Dissertations / Theses on the topic 'Label-free quantitative proteomic'

The aim of this Ph.D. was to develop advanced methods for quantitative proteomics and use these methods to investigate the presence of protein biomarkers of sperm performance, differential expression of sperm membrane proteins and differential expression of E.coli proteins. Quantitative analysis of E.coli generated analytical samples that were analysed with multiple mass spectrometers and with multiple software packages. Through these samples an optimal label free quantitative proteomic workflow was generated and software was thoroughly tested to determine the optimal software to be used for data analysis on varying biological questions. Identification of protein(s) that correlate with increased or decreased fertility would be economically beneficial. Currently semen samples are subject to quality control where general movement and morphological defects are studied, but this does not always correlate with the ejaculate passing a post cryopreservation quality control check or that specific bull generating offspring. Identification of a protein or set of proteins with abundance variation in bulls of known high or low fertility would allow lower fertility bulls to be removed from the breeding programme at an early age, reducing rearing costs, and would allow longitudinal health monitoring of individual bulls. Discovery of differentially expressed proteins in the membrane of sperm with the X or Y chromosome would allow the generation of a method to separate the two sperm populations. This will be beneficial as most livestock farmers would prefer offspring of a specific sex, either to sell or replenish animal stock. Quantitative analysis of proteins present in bovine seminal plasma led to the identification and quantitative comparison of the seminal plasma proteins present in two breeds of bull, Holstein and Belgian Blue and a quantitative comparison of the seminal plasma from two domestic farm animal species, bovine and porcine. Intra species comparisons determined no quantitative variation between the two breeds, while the inter species comparison determined variation between the proteins present in both species seminal plasma and the corresponding amounts of proteins present in both species. A quantitative comparison was performed to determine the expression of proteins from two strains of E.coli, a wild type strain (MG1655) and a genome depleted strain (MDS66), this led to the confirmation of gene deletions in the genome depleted strain due to their lack of protein products in mass spectrometric analysis, and the identification of proteins that were differentially expressed due to pleiotropic effects of these genome deletions. To investigate the proteins expressed in the sperm membrane a mass spectrometer compatible enrichment method was generated and membrane proteins were identified, quantified and compared between sperm expressing X and Y chromosomes. This study did not lead to the determination of any proteins with differential expression in the X or Y bearing sperm.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
As sessile organisms, plants are exposed to a plethora of environmental stresses to which they must respond to maintain efficient growth and survival. Therefore, in order to improve our understanding on the complex mechanisms involved in the cowpea response to salt stress and to a compatible interaction with the cowpea severe mosaic virus (CPSMV), we used a label-free quantitative proteomic approach to identify the salt and virus responsive proteins in the leaves of the Pitiuba (CE-31) cultivar. The proteins extracted from the leaves (control and treated) 2 and 6 days post-treatment only with salt (DPS), only infected with CPSMV (DPV) or both of them (DPSV) were analyzed using mass spectrometry. At 2 DPS, 350 proteins with at least two-fold differences in abundance, in comparison with controls, were differentially accumulated in the leaves of the salt-treated (80% up and 20% down-accumulated), 281 at 2DPV (25% up and 75% down-accumulated) and 321 at 2 DPSV (45% up and 55% down-accumulated) plants. At 6 DPS, 350 proteins were differentially accumulated in the leaves of the salt-treated (90% up and 10% down-accumulated), 225 at 6 DPV (80% up and 20% down-accumulated) and 315 at 6 DPSV (94% up and 6% down-accumulated) plants. The qualitative analysis showed biochemical differences when the cowpea plants were challenged concurrently with both stresses. To cope with salinity, cowpea increased the abundance of proteins directly involved with the salt tolerance mechanisms. The results indicated that the CPSMV induce the down-accumulating of several proteins to invade and spread in host at early infection period (2 DPV), but at 6 DPV plant can induce accumulation of diverse proteins related with defense, although these strategies canât avoid the negatives effects of disease. When exposed simultaneously to salt/CPSMV stresses, a balance in protein accumulation involved in many biological process. This is the first work employing this approach in cowpea and providing evidences of the plant biochemical mechanisms involved in the responses of cowpea to these stresses.
Como organismos sÃsseis, as plantas sÃo expostas a uma variedade de estresses ambientais aos quais devem responder para sobreviverem e se desenvolverem. A fim de melhorar a nossa compreensÃo sobre os mecanismos complexos envolvidos na resposta do feijÃo-de-corda ao estresse salino e na interaÃÃo compatÃvel com o vÃrus do mosaico severo do caupi (CPSMV), foi utilizada uma abordagem proteÃmica quantitativa, livre de marcaÃÃo, para identificar proteÃnas, responsivas a essess estresses em folhas de feijÃo-de-corda, cv. CE-31. As proteÃnas extraÃdas a partir de folhas primÃrias, 2 e 6 dias apÃs o tratamento sà com o sal (DPS), somente infectadas (DPV), ou sob aÃÃo combinada dos dois (DPSV) foram analisadas, usando espectrometria de massas e comparadas com grupo controle. No 2 DPS, foram identificadas 350 proteÃnas diferencialmente acumuladas (80% aumentaram em abundÃncia e 20% diminuÃram), no 2 DPV 281 (25% aumentaram em abundÃncia e 75% diminuÃram) e no 2 DPSV 321 (45% aumentaram em abundÃncia e 55% diminuÃram). Jà no 6 DPS, foram identificadas 350 proteÃnas diferencialmente acumuladas (90% mostraram aumento em abundÃncia e 10% diminuiÃÃo), no 6 DPV 225 (80% aumentaram em abundÃncia e 20% diminuÃram) e no 6 DPSV 315 proteÃnas(94% aumentaram em abundÃncia e 6% diminuÃram). Para lidar com a salinidade, o cv. CE-31 aumentou a abundÃncia de proteÃnas envolvidas diretamente com os mecanismos de tolerÃncia ao sal. Em relaÃÃo à infecÃÃo da planta pelo CPSMV, os resultados obtidos indicaram que o vÃrus induz reduÃÃo na abundÃncia de vÃrias proteÃnas nos tempos iniciais de infecÃÃo, provavelmente favorecendo a invasÃo e propagaÃÃo na planta, mas, no 6 DPSV, a planta recupera sua capacidade de acionar mecanismos de defesa, embora esses jà nÃo sejam mais efetivos para evitar o estabelecimento da doenÃa viral. Durante exposiÃÃo simultÃnea da planta ao sal e ao vÃrus, ocorreu um equilÃbrio entre o aumento e diminuiÃÃo em abundÃncia de proteÃnas envolvidas em diversos processos metabÃlicos. Esse trabalho à pioneiro nessa abordagem em feijÃo-de-corda e fornece evidÃncias dos mecanismos bioquÃmicos envolvidos nas resposta da planta a esses estresses.

L’objectif de cette thèse était de développer et d’optimiser de nouvelles méthodologies et approches analytiques afin d’améliorer le potentiel de l’analyse protéomique pour les études biologiques.La première partie de ce travail est consacrée à la détermination massive et exacte de la position N-Terminale des protéines (N-Terminome). Pour cela, nous avons utilisé et développé une approche basée sur une dérivation N-Terminale au TMPP. Cette méthodologie de marquage de la position N-Terminale a permis d’aborder l’étude des clivages protéolytiques des protéines exportées par le parasite P. falciparum (pathogène de la malaria) dans le globule rouge.Afin de permettre une exploitation automatique à haut débit des données de MS/MS, nous avons élaboré une nouvelle méthodologie (dénommée dN-TOP). Celle-Ci repose sur l’utilisation de TMPP portant des isotopes stables et permet ainsi d’accéder à la détermination des positions N-Terminales pour des études de N-Terminome à large échelle.La seconde partie est dédiée aux développements de différentes stratégies analytiques de quantification, aussi bien au niveau peptidique qu’au niveau protéique, appliquées à une série de problématiques biologiques. Ces optimisations ont été réalisées dans le contexte de l’étude des complexes protéiques, du dosage de prion par SRM, de quantification des glycations d’anticorps monoclonaux thérapeutiques et de l’hémoglobine HbA2 pour la standardisation des méthodes de référence
The objective of this Ph.D. thesis was to develop and optimize new methodologies and analytical approaches to improve the potential of the mass spectrometry based proteomics.The first part of this work focused on the development of the N-Termini proteomics. This topic was addressed with a specific N-Termini chemical derivatization based on TMPP. We have shown that our method allowed both specific N-Terminomics and classical proteomics studies in the same experiment.This N-Terminus methodology was applied to study the proteolytic cleavages of the exported proteins in P. falciparum, a parasite responsible for the malaria.In order to automatize the complex and tedious informatics processsing of the MS/SM data of ourTMPP based N-Terminomics method, we have introduced a new approach (named dN-TOP), based on the use of a stable isotope labeled TMPP which made now N-Terminome proteomics compatible with high throughput studies.The second part addresses quantitative aspects of proteomics. It describes the optimization of quantitative methods at the peptide level or at the protein level for five different proteomic studies in the context of protein complex subunits, targeted SRM based prion, quantification of monoclonal antibodies glycation and hemoglobin HbA2 for reference measurement methods standardization

BACKGROUND: The cysteine (Cys) proteome includes 214.000 Cys with thiol and other forms. Of these, only a relatively small subset functions in cell signalling. Redox-active Cys are more susceptible to oxidation, and their oxidized form is more susceptible to reduction. Specific proteomic techniques are required to identify these modifications and to study their regulation in different cell processes that are collectively known as redox proteomics. Thus, it is of interest to be able to identify both the proteins and the cysteine residues affected, and to quantify the extent of the modification involved.The quantification of differences between two or more physiological states of a biological system is among the most challenging technical tasks in proteomics: liquid chromatography coupled to mass spectrometry (LC-MS) based quantification methods have gained increasing robustness and reliability over the past five years. Many authors still share a view of redox signalling in which the fate of the cell is dependent mainly on the intensity and duration of pro-oxidant stimulus: here we sustain the involvement of an equilibrium encompassing the action of both nucleophiles and electrophiles at the same time. AIM: The dual aim of my PhD work has been both to develop suitable methodology to identify and quantify redox-active proteins in complex samples and to apply it to the study of a cellular model of breast cancer (MCF10A) engineered to reproduce malignancy. METHODS: In order to pursue this aim, I took advantage of an approach integrating differential chemical sample labelling (non-isotopic) with Cys reactive probes (NEM, IAM, HPDP) and chromatographic purification of redox-sensitive proteins, with subsequent LC-MS/MS analysis and computational data handling for OpenMS-based label-free quantification. All the steps of this methodology have been developed and validated in close collaboration with experts from both the biochemistry and bioinformatics field. RESULTS: We obtained an efficient cost-effective and isotopes-free methodology to characterize the redoxome in complex protein samples. Application of our quantification protocol to benchmark dataset leads to 100% correct estimates of under/over expression of the protein moiety. Application of the methodology to the breast cancer cellular model lead to identification of more than 300 proteins and allowed us to group-up unchanged and differentially oxidized redox-sensitive proteins in the more malignant cells in respect to their less aggressive counterpart. CONCLUSION: Despite the commonly accepted association between cancer and higher oxidative-stress, this study links higher breast cancer cells malignancy to a finely tuned dynamic equilibrium in which selected protein targets are oxidized in the context of a more reduced cell environment. Preliminary results point at the enzyme G6PDH as a crucial regulator of this redox process.
STATO DELL’ARTE: Il proteoma include 214.000 cisteine in forma di gruppi tiolici liberi od altra forma. Di queste, solamente un insieme relativamente ristretto ha un ruolo nella mediazione di segnali cellulari. Tali cisteine, attive dal punto di vista dell’ossido-riduzione, sono più sensibili all’ossidazione e la loro forma ossidata è più facilmente riducibile. Sono dunque necessarie specifiche tecniche di proteomica, globalmente indicate con il termine proteomica delle ossido-riduzioni, per identificare tali modifiche e studiarne la regolazione in diversi processi cellulari. Risulta quindi determinante la capacità di identificare sia le proteine che i residui coinvolti e di quantificarne il grado di modificazione. E proprio la quantificazione delle differenze tra due o più stati di un sistema biologico, si colloca tra gli obiettivi tecnicamente più sfidanti della proteomica: nel corso degli ultimi cinque anni, tecniche basate sulla spettrometria di massa associata a cromatografia in fase liquida hanno progressivamente guadagnato affidabilità e robustezza. Molti autori condividono tuttora una visione delle ossido-riduzioni nella mediazione del segnale in cui il destino cellulare dipende principalmente dall’intensità e dalla durata degli stimoli ossidanti: nel presente lavoro si vuole invece sostenere il coinvolgimento di un equilibrio che includa l’azione concomitante sia di specie nucleofile sia di specie elettrofile. OBIETTIVO: Il duplice obiettivo del mio lavoro di Dottorato è stato sia lo sviluppo di una metodologia idonea all’identificazione e quantificazione di proteine, attive dal punto di vista delle ossido-riduzioni, in campioni complessi, sia l’applicazione di tale metodologia allo studio di un sistema cellulare ingegnerizzato di carcinoma mammario (MCF10A) caratterizzato da diversi gradi di malignità. METODI: Al fine di perseguire tale obiettivo ho tratto vantaggio da un approccio che integra la marcatura chimica differenziale (non-isotopica) per mezzo di sonde reattive con i residui di cisteina (NEM, IAM, HPDP) e la purificazione cromatografica delle proteine attive dal punto di vista ossido-riduttivo, alla successiva analisi LC-MS/MS ed elaborazione informatizzata dei dati mediante OpenMS per una quantificazione label-free. Tutti i passaggi di tale metodologia sono quindi stati messi a punto e validati in stretta collaborazione con esperti biochimici e bioinformatici. RISULTATI: E’ stato sviluppato un metodo efficiente ed economico, non basato sull’utilizzo di marcatori isotopici, per la caratterizzazione delle proteine attive dal punto di vista ossido-riduttivo in campioni proteici complessi. L’applicazione del protocollo di quantificazione ad un campione test ha dato il 100% di stime corrette di sovra/sotto-espressione della miscela proteica. L’applicazione del metodo allo studio del modello cellulare di carcinoma mammario ha portato all’identificazione di più di 300 proteine ed ha permesso il raggruppamento di quelle sensibili dal punto di vista ossido-riduttivo in gruppi non differenziali e sovra- o sotto-ossidate nelle cellule più maligne rispetto alla loro controparte meno aggressiva. CONCLUSIONI: Nonostante sia comunemente riconosciuta l’associazione tra fenomeni neoplastici ed uno stress ossidativo, questo studio collega la maggiore malignità di un modello cellulare di carcinoma mammario ad un complesso equilibrio ossido-riduttivo. In questo contesto, specifici bersagli proteici sono ossidati mentre viene mantenuto un ambiente cellulare complessivamente ridotto. Risultati preliminari evidenziano poi l’enzima G6PDH come possibile elemento chiave nella regolazione di tale equilibrio.

Les stratégies de protéomique quantitative sans marquage sont très attractives dans le domaine de la recherche de biomarqueurs de pathologies. Cependant, elles requièrent une pleine maîtrise du schéma analytique et de sa répétabilité. Plus particulièrement, la préparation d’échantillons nécessite d’être suffisamment répétable pour ne pas impacter la qualité et la fiabilité des résultats. Les objectifs de cette thèse étaient de développer et d’optimiser des méthodes analytiques pour la protéomique quantitative, en particulier pour l’étape de préparation d’échantillons. Ainsi, un protocole innovant, simple, rapide et permettant l’analyse quantitative sans marquage d’un grand nombre d’échantillons avec une haute répétabilité a été développé et optimisé : le « Tube-Gel ». Par ailleurs, des préparations d’échantillons adaptées à différentes matrices biologiques pour la recherche de biomarqueurs ont été élaborées. Les méthodes mises au point et leur application ont permis de proposer des candidats biomarqueurs pour plusieurs pathologies : le glioblastome, les lymphomes B diffus à grandes cellules, et les complications survenant sur les greffons rénaux
Label-free quantitative proteomics strategies are very attractive for diseases biomarkers researches. These approaches require the full control and the repeatability of the analytical workflow. In particular, the sample preparation has to be repeatable enough to ensure the quality and reliability of the results. Objectives of this work were to optimize and develop analytical methods for quantitative proteomics, with a special focus on the sample preparation step. Thus, an innovative, easy and fast protocol allowing the analysis of high sample numbers with high repeatability was developed and further optimized: the “Tube-Gel” protocol. Besides,sample preparations adapted to a variety of biological matrices were developed for the search of biomarkers. The developed methods and their application allowed the identification of potential biomarkers for a variety of diseases: glioblastoma, diffuse large B-cell lymphomas and renal transplants failures

Vascular dysfunction is one of the primary factors in the onset and progression of atherosclerosis and other vascular-related diseases such as acute myocardial infarction (AMI) and chronic thromboembolic pulmonary hypertension (CTEPH). Emerging evidence indicates that pathological blood vessel responses and endothelial dysfunction are associated with metabolic alterations in endothelial cells (ECs). The identification of the insights and causes resulting in dysfunctional ECs is crucial for the understanding of the disease and to the development of new therapeutic tools. Therefore, to study the characteristics of such EC dysfunction in AMI and CTEPH diseases, we aim to determine the protein profile and/or the altered redox status of patient-derived EC lines by using mass spectrometry-based label-free quantitative proteomic and/or, spectrophotometric and fluorometric approaches. Moreover, quantitative proteomic approach was used to explore the underlying mechanisms of 3PO [(3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one), PFKFB3 inhibitor] at cellular level. 3PO is a compound able to inhibit the glycolytic flux partially and transiently and to reduce pathological angiogenesis in a variety of disease models. Bioinformatic and network analyses performed on pathologic HCAEC-AMI cells revealed the alteration of a) metabolism of RNA, b) platelet activation, signaling and aggregation, c) neutrophil degranulation, d) metabolism of amino acids and derivatives, e) cellular responses to stress and, f) response to elevated platelet cytosolic Ca2+ pathways. Similarly, network analysis in pathologic CTEPH-ECs, revealed the differentially regulation of pathways related to a) neutrophil and platelet degranulation, b) metabolism of lipids, amino acids and selenoamino acids, c) response to elevated cytosolic Ca2+, d) detoxification of reactive oxygen species. In addition, the main parameters, indicators of the redox status of both HCAEC-AMI and CTEPH-ECs were significantly increased: advanced oxidation protein products (AOPPs), protein carbonyls (PCO) content, and intracellular reactive oxygen species (ROS). Interestingly, the amount of GSH/GSSG (reduced glutathione/oxidized glutathione) and NADPH/NADP (reduced/oxidized form of nicotinamide adenine dinucleotide phosphate) ratios in dysfunctional ECs were reduced, a clear indication of oxidative stress involvement in both the pathological ECs. Finally, 3PO has multiple targets in the ECs, targeting mitochondrial inner membrane and it inhibits the important cellular pathways including the tricarboxylic acid cycle, the mitochondrial respiratory chain, and vasculogenesis, that may be useful for understanding the inhibitory effect of 3PO on EC proliferation and migration. Therefore, the present data suggest a potential application of this molecule as a starting point in designing novel molecules to prevent diseases where inflammatory reactions are involved, such as in atherosclerosis, cancer or neurodegenerative diseases.

The large amounts of data generated by modern proteomics experiments necessitates the use of software pipelines to conduct the bulk of the post-processing. While many software packages (both commercial and open-source) are available to perform some or all of the necessary post-processing steps, it is usual for each research group to use only the instrumentation and software packages with which they are most familiar and/or which are available to analyse their unknown data. The intention of the studies presented within this thesis was to assess the correlation between the experimental results obtained when; - a single result dataset is obtained and post-processed in parallel using four separate software pipelines - a single sample is analysed on two different mass spectrometers and post-processed in parallel and; - when different identification thresholds are applied to a dataset prior to parallel quantitation of the resultant data sets Correlation between different mass spectrometry instruments was assessed and found to yield high r values, especially at the protein level, and was also found to improve following the application of abundance thresholds, however the result of applying score thresholds was unpredictable. The use of manual FDR thresholds prior to importing data into Progenesis LC-MS yielded interesting results, which suggest that a threshold of 1% peptide FDR and 1 or 2% protein FDR is most effective in terms of yielding accurate ratios while maintaining acceptable sensitivity. In addition, a consensus method is suggested to utilise the results from multiple software pipelines in order to increase sensitivity and reduce the FDR, through the use of the QPROT tool and manual post-processing.

In the last decade omics technologies enabled detailed and system-wide analysis of complex biological samples. Genomics, transcriptomics and metabolomics all benefited tremendously from technological advances in their respective fields. Proteomics was revolutionised by mass spectrometry, which allowed simultaneous identification of thousands of proteins in cells, tissues and organisms. And this mainly qualitative revolution, quickly turned quantitative. This work had two main objectives. Firstly, to apply the state of the art instrumentation, data analysis and bioinformatics methods to better our understanding of basic cell biology in a model organism Saccharomyces cerevisiae. Specifically, to quantitatively describe the effects of perturbations, such as adverse environmental conditions or chaperone gene deletions, on protein abundances in the cell. Additionally, it was aimed to demonstrate and evaluate the ability of a new timeof-flight mass spectrometer to perform large-scale absolute quantification. First, it was found that yeast cells are remarkably robust to deletions of major chaperone hub proteins (Ssa1p or Ssb1p deletions). This ability was attributed to network structure and redistribution of folding workload among other related chaperones rather than simple functional redundancy. Second, to build on the first set of results, a detailed time resolved description of yeast proteome dynamics in response to heat stress was provided for the wild type and Ssb1p chaperone mutant strains. In this study, for the first time in the literature, temporal expression patterns of many hallmark heat shock proteins were elucidated. Globally, a slow and sustained proteome remodelling or 'buffering' was revealed in both strains. However, it was also shown that the cells knocked out for the Ssb1p chaperone respond to heat in a distinctly different manner to the wild type strain. Finally, consistent and reproducible absolute quantification of multiple yeast proteomes was demonstrated using a new commercial time-of-flight mass spectrometer with ion mobility separation capabilities. The data obtained revealed global differences in cellular protein content between various chaperone prefoldin mutants as well as differential expression of a set of proteins promising to be interesting targets for further investigations.

Les maladies des petits vaisseaux cérébraux sont responsables de lésions de la substance blanche du cerveau et d'infarctus cérébrauxprofonds multiples. C'est un ensemble de processus pathologiques, qui affectent les petites artères, les artérioles, veinules oucapillaires cérébraux de moins de 400µm. Le matrisome cérébrovasculaire semble être une voie pathologique convergente entre lesdifférentes maladies des petits vaisseaux de type génétique et de type sporadique. La diversité structurale et physico-chimique desprotéines du matrisome rend cependant leur analyse particulièrement délicate. En effet, certaines protéines du core matrisome,protéines de haut poids moléculaire, sont particulièrement insolubles et d’autres protéines du matrisome associé sont généralementplus petites et moins abondantes. Dans le cadre de cette thèse nous nous proposons de caractériser de manière quantitative etqualitative le matrisome microvasculaire dans les maladies des petits de vaisseaux, ainsi que d'identifier des anomalies communes ouspécifiques à chaque maladie. Pour cela nous avons, dans un premier temps, développé une approche protéomique quantitative enspectrométrie de masse de type label-free sur des vaisseaux cérébraux et périphériques isolés, et nous l’avons validée dans unepremière étude sur un modèlemurin préclinique de CADASIL. Ensuite nous avons appliqué cette approche sur deux autres modèlesmurins pour les maladies génétiques CARASIL et la maladie du collagène de type IV, et deux modèles murins pour l'hypertensionetl'âge, modèle pour leur caractère sporadique. Nous avons développé et validé une nouvelle méthode robuste et sensible pourl’analyse quantitative sans marquage des changements du matrisome des artères cérébrales de souris. En effet, nous avons mis enévidence une protéine commune à toutes ces formes de cSVDs, PRSS23, une serine protéase dans les 5 modèles étudiée et HTRA1,une autre serine protéase, dans CADASIL, la maladie du collagène de type IV et l’âge
Diseases of the small vessels of the brain are responsible for damage to the white matter of the brain and multiple deep braininfarctions. They are the cause of more than 25% of strokes and are the second cause of dementia after Alzheimer's dementia. It is aset of pathological processes, which affect small arteries, arterioles, cerebral venule or capillary of less than 400µm. Thecerebrovascular matrisome seems to be a converging pathological pathway between the various diseases of the small vessels of thegenetic type and of the sporadic type. The matrisome is the set of proteins constituting the extracellular matrix (ECM) as well as theassociated proteins, their roles consist not only in the support and the anchoring of the cells but also in various fundamental processessuch as differentiation, proliferation, Survival or migration of cells. The structural and physico-chemical diversity of these proteins,however, makes their analysis particularly delicate. Within the framework of this thesis we propose to characterize in a quantitativeand qualitative way the microvascular matrisome in the diseases of the small vessels, as well as to identify commonabnormalities orspecific to each disease. For this we have developed a label-free quantitative proteomic approach on cerebral and peripheral vesselsisolated from three preclinical genetic murine models and two murine models for the sporadic character. We have developed andvalidated a new robust and sensitive method for the quantitative non-labeling analysis of changes in the matrisome of mouse cerebralarteries and the application of our method on the arteries of the different mouse models studied has allowed us to identify someavenues. Interesting for each disease independently but also highlighted some common signatures between the different studies

Many diseases are caused by proteins or peptides folding incorrectly and aggregating into fibrils or plaques, including Alzheimer’s disease, Parkinson's disease and type II diabetes. Amyloid formation in the human pancreas occurs via the aggregation of a 37 amino acid peptide called amylin or IAPP which is shown to be toxic to pancreatic β cells. Amylin (IAPP) aggregation initiates a large number of events, leading ultimately to cell death. However the exact cytotoxic action of human IAPP and also the underlying molecular events leading from amylin (IAPP) aggregation to β cell death is still unknown. The toxic effect of human amylin (IAPP) is thought to involve changes in the expression of several genes and proteins. Further transcriptional and proteomics studies in this field can therefore facilitate the identifications of new targets whose expression are affected by amylin (IAPP). These information could be further used to construct an integrated model of the signalling and regulatory pathways through which amylin (IAPP) interacts with cellular metabolism.To investigate the effects of amylin (IAPP) aggregation on the islets proteome in this study, rat Rin-5F cell line, reported as a model of pancreatic β cell, was used. MTT assay was initially performed to determine the effect of IAPP on the cell viability at different time points. The isolated proteins form the untreated and IAPP treated Rin-5F cells were then fractionated by off gel electrophoresis and analysed by quantitative label free LC- MS/MS approach.Label free quantification of IAPP treated Rin-5F cells has identified the altered expression of many proteins, some of which were previously suggested in the literature to be involved in the pathogenesis of type 2 diabetes. These proteins were map to several pathways (including glycolysis and proteasome) whose expressions were significantly affected upon amylin (IAPP) exposure. The IAPP responsive proteins were also structured into a well connected network. Some of the hub proteins identified in this network were greatly affected as the result of IAPP treatments of RIN-5F cells. Our data therefore revealed the effect of IAPP on several proteins and pathways that might be important in the pathogenesis of type 2 diabetes.

Há muito tempo os venenos de abelhas se tornaram objeto de interesse de muitos cientistas, principalmente os venenos daquelas linhagens do gênero Apis, chamadas abelhas europeias e as conhecidas abelhas africanizadas. O foco no desenvolvimento de terapias eficazes que pudessem prevenir ou frear as reações desencadeadas pelas toxinas dos venenos desses insetos foi o principal estimulador do surgimento dessa grande área da pesquisa, uma vez que esses animais causam um grande número de acidentes em animais e seres humanos e os acidentes podem desencadear graves consequências, inclusive óbito. Sendo assim, desenvolvemos o presente trabalho baseado na caracterização da composição proteica dos venenos de abelhas europeias e africanizadas, na análise quantitativa diferencial entre os venenos e, na investigação de fosforilações e a possível relação das mesmas com as funções biológicas. Reunindo todos os elementos que estavam ao nosso alcance para compor o melhor conjunto de etapas para a realização do estudo proteômico de venenos de abelhas, nós atingimos todos os objetivos propostos. Utilizando uma abordagem baseada em espectrometria de massas, consistindo de análise shotgun seguida de LC-MS/MS realizada em um dos mais modernos espectrômetros de massas, nós fomos capazes de obter resultados extremamente confiáveis e interessantes. Comparando-se o efeito da extensão do gradiente de separação na cromatografia líquida, nós fomos capazes de atingir uma maior cobertura da amostra, uma vez que identificamos um maior número de proteínas após a utilização do gradiente mais longo. A identificação de fosforilações foi favorecida pela combinação de fragmentação por \"Colisão Induzida por Dissociação\" e medidas de massa de alta acurácia realizadas no instrumento LTQ-Orbitrap-Velos. Enquanto apenas 29 proteínas foram identificadas após 120 minutos de separação cromatográfica, um total de 51 proteínas foram identificadas aplicando-se gradiente mais longo. Dentre as 51 proteínas totais, 42 são comuns aos venenos das três abelhas. A comparação em pares mostrou que os venenos das abelhas europeias compartilham 44 proteínas e os venenos das abelhas africanizadas compartilham 43 proteínas tanto com o veneno de A. m. carnica quanto com o de A. m. ligustica. Além disso, nós revelamos que existem diferenças quantitativas entre algumas das proteínas dos venenos, sendo muitas dessas proteínas diferenciais toxinas com funções conhecidamente relevantes. A investigação de fosforilação mostrou que duas toxinas apresentam-se na forma fosforilada: melitina e icarapina. Melitina é considerada a principal toxina de venenos de abelhas, sendo bem conhecida por sua ação altamente tóxica e alergenicidade. Este peptídeo foi identificado com fosforilação ocorrendo no sítio Ser18 em todas as amostras de venenos, enquanto somente no veneno da abelha africanizada foi também identificado com sítio de fosforilação no resíduo de Thr10. Icarapina, também já descrita como um alérgeno do veneno, apresentou sítio de fosforilação no resíduo Ser205. Por fim, nós demonstramos o efeito da fosforilação presente em melitina (Ser18) realizando ensaios de atividade biológica do peptídeo fosforilado e nativo, como: hemólise, lise celular e desgranulação de mastócitos e atividade quimiotáctica. Foi observado que a toxicidade do peptídeo fosforilado é reduzida em comparação ao do peptídeo nativo. Sendo assim, nós podemos concluir que a combinação de metodologias eficientes e a utilização de moderna instrumentação nos levou a resultados surpreendentes, os quais se somam a todo o conhecimento já existente acerca de venenos de abelhas
Honeybee venom toxins disturb the activity of critical cellular processes, triggering immunological, physiological, and neurological responses within victims. Studies on venom toxins have provided invaluable knowledge towards elucidating the molecular and functional details of their biological targets, yet there has been no report of a full proteome/phosphoproteome profile of honeybee venom. In this study, we focused on Apis mellifera honeybee venom characterization, including proteins identification, label-free quantitative analysis and phosphorylation identification. Making use of a MS-based proteomic approach, consisting on in-solution digestion followed by LC-MS/MS analysis, we were able to compare the effect of the liquid chromatography gradient length on the sample coverage, consequently, to identify a higher number of proteins using longer separation gradient of the tryptic peptides. Favorable identification of phosphorylations was achieved by the application of a long separation gradient combined with CID fragmentation and high accuracy mass measurement using an LTQ Orbitrap Velos. Here we report on the comparative shotgun proteomics study of the venoms of two Apis mellifera subspecies, A. m. carnica and A. m. ligustica, and the hybrid known as Africanized honey bee (AHB). We identified 51 proteins in total, with 42 of them being common among the three venoms, including many previously unidentified entries. Performing label-free quantification, we observed that few proteins were found with different relative amounts. Additionally, we revealed the phosphorylation of two proteins in all the samples, with two of them being HBV toxins/allergens: melittin and icarapin. Icarapin was identified as phosphorylated at 205Ser. Melittin was identified as phosphorylated at the 18Ser and 10Thr positions in all venoms, as well. Given these novel findings, we then chose to compare the toxicity of the phosphorylated/unphosphorylated forms of the major venom toxin, melittin, considering the most prominent phosphorylation event, the phosphorylated 18Ser position. We showed that the toxicity is in fact decreased when the peptide is phosphorylated. Based on a combination of efficient methodology and state-of-the-art instrumentation, delineated by our Shotgun-NanoESI-Long Gradient-LTQ Orbitrap Velos analysis, we achieved proteomic coverage far surpassing any previous report. Together, these discoveries pave the way for future phosphovenomic studies

PAIVA, Ana Luiza Sobral. Respostas bioquímicas do feijão-de-corda [Vigna unguiculata L. (Walp.)] ao estresse salino e infecção pelo vírus do mosaico severo do caupi (CPSMV) reveladas pela proteômica quantitativa livre de marcação. 2015. 200 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2015.
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As sessile organisms, plants are exposed to a plethora of environmental stresses to which they must respond to maintain efficient growth and survival. Therefore, in order to improve our understanding on the complex mechanisms involved in the cowpea response to salt stress and to a compatible interaction with the cowpea severe mosaic virus (CPSMV), we used a label-free quantitative proteomic approach to identify the salt and virus responsive proteins in the leaves of the Pitiuba (CE-31) cultivar. The proteins extracted from the leaves (control and treated) 2 and 6 days post-treatment only with salt (DPS), only infected with CPSMV (DPV) or both of them (DPSV) were analyzed using mass spectrometry. At 2 DPS, 350 proteins with at least two-fold differences in abundance, in comparison with controls, were differentially accumulated in the leaves of the salt-treated (80% up and 20% down-accumulated), 281 at 2DPV (25% up and 75% down-accumulated) and 321 at 2 DPSV (45% up and 55% down-accumulated) plants. At 6 DPS, 350 proteins were differentially accumulated in the leaves of the salt-treated (90% up and 10% down-accumulated), 225 at 6 DPV (80% up and 20% down-accumulated) and 315 at 6 DPSV (94% up and 6% down-accumulated) plants. The qualitative analysis showed biochemical differences when the cowpea plants were challenged concurrently with both stresses. To cope with salinity, cowpea increased the abundance of proteins directly involved with the salt tolerance mechanisms. The results indicated that the CPSMV induce the down-accumulating of several proteins to invade and spread in host at early infection period (2 DPV), but at 6 DPV plant can induce accumulation of diverse proteins related with defense, although these strategies can’t avoid the negatives effects of disease. When exposed simultaneously to salt/CPSMV stresses, a balance in protein accumulation involved in many biological process. This is the first work employing this approach in cowpea and providing evidences of the plant biochemical mechanisms involved in the responses of cowpea to these stresses.
Como organismos sésseis, as plantas são expostas a uma variedade de estresses ambientais aos quais devem responder para sobreviverem e se desenvolverem. A fim de melhorar a nossa compreensão sobre os mecanismos complexos envolvidos na resposta do feijão-de-corda ao estresse salino e na interação compatível com o vírus do mosaico severo do caupi (CPSMV), foi utilizada uma abordagem proteômica quantitativa, livre de marcação, para identificar proteínas, responsivas a essess estresses em folhas de feijão-de-corda, cv. CE-31. As proteínas extraídas a partir de folhas primárias, 2 e 6 dias após o tratamento só com o sal (DPS), somente infectadas (DPV), ou sob ação combinada dos dois (DPSV) foram analisadas, usando espectrometria de massas e comparadas com grupo controle. No 2° DPS, foram identificadas 350 proteínas diferencialmente acumuladas (80% aumentaram em abundância e 20% diminuíram), no 2° DPV 281 (25% aumentaram em abundância e 75% diminuíram) e no 2° DPSV 321 (45% aumentaram em abundância e 55% diminuíram). Já no 6° DPS, foram identificadas 350 proteínas diferencialmente acumuladas (90% mostraram aumento em abundância e 10% diminuição), no 6° DPV 225 (80% aumentaram em abundância e 20% diminuíram) e no 6° DPSV 315 proteínas(94% aumentaram em abundância e 6% diminuíram). Para lidar com a salinidade, o cv. CE-31 aumentou a abundância de proteínas envolvidas diretamente com os mecanismos de tolerância ao sal. Em relação à infecção da planta pelo CPSMV, os resultados obtidos indicaram que o vírus induz redução na abundância de várias proteínas nos tempos iniciais de infecção, provavelmente favorecendo a invasão e propagação na planta, mas, no 6° DPSV, a planta recupera sua capacidade de acionar mecanismos de defesa, embora esses já não sejam mais efetivos para evitar o estabelecimento da doença viral. Durante exposição simultânea da planta ao sal e ao vírus, ocorreu um equilíbrio entre o aumento e diminuição em abundância de proteínas envolvidas em diversos processos metabólicos. Esse trabalho é pioneiro nessa abordagem em feijão-de-corda e fornece evidências dos mecanismos bioquímicos envolvidos nas resposta da planta a esses estresses.

Plusieurs évolutions sont constatées dans la recherche en biologie. Tout d’abord, les études menées reposent souvent sur des approches expérimentales quantitatives. L’analyse et l’interprétation des résultats requièrent l’utilisation de l’informatique et des statistiques. Également, en complément des études centrées sur des objets biologiques isolés, les technologies expérimentales haut débit permettent l’étude des systèmes (caractérisation des composants du système ainsi que des interactions entre ces composants). De très grandes quantités de données sont disponibles dans les bases de données publiques, librement réutilisables pour de nouvelles problématiques. Enfin, les données utiles pour les recherches en biologie sont très hétérogènes (données numériques, de textes, images, séquences biologiques, etc.) et conservées sur des supports d’information également très hétérogènes (papiers ou numériques). Ainsi « l’analyse de données » s’est petit à petit imposée comme une problématique de recherche à part entière et en seulement une dizaine d’années, le domaine de la « Bioinformatique » s’est en conséquence totalement réinventé. Disposer d’une grande quantité de données pour répondre à un questionnement biologique n’est souvent pas le défi principal. La vraie difficulté est la capacité des chercheurs à convertir les données en information, puis en connaissance. Dans ce contexte, plusieurs problématiques de recherche en biologie ont été abordées lors de cette thèse. La première concerne l’étude de l’homéostasie du fer chez la levure pathogène Candida glabrata. La seconde concerne l’étude systématique des modifications post-traductionnelles des protéines chez la levure pathogène Candida albicans. Pour ces deux projets, des données « omiques » ont été exploitées : transcriptomiques et protéomiques. Des outils bioinformatiques et des outils d’analyses ont été implémentés en parallèle conduisant à l’émergence de nouvelles hypothèses de recherche en biologie. Une attention particulière et constante a aussi été portée sur les problématiques de reproductibilité et de partage des résultats avec la communauté scientifique
Biological research is changing. First, studies are often based on quantitative experimental approaches. The analysis and the interpretation of the obtained results thus need computer science and statistics. Also, together with studies focused on isolated biological objects, high throughput experimental technologies allow to capture the functioning of biological systems (identification of components as well as the interactions between them). Very large amounts of data are also available in public databases, freely reusable to solve new open questions. Finally, the data in biological research are heterogeneous (digital data, texts, images, biological sequences, etc.) and stored on multiple supports (paper or digital). Thus, "data analysis" has gradually emerged as a key research issue, and in only ten years, the field of "Bioinformatics" has been significantly changed. Having a large amount of data to answer a biological question is often not the main challenge. The real challenge is the ability of researchers to convert the data into information and then into knowledge. In this context, several biological research projects were addressed in this thesis. The first concerns the study of iron homeostasis in the pathogenic yeast Candida glabrata. The second concerns the systematic investigation of post-translational modifications of proteins in the pathogenic yeast Candida albicans. In these two projects, omics data were used: transcriptomics and proteomics. Appropriate bioinformatics and analysis tools were developed, leading to the emergence of new research hypotheses. Particular and constant attention has also been paid to the question of data reproducibility and sharing of results with the scientific community

碩士
國立暨南國際大學
應用化學系
104
Dopamine (DA) acts as a key neurotransmitter in the brain and plays a vital role is sexual motivation in old male drosophila melanogaster. In order to understand how dopamine (DA) affects sexual behavior, quantitative MS-based proteomic analysis and RNA microarray analysis were used to explore the mechanisms of dopaminergic controls at the molecular level. Our results shows that increasing DA levels in the PPL2ab neurons resulted in the significant alternation of 1870 transcript level (909 up-regulated and 961 down-regulated) and 100 protein levels (75 up-regulated and 58 down-regulated). The identified dys-regulated proteins and RNAs are mainly involved in the metabolic process and biosynthetic process. Gene overlap analysis identified six promising candidate genes that could play a role in DA-associated hyper-sexuality such as Fat body protein, Cuticular protein, Larval serum protein, Yolk protein, Heat-shock-protein, and Plasma membrane calcium ATPase. Further investigation will be pursued to investigate the correlation of these candidate genes with hyper-sexuality of old male drosophila melanogaster.

碩士
國立中興大學
分子生物學研究所
101
Proteins are the final pigment of central dogma, they can strongly describe the phenotype of organism as such. Nowadays, some of scientist focused on protein research, and the disciplines to study protein called proteomics. Traditionally, the two-dimensional gel electrophoresis couple with gel image quantification is widely used to adapt quantitative proteomics, though it can provide large-scale and visible information, however, it still have some challenge and drawback. Multidimensional protein identification technology is the alternative way in quantitative proteomics research; it uses multidimensional liquid chromatography and mass spectrometry to separate and identify analyte. In other hand, photosynthesis is the only way to convert and storage light to other energy formation and it widely exist in plant leaf and bacteria. Chlorophyll is the core compound of photosynthesis reaction, it reflect green light instead of absorbing, because of it, most of plant leaf are green. However, the abnormal leaf color yellow or white occurred in rice mutant SA0405, SA0407 and SA0408 different phenotype from others plant leaf. In this study, hydrophilic interaction liquid chromatography and ultra performance liquid chromatography mass spectrometry were used in peptides separation and protein identification, and MASCOT Distiller software were used in label-free quantification. Here, we demonstrated hydrophilic interaction liquid chromatography was able to separate peptides, and MASCOT Distiller was able to using label-free quantification. We also applied in rice mutants, total of 205, 250 and 279 proteins were quantified successfully in SA0405, SA0407 and SA0408. According to protein quantification result, we speculated the mutant of MGD, TRX and GGT genes may indirectly cause abnormal leaf color.

碩士
國立臺灣海洋大學
生物科技研究所
96
Human colorectal cancer is one of the common cancer-related death diseases due to absence of a fast and specified diagnostic biomarker. Abnormal expressions of membrane proteins, such as receptors, have been linked to diseases and drug targets. To decipher the key modulators of tumorigenesis and the potential candidate proteins as biomarkers or therapeutic targets for colorectal cancer, we have developed a label-free quantitative membrane proteomic strategy for analysis of paired normal and tumor colorectal cancer tissues from 27 patients in different colon cancer stages. By four-dimensional alignment algorithm, termed SEMI, on Sequence, Elution time, Mass to charge and Internal standard, the unidentified peptide can be computationally located and thus increase the numbers of quantifiable proteins. Without conventional isotopic labeling, we used the integrated chromatographic peak areas for peptide quantification. By addition of internal standard protein before gel-assisted digestion, the quantitation deviation during tryptic digestion and LC-MS/MS can be normalized. Using HeLa cell lysate containing different amount of BSA as a model system, reliable linear correlation can be obtained (R2=0.9972) with 40-fold dynamic concentrations of bovine serum albumin (BSA). High quantitation accuracy and reasonable standard deviation (15.5% ~17%) were demonstrated in cell and tissues models. Furthermore, we applied this strategy to quantitative membrane proteomic analysis of paired normal and colorectal cancer tissues. On contrast to most previous studies using pooled samples, the high throughput strategy offers advantage for providing individual proteomic pattern of patients. A total of 868, 2017, 2040 and 1127 proteins were identified from colorectal cancer patients of Duke’ A、B、C、D stage, respectively. We identified 120 up-regulated (> 2-fold) and 37 down-regulated proteins (< 0.5-fold) in stage A, 6 up-regulated and 30 down-regulated proteins in stage B, 4 up-regulated and 4 down-regulated proteins in stage C and 3 up-regulated and 4 down-regulated proteins in stage D. In this study, we also found that six proteins, CEACAM5、CEACAM6、C6、DEFA1、RPH3Al and SEC61B, show overexpression in 60% patients. In previous literatures, CEACAM has been found to be associated with human colorectal cancer. We expect that the these differentially express proteins may provide insight into the molecular mechanism underlying the development and progression of colon cancer as well as an opportunity to provide panel of membrane proteins as biomarker candidates, enabling early diagnosis of human colorectal cancer to decrease the mortality.

Yes
It has long been known that Tissue Factor (TF) plays a role in blood coagulation and has a direct thrombotic action that is closely related to cardiovascular risk, but it is becoming increasingly clear that it has a much wider range of biological functions that range from inflammation to immunity. It is also involved in maintaining heart haemostasis and structure, and the observation that it is down-regulated in the myocardium of patients with dilated cardiomyopathy suggests that it influences cell-to-cell contact stability and contractility, and thus contributes to cardiac dysfunction. However, the molecular mechanisms underlying these coagulation-independent functions have not yet been fully elucidated. In order to analyse the influence of TF on the cardiomyocitic proteome, we used functional biochemical approaches incorporating label-free quantitative proteomics and gene silencing, and found that this provided a powerful means of identifying a new role for TF in regulating splicing machinery together with the expression of several proteins of the spliceosome, and mRNA metabolism with a considerable impact on cell viability.

Bladder cancer is the fourth most common neoplasia in more developed countries and the seventh worldwide, in the male gender. On cost per patient, it is also one of the most expensive malignancies at patient level, because current diagnostics, follow-up, and treatment are not cost-effective. Cystoscopy, the regular method to diagnose bladder cancer, is invasive, causes pain and it has a low sensibility. Furthermore, it is used to monitor recurrent urothelial carcinomas, contributing significantly to rise bladder cancer costs. Therefore, new non-invasive and more reliable methods of diagnosis and prognosis are needed. Genetic mutations in bladder tumour cells, as well as tumour response from neighbouring cells and from the immune system, implicate protein expression and activation different from healthy people, therefore originating new features in the urine proteome. As urine is in direct contact with these tumour and urothelial cells, it is expected that changes in the bladder are reflected in the urine content. Therefore, the urinary proteome is an excellent biopsy for finding protein biomarkers of diagnosis and prognosis. This work has the primary goal of finding a urine-based panel of biomarkers of diagnosis and staging for bladder cancer. The Hospital São José provided urine samples from patients who had Bladder Cancer as follows: (i) non-muscle invasive stages Ta and T1, and (ii) muscle invasive T2-T4 (T2+) and (iii) from other patients used as controls. So far, five groups were formed as indicated next: (a) bladder cancer-Ta; (b) T1; (c) T2+ (d) volunteers with no urothelial conditions and (e) volunteers presenting lower urinary tract symptoms. The methodology selected to find the biomarkers was free-label quantification of peptides by High-Resolution Mass Spectrometry. To this end, the urine proteome was first separated and then digested using the Filter Aided Sample Preparation -FASP- method. The pools of peptides obtained were used to identify and quantify the proteins present in the urine samples. Then, using bioinformatics, data was interpreted, and two biomarker panels were obtained. The first panel consists of 35 proteins to diagnostic bladder cancer. The second panel consists of 76 proteins to stage bladder cancer.

Vitreous, also termed vitreous body or vitreous humor, is a transparent fluid that fills the posterior cavity of the eye, surrounded by the neurosensorial retina, and lens. For a long time, the vitreous was not appreciated for its role in health and disease, and its function was thought to be merely structural. Nevertheless, the analysis of vitreous proteome has gained a growing interest in recent years. These studies proved that vitreous is highly complex and biologically more active than initially thought. As a matter of fact, changes in vitreous proteome reflect the physiological and pathological state of the eye, and, therefore, it is the ideal matrix for studying vitreoretinal diseases. Although the search for sensitive and specific vitreous biomarkers in ocular disease has not been successful so far, the analysis of vitreous proteome has been seen to be promising in elucidating some of the pathological mechanisms underlying vitreoretinal diseases. In this project, several gel-based and gel-free techniques were developed and applied for the analysis of vitreous proteome in retinal detachment (RD), diabetic retinopathy (DR), and age-related macular degeneration (AMD). Since the early proteomic studies, two-dimensional gel electrophoresis (2DE) has been the preferential method for the separation and identification of vitreous proteins. If combined with more sensitive detection techniques, refined gel image processing, and proper sample preparation, 2DE is still a valuable tool for high-resolution separation and routine analysis of proteoforms. Despite technological advances, 2DE of biological fluids, such as vitreous, remains a major challenge. Therefore, in the first part of this work, an artificial neural network was applied to optimize the recovery of vitreous proteins and their detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer) and physical parameters (temperature and total voltage). Using a mathematical model created by ANN, both the protein recovery and the number of spots detected in 2DE gels were significantly improved. The optimized response (580 spots) represents a 2.4-fold improvement over the standard conditions applied for vitreous analysis by 2DE. Our results clearly indicate that it is crucial to combine appropriate amounts of solubilizing agents to improve the extraction, solubilization, and detection of vitreous proteins, and to obtain well-resolved gels. Beyond that, our results also indicate that physical parameters have a significant influence on isoelectric focusing and, thereby, should be adjusted and monitored. When working with biological fluids, it is also important to reduce their complexity before 2DE analysis to facilitate the detection of low-abundant proteins, and to increase the detected in the gel increased 1.3-fold over the optimal output refined by the ANN model, with an average of 761 spots detected in vitreous from different vitreoretinopathies, including rhegmatogenous retinal detachment (RRD) and the proliferative diabetic retinopathy (PDR). In the second task of this Ph.D. project, the performance of gel-free proteomic techniques combined with stable-isotope labeling was tested for the analysis of vitreous samples in RRD. RRD is a potentially blinding condition characterized by a physical separation between the neurosensory retina and retinal pigment epithelium. Vitreous has a central role in the onset of RRD, which may be triggered by vitreous liquefaction. It reduces the vitreoretinal adhesion, leading to the accumulation of vitreous fluid in subretinal space, and, subsequently, to the physical separation between the neuronal retina and the retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur in the eye, providing additional information about the molecular mechanisms underlying RRD pathogenesis. In this study, the proteome of vitreous collected from patients with RRD was analyzed and compared to epimacular membranes (MEM) using iTRAQ reagents (Isobaric tags for relative and absolute quantitation) combined with analysis by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS). Using this strategy, we identified 6078 peptides corresponding to 1030 proteins, with 2613 out of these corresponded to unique peptides. Overall, 150 proteins were found differentially expressed in the RRD vitreous, including 96 overexpressed and 54 underexpressed. Among overexpressed proteins, several glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), and protease inhibitors (metalloproteinase inhibitor 1, plasminogen activator inhibitor 1) are regulated by hypoxia-inducible factor-1 (HIF-1), which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the overexpression of proteins of carbon metabolism or molecular chaperones or, among others, suggests that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses, e.g. the activation of the HIF-1 signaling pathway. In the third task, a label-free quantitative (LFQ) method was applied to analyze the vitreous proteome in PDR and dry AMD. DR and AMD are leading causes of visual impairment and blindness in people aged 50 years or older in middle-income and industrialized countries. Although Anti-VEGF therapies have improved the management of neovascular AMD (nAMD) and PDR, no treatment options exist for dry AMD. Therefore, quantitative proteomics can help to recognize the biological mechanisms underlying these pathologies and to find new potential biomarkers and/or pharmaceutical targets. For this purpose, the proteome of vitreous collected from patients with PDR (n=4) were compared to dry AMD (n=4) and epiretinal membranes (ERM) (n=4) using an LFQ method that combines a fractionation by short SDS–polyacrylamide gel electrophoresis and analysis by LC-MS/MS. A total of 680 proteins were identified, of which 586 were identified using the software search engine MASCOT and 580 using MaxQuant. Subsequently, post hoc tests, hierarchical clustering, and multiple t-tests were performed for differentiating the three disease groups in terms of protein expression based on their intensity. Post hoc tests revealed that 96 proteins are capable of differentiating among the different groups, whereas 118 proteins (17 up- and 101 down-regulated) were found differentially regulated in PDR compared to ERM and 95 proteins (10 up- and 85 down-regulated) in PDR compared to dry AMD. Functional enrichment analysis indicates that these underexpressed proteins are correlated to pathways/ biological processes, such as extracellular matrix (ECM) disassembly and organization, platelet degranulation, lysosomal degradation, cell adhesion, and central nervous system development. In turn, mediators of complement and coagulation cascades and acute-phase inflammatory responses were found enriched in PDR vitreous, reinforcing the role of these pathways in its pathogenesis of PDR. For last, some potential biomarkers were selected according to iTRAQ and LFQ experiments and validated by multiple reaction monitoring (MRM) in a larger set of vitreous samples. Therefore, we develop a scheduled MRM method for the analysis of 35 proteins in vitreous samples collected from patients with ERM (n=21), DR/PDR (n=20), AMD (n=11), and RRD (with and without proliferative vitreoretinopathy) (n=13). Of these, 26 proteins have been shown the potential to differentiate between different disease groups according to MRM results and respective receiver operating characteristic curves. Complement and coagulation components (C6, C8B, prothrombin), acute-phase proteins (alpha-1-antichymotrypsin), adhesion molecules (galectin-3-binding protein), ECM components (opticin), and neurodegeneration biomarkers (beta-amyloid, amyloid-like protein 2) stand out as the more efficient biomarkers to discriminate among the different disease groups. In conclusion, several gel-based and gel-free strategies were develop ed and implemented for the preparation and analysis of the proteome of vitreous in different vitreoretinal diseases. Concerning the gel-based method, a mathematical model created by ANN provided an effective 2DE protocol for high-resolution analysis of vitreous proteome, which can be advantageous for analysis of specific proteoforms, including different isoforms and post-translational modified proteins. On the other hand, high-throughput methods, such as iTRAQ and LFQ, provided a more in-depth analysis of vitreous proteome. In these techniques, we identified 1030 proteins by iTRAQ and 680 by LFQ, some of them have not been previously identified. Even more relevant is the fact that vitreous analysis using these techniques provided new insights on the pathogenesis of RRD, PDR, and AMD. Beyond that, they provided fundamental information regarding potential biomarkers, which enabled the successful validation of 26 proteins by MRM. Nevertheless, it must be taken into consideration that vitreous biomarkers cannot be used for regular diagnosis due to invasive sampling. However, they can be candidates for new pharmaceutical targets and, when the samples are obtained as part of the clinical routine, be used for the prognosis of the patient's disease evolution and/or to predict the proper response to treatment.
O vítreo, também denominado por corpo vítreo ou humor vítreo, é um fluido transparente que preenche a cavidade posterior do olho entre a retina neurossensorial e o cristalino. Durante muitos anos, o papel do vítreo na saúde e na doença foi negligenciado, pensando-se que a sua função era meramente estrutural. No entanto, tem-se registado um crescente interesse pela análise do proteoma vítreo nos últimos anos. Estes estudos comprovaram que o vítreo é altamente complexo e biologicamente mais ativo do que se pensava inicialmente. De facto, alterações a nível do proteoma do vítreo refletem o estado fisiológico e patológico do olho e, portanto, esta é a matriz ideal para o estudo das doenças vitreorretinianas. Embora a procura de biomarcadores no vítreo, mais sensíveis e específicos para cada patologia ocular, não tenha sido bem-sucedida até o momento, a análise do proteoma vítreo mostrou-se promissora na elucidação de alguns dos mecanismos patológicos subjacentes a estas patologias. Neste projeto, diversas técnicas proteómicas baseadas na separação de proteínas em gel de poliacrilamida (gel-based proteomics) ou no fracionamento de péptidos por cromatografia líquida (gel-free proteomics) foram desenvolvidas e aplicadas para a análise do proteoma do vítreo no descolamento da retina (RD), na retinopatia diabética (DR) e na degeneração macular relacionada com a idade (AMD). Desde os primeiros estudos em proteómica, a eletroforese bidimensional do gel (2DE) foi o método preferencial para a separação e a identificação de proteínas do vítreo. A 2DE é uma ferramenta valiosa para a separação com elevada resolução e análise de rotina de proteoformas, principalmente se for combinada com técnicas de deteção mais sensíveis, com um processamento de imagem mais refinado e com uma preparação adequada das amostras. Portanto, na primeira parte deste trabalho, aplicou-se uma rede neural artificial (ANN) para a otimização da extração de proteínas do vítreo e da sua análise por 2DE, através da combinação de vários agentes solubilizantes (CHAPS, Genapol, DTT, tampão IPG) e parâmetros físicos (temperatura e voltagem total). Pela aplicação de um modelo matemático criado por ANN, a extração de proteínas e o número de spots detetados após a sua análise por 2DE melhoram significativamente. A resposta otimizada (580 spots detetados) representa um incremento melhoria de 2,4 vezes comparando com as condições padrão utilizadas no desenho experimental inicial. Os resultados alcançados indicam claramente que é crucial combinar as concentrações adequadas de agentes solubilizantes para melhorar a extração, solubilização e a deteção das proteínas do vítreo, assim como para obter géis bem resolvidos. Para além disso, os nossos resultados também indicam que os parâmetros físicos têm uma influência significativa na focagem isoelétrica e, por esta razão, devem ser ajustados e monitorizados neste tipo de análise. Quando se trabalha com fluidos biológicos também é importante reduzir a sua complexidade antes da análise por 2DE, de modo a facilitar a deteção de proteínas pouco abundantes e a aumentar a cobertura do proteoma. Após a remoção da albumina e da imunoglobulina G, o número de proteínas detetadas no gel aumentou 1,3 vezes quando comparado com o ponto ótimo do modelo proposto por ANN, com uma média de 761 spots detetados no vítreo em doenças vitreorretinianas, como, por exemplo, o descolamento regmatogénico da retina (RRD) ou a retinopatia diabética proliferativa (PDR). Na segunda tarefa deste projeto de doutoramento, testou-se a performance das técnicas de marcação isobárica, na análise de amostras de vítreo de RRD. O RRD é uma das causas de cegueira e é caracterizado por uma separação física entre a retina neurossensorial e o epitélio pigmentar da retina (RPE). O vítreo tem um papel central no aparecimento do RRD, que pode ser provocado pela liquefação do vítreo. Esta reduz a adesão vitreorretiniana, conduzindo assim à acumulação de líquido do vítreo no espaço subretinal, e, consequentemente, à separação física entre a retina e o RPE. Assim, a proteómica quantitativa pode contribuir para a compreensão das alterações que ocorrem no olho, providenciando uma informação complementar sobre os mecanismos moleculares subjacentes à patogénese do RRD. No presente estudo, o proteoma do vítreo recolhido de doentes com RRD foi analisado e comparado com amostras de vítreo de membranas epimaculares (MEM) usando reagentes iTRAQ (Isobaric tags for relative and absolute quantitation) em combinação com análise por Cromatografia Líquida Bidimensional acoplada à Espectrometria de Massa em Tandem (2D-LC-MS/MS). A análise destas amostras por LC-MS/MS resultou na identificação de 6078 péptidos relativos a 1030 proteínas, 2613 dos quais correspondem a péptidos únicos. Das proteínas identificadas, um total de 150 estava diferencialmente expressa no vítreo de doentes com RRD, incluindo 96 proteínas sobreexpressas e 54 subexpressas. Entre as sobreexpressas encontraram-se várias enzimas glicolíticas (frutose-bifosfato aldolase A, gama-enolase e fosfoglicerato cinase 1), transportadores de glicose (GLUT-1), e inibidores de proteases (inibidor da metaloproteinase 1, inibidor do ativador de plasminogénio 1) que são regulados pelo fator induzido por hipóxia (HIF-1), o que sugere que a via de sinalização HIF-1 pode ser activada em resposta à RRD. Além disso, a acumulação no vítreo de proteínas intracelulares dos fotorreceptores, incluindo fosducina, rodopsina e S-arrestina, ou da vimentina revela que a RRD leva a uma degeneração significativa das células fotorreceptoras. No entanto, a sobreexpressão de proteínas envolvidas no metabolismo do carbono ou chaperones moleculares, entre outras, indica que diversos mecanismos são ativados em resposta ao RRD de forma a promover a sobrevivência das células retinianas através de respostas celulares complexas, como por exemplo, a ativação da via de sinalização HIF-1. Na terceira tarefa, aplicou-se um método quantitativo label-free (LFQ) para analisar o proteoma do vítreo na PDR e na forma seca da AMD (dry AMD). DR e AMD são as principais causas de deficiência visual e cegueira em indivíduos com idade igual ou superior a 50 anos, em países industrializados ou de rendimento médio. Embora as terapias direcionadas à inibição do fator de crescimento vascular (VEGF) tenham melhorado o tratamento da forma neovascular da AMD (nAMD) e da PDR, neste momento não existem opções terapêuticas para a AMD seca. Portanto, a proteómica quantitativa pode contribuir para o conhecimento dos mecanismos biológicos subjacentes a estas patologias e a encontrar novos potenciais biomarcadores e/ou alvos terapêuticos. Com esta finalidade, o proteoma do vítreo recolhido de doentes com PDR (n=4) foi comparado com o de doentes com AMD seca (n=4) e com membranas epiretinianas (ERM) (n=4) utilizando um método LFQ, que combina o fracionamento “curto” por eletroforese desnaturante em gel de poliacrilamida e análise por LC-MS/MS. Foram identificadas 680 proteínas, das quais 586 foram identificadas com recurso ao software MASCOT e 580 com o software MaxQuant. Posteriormente, foram realizados testes post hoc, métodos hierárquicos para análise de agrupamento de dados e testes t múltiplos para diferenciar os três grupos de doenças em termos de expressão proteica com base na sua intensidade. Os testes post hoc revelaram que 96 proteínas são capazes de diferenciar entre os diferentes grupos, enquanto 118 proteínas (17 para sobreexpressas e 101 subexpressas) foram identificados como diferencialmente expressas na PDR em comparação com doentes com ERM e 95 proteínas (10 sobreexpressas e 85 subexpressas) em comparação com os doentes com AMD seca. A análise de enriquecimento funcional indica que estas proteínas subexpressas estão correlacionadas com vias/processos biológicos, como reorganização da matriz extracelular (ECM), desgranulação das plaquetas, digestão intracelular nos lisossomas, adesão celular e desenvolvimento do sistema nervoso central. Por sua vez, os resultados indicam que o vítreo de doentes com PDR é enriquecido em mediadores dos sistemas de complemento e coagulação e da fase aguda da inflamação, reforçando o papel destas vias na sua patogénese. Por último, alguns potenciais biomarcadores foram selecionados de acordo com os resultados obtidos na quantificação do proteoma do vítreo por iTRAQ e LFQ e validados pela monitorização de múltiplas reações (MRM) num maior número de amostras de vítreo. Assim, desenvolveu-se um método de MRM scheduled para a análise de 35 proteínas, em amostras de vítreo recolhidas de doentes com ERM (n=21), DR/PDR (n=20), AMD (n=11) e RRD (com e sem vitreorretinopatia proliferativa) (n=13). Desta forma, 26 proteínas demostraram potencial para discriminar entre os diferentes grupos de doenças de acordo com os resultados obtidos no MRM e as respectivas curvas ROC (receiver operating characteristic curve). Componentes das cascatas do complemento e coagulação (C6, C8B, protrombina), proteínas de fase aguda (alfa-1-antiquimotripsina), moléculas de adesão (proteína galectina-3), componentes da ECM (opticina) e biomarcadores de neurodegeneração (beta-amilóide, proteína tipo-precursora amilóide 2) destacam-se como os biomarcadores mais eficientes para discriminar entre os diferentes grupos de doenças. Em conclusão, foram desenvolvidas e implementadas diversas estratégias para a preparação e análise do proteoma do vítreo em diferentes doenças vitreorretinianas, baseadas na separação por 2DE ou por LC. Em relação ao método baseado na separação em gel, um modelo matemático criado por ANN permitiu o desenvolvimento de um protocolo altamente eficiente para a análise de elevada resolução do proteoma do vítreo por 2DE, o que pode ser vantajoso para a detecção de proteoformas específicas, incluindo diferentes isoformas e proteínas com modificações pós-traducionais. Por outro lado, métodos de alta produtividade, como iTRAQ e LFQ, proporcionaram uma análise mais aprofundada do proteoma do vítreo. Nestas técnicas, foram identificadas 1030 proteínas pela técnica de iTRAQ e 680 por LFQ, sendo que algumas não tinham sido identificadas anteriormente. Ainda mais relevante é o fato de que a análise do proteoma do vítreo, com base nestas técnicas, forneceu novos perspetivas sobre a patogénese do RRD, PDR e AMD. Além disso, estes estudos forneceram informações fundamentais sobre potenciais biomarcadores, o que permitiu a validação de 26 proteínas por MRM. No entanto, deve ter-se em consideração que os biomarcadores encontrados no vítreo não podem ser utilizados para um diagnóstico médico regular, devido ao modo de recolha invasivo deste tipo de amostras. Porém, estes podem ser candidatos a novos alvos farmacêuticos e, quando as amostras são obtidas como parte da rotina clínica, podem ser usados para o prognóstico da evolução da doença e/ou para prever a resposta adequada ao tratamento.

With 2.1 million new cases, breast cancer is the most common cancer in women. Triple negative breast cancer (TNBC), which is 15-20% of these breast cancer cases is clinically negative for expression of estrogen and progesterone receptors (ER/PR) and human epidermal growth factor receptor 2 (HER2) receptors. It is characterized by its unique molecular profile, aggressive behavior, distinct patterns of metastasis, and lack of targeted therapies. TNBCs utilize glycolysis for growth, proliferation, invasiveness, chemotherapeutic resistance and hence has poor therapeutic response. There is an urgent need for novel/alternate therapeutic strategies beyond current standard of treatment for this subset of high-risk patients. Electrical pulse-based chemotherapy, known as electrochemotherapy (ECT) could be a viable option for TNBC therapy. ECT involves the local application of precisely controlled electrical pulses to reversibly permeabilize the cell membrane for enhanced uptake. ECT can increase the cytotoxicity of the chemotherapeutics up-to 1000 times, facilitating a potent local cytotoxic effect.

The high cost and severe side-effects of conventional chemotherapeutics motivate the application of effective natural compounds. Combining electrical pulses with natural compounds will enhance the treatment efficacy. This dissertation focuses on curcumin, the yellow pigment of natural herb turmeric, that has been used for over 5000 years for its excellent anticancer properties. Previous studies have demonstrated the effectiveness of curcumin for treating multiple cancers, including TNBC, with limited side effects. The potency of curcumin can be enhanced further by combining it with ECT to provide an attractive and cost-effective alternative for TNBC treatment.

Towards this we studied the effect of ECT with curcumin on MDA-MB-231 cell line, a human adenocarcinoma epithelial TNBC cell line. We performed various assays, including cell viability, colony forming, cell cycle, apoptosis, H₂O₂ reactive oxygen species (ROS), immunoblotting, real time quantitative PCR (qPCR), and cellular metabolites detection to study the impact of ECT with curcumin on MDA-MB-231 cells. In addition, to better understand the underlying mechanisms, we used high throughput, label-free quantitative proteomics. While several studies have attempted to define the mechanism of action of curcumin on cancer cells, little is known on the action mechanism of the curcumin delivered with electrical pulses. This work unravels the molecular mechanism behind the enhanced effects observed under the ECT-based curcumin therapy in TNBC cells, employing a high-throughput, quantitative, label-free mass spectroscopy-based proteomics approach. The proteomics approach provides information on the thousands of cellular proteins involved in the cellular process, allowing a comprehensive understanding of the electro-curcumin-therapy mechanism. Similar studies were also performed for ECT with cisplatin to compare the efficacy of the electro-curcumin-therapy to the standard stand-alone cisplatin-based therapy.

Our results revealed a switch in the metabolism from glycolysis to mitochondrial metabolic pathways. This metabolic switch caused an excessive production of H₂O₂ ROS to inflict apoptotic cell death in MDA-MB-231 cells, demonstrating the potency of this ECT based curcumin therapy. These results encourage further studies to extend the application of ECT for clinical practice.