Dissertations / Theses on the topic 'ITRAX'

The Itrax core scanner provides rapid, high resolution, non-destructive sediment core analysis using x-ray fluorescence (XRF) spectrometry and x-radiography. The effect of varying instrument settings are explored. Effects of sample properties on XRF data are tested and the uncertainty in XRF data is considered. Existing methods of quantifying XRF data are evaluated. Comparisons are made with other XRF micro-scanners. Finally, the x-radiographic capability of the Itrax core scanner is compared to x-ray computed tomography. Itrax XRF data is generally optimised by use of a 30kV x-ray tube voltage. Current should be set as high as possible without generation of sum peaks (30mA is often a good value). A chromium anode tube is suitable for use with most samples. Water content has a diluting effect on detected peak areas, but it is shown that the effect can be corrected, removing an obstacle to quantification of Itrax data. Water content can be determined non-destructively from the ratio of incoherent to coherent scatter of characteristic radiation from the x-ray tube anode. Surface slope can change recorded peak areas, but a simple model is developed to correct for this effect. Surface roughness increases variability in data and, if the scale of roughness is similar to the beam size, elemental peak areas may be reduced. The presence of a mixture of grain sizes greatly reduces peak areas for elements in the larger grains. The uncertainty in Itrax data is found to be higher than suggested by the conventional estimate that uncertainty is equal to the square root of the peak area. This information is vital for researchers to decide what significance they should attach to variations in Itrax elemental profiles. Quantification methods for core scanner XRF data are compared and an approach using log-ratio transformations determined to be the best. Additionally, an improved entirely non-destructive, quantification approach is presented in which explicit corrections are made for the diluting effect of water (water content may determined from the ratio of incoherent and coherent scatter of the tube anode characteristic radiation) . Compared to similar instruments, the Itrax core scanner is more tolerant of surface imperfections. Its x-radiographic scanning helps to determine the significance and extent of features revealed in XRF data. Itrax x-radiography provides no match for the level of detail that can be obtained using x-ray computed tomography and is not readily quantified. It does however provide information on features below the sample surface and, in masking small variations, can make the main core features more apparent. Users of the Itrax core scanner are provided with quantification of known effects (water content, surface slope; x-ray tube, current and voltage) and are alerted to issues that were not previously widely known (mixing of grain sizes, size of uncertainty in data). The areas of effective use and limitations of the Itrax core scanner are set out and recommendations made for optimising results. An optimal quantification method is identified. Many of the conclusions may have relevance to other XRF core scanners.

En transplantation d’organe solide, les Inhibiteurs de la Calcineurine (ICN), Cyclosporine A et Tacrolimus, ont permis un amélioration significative de la survie à court terme du greffon en prévenant le rejet d’allogreffe. Cette évolution positive est contrebalancée par une néphrotoxicité susceptible de contribuer au développement complexe et multifactoriel de la dysfonction chronique du greffon, facteur pronostique majeur d’une insuffisance rénale terminale. L’objectif principal de ce travail a été de combiner deux approches expérimentales complémentaires, afin de mettre en lumière des aspects inédits de la physiopathologie des ICN. La première approche repose sur l’application de la technique de protéomique quantitative« shotgun » iTRAQ (« isobaric Tags for Relative & Absolute Quantitation ») à l’analyse non ciblée du protéome de cellules tubulaires proximales. La seconde approche applique de manière ciblée les outils classiques de biologie moléculaire à l’étude du cytosquelette d’Actine de cellules tubulaires proximales. La combinaison de ces deux stratégies complémentaires a permis de mettre en lumière un rôle inédit du cytosquelette d’Actine dans les effets physiopathologiques de la Cyclosporine A en apportant des éléments en faveur d’un mécanisme reposant sur une régulation originale de la dynamique intracellulaire de l’Actine
In solid organ transplantation, Calcineurin Inhibitors, Cyclosporin A and Tacrolimus, prevent allograft rejection and ensure short-term allograft survival. However, CNI elicit nephrotoxic side effects whose mechanisms remain widely unsolved and are thought to participate to the multifactorial development of chronic kidney disease, leading to renal failure. The aim of thiswork was to combine targeted and untargeted experimental strategies to better understand CNI-induced physiopathological mechanisms.The first approach was based on the untargeted monitoring of the proximal tubular proteome by the quantitative shotgun proteomic technique, iTRAQ (« isobaric Tags for Relative & Absolute Quantitation »). The second approach consisted in the study of the Actin cytoskeleton of proximal tubular cells by classical molecular biology techniques. In the light of results from both approaches, this work reported that the Actin cytoskeleton of proximal tubular cells may play a part in the pathophysiology of CsA thanks to a mechanism based on an original regulation of the intracellular dynamics of Actin

p53 is a critical tumour suppressor which acts to repair or remove abnormal cells and thus prevent cancer. Aberrant function of p53 is therefore a critical step in tumourigenesis and p53 is mutated in half of all cancers. Mutation of p53 leads to both a loss of normal wildtype function as well as the gain of oncogenic function. p53 is considered to be a promising therapeutic target and therapeutic strategies for targeting of the p53 pathway include: 1. Activation of wild-type p53 (wtp53) protein function, 2. Refolding of mutant p53 (mtp53) into the wtp53 conformation, 3. Reduction of mtp53 protein levels. In this work a number of small molecule screening assays were used to identify potentially novel regulators of both wtp53 and mtp53. Screening of a protein kinase inhibitor library for small molecules which can stimulate wtp53 activity identified the GSK3 pathway and a CDK pathway as dominant suppressors of wtp53 function. Screening of the library for inhibitors which reduce mtp53 protein levels led to the identification of two IKKβ inhibitors. The work then focused on investigating the effects of one of these compounds, IMD0354, on the mutant p53 pathway; with a specific focus on MDM2 as the most rapidly responding biomarker. IMD0354 is a well characterised inhibitor which has been shown to specifically inhibit IKKβ leading to the repression of the Nf-κB pathway. This study shows that IKKβ inhibition leads to the loss of a number of oncogenic proteins including mtp53, MDM2 and cyclin D. Mass-spectrometry based (ITRAQ) proteomic analysis was then employed to identify potential mediators and/or co-regulated factors in response to IKKβ-inhibition via IMD0354 treatment. This led to the identification of RPS3 as a potential negative regulator of MDM2 protein expression; the reduction in MDM2 protein in response to IMD0354 treatment is shown to be partially dependent on RPS3. Together this data has identified, using small molecule kinase inhibitor libraries: (i) dominant kinase signalling pathways that suppress wt-p53 and (ii) a dominant kinase signalling pathway that sustains expression of mutant p53 and MDM2 in cancer cell lines. This latter data supports further investigation to aid understanding of how the IKK signalling pathway cross-talks to the p53-MDM2 axis.

La cicatrisation cornéenne représente un processus de réparation complexe qui vise à restaurer l'intégrité, la structure et la transparence de la cornée. Cependant, dans un certain nombre de cas, ce processus peut évoluer de façon anormale et se stabiliser en entraînant la formation d'une opacité cornéenne installée. Les mécanismes impliqués dans la formation de ces cicatrices persistantes ne sont pas encore complètement élucidés, mais il est établi que la composition et l'organisation de la matrice extracellulaire du stroma jouent un rôle majeur dans la restauration de la transparence de la cornée. Ce projet s’est concentré sur la métalloprotéase extracellulaire BMP-1 (Bone Morphogenetic Protein 1), déjà connue pour son rôle dans l'assemblage de la matrice extracellulaire et l'activation du TGF-bêta. Afin d’identifier les processus contrôlés par BMP-1 dans la cornée, nous avons d’abord effectué une comparaison systématique des inhibiteurs de BMP-1 connus ou potentiels, de différentes origines, pour caractériser leurs propriétés à la fois in vitro et dans des cultures cellulaires. Ensuite, nous avons mené une étude approfondie du sécrétome des cellules stromales de la cornée humaine (kératocytes), et des conséquences de la différenciation de ces cellules en myofibroblastes. Enfin, nous avons analysé les événements protéolytiques médiés par BMP-1 dans le sécrétome des kératocytes en utilisant principalement une approche de protéomique quantitative basée sur le marquage iTRAQ des protéines entières (technique TAILS). La comparaison des inhibiteurs disponibles de BMP-1 a permis de mettre en évidence différents profils d’efficacité, de spécificité et de toxicité et a conduit à l’identification d’un inhibiteur hydroxamate et d’un inhibiteur protéique efficaces, peu toxiques et très spécifiques de BMP-1. Le sécrétome des kératocytes s’est avéré être un modèle adéquat pour l’étude des activités de BMP-1 dans le contexte cornéen. Plus de 2022 protéines ont été identifiées, dont la métalloprotéase BMP-1 et 16 de ses 33 substrats connus jusqu’à présent. Enfin, 76 protéines modifiées par l’activité de BMP-1 ont été identifiées dans le sécrétome des kératocytes. Ces résultats confirment les liens forts entre BMP-1, l'assemblage de la matrice extracellulaire et le TGF-bêta, mais suggèrent également de nouveaux rôles pour la protéase dans l'inflammation. Certains des substrats nouvellement identifiés (TGFBI, HSP47 et collagène VI) sont très pertinents dans le contexte de la cicatrisation de la cornée et ont été validés d’un point de vue biochimique. En conclusion, BMP-1 est confirmée comme une cible potentielle intéressante pour traiter ou prévenir la formation des opacités cornéennes et la caractérisation des inhibiteurs disponibles ouvre des perspectives importantes pour des études précliniques chez l’animal
When the cornea is injured, a complex multi-step healing process is triggered which aims at restoring corneal integrity, structure and transparency. However, in some cases, corneal healing results in the formation of a stable scar associated with a prolonged loss of corneal transparency and with functional blindness. The mechanisms involved in the formation of these persistent scars are still not fully understood but it is known that the composition and organization of the extracellular matrix significantly contributes to the maintenance of corneal transparency. This work focused on the extracellular metalloproteinase called BMP-1 (Bone Morphogenetic Protein 1), a major player in the control of extracellular matrix assembly and TGF-beta activation, which was previously shown to be up-regulated in corneal healing and scarring. In order to further probe BMP-1 functions in corneal healing, we first performed a systematic comparison of known or potential BMP-1 inhibitors from different origins to characterize their properties both in vitro and in cell cultures. We then carried out an in-depth study of the secretome of human corneal stromal cells (keratocytes) and of the consequences of the differentiation of these cells into myofibroblasts. Finally, we analyzed BMP-1-mediated proteolytic events in keratocyte secretomes, mainly using a quantitative proteomic approach based on iTRAQ labeling of proteins (TAILS technique). The comparison of BMP-1 available inhibitors revealed different profiles of efficacy, specificity and toxicity and led to the identification of one hydroxamate inhibitor and one protein inhibitor, which were very efficient, non-toxic and very specific of BMP-1. The keratocyte secretome was shown to be a suitable model for the study of BMP-1 activities in the corneal context. More than 2022 proteins were identified, including the BMP-1 metalloprotease and 16 of its 33 already known substrates. Finally, 76 proteins modified by BMP-1 activity were identified in the keratocyte secretome. These results confirm the strong links between BMP-1, extracellular matrix assembly and TGF-beta, but also suggest new roles for this protease in cell proliferation and inflammation. Some of the newly identified substrates (TGFBI, HSP47 and collagen VI) are highly relevant in the context of corneal healing and were validated at the biochemical standpoint. In conclusion, BMP-1 is confirmed as a potential target to treat or prevent the formation of corneal opacities and the characterization of available inhibitors opens up important perspectives for preclinical studies in animals

Positional proteomics is emerging as an attractive technique to characterize protein termini, which play important biological roles in cells. Even with the advances in past decades, there still are areas for improvement. This thesis focuses on improving data quality and assignment confidence in positional proteomics. A novel workflow was designed for the large-scale identification of protein N-terminal sequences. 4-sulfophenyl isothiocyanate (SPITC) is used for N-termini sulfonation; Upon higher energy collisional dissociation (HCD), SPITC peptides in electrospray ionization ESI generate predominately y-type ion series; such simplification of spectra enables the identification of N-termini with high fidelity. The presence of b1 + SPITC product ions upon HCD furthers the confidence for N-terminal identifications. Secondly, sulfonated N-terminal peptides possess one negative charge site at low pH, which was exploited to enrich the SPITC modified N-terminal peptides by electrostatic repulsion hydrophilic interaction (ERLIC) chromatography. Such enrichment process allows both N-termini enriched and N-termini deficient fractions to be collected and analyzed by LC-MS/MS. This method was applied to an E. coli cell lysate, identifying approximately 350 N-terminal peptides (85% represented neo-N-termini from protein degradation and 15% from leading methionine excision). These N-terminal peptides represented 274 distinct E.coli proteins, 224 of which were also identified in the analysis of flow-through fractions from internal peptides. Another approach we took to boost the identification confidence is by exploiting iTRAQ (isobaric tag for relative and absolute quantitation) in the positional proteomics workflow. This approach allows for multiplexed comparison between different samples, and thus is well-suited for degradadomics analyses where degraded samples are compared to control samples. Both control and protease treated sample are labeled by different tags which allows direct comparison of protein N-termini with neo-N-termini. In addition, samples are analyzed duplicate by labeling with two tags, aiming for quick validation of peptides by internal replicates. In this study, Asp-N digested E.coli cell lysate is taken as a model system. A total of 500 N-terminal peptides, corresponding to 370 proteins, were identified with high confidence in one experiment, with 87% of those proteolytic products matching the expected protease digestion specificity, validating the assignment accuracy of this approach.

Very little is known about the hydrophobic proteins of psychrophiles and their roles in cold adaptation. In light of this situation, methods were developed to analyse the hydrophobic proteome (HPP) of the model psychrophilic archaeon Methanococcoides burtonii. Central to this analysis was a novel differential solubility fractionation procedure, which resulted in a significant increase in the efficiency of resolving the HPP. Over 50% of the detected proteins were not identified in previous whole cell extract analyses, and these underwent an intensive manual annotation process producing high quality functional assignments. Utilising the functional assignments, biological context analysis of the HPP was performed, revealing novel and often unique biology. The analysis acted as a platform for differential proteomics of the organism???s response to both temperature and substrate using stable isotope labelling. The results of which revealed that low temperature growth was associated with an increase in the abundance of surface and secreted proteins, and translation apparatus. Conversely, growth at a higher temperature was associated with an increase in the abundance of general protein folding machinery and indications of an oxidative stress response, emphasising that the temperature for maximum growth rate is stressful. Through investigation of the response of M. burtonii to substrate it was found that growth on methanol was stressful, and its low energy yield resulted in an increase in the abundance of energy conserving systems. The extracellular polymeric substance (EPS) and morphology of M. burtonii was also investigated with respect to both temperature and substrate, using a number of techniques in microscopy. It was found that the EPS was comprised of proteins, sugars and RNA, and that growth at different temperatures resulted in the production of EPS that displayed significantly different properties on dehydration, thus indicating compositional variation. When cells were grown on methanol they took on highly irregular shapes and had electron transparent inclusions. The observations from the ultrastructural analysis were contemplated with respect to the proteomic findings, revealing novel avenues of research. This study has highlighted the roles of hydrophobic proteins in cold adaptation biology, and the value of comprehensive proteomics for the examination of adaptation in microorganisms

O crescimento, desenvolvimento e manutenção do tecido ósseo são processos altamente regulados. Diversas proteínas como hormônios, fatores de crescimento e citocinas estão envolvidas nestes processos e exercem atividade direta sobre células osteoblástica e osteoclástica, atuando em sua diferenciação e ativação metabólica. O processo de regeneração óssea é iniciado por fatores estimuladores locais como as proteínas morfogenética óssea (BMP Bone Morphogenetic Proteins). As BMPs são um produto do metabolismo dos osteoblastos, odontoblastos e de várias células tumorais, sendo armazenadas na forma de concentrados no osso, dentina e em células neoplásicas do osteossarcoma e de certos tumores odontogênicos, tais como: fibroma cementificante, cementoblastoma benigno, dentinoma, fibroma odontogênico e odontoma. Esclarecer os mecanismos que controlam a remodelação óssea é uma questão bastante relevante. Nesse sentido, as células-tronco mesenquimais têm despertado grande interesse devido ao seu potencial envolvimento no processo de reparo tissular. A obtenção de osteoblastos funcionais a partir de células-tronco mesenquimais tem sido utilizada na engenharia de tecidos e terapia celular. Desse modo, no presente trabalho foi realizada uma análise proteômica das proteínas envolvidas nas diversas fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea de rato Wistar e humana, no sentido de obter maiores informações sobre a diferenciação celular e a biologia do tecido ósseo. Células-tronco mesenquimais obtidas de medula óssea foram cultivadas em meio osteogênico por diferentes períodos para obter células em diversas fases da diferenciação osteoblástica. Para análise proteômica foram utilizadas ferramentas como a estratégia de shotgun proteomics e quantificação relativa (iTRAQ - Isobaric Tag for Relative and Absolute Quantitation) para separação de proteínas e a espectrometria de massas para a identificação e quantificação relativa de proteínas e peptídeos. Neste contexto, os nossos resultados nos levam a concluir que: as CTMs de medula óssea de rato Wistar expressam genes que estão envolvidos na diferenciação osteogênica quando estimuladas in vitro formando matriz óssea no período de 14 dias, ou seja, o fator estimulante no microambiente é de fundamental importância; as CTMs de medula óssea humana apresentaram resultados semelhantes com as CTMs de ratos em nível genômico durante a diferenciação osteogênica, entretanto quando estimuladas in vitro formaram a matriz óssea no período de 21 dias; utilizando duas abordagens proteômicas, foi possível identificar proteínas importantes que estão envolvidas no processo de diferenciação. Mas cabe salientar que, embora tenham sido detectados genes que parecem envolvidos no processo de diferenciação, isso não teve reflexo no proteoma dessas células nos períodos de 7 e 14 dias da indução de diferenciação à osteogênese, o que indica que a maior parte da funcionalidade dessas células quanto aos outros processos biológicos estão preservados, como por exemplo a proliferação celular permaneceu sem grandes alterações. Isso indica que manipulações de isolamento, cultivo e indução da diferenciação dessas células não afetaram o proteoma, com aspectos positivos para a utilização de células-tronco mesenquimais em terapia celular. Do ponto de vista metodológico, esse trabalho abre perspectivas da utilização de estratégias proteômicas baseadas na marcação por isóbaros em combinação com separação de proteínas por eletroforese unidimensional SDS-PAGE para a análise de amostras biologicamente complexas e de quantidades limitadas de obtenção como células-tronco mesenquimais. O estudo da expressão de proteínas durante as fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea deve refletir seu estado funcional e contribuir para o entendimento das diversas vias envolvidas no processo de diferenciação.
The growth, development and maintenance of bone tissue are highly regulated processes. Several proteins such as hormones, growth factors and cytokines are actively involved in these processes and exert direct activity on osteoblastic and osteoclastic cells, acting in their differentiation and metabolic activation. The process of bone regeneration is initiated by local stimulating factors as bone morphogenetic proteins (BMP). BMPs are a product of the metabolism of osteoblasts, odontoblasts and various tumor cells and is stored in the form of concentrates in bone, dentin and neoplastic cells of osteosarcoma and certain odontogenic tumors such as fibroma cementifying, cementoblastoma benign dentinoma, odontogenic fibroma and odontoma. Clarify the mechanisms that control bone remodeling is a very relevant issue. Accordingly, the mesenchymal stem cells have attracted great interest because of its potential involvement in the process of tissue repair. Obtaining functional osteoblasts from mesenchymal stem cells has been used in tissue engineering and cell therapy. Thus, this present work performed a proteomic analysis of proteins involved in various stages of osteoblast differentiation of mesenchymal stem cells from bone marrow of Wistar rat and human, in order to obtain more information on the biology of cell differentiation and bone tissue. Mesenchymal stem cells obtained from bone marrow were cultured in osteogenic medium for different periods to obtain cells at different stages of osteoblast differentiation. For proteomics analysis tools were used as the strategy of shotgun proteomics and relative quantification (iTRAQ - isobaric Tag for Relative and Absolute quantitation) for protein separation and mass spectrometry to identify proteins. In this context, our results take us to conclude that the MSCs of Wistar rat bone marrow express genes that are involved in osteogenic differentiation in vitro when stimulated to form bone matrix during the 14 days, ie stimulating factor in the microenvironment is of fundamental importance, the MSCs from human bone marrow showed similar results with rat MSCs at the genomic level during osteogenic differentiation, however, when stimulated in vitro formed bone matrix within 21 days, using two proteomic approaches, we could identify proteins important that are involved in the process of differentiation. But it should be noted that although it has been identified genes that seem involved in the process of differentiation, it was not reflected in the proteome of these cells at 7 and 14 days after induction of the osteogenic differentiation, indicating that most of the functionality of these cells and other biological processes are preserved, such as cell proliferation remained without major changes. This indicates that manipulations of isolation, culture and induction of differentiation of these cells did not affect the proteome, with positive aspects to the use of mesenchymal stem cells in cell therapy. From the methodological point of view, this work opens up the use of proteomic strategies based on the score for isobars in combination with protein separation by electrophoresis, one-dimensional SDS-PAGE for the analysis of complex biological samples and limited quantities of production as mesenchymal stem cells. The study of protein expression during stages of osteoblast differentiation of mesenchymal stem cells from bone marrow should reflect their functional status and contribute to the understanding of pathways involved in the process of differentiation.

The main approach to studying the proteome is a technique called data dependent acquisition (DDA). In DDA, peptides are analyzed by mass spectrometry to determine the protein composition of a biological isolate. However, DDA is limited in its ability to analyze the proteome, in that it only selects the most abundant ions for analysis, and different protein identifications can result even if the same sample is analyzed multiple times in succession. Data independent acquisition (DIA) is a newly developed method that should be able to solve these limitations and improve our ability to analyze the proteome. We used an implementation of DIA (SWATH) to perform relative protein quantitation in the model bacterial system, Clostridium stercorarium, using two different carbohydrate sources, and found that it was able to provide precise quantitation of proteins and was overall more consistent in its ability to identify components of the proteome than DDA. Relative quantitation of proteins is an important method that can determine which proteins are important to a biochemical process of interest. How we determine which proteins are differentially regulated between different conditions is an important question in proteomic analysis. We developed a new approach to analyzing differential protein expression using variation between biological replicates to determine which proteins are being differentially regulated between two conditions. This analysis showed that a large proportion of proteins identified by quantitative proteomic analysis can be differentially regulated and that these proteins are in fact related to biological processes. Analyzing changes in protein expression is a useful tool that can pinpoint many key processes in biological systems. However, these techniques fail to take into account that enzyme activity is regulated by other factors than controlling their level of expression. Activity based protein profiling (ABPP) is a method that can determine the activity state of an enzyme in whole cell proteomes. We found that enzyme activity can change in response to a number of different conditions and that these changes do not always correspond with compositional changes. Mass spectrometry techniques were also used to identify serine hydrolases and characterize their expression in this organism.
February 2016

Mestrado em Finanças
Despite the absence of good theoretical models to cope with credit portfolio issues, the development of credit derivative markets and the popularity of portfolio credit derivatives have created the need of handling the issue of default correlations in some way. In that context the copula models emerged and became extremely popular within the industry. In recent studies copula models have been criticized for not being flexible enough and for being a static approach. The recent turmoil on the Asset Backed Security market and the failure of Lehman Brothers, Inc brought to discussion the accuracy of these models. Based on data provided by two banks, on default correlation implied from CDO tranche market quotes, we try to draw conclusions about: 1)The credibility of the HLPGC copula model; 2) The power that correlations between single name CDS spreads have to explain those implied by market data, specially during the current. For the empirical study we will use the most popular and liquid portfolio credit derivatives: Collateralized Debt Obligations (CDO based on the iTraxx credit index for 5 years maturity), and implied correlations of CDO tranches written on the same index. The data source will be Bloomberg for single name CDS spreads and Calyon and JP Morgan for implied correlations from a Copula model.
Apesar da inexistência de modelos teóricos robustos para lidar com carteiras de risco de crédito, o desenvolvimento e a popularidade dos mercados de derivados de crédito criaram a necessidade de lidar com a questão das correlações de probabilidades de incumprimento de uma forma simples. Foi neste contexto que surgiram os modelos de cópula associados à indústria do risco de crédito. Estudos recentes criticam os modelos de cópula pela sua falta de flexibilidade e por assumirem uma abordagem estática. A recente crise no mercado de titularizações de hipotecas bem como a falência do Lehman Brothers, Inc reacenderam a discussão sobre a eficácia destes modelos. Com base em informação cedida por dois bancos de investimento sobre correlações implícitas nas cotações de mercado de tranches de CDOs, procurar-se-á concluir acerca da: 1) Credibilidade do modelo de cópula HLPGC; 2) Capacidade que as correlações entre spreads dos CDS individuais têm, na actual crise, para explicar as correlações essas correlações implícitas. Para a análise empírica usamos a carteira mais líquida de derivados de crédito: o índice iTraxx com maturidade de 5 anos e as correlações implícitas para as tranches emitidas sobre esta carteira. As fontes de informação utilizadas são, a Bloomberg para os prémios de risco dos nomes que constituem o iTraxx e JP Morgan e Calyon para correlações implícitas geradas pelos seus modelos de cópula.

L'oxaliplatine est un médicament de référence dans les traitements des cancers colorectaux métastatiques. Toutefois, l'oxaliplatine occasionne une toxicité d'ordre neurologique qui retentit sur la qualité de vie de certains patients prédisposés. Nous avons analysé pour la première fois la neurotoxicité de l'oxaliplatine par une approche protéomique. Un protocole expérimental, associant marquage iTRAQ et fractionnement par IEFOFFGEL a tout d'abord été mis en place afin d'améliorer la couverture protéomique d'échantillons complexes. Puis, l'expression protéique différentielle après traitement par I'oxaliplatine a été analysée globalement, à la fois dans le protéome intracellulaire et dans le sécrétome d'un modèle cellulaire humain de nerotoxicité. L'analyse du protéome intracellulaire a permis l'identification de plus de 2700 protéines et offre de nouvelles perspectives quant aux mécanismes moléculaires de la neurotoxicité de I ' o x aliplatine. Le médicament aItère l'expression de protéines impliquées dans la réponse aux dommages à l'ADN, le contrôle du cycle cellulaire, le stress oxydatif , le métabolisme énergétique, le stress protéotoxique, la résistance multidrogue, la plasticité neuronale et l'homéostasie calcique. L'analyse du sécrétome a mis en évidence la sur-expression, suite au traitement par l'oxaliplatine, de 23 protéines sécrétées. Nous proposons pour la première fois les protéines Calmoduline, Thymosine beta-10 et le facteur neurotrophique Neudésine comme candidats biomarqueurs de la neurotoxicité des traitements anticancéreux à base d'oxaliplatine.

Normally present in water, soil and waste water, Acinetobacter baumannii has become an important nosocomial pathogen, as causal agent of pneumonias, septicemias and urinary tract infections, among other complications in compromised patients from hospital’s intensive care units. One of its last acquired abilities is the resistance to colistin (polymixin E), the last therapeutic option for its infections. In this thesis, descriptive and quantitative differential expression proteomics is used in the study of acquired colistin resistance. As result of this research, 1,097 proteins belonging to the Acinetobacter genus have been identified by combined application of bidimensional gel electrophoresis (2DE), differential gel electrophoresis (DIGE), and peptide labeling with stable isobaric isotopes tags (iTRAQ). Analyses have been performed on the global expressed proteome of a reference, colistin-sensible strain (A. baumannii ATCC 19606) and, for comparative purposes, on a derived strain on which colistin resistance has been induced in vitro. The resistant phenotype shows reduced fitness, with significant differences in expression found in outer membrane proteins, membrane active transporters, diverse metabolic enzymes (fatty acids, citrate, phenylacetate, piruvate and nitrogen), proteins involved in stress response and biofilm formation, as well as in protein synthesis and folding pathways. The work has allowed to assess the strengths and weaknesses of the different techniques currently used in this type of proteomic analysis.
Acinetobacter baumannii, normalmente aislado en suelos y aguas (corrientes o residuales), se ha convertido en importante patógeno nosocomial, siendo agente causal de, entre otras complicaciones, neumonías, septicemias e infecciones del tracto urinario de pacientes comprometidos en unidades hospitalarias de cuidados intensivos. La más reciente de sus capacidades adquiridas es la resistencia a colistina (polimixina E), antibiótico peptídico considerado la última opción terapéutica en contextos clínicos. Esta tesis doctoral emplea la proteómica descriptiva y de expresión diferencial cuantitativa para investigar la resistencia adquirida por A. baumannii a dicho antibiótico. Los resultados han supuesto la identificación de 1.097 proteínas de Acinetobacter mediante el empleo combinado de electroforesis bidimensional convencional (2DE), 2DE diferencial (DIGE) y marcaje peptídico mediante isótopos isobáricos estables (iTRAQ). Los análisis se han realizado en el proteoma expresado por una cepa de referencia sensible a colistina (A. baumannii ATCC 19606), así como en una cepa derivada de ésta en la que se ha inducido, a efectos comparativos, resistencia a colistina in vitro. El fenotipo resistente manifestó reducida adaptabilidad biológica, encontrándose las principales diferencias en la estructura de la membrana externa, en la expresión de transportadores activos de membrana, en diversos enzimas metabólicos (ácidos grasos, citrato, fenilacetato, piruvato, nitrógeno) y de respuesta a condiciones de estrés, así como en la expresión de proteínas participantes en la formación de biopelículas y en el proceso de síntesis y plegamiento de proteínas. Además, el trabajo ha permitido evaluar los puntos fuertes y débiles de las técnicas empleadas actualmente en este tipo de análisis proteómicos.

Global bond market capitalization amounts to approximately $100 trillion, compared to $60 trillion in the equity markets. Despite debt financing being a large part of the global financial market, the measurements and greenhouse gas reduction investment strategies to date are not nearly as thorough as for equity financing. More recently, the problem has been brought into light by the World Bank, expressing concerns about the crucial role of debt financing activities in the current and upcoming threats caused by climate change. A commonly used credit derivative in debt financing is credit default swaps (CDS), which is an agreement between two parties to exchange the credit risk of a reference entity. The buyer of the contract makes fixed periodic payments to the seller of the contract, who collects the premiums in exchange for making the protection buyer whole in the case of a defaulting reference entity. This thesis aims to minimize the greenhouse gas emission exposure for two CDS indices, iTraxx Main and CDX.IG, each consisting of 125 equally weighted constituents, or companies. The CDS indices are widely used high liquid fixed income instruments. In 2017, iTraxx Main had a monthly trading volume of $330-440 billion notional, and CDX.IG a corresponding volume of $200-275 billion. In order to rate the greenhouse gas emissions of the constituents, the ECOBAR model was used. The model utilizes a discrete ranking score system, where the aim is to obtain as low score as possible. To minimize the ECOBAR score for the baskets, Markowitz Modern Portfolio Theory was used, implemented by using a quadratic programming algorithm. By optimizing the portfolios while retaining a low tracking error and high correlation toward the CDS indices, underlying investment properties were retained. We show that one can construct replicated portfolios of the CDS indices that have significantly lower ECOBAR scores than the indices themselves, whilst still maintaining a low tracking error and high correlation with the actual indices. When constructing baskets of fewer constituents, one can replicate the indices with merely 10-30 constituents, without worsening the tracking error or correlation substantially, and obtain an even lower ECOBAR score for the respective portfolios.

Mestrado em Finanças
Este estudo tem como objetivo estudar o mercado europeu de CDS e CDO. Através de uma análise econométrica estimaremos a relevância de diversas variáveis para explicar o logaritmo das primeiras diferenças dos spreads das tranches do CDO baseado no iTraxx Europe 5-year. Assim, a nossa amostra é composta por dados diários desde Fevereiro de 2005 até Fevereiro de 2012 das tranches do iTraxx Main 5-year e de proxies para os riscos de crédito, taxa de juro, liquidez e para a volatilidade de mercado e rendibilidades do mercado acionista. Para aferir se houve alterações significativas no mercado Europeu de CDO depois da crise financeira, estimaremos duas regressões adicionais, onde na primeira utilizaremos uma dummy temporal para isolar os períodos antes e depois da crise e na segunda outra dummy temporal para isolar o período após a falência do Lehman Brothers. As nossas principais conclusões são que as proxies para os riscos de crédito e de taxa-de-juro, bem como a volatilidade de mercado são relevantes em todas as tranches para a totalidade do período da amostra. Além disso, as rendibilidades do mercado acionista e o declive da estrutura temporal parecem assumir uma maior relevância para explicar as tranches do CDO depois da crise financeira de 2007.
The focus of this study is the European CDS and CDO markets. Using a regression-based approach we estimated the relevance of market-based proxies for explaining the first differences of the logarithm of European CDS Index tranches premia (iTraxx Europe 5-year index). Therefore, our sample is comprised by daily data since February 2005 to February 2012, of iTraxx Main 5-year tranche premia and proxies for credit risk, interest rate risk, liquidity risk, equity returns and market volatility. In order to understand if there were significant changes in the CDO market after the financial crisis, we run two additional regressions, where first, we add a time dummy to isolate the periods before and after the turmoil and, after that we include a time dummy to isolate the period after the Lehman Brothers´ collapse. Our main findings are that proxies for credit risk, risk-free rate risk and market volatility are significant in all tranches when we consider the entire sample. Moreover, equity returns and the slope of the term structure seem to play a more important role in pricing tranche premia, since the start of the financial crisis of 2007.

Les avancées récentes dans l'étude des phénomènes aboutissant au cancer montrent que chaque tumeur possède des caractéristiques physiopathologiques qui lui sont propres. L'apport de biomarqueurs, permettant de réaliser des diagnostics moléculaires précis est donc un enjeu primordial. Une analyse protéomique quantitative globale basée sur l'utilisation du marquage iTRAQ TM a été réalisée sur des tumeurs colorectales congelées. Ces tumeurs ont été analysées en fonction de leur stade TNM d'une part, et de leur statut concernant l'oncogène KRAS d'autre part. Afin de compléter notre analyse nous avons également analysé le protéome de lignées cellulaires colorectales. Nous avons également examiné le stroma de ces tumeurs, et nous avons développé une technique d'analyse d'un sous-protéome : le glycoprotéome. L'ensemble de ces résultats nous a conduit à proposer une base de données des protéines identifiables dans ce modèle constituée de 3 168 protéines. 513 des protéines que nous avons identifiées sont susceptibles d'être sécrétées par les tumeurs et constituent une base de données de candidats biomarqueurs pour le cancer colorectal. Nous avons également validé l'un d'entre eux : l'OLFM4. Il s'agit d'un candidat biomarqueur potentiel pour la détection précoce des cancers colorectaux, et nous avons également mis en évidence que son niveau d'expression était modifié lorsque la tumeur était mutée sur l'oncogène KRAS. Nous nous sommes également intéressés à ses fonctions cellulaires, et nous avons montré qu'elle participait à la résistance des tumeurs aux traitements, et il est probable qu'elle joue un rôle dans la dissémination tumorale.

Mass spectrometry (MS) is a power analytical tool which is capable of analyzing biomolecules in great detail, both structurally and quantitatively. With regards to glycans, special considerations regarding sample preparation are necessary in order to achieve reproducible identification and relative quantification of these analytes. A workflow for isolation at the glycopeptide level and subsequent detection at the glycan level with phenylhydrazine, demonstrated that monoclonal antibodies (mAbs) containing a specific amino acid mutation were able to express approximately an additional 50% of the α2,6 disialylated glycan compared to their non-mutant analogues. In a second experiment using mAbs, an azide modified glycan (Ac4ManAz) was introduced both metabolically and enzymatically during mAb production. This glycan is a precursor in the sialic acid pathway and the azide moiety allows for specific chemistry post-production including the potential for highly specific enrichment. The results of this workflow demonstrated that [100 μM] of Ac4ManAz precursor added to the cell media was necessary for metabolic expression. More complex samples however, may contain multiple sites of glycosylation. To conserve the site of attachment, these molecules are often studied at the glycopeptide level, and require enrichment of glycopeptides to improve the lower signal intensity observed in the presence of co-eluting peptides. Carboxymethyl chitosan (CMCH) as well as amine-functionalized magnetic-nanoparticles (MNP) were developed as novel materials for this purpose. CMCH is naturally occurring, and therefore is cost-effective and readily available. In a 12 protein mixture CMCH demonstrated the bulk enrichment of glycopeptides yielding an approximately 20% higher enrichment of sialylated species as compared to a commercially available glycopeptide kit through the use of tandem mass tags for relative quantification. In the same approach, amine functionalized MNP were produced and used to enrich glycopeptides from tryptic digests. This approach was fast (about 10 mins) and quantitatively demonstrated improved retention for sialylated species. Examples of these techniques and their applications are reported in this work.
October 2015

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

Pseudoexfoliation (PEX) syndrome is an age-related condition characterized by the production and accumulation of extracellular fibrillary material in the anterior segment of the eye. PEX predisposes for several pathological conditions, such as glaucoma and complications during and after cataract surgery. The pathogenesis of PEX is not yet fully understood. It is multifactorial with genetics and ageing as contributing factors. We aimed to study the proteome in aqueous humor (AH) in PEX in order to increase the knowledge about its pathophysiology. Therefore, we developed sampling techniques and evaluated separation methods necessary for analyzing small sample volumes. Other objectives were to study the lens capsule in eyes with PEX regarding small molecules, and to investigate the association between PEX and cataract surgery in a population-based 30-year follow-up study. Samples of AH from eyes with PEX and control eyes were collected during cataract surgery. In pooled, and individual samples, various liquid based separation techniques and high resolution mass spectrometry were utilized. For quantitation, various methods for labeling, and label free techniques were applied. Lens capsules were collected from some of the patients, and analysed by imaging mass spectrometry. A cohort of 1,471 elderly individuals underwent a comprehensive ophthalmological examination at baseline. Medical information was obtained by questionnaires, and from medical records. Incident cases of cataract surgery were identified by review of medical records. In the initial study, several techniques were explored for protein detection, and a number of proteins were identified as differentially expressed. In the individually labelled samples, changes in the proteome were observed. Eyes with PEX contained higher levels of proteins involved in inflammation, oxidative stress, and coagulation, suggesting that these mechanisms are involved in the pathogenesis in PEX. The levels of β/γ-crystallins were significantly increased in PEX, which is a novel finding. In the lens capsules from individuals with PEX, changes in the lipid composition was observed with time-of-flight secondary ion mass spectrometry. These changes remain to be elucidated. By multivariate analysis, lens opacities were the first, and PEX the second most important predictor for cataract surgery, the later accounting for a 2.38-fold increased risk for cataract surgery.

碩士
國立暨南國際大學
財務金融學系
96
This paper examines the long-term relationships between iTraxx index and stock index. By using Engle-Granger test, we can find that there exist cointegration between iTraxx index and stock index in financial area, including Asia ex Japan, Europe and Australia. Only the market of Japan doesn’t have cointegration. In the result of causality test, we also find that there exist causality from stock index to iTraxx index in the financial markets of Australia, while in Asia ex Japan and Europe exist reciprocal causality.

iTrak is a combined mobile and web application that takes advantage of the GPS to allow travelers to share their experience while travelling. The application gathers GPS data and broadcasts it via a web interface or social networks such as Facebook to update user’s status during a trip. iTrak is also equipped with other features such as writing notes or recording video journals to offer a rich experience and provide an interactive diary, along with a real-time tracking ability, for travelers.
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碩士
國立暨南國際大學
財務金融學系
100
The purpose of this study is to explore effects between the credit default swap （CDS） market and other market. This paper examines the spillover effect between the changes in the iTraxx 5-year Europe Credit Default Swaps （CDS） index and its determinants by using the vector auto regression （VAR） model. We find that iTraxx could affect other indexes, but other indexes could not affect iTraxx. To test the explanatory power of subprime mortgage crisis, we study both of pre-crisis and crisis period. The result shows that no mater pre-crisis or crisis period, iTraxx a will affect S&P, SX5P and VSTOXX. In particular, we show that iTraxx CDS indexes have large effect during crisis period then pre-crisis. And then we examine European sovereign debt crisis, we found the same result. Our results show that credit default swaps have a significant effect on market, and it is an important factor on economics.

This thesis provides an update to the Aboriginal and environmental histories of the ‘high country’ in southeastern Australia from the terminal Pleistocene to the recent past. Its focus is the Namadgi Ranges, representing the northern-most outliers of the Australian Alps. The study combines archaeological excavations of rock shelter sites – from Wee Jasper in the north to the southern Namadgi valleys – with environmental reconstructions from adjacent peatlands. In a context of changing local environments, the findings provide new perspectives on when and how Aboriginal people were active in the mountains, and allow for a re-evaluation of existing archaeological models of occupation and technological change. AMS radiocarbon dates, sediment geochemistry, quantitative stone artefact analyses and other proxies contribute to solidifying the chronology and characteristics of high country habitation. Evidence of terminal Pleistocene activities is found at Wee Jasper in the Namadgi foothills, but remains elusive at higher elevations (> 1000 m). The revised datasets also reveal a previously unidentified period of low-intensity habitation across the ranges from the early to mid Holocene period (8000 to 5000 BP), possibly in response to a Holocene ‘climatic optimum’. The new evidence suggests that people may have largely abandoned the high country from 5000 or 4500 BP. From 2000 BP, however, evidence of habitation reappears, culminating in evidence for a maximum of occupation during the past 1000 years. In combination with an evaluation of known archaeological data from the high country and around its margins, the findings presented herein contradict several existing occupational and technological models, and also caution against the application of broad-scale cultural models across southeastern Australia. A regional environmental history is constructed by analyses of sediments that started to build up 16,000 years ago as the climate warmed. Fire event reconstruction based on charcoal, stratigraphic clues in peat sediments, geochemical signatures of landscape productivity and instability, and a faunal record from Wee Jasper provide a detailed record of change. Comparison of the archaeological and palaeoenvironmental datasets reveals a potential link in these two records, in the form of a tentatively identified signal of anthropogenic burning during the early to mid Holocene. More generally, the environmental history provides a backdrop of changing climates and landscape processes to which Aboriginal people adapted and responded over thousands of years.

碩士
長庚大學
生物醫學研究所
97
Base on the Statistic of Department of Health, Executive Yuan, R.O.C., bladder cancer is the secondarily malignant cancer in urinary tract cancer. The body count is increasing in bygone years. As yet many biomarkers for detecting bladder cancer have been tested and reported, but none of them have shown sufficient sensitivity and specificity. During recent years, discovering new biomarkers of varied diseases in body fluid is prevalent. Urine is a non-invasive specimen and represents powerful potential in discovery of tumor-derived molecules released directly from urinary tract system. In our study, we combined the 14 abundant proteins depletion system, isobaric tagging for relative and absolute quantitation (iTRAQTM) approach and LC-MS/MS to discover new bladder cancer biomarkers in urine. The Multiple Affinity Removal System- Human 14 (MARS-Hu14) can specifically deplete the 14 abundant proteins in urine and decrease the masked effect of high abundant proteins in detection. It is expected to enrich the pool of low-abundant proteins for enhancement of detection by LC-MS/MS. In the preliminary results, the significance of 14 proteins depletion has been shown on urine specimens, particularly the urine of bladder cancer patients. Furthermore, the iTRAQTM approach coupling with the LC/MS/MS technique has been applied to study differential proteomics in urine specimens of control and bladder cancer patients for the elucidation of disease progression at the proteome level. We have identified total 644 proteins from the urine proteome. 152 proteins were up-expressed and 130 proteins were down-expressed in the urine of bladder cancer patients. We have selected several candidates from iTRAQTM results for validation by western blotting in large number of individual urine samples currently. The roles of the urine candidates in bladder cancer have not been reported. The results will provide new insights into the molecular nature of this disease. Moreover, the biomarker candidates discovered in this study will led to new opportunities for the understanding detection, treatment and prevention of bladder cancer.

碩士
國立臺灣師範大學
化學系
102
Hepatocellular carcinoma (HCC) is one of the most prevalent and mortal cancer in the world, and 20% to 30% of patients with chronically hepatitis C virus lead to liver cirrhosis and liver cancer. Due to genetic variability of hepatitis C virus, the development of antiviral drugs and vaccines becomes a real challenge. In this study, naïve Huh7.5-SEAP cells were established and infected by hepatitis C virus, then isobaric tags for relative and absolute quantitation (iTRAQ) was applied to investigate protein profiles in both acute and chronic infections. The iTRAQ labeled peptides were fractionated by solution isoelectric focusing (sIEF) or hydrophilic interaction liquid chromatography (HILIC), followed by reversed phase nano-LC tandem mass spectrometry analysis. A total of 2615 proteins were identified, and 1816 of them were also quantified. Two-dimensional liquid chromatography technique that employed on iTRAQ labeled peptides provided results with excellent complementarity and orthogonality. Moreover, 78 and 140 differentially expressed proteins were selected in acute and chronic infection cases for GeneGo analysis. As a result, these proteins were found to be associated with cytoskeleton remodeling and cell adhesion. Besides, proteins correlated with insulin resistance, vesicular traffic, actin remodeling and secretory pathway, will be further validated by Western blot, and could serve as novel diagnostic biomarkers for hepatitis C virus infection.

博士
國立臺灣師範大學
化學系
103
Proteomics is a large-scale comprehensive study of a specific proteome, including information on the levels of different types of proteins, their modifications and variations, as well as their interactions and networks, in order to understand biological processes. Recent successes clearly show that mass spectrometry-based proteomics as an essential tool for molecular and cellular biology and for the rising field of systems biology. Two-dimensional fractionation is a useful tool to increase proteome coverage and the dynamic range than single-dimensional LC. In part I of this dissertation, various peptide fractionation strategies that are used for 2D (two-dimensional) separations were evaluated. The use of SCX x RPLC for desalted samples provided superior results in protein identification. These approaches are complementary and allowed 43% more peptides to be identified, when compared with a single fractionation strategy. In part II, LTQ-PQD parameters were optimized in order to used isobaric tags technology for quantitative proteomics. The number of microscans and the target value are the most critical factors in producing intense reporter ions for quantitation. The appropriate normalized collisional energy range for PQD could be very narrow and must be carefully determined. The optimized LTQ-PQD parameters were introduced to a murine air pouch membrane in part III. iTRAQ-based approach coupled with offline 2D LC-MS/MS proteomics technology was applied to analyze the protein expression profile using an inflamed murine air pouch membrane as a model. Statistical analyses revealed that 317 proteins are differentially expressed, at least at one time point, after the MSU treatment, that they are mainly involved in the complement system and activation of NALP3 inflammasome. Moreover, the TCA cycle was found to be down-regulated at both the translational and transcriptional levels. Lastly, pyruvate carboxylation was found to be a potential target for an anti-gout treatment. These results provide novel insights into the nature of gouty inflammation.

碩士
國立臺灣師範大學
化學系
105
Hepatocellular carcinoma (HCC) is one of the most prevalent and mortal cancer in the world, and 20% to 30% of patients with chronically hepatitis C virus (HCV) lead to liver cirrhosis and liver cancer. Most infected acute HCV people develop chronic HCV easily. Due to genetic variability of HCV, the development of antiviral drugs and vaccines becomes a real challenge. In this study, naïve Huh7.5-SEAP cells were established and infected by hepatitis C virus, then isobaric tags for relative and absolute quantitation (iTRAQ) was applied to investigate protein profiles in acute and chronic infections. The iTRAQ labeled peptides were fractionated by solution isoelectric focusing (sIEF), strong cationic exchange chromatography (SCX) and basic reverse phase chromatography (bRP) column, followed by nano-LC tandem mass spectrometric analysis. Two-dimensional liquid chromatography technique that employed on iTRAQ labeled peptides provided results with excellent complementarity and orthogonality. In the study, the differentially expressed proteins were found to be associated with RNA binding, extracellular exosome, melanosome and ribonucleoprotein complex binding. Besides, selected correlated proteins will be confirmed and validated by Western blot in the future, they could serve as novel diagnostic biomarkers for hepatitis C virus infection.

Influenza viruses cause significant mortality and morbidity worldwide due to seasonal oubreaks as well as occassional, and sometimes devastating, pandemics. Estimates state that approximately 5% of the adult and 20% of the child population is infected yearly, leading to approximately a half-million deaths and three million severe infections in non-pandemic years. Aside from globally-circulating strains, zoonotic outbreaks caused by avian strains are a constant threat. In 1997, the first human cases of H5N1 infections occurred and since then strains of this subtype have killed approximately 700 people causing a severe disease with as high as 60% lethality rate. In March 2013, a strain of the H7N9 subtype started an epizootic in China causing a severe respiratory disease reminiscent of H5N1 infections and with a 20% case fatality rate. In this thesis, we have studied the host responses as well the viral replication kinetics of H5N1 and H7N9 strains and compared then to those of mild H1N1 seasonal and 2009 pandemic strains. During early infections of A549 cells, we have shown that the H5N1 virus induced a more profound and functional change to the host proteome. All viruses induced the NRF2-mediated oxidative stress responses and the H7N9 and H5N1 strains downregulated fibronectin, a host protein vital to infection for human strains. Using mathematical modeling and extensive growth kinetic analysis, we showed that the H5N1 and H7N9 strains had higher peak titers and faster growth kinetics. This was due to an higher infection rate for the H7N9 strain and an higher production rate for the H5N1 strain, compared to the human viruses. Conversely, the 2009 pandemic H1N1 strain had the poorest replication kinetics, longest eclipse phase and lowest infection rates. These results point towards the higher level of cellular disruption during infection with highly pathogenic strains of influenza, which may be indicative of the more profound changes required to support growth of viruses with faster kinetics to higher titers. Furthermore, the greater changes in the cellular proteome that we have characterized in vitro may be connected to the significantly greater virulence associated with infection by avian viruses in vivo, opening a novel and productive avenue of investigation to understand viral virulence mechanisms.
October 2015

Proteomic analysis of breast cancer tissue has proven difficult due to its inherent histological complexity. This pilot study presents preliminary evidence for the ability to differentiate adenoma and invasive carcinoma by measuring changes in proteomic profile of matched normal and disease tissues. A dual lysis buffer method was used to maximize protein extraction from each biopsy, proteins digested with trypsin, and the resulting peptides iTRAQ labeled. After combining, the peptide mixtures they were separated using preparative IEF followed by RP nanoHPLC. Following MALDI MS/MS and database searching, identified proteins were combined into a nonredundant list of 481 proteins with associated normal/tumor iTRAQ ratios for each patient. Proteins were categorized by location as blood, extracellular, and cellular, and the iTRAQ ratios were normalized to enable comparison between patients. Of those proteins significantly changed (upper or lower quartile) between matched normal and disease tissues, those from two invasive carcinoma patients had >50% in common with each other but <22% in common with an adenoma patient. In invasive carcinoma patients, several cellular and extracellular proteins that were significantly increased (Periostin, Small breast epithelial mucin) or decreased (Kinectin) have previously been associated with breast cancer, thereby supporting this approach for a larger disease-stage characterization effort.

The study of leptospiral pathogenesis is hampered by the lack of efficient mutagenesis methodologies. Thus research has focused on alternative approaches including genome sequencing, comparative genomics, transcriptomics and proteomics. In this thesis a comparative proteomic approach was used to identify leptospiral proteins with a potential role in the leptospiral infection process. Identification of proteins was followed by characterization of target proteins with potential roles in the infection process and ultimately led to the identification of a novel leptospiral virulence factor. Specifically, comparative proteomics using isobaric tags for relative and absolute quantitation complemented with two-dimensional gel electrophoresis were used for mass spectrometry-based protein identification and quantitation. These methodologies were utilised to identify and quantitate leptospiral proteins altered in expression in response to growth media limited in iron supply and/or supplemented with serum. These conditions were designed to mimic a subset of variables encountered by the bacteria within the host. These experiments led to the identification of five proteins with potentially novel roles in the leptospiral infection process. One of these proteins was further characterized as a periplasmic catalase, KatE. Using insertion mutagenesis it was demonstrated that KatE enhances extracellular H2O2 resistance and is required for virulence in guinea pigs and hamsters. Proteomic analyses also led to the identification of glutamic acid methylation of a protein that was further characterised to be surface exposed and expressed during leptospiral colonization of hamster liver and kidneys. This was the first description of glutamic acid methylation of a surface exposed protein in Leptospira.
Graduate

Glaucoma is a leading cause of irreversible blindness worldwide. Primary Open Angle Glaucoma is the most common form of the disease and can be characterized by the slow and irreversible apoptotic death of retinal ganglion cells, a unique optic nerve neuropathy resulting in loss of vision. Increased intra-ocular pressure is known to be a leading risk-factor for glaucoma, and lowering IOP is currently the only evidence based method for the clinical management of the disease. However the exact mechanism by which an elevated IOP leads to the death of the retinal ganglion cells is still poorly understood. By using previous finite element models of glaucoma to quantify the biomechanical environment within the optic nerve head we have built human primary cell culture models in an attempt to replicate aspects of early glaucomatous optic neuropathy. In these models we mimic the in vivo biomechanical environment in the lamina cribrosa by growing human optic nerve head astrocytes and lamina cribrosa cells on compliant substrates and subjecting the cells to deformation. Specifically, a global protein scan using isobaric tags for relative and absolute quantitation (iTRAQ) was performed on all the experiments to identify potential biomarkers for glaucoma. A secondary analysis using enzyme-linked immunosorbent assay (ELISA) identified extracellular proteins of interest. Over 520 proteins were identified in response to biomechnical strain from both cell types. Many of these proteins centred on TGF-, p53 and TNF, which have previously been shown to play a role in the pathogenesis of glaucoma. Proteins found in astrocytes were astrocytic phosphoprotein (PEA15), UDP-glucose dehydrogenase (UGDH), and annexin A4 (ANXA4). LC proteins were bcl-2-associated athanogene 5 (BAG5), nucleolar protein 66 (NO66) and Eukaryotic translation initiation factor 5A (eIF-5A). These proteomic results will enable a series of functional studies looking into the role select markers play in ONH glial cell activation, a process still not well understood. Candidates for this work will be prioritized based on novelty and relevance to mechanisms of cellular stress and death. We hypothesize that study of these molecular pathways will provide insight into this process, as well as improve our understanding of how glial activation contributes to the development of glaucomatous optic neuropathy.

The overall goal of this research was to investigate oxidative stress related changes to the proteomes of 3T3-L1 adipocytes and an inducible CYP2E1 expressing HepG2 cells. Enhanced oxidative stress in hypertrophic adipocytes is associated with metabolic dysregulation and insulin resistance. Because mitochondria generate reactive oxygen species (ROS), we monitored changes to the adipocyte mitochondrial proteome during differentiation and enlargement. We labeled mitochondrial extracts from 3T3-L1 cells that were 0, 4, 7, 10, 14, and 18 days post differentiation with iTRAQ, followed by MS based identification. We found citric acid cycle proteins such as pyruvate carboxylase, citrate synthase, as well as beta-oxidation enzymes; cartinine acyl transferase and long-chain enoyl-CoA hydratase up-regulated from 7 through 18 days post differentiation onset. These data indicate TCA up-regulation for enhanced metabolic and citrate output necessary for lipid synthesis in adipocytes. Paradoxically, the data also show the simultaneous increase in the fatty acid oxidation, indicating a metabolic overdrive state. Biochemical assays showing peaks in ATP and ROS generation in 3 day old adipocytes provide further evidence of this overdrive state. A second peak in ROS generation occurred in 10 day old adipocytes; concurrent ATP generation reduced to near pre-adipocyte levels and this may indicate a metabolic shift that may be responsible for increased oxidative stress in hypertrophic adipocytes. We developed a doxycycline inducible CYP2E1 expressing HepG2 cell line using the pTet-On/pRevTRE expression system to allow greater control and sensitivity in the generation CYP2E1 mediated oxidative stress. Our cell line (RD12) demonstrated stability and tight expression control. After induction, RD12 cells showed 30 percent higher CYP2E1 activity when compared to the constitutive E47 cell line. RD12 cells showed 30 percent greater toxicity than E47 cells and 25 percent less free glutathione when exposed to 20 mM acetaminophen, indicating RD12 cells are more sensitive to the effects reactive intermediates and oxidative stress generated by CYP2E1. We conducted a survey of the toxicity of dietary fatty acids (oleic, linoleic, and palmitic) on HepG2 cells to determine fatty acid doses that induced metabolic changes, but did not cause excessive cell death. The dose of 0.20 mM linoleic and palmitic acid for 48 hours produced low toxicity, but oleic acid actually produced lower toxicity than untreated cells. After exposure cells were treated with a pro-oxidant to determine which fatty acid increased the susceptibility to protein carbonylation. The carbonylated protein isolation procedure indicated the palmitic acid may induce more carbonylation than oleic acid, but greater efficiency in the isolation procedure is required for a confidant determination.

Yes
Matched healthy and diseased tissues from breast cancer patients were analyzed by quantitative proteomics. By comparing proteomic profiles of fibroadenoma (benign tumors, three patients), DCIS (noninvasive cancer, three patients), and invasive ductal carcinoma (four patients), we identified protein alterations that correlated with breast cancer progression. Three 8-plex iTRAQ experiments generated an average of 826 protein identifications, of which 402 were common. After excluding those originating from blood, 59 proteins were significantly changed in tumor compared with normal tissues, with the majority associated with invasive carcinomas. Bioinformatics analysis identified relationships between proteins in this subset including roles in redox regulation, lipid transport, protein folding, and proteasomal degradation, with a substantial number increased in expression due to Myc oncogene activation. Three target proteins, cofilin-1 and p23 (increased in invasive carcinoma) and membrane copper amine oxidase 3 (decreased in invasive carcinoma), were subjected to further validation. All three were observed in phenotype-specific breast cancer cell lines, normal (nontransformed) breast cell lines, and primary breast epithelial cells by Western blotting, but only cofilin-1 and p23 were detected by multiple reaction monitoring mass spectrometry analysis. All three proteins were detected by both analytical approaches in matched tissue biopsies emulating the response observed with proteomics analysis. Tissue microarray analysis (361 patients) indicated cofilin-1 staining positively correlating with tumor grade and p23 staining with ER positive status; both therefore merit further investigation as potential biomarkers.
Cyprus Research Promotion Foundation, Yorkshire Cancer Research

This thesis is focused on the credit derivatives market. The aim is to identify and quantify the causal dependence of the development of credit derivatives market in relation to the dynamics of macroeconomic indicators and on this basis the possible prediction. At first, it shows with the help of literature review of their present and history and gives a better look to the different types of credit derivatives. The following section deals with the use of these underlying instruments in practice, their possible trading, insurance, or speculation, whether on the OTC markets or organized exchanges. The following describes the events in the capital and commercial markets during the financial crisis that is between the years 2005-2010, from which are taken the data for the empirical part. The empirical part is based on correlation analysis (multiple regression model) of a few selected and described macroeconomic indicators enriched with Granger causality test. In the conclusion may be find the discussion of the results and possible recommendations for potential investors.

O presente estudo investiga o impacto das alterações da taxa de juro interbancária, Euribor, no risco dos activos bancários. Com a recente crise financeira, o efeito da turbulência no mercado interbancário criou uma necessidade de explorar as forças que foram causa de vários fenómenos incomuns nesse mercado. Por um lado, a Euribor testemunhou valores anormalmente baixos, chegando mesmo a atingir valores negativos. Por outro lado, os spreads dos activos bancários dispararam. Tendo como base a vasta revisão da literatura, foram utilizados dados da Euribor a três meses retirados da Thomson Datastream entre 2000 e 2015 e dados do índice iTraxx fornecidos pela Markit de forma a construir um modelo auto-regressivo com a possibilidade de quebras de estrutura (Bai e Perron, 1998; 2003a). Concluiu-se que (i) a Euribor não é o principal instrumento de explicação para o comportamento dos spreads nos mercados financeiros; e que (ii) com o nosso modelo econométrico, não é possível obter inferência estatística sobre a previsão da Euribor adicionando a componente do risco bancário, iTraxx. É evidenciado na nossa análise o problema de co-breaking.
This study investigates how Euribor rate affects the risk on banks’ assets. With the recent financial crisis, the effect of turbulence on interbank markets has created a need to explore forces that have caused multiple uncommon phenomena in markets. On the one hand, Euribor rate witnessed drastic values, specifically reached values below zero. On the other hand, the volatility of banks’ risk spreads soared. Considering the vast literature review, we use three-month Euribor dataset from Thomson Datastream between 2000 and 2015, and iTraxx dataset from Markit to construct an autoregressive model, addressing the possibility of structural breaks (Bai and Perron, 1998; 2003a). We concluded that (i) the Euribor is not the main instrument to explain the behavior of spreads in the financial markets; and (ii) with our econometric model we are unable to obtain statistical inference to draw conclusions about Euribor predictions based on iTraxx series. The co-breaking problem stands out in our analysis.

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.