell variants; whereas, only one type was detected in MT51 biofilms. STXM and IR spectral analyses revealed that CHX-susceptible MT51 cells accumulated the highest levels of CHX, an observation supported by TEM wherein prominent changes in the cell envelope of CHX-susceptible MT51 cells were observed. DIGE analysis demonstrated that numerous changes in protein abundance occurred in biofilm cells following CHX exposure and that most of these proteins were associated with amino acid and lipid biosynthesis, protein translation, energy metabolism and stress-related functions. Overall, these studies indicate the probable role of the cell membrane and TolQ protein in CHX tolerance in D. acidovorans biofilms, in association with various proteins that are differentially-expressed.

Le remodelage ventriculaire gauche (VG) en post-infarctus du myocarde (IDM) est associé à une augmentation du risque d’insuffisance cardiaque et de décès, mais il demeure difficile à prédire en pratique clinique.L’objectif principal de ma thèse était la recherche de biomarqueurs circulants du remodelage VG par l’approche protéine candidate et par protéomique différentielle dans la population REVE-2.Par l’approche protéine candidate, nous avons confirmé que le peptide natriurétique detype B (BNP) était un puissant facteur prédictif du remodelage VG en post-IDM. La métalloprotéase matricielle-8 (MMP-8), la MMP-9, l’hepatocyte growth factor (HGF), la Créactive protéine (CRP), la troponine I ont également fait la preuve de leur association.Par l’approche protéomique différentielle, en électrophorèse 2D différentielle en fluorescence (2D-DIGE), la clusterine a été identifiée comme biomarqueur potentiel,positivement associée au remodelage VG, nécessitant toutefois des travaux de confirmation.Par SELDI TOF MS, nous avons sélectionné 26 pics définis par leur rapport m/z, commebiomarqueurs potentiels du remodelage VG, dont 12 ont pu être identifiés et devrontdésormais être validés : le pic de m/z 2777 a été identifié comme le peptide N-terminal issu del’albumine après clivage par la pepsine. Les autres pics correspondraient à des fragments protéolytiques de protéines que sont le fibrinogène, le complément C3, C4 et C1q.La découverte de nouveaux biomarqueurs du remodelage VG devrait permettre d’améliorer la stratification du risque en post-IDM afin d’identifier les patients devant bénéficier d’un suivi plus rapproché et peut-être d’une prise en charge thérapeutique plus agressive / Left ventricular (LV) remodelling after myocardial infarction (MI) indicates a high risk of heart failure and death but remains difficult to predict in clinical practice. Biomarkers may help to refine risk stratification. The main purpose was to find circulating biomarkers of LV remodelling after MI, using two strategies : candidate protein approach and differential proteomic approach, working on a population with a clearly defined phenotype, the REVE-2 study, a prospective multicenter study including 246 patients with a first anterior Q-wave MI. Blood samples were obtained at hospital discharge, at 1 month, 3 months and 1 year. An echocardiography was performed at the same time except for the 1st month to assess LVR.By candidate protein approach, we confirmed that B-type natriuretic peptide (BNP) was a powerful predictor of LV remodelling after MI. Additional biomarkers, such as matrix metalloproteinase-8 (MMP-8), MMP-9, hepatocyte growth factor (HGF), C-reactive protein (CRP) and cardiac troponin I were found to be associated with LV remodelling, highlighting several pathways implicated in pathophysiology of LV remodelling. We have also shown that biomarkers in association (BNP and cardiac troponin I, BNP and MMP-8, BNP and MMP-9) could improve risk stratification in post-MI by selecting groups of patients at higher risk.As the ideal biomarker was still not identified, we applied a differential proteomic approach, with no a priori hypothesis, in order to characterize proteomic signature of LV remodelling. The use of a protein enrichment kit, consisting of a library of combinatorial hexapeptide ligands, compressed the protein concentration range of plasma and serum, through the simultaneous onestep dilution of high-abundance and concentration of lowabundance proteins. Protein enrichment kit prior to two-dimensional (2D) electrophoresis or SELDI TOF MS (surface-enhanced laser desorption–ionization time of flight) analysis enabled the detection of proteins that were not detected in native blood sample and the accessibility to proteolytic fragments obtained from major proteins. Clusterin (apolipoprotein J) was identified as a potential biomarker of LV remodelling by 2D-DIfferential Gel Electrophoresis (2D-DIGE). Clusterin was quantified by Western blot and ELISA and was found to be positively associated with LV remodelling. However, this association was not found with all LV remodelling parameters nor at each time during the year following MI, requiring further analysis. Differential proteomic approach by SELDI TOF MS selected 26 m/z peaks, as potential biomarkers of LV remodelling. Of them, 12 were identified by mass spectrometry. The 2777 m/z peak was identified directly from the ProteinChip array as being the N-terminal peptide (24–48 aa) generated from albumin by pepsin cleavage. Other peaks were identified after purification using chromatographic columns or liquid-phase isoelectric focusing : most of them were found to be proteolytic fragments of proteins like fibrinogen, C3, C4 and C1q complement. Identifications have now to be validated with specific techniques, usually by immmunoprecipitation and Western blot analysis.Finding new biomarkers of LV remodelling could help refine risk stratification and identify patients in whom more aggressive therapy and/or more frequent follow-up could be needed.

Work related musculoskeletal disorders are the dominating causes of reported ill-health in industrialized countries. These chronic pain conditions are one of the most costly public health problems in Europe and North America. When work related musculoskeletal disorders are considered to be of muscular origin and the trapezius muscle is affected, the common appellation is trapezius myalgia. Since little is known about the genesis or how it is maintained, it is of great importance to better understand the pathophysiology of trapezius myalgia; doing so will better enable recommendations for prevention, treatment and rehabilitation. Several hypotheses have been presented based on biochemical alterations in the muscle, suggesting increased signaling of inflammatory substances and altered metabolism. Previous research has not been able to present the comprehensive picture of the muscle in pain. Thus there is a demand for more comprehensive research regarding the biochemical milleu of the chronic trapezius muscle. Proteomic and metabolomic methods allow non-targeted simultaneous analyses of a large number of proteins and metabolites. The main emphasis in this thesis is on a proteomic method, two-dimensional differential gel electrophoresis (2D-DIGE). The method is validated to human skeletal muscle biopsy research with laboratory specific settings. In the baseline study, there were 14 metabolic, contractile, structural and regulatory proteins that differed significantly in abundance when trapezius and vastus lateralis muscles were compared. Using the validated 2D-DIGE method and the baseline study, a comparison between healthy and myalgic muscles was made. Biopsies from female cleaners with and without myalgia were compared to obtain results from women with the same type of work exposure. In the multivariate model, 28 identified unique proteins separated healthy and myalgic muscle and were grouped according to function: metabolic (n=10), contractile (n=9), regulatory (n=3), structural (n=4), and other (n=2). Finally, a second screening method, metabolomics, was introduced to analyze differences in metabolite content as a complement to and verification of the proteomic results. Gas chromatography-mass spectrometry (GC-MS) was performed on muscle interstitial fluid samples obtained with microdialysis, and differences in the abundance of extracellular metabolites were revealed.  The 2D-DIGE method is a reliable method to analyze human skeletal muscle. The outcomes of the proteomic analyses were dependant on the statistical approach. Systematic differences in protein and metabolite content were detected using a multivariate approach. Univariate analyses were used to analyze individual proteins for their significance. The significant proteins in the baseline study were predominately related to muscle fiber type which correlated with the differences in fiber type content between trapezius and vastus lateralis. The proteomic and metabolomics studies where myalgic and healthy muscles were compared provide us with new clues and new aspects regarding the pathophysiology of the myalgic muscle. Technically advanced methods employed in the thesis enabled an explorative screening of proteins of relevance for the pathophysiology of the myalgic muscle. The results of these analyses may contribute to the formulation of future hypothesis that need to be further evaluated.

WWOX (WW domain-containing oxidoreductase) is a candidate tumor suppressor gene that has been shown to be involved in various cancers including breast, lung, prostate, gastric and hepatic. The Drosophila ortholog Wwox was identified and subjected to targeted ‘loss of function’ mutagenesis. The resulting mutants were found to be viable when homozygous with no obvious defects in the adult fly. As Wwox mutant flies were found to exhibit an increased sensitivity to ionising radiation (IR), a number of Wwox proteins specifically deleted or mutated at positions consisting of conserved functional protein motifs, or regions that are highly conserved among WWOX / Wwox homologs. The Wwox variants were tested for their ability to modify the IR sensitivity phenotype. In the course of this study, it was found that background mutations introduced during the generation of the mutant flies was responsible for the IR sensitivity phenotype. As a result, proteomic alterations resulting from changes in Wwox protein levels in Drosophila were investigated in order to ascertain the possible molecular functions of the Wwox protein. 2D-DIGE analysis was conducted on a number of different fly genotypes expressing differing levels of Wwox protein in both adult and embryonic flies. The proteomic changes resulting from lack of Wwox function as well as Wwox over-expression were detected with the proteins of interest identified by mass spectrometry (MS) using both MALDI-TOF/TOF-MS and LC-ESI-MS/MS. Label free quantitative MS analysis was also performed in order to determine the most abundant protein(s) in those spots found to contain multiple proteins. These proteomic studies identified changes in a wide variety of proteins with a significant number of metabolic proteins as well as proteins involved in oxidative stress response as a result of different levels of Wwox expression. Of particular interest, consistent changes in different isoforms of superoxide dismutase 1 (Sod1) were identified. Due to the known roles these proteins play in pro and anti-apoptotic pathways, it is possible that Sod1 and Wwox may work in concert to regulate the delicate balance of defence mechanisms in response to environmental stresses, particularly oxidative stress. The protein/gene targets identified in this work therefore offer some insights into normal Wwox function. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

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