Thèses sur le sujet « Proteomic studie »

Tato práce se zabývá proteomickými studiemi ječmene v souvislosti s výrobou piva. Ječmen patří mezi nejvýznamnější plodiny na světě a je využíván hlavně pro sladovnické účely, nejčastěji pro pivovarnictví. Studium proteinů ječmene během sladování a výroby piva poskytuje informace o změnách v proteinovém složení nebo jejich posttranslačních modifikacích. Jelikož jsou proteiny v ječmeni a jejich změny zásadní pro kvalitu sladu a piva, proteomické studie ječmene mají potenciál pro zlepšení procesu sladování a pivovarnictví. Hlavním cílem této práce je studium ve vodě rozpustných proteinů ječmene a jejich změn, ke kterým dochází během sladování a výroby piva. Rozdíly v proteinovém složení byly sledovány pomocí gelové elektroforézy, kapalinové chromatografie na reverzní fázi, gelové chromatografie a MALDI-TOF hmotnostní spektrometrie. Během sladování se vlivem klíčení zrna zvyšuje množství některých proteinů a také jsou tvořeny nové proteiny. V průběhu vaření piva se naopak v důsledku vysoké teploty a enzymatické aktivity proteáz mnoho proteinů rozkládá. Těmto drsným podmínkám odolají jen některé proteiny, které přechází až do piva a mohou ovlivnit jeho kvalitu. Dále byly zkoumány různé odrůdy ječmene a jejich rozdíly. Byly porovnány odrůdy povolené pro výrobu certifikovaného Českého piva s jednou osvědčenou sladovnickou odrůdou a jednou nesladovnickou odrůdou ječmene. Kromě toho byly studovány v alkoholu rozpustné proteiny ječmene a jejich změny v průběhu sladování. Zvláštní pozornost byla věnována vybrané skupině posttranslačních modifikací proteinů: glykosylacím. Neenzymaticky glykosylované proteiny ječmene (neboli glykované proteiny) jsou tvořeny v průběhu sladování kvůli přítomnosti velkého množství glukózy uvolněné z rozkladu škrobu. Glykované proteiny ovlivňují stabilitu proteinů a kvalitu piva, obzvlášť pěnotvorný účinek. Enzymatické N-glykosylace představují nejčastěji studované posttranslační modifikace u rostlin, protože glykoproteiny hrají klíčovou roli v různých biologických funkcích. Glykoproteiny jsou často přítomny v malém množství, a proto je pro jejich analýzu potřebné obohacení glykoproteinů z komplexní směsi. Pro studium glykoproteinů byla využita afinitní chromatografie s lektinem concanavalin A. Kromě toho byla také optimalizována analýza sacharidové části glykoproteinů. Tato disertační práce přináší důležité informace o proteinech ječmene, jejich změnách a analýze, které budou užitečné pro další studium.

This thesis provides the basis for a label-free bioanalytical platform using electrochemical analysis at liquid –liquid interfaces. The possibility to detect biomolecules such as proteins in a label-free manner via adsorption and ion-transfer was achieved. Several pre-treatment steps used in proteome analysis, such as protein pre-concentration and digestion, were studied. The results demonstrate the promise of this strategy for the detection and identification of proteins.

Contesto scientifico. I glicani rivestono un ruolo cruciale nel sistema nervoso centrale (SNC) e il loro studio è fondamentale per un'accurata comprensione della neurochimica, ma la conoscenza scientifica riguardo ai glicani presenti nel SNC è ancora limitata. Lo scopo di questa tesi è fornire nuovi strumenti al glicochimico e al glicoanalista per condurre studi di neurochimica, funzionali all'esplorazione del ruolo dei glicani nel SNC. Questa tesi presenta un nuovo metodo analitico per lo studio degli N-glicani da tessuti cerebrali (LSD); lo stato dell'arte di un lavoro in corso riguardante l'investigazione di N-glicani e N-glicoproteine differentemente espresse in tessuti cerebrali appartenenti a diverse specie; un efficiente metodo di marcatura chimica per (glico)proteine, applicato con successo a una N-glicoproteina, Neuroserpina (NS), che polimerizza nel SNC con conseguenze patologiche; e le sintesi di glicosidi e glicodendrimeri con potenziali applicazioni in studi neurochimici. Metodi. LSD si sviluppa a partire dalla lisi chimica di tessuti cerebrali (tc), con conseguente precipitazione del proteoma (i.e., metanolo/cloroformio), deglicosilazione enzimatica (i.e., PNGase F), purificazione degli N-glicani, marcatura chimica (i.e., amminazione riduttiva su N-acetilglucosammina terminale) e analisi tramite approcci LC-MS. LSD è stato ottimizzato su campioni di tc e accuratamente validato (i.e., sensibilità, precisione, linearità, range dinamico, selettività, robustezza). L'analisi degli N-glicani è stata anche effettuata tramite deglicosilazione enzimatica in-gel da elettroforesi di proteine, mentre un protocollo di digestione con Tripsina in-gel è stato usato per l'identificazione di N-glicoproteine e siti di N-glicosilazione tramite approcci LC-MS su peptidi. NS è stata dimetilata chimicamente (i.e., amminazione riduttiva su lisina), nella sua forma monomerica (mhNS) e polimerica (phNS) e l'esito della reazione è stato valutato tramite MS, per l'investigazione dei determinanti molecolari coinvolti nel meccanismo di polimerizzazione. I glicosidi sono stati sintetizzati usando una reazione di glicosilazione di tipo Fischer, condotta su monosaccaridi deprotetti, usando alcol allilico e decenolo come accettori glicosidici, mentre i glicodendrimeri sono stati ottenuti sfruttando la chimica delle ossime per la decorazione con gruppi maltosilici di dendrimeri sintetizzati tramite metatesi delle olefine. Risultati. LSD presenta il più basso limite di rivelabilità (1 mg di tc) rispetto a molti altri lavori riportati in letteratura ed è attualmente il metodo di neuro-N-glicomica più accuratamente validato. La deglicosilazione in-gel per l'analisi degli N-glicani cerebrali ha prodotto cromatogrammi informativi per ogni frazione del proteoma con elevata risoluzione (e.g., sensibilità fino a 100 EU da una singola banda di gel), permettendo l'analisi dei peptidi deglicosilati dallo stesso campione (i.e., un totale di 1200 peptidi, 570 proteine, 57 N-glicoproteine, e nuovi siti di N-glicosilazione identificati). La marcatura chimica di NS si è dimostrata efficiente (i.e., 80-90% di resa), compatibile con la struttura nativa della proteina, e funzionale all'uso designato, in quanto capace di evidenziare differenze statisticamente significative nel profilo di marcatura di mhNS e phNS (i.e., 9 lisine). La sintesi di glicosidi ha fornito prodotti con buona resa (e.g., 70%) e alfa-stereoselettività, mentre quella dei glicodendrimeri ha generato molecole esponenti gruppi maltosilici, utilizzabili nel contesto di studi di neurochimica. Conclusioni. I metodi e le molecole contenuti in questa tesi apporteranno beneficio alla comunità scientifica glicochimica, incrementando il numero di strumenti che il glicochimico e il glicoanalista possono utilizzare per portare avanti lo studio degli effetti dei glicani in contesto neurologico e neuromedico.
Background. Glycans play crucial roles within the central nervous system (CNS) and their study is essential for a thorough comprehension of neurochemistry, but the scientific knowledge about CNS glycans remains scarce. The aim of this thesis is to provide the glycochemist and glycoanalyst with novel tools for neurochemistry studies, towards the exploration of glycan roles in the CNS. This thesis presents a novel analytical method for brain N-glycans investigation (LSD); the state of the art of an ongoing work for the investigation of N-glycans and N-glycoproteins differentially expressed in brain tissues of different species; an efficient chemical labelling method for (glyco)proteins, successfully applied on Neuroserpin (NS), a pathologically-polymerising CNS N-glycoprotein; and the syntheses of glycosides and glycodendrimers with potential room for neuromedical studies. Methods. LSD comprised brain tissue (bt) chemical lysis, proteome precipitation (i.e., methanol/chloroform), enzymatic deglycosylation (i.e., PNGase F), N-glycans purification, chemical labelling (i.e., reductive amination on terminal N-acetylglucosamine), and LC-MS bioanalysis. The method has been optimised on bt and thoroughly validated (i.e., sensitivity, precision, linearity, range, selectivity, robustness). N-glycans analysis has also been carried out through protein electrophoresis in-gel deglycosylation, while in-gel trypsinisation was used for the LC-MS identification of N-glycoproteins and N-glycosylation sites. NS has been dimethylated (i.e., reductive amination on lysine) in its monomeric (mhNS) and polymeric (phNS) forms, and the reaction outcome has been evaluated using MS, towards the investigation of NS polymerisation-driving molecular features. Glycosides were synthesised with a Fischer- type glycosylation reaction on unprotected monosaccharides using either allyl alcohol or decenol as glycosyl acceptors, while glycodendrimers were obtained decorating olefin-metathesis-synthesised dendrimers with maltose moieties, exploiting oxime chemistry. Results. LSD displayed the lowest detection limit (1 mg of bt) in comparison to many other works reported in the literature and is the most thoroughly validated neuro-N-glycomic method reported to date. In-gel deglycosylation for brain N-glycans analysis furnished informative chromatograms for every proteome fraction with high resolution (e.g., sensitivity up to 100 EU from a single gel band), permitting the analysis of deglycosylated peptides from the same sample (i.e., a total of 1200 peptides, 570 proteins, 57 N-glycoproteins, and novel N-glycosylation sites identified). NS chemical labelling displayed high efficiency (i.e., 80-90% yield), compatibility with the protein folding, and suitability towards the intended purpose, being able to highlight statistically significant differences in mhNS and phNS labelling patterns (i.e., 9 lysines). The syntheses of glycosides furnished products with good yield (i.e., 70%) and a- stereoselectivity, while that of glycodendrimers afforded molecules exposing several maltose moieties, employable in the context of neurochemistry studies. Conclusions. Methods and molecules delivered within this thesis will benefit the glycochemistry community, by enlarging the glycochemist and glycoanalyst toolkits to carry on the investigation of glycans- related effects in neurological and neuromedical context.

Pierce’s disease (PD), caused by bacterium Xylella fastidiosa, seriously hampers the cultivation of Vitis vinifera also known as bunch grapes, in different parts of the world. The bacterium clogs xylem vessels and forms a biofilm, resulting in the wilting of the plant. Bunch grape cultivars exhibit certain degree of tolerance to PD, however most commercial cultivars suffer heavy loss due to this devastating disease. Therefore, studies on genetic variation for disease tolerance will assist in identification of key molecular components that confer tolerance to PD. Vitis species, such as, Florida hybrid bunch (FH) and muscadine grape ( Vitis rotundifolia) are widely cultivated in southeastern United States, and are known for their tolerance to PD. A detailed proteomic profile study of contrasting grape species is vital to understand the biological molecules associated with the PD tolerance. However information on total protein composition of Vitis xylem and sap is limited. The overall goals of this study are to determine the signal sequences associated with xylem and sap for the delivery of therapeutic proteins to control Xylella fastidiosa. The specific objectives of this research project are: 1) to compare the proteome profiles of xylem tissue and xylem sap from PD tolerant and -susceptible grapevine cultivars, and 2) to determine the role of proteins in the tissue and sap associated with PD tolerance mechanism. In this study, we used Bunch, FH, and Muscadine grape cultivars to characterize differentially expressed and unique proteins. Differentially expressed proteins were identified using LC MS/MS spectrometry searched against Vitis database. A total of 2519 and 402 proteins were identified in xylem and sap respectively, of which 151 proteins were common to both tissues. Bunch, FH, and muscadine sap showed 52, 53, and 30 unique proteins respectively. The cluster dendrogram analysis of the sap proteome showed that all of the Vitis species are bifolious. Based on the aforementioned, Florida hybrid bunch and muscadines are more closely related to each other than to bunch grape. Functional analysis and gene ontology revealed that proteins involved in carbohydrate metabolic process are more abundant in bunch grape, while FH and muscadine grape have more defense related proteins. Therefore, it is plausible to conclude that major functions of sap proteins in Bunch, FH, and Muscadine grapes are carbohydrate metabolic process and proteolysis (23%), protein phosphorylation (38%), and oxidation and reduction process (16%), respectively. Proteins involved in the defense and peroxidase activity are abundantly present in xylem and sap of FH and muscadine, and these proteins are relatively in reduced levels in bunch xylem and sap. Together, our findings highlight the possible roles of the identified unique proteins towards PD tolerance to Florida hybrid bunch and muscadine cultivars.

The principal objectives of the study presented in this thesis were to study the changes of milk proteomes, peptidomes and metabolomes during the course of bovine mastitis in comparison with normal milk samples and to discover new bovine mastitis biomarkers using various modern and up-to-date methodologies such as proteomics, peptidomics and metabolomics. Bovine mastitis caused by bacterial infection of the mammary gland of dairy cows is often associated with loss of milk production due to a reduction in milk composition and quality which in turns, lead to negative economic impact on dairy industry. Two important acute phase proteins (APPs) which serve as valuable biomarkers in bovine mastitis were investigated in every chapter using developed and validated enzyme linked immunosorbent assay (ELISA) for bovine milk haptoglobin and commercially available ELISA for bovine milk serum amyloid A3 (M-SAA3). These APPs were quantified alongside somatic cell counts (SCC) and California Mastitis Test (CMT) to confirm the disease status of each animal used in this study. Proteomic methodologies were applied including 1D gel electrophoresis, 2D gel electrophoresis, MALDI-TOF analysis and difference gel electrophoresis to investigate the changes of milk proteome in both subclinical and clinical mastitic milk samples in comparison with healthy milk samples. However these investigations did not reveal novel biomarkers for mastitis. Next, peptidomic methodologies were used to study the changes in milk peptidome and to detect the presence of any significant disease biomarkers in the presence of bovine mastitis by using CE-MS and LC-MS/MS. A total of 31 and 14 polypeptides can be used to discriminate control from infected groups and E. coli from S. aureus infected groups respectively. Lastly, metabolomic methodology was applied with an intention to study the changes in milk metabolome and ultimately to detect the presence of novel biomarkers in bovine mastitis. Di- and tri-peptides were found higher in S. aureus than in E. coli infected groups and based on metabolic pathways, arachidonic, arginine and galactose metabolites were seen increased in mastitic milk samples in comparison to healthy milk samples. Overall, the findings detailed in this thesis indicate that the use of advanced proteomic and metabolomic methodologies could deliver on their promise of the discovery of potential significant bovine mastitis biomarkers. Further studies are needed for validation of these proposed biomarkers and it was hoped that better prevention and treatment methods for bovine mastitis can be achieved in the future.

Background: Enamel Renal Syndrome is an autosomal recessive disorder that is characterised by nephrocalcinosis and amelogenesis imperfecta where the causative gene is FAM20A. Unlike most cases of nephrocalcinosis where hypercalciuria is its instrumental cause, patients with ERS have normocalciuria, suggesting an atypical mechanism in calcium handling by the kidneys. Methods: Detection of recombinant FAM20A was performed on HeLa cells and its culture media by Western analysis. Plasma and gingival fibroblasts were used for the detection of FAM20A in control and ERS samples by two-dimensional gel electrophoresis. Human milk was utilised for the quantitation of FAM20A using both shotgun and targeted proteomics approach. Mouse kidneys were used for differential protein expression and functional proteomic analysis to help understand how the absence of FAM20A can severely impact calcium homeostasis. Results: A low amount of FAM20A was detected in human milk. Results from plasma samples and gingival fibroblasts were inconclusive. Differential protein expression profiling of kidneys revealed S100 calcium-binding protein A9 to be 2.9 fold up-regulated in the Fam20a knockout mouse. This protein is a major determinant of arterial calcification. Functional analysis indicated disturbed calcium-regulation mediated by mitochondria and peroxisomes in the absence of Fam20a. Conclusion: The removal of FAM20A triggers a disruptive ripple in the finely tuned intricate pathway of proteins all entangled to regulate calcium homeostasis; a consequence of which leads to ectopic calcification within the interstitium.

Mass spectrometry has emerged as having a vital role in various applications to biochemical fields. In this thesis, we have utilized a variety of mass spectrometry techniques for both bacteriophage proteomics and colostrum and milk lipidomics studies. Our first study was the proteome characterization of Great Salt Lake bacteriophage NS01 with SDS-PAGE GEL to separate the viral proteins and high performance liquid chromatography (HPLC) coupled with an LTQ Orbitrap to identify the proteins after in-gel digestion. In this project, we have successfully identified 11 proteins with high confidence, p-values < 0.01, including coat protein gp88 with a coverage of 91% and tail protein gp86 with a coverage of 40.96%, which facilitated the classification of NS01 as a T7-like phage. Our second study was the discovery of colostrum and milk biomarkers that can be used to predict the likelihood of development of production-related metabolic diseases (PRMDs) in dairy cows through a lipidomics approach. In this study, an electrospray ionization, time-of-flight mass spectrometer was applied to lipid profiling, quantification and significant biomolecule selection. A Q-Star quadrupole, orthogonal time-of-flight mass spectrometer and an Agilent 6530 accurate-mass quadrupole/time-of flight mass spectrometer were both used for lipid biomarker fragmentation and identification. According to linear discriminative statistical modeling, three panels of biomarkers were defined. A combination of 2 milk lipid predictors, including DG18:0/18:0 and TG 18:0/18:0/18:1, provided PRMD predictions with 75.0% sensitivity at 90.0% specificity. A combination of 3 colostrum lipid predictors, including TG16:0/18:1/18:3, DG16:0/16:0 and C40H60NO, provided PRMD prediction with 90.0% sensitivity at 86.4% specificity. Furthermore, a combination of 7 colostrum and milk biomarkers, including calculated differences between 'shared' markers found to be significantly different in both colostrum and milk, provided a predictive sensitivity of 87.5% at a specificity of 100%. Thus, three panels of lipid biomarkers have been discovered in 1-4 day postparturient dairy cow colostrum and milk that can be used to predict resistance or susceptibility prior to onset of clinically apparent PRMDs. These novel lipids could be used as important diagnostic predictors in the future. Therefore, mass spectrometry based proteomics and lipidomics approaches have been efficient tools in the biochemical research described in this thesis.

Dystroglycan is a ubiquitous protein that links the extracellular matrix to the cytoskeleton and is the central unit of the dystrophin glycoprotein complex (DGC), a membrane complex that connects the cytoskeleton to the extracellular matrix (ECM). Dystroglycan is composed of two subunits that are tightly but non-covalently linked. alpha Dystroglycan (alpha DG) is located extracellularly and it is the only component of the DGC linked to the ECM, while beta Dystroglycan (beta DG) spans the plasma membrane and has both an extracellular and a cytoplasmic domain. The DGC is involved in skeletal muscle maintenance and viability, and in the organization and stabilization of the neuromuscular junction, but its function in brain is poorly understood. DGC components are target of several protein kinases, indicating that they are involved in cell signalling pathways. The finding of new dystroglycan interacting proteins could help to obtain some insights in its function in brain tissues. Previous immunoprecipitation and pull down experiments have been used to identify proteins interacting with the cytoplasmic tail of beta DG in brain tissues. Here, we attempt to extend the use of these techniques by using pull down experiments performed with the Glutathione-S-transferase (GST) fusion expression system as a tool for the proteomic analysis of Dystroglycan interacting proteins in the brain.

Schizosaccharomyces pombe est devenu un important système modèle pour étudier les processus physiologiques, biochimiques et génétiques chez l'homme. Ces travaux appartiennent à un projet sur la façon dont la fonction altérée de l'appareil de Golgi contribue à des maladies comme le cancer ou cause des anormalités génétiques. La protéine HID-1 de C. elegans et des humains est une protéine de l'appareil de Golgi qui appartient à la superfamille de protéines DYMECLIN. Les animaux ont à la fois un gène HID1 et un gène DYM. Chez les humains, une expression réduite de HID1 est impliquée dans la prolifération de tumeurs. La perte de DYM chez les humains mène à une déformation squelettique. S. pombe a trois gènes orthologues HID-1, mais pas de DYM. Par contraste, de nombreux eucaryotes unicellulaires et pluricellulaires n'ont qu'un gène DYM. Les mutants de S. pombe sans Hid1 et Hid3 étaient sensibles au stress oxydatif et la croissance de hid3Δ a été stoppée dans des milieux de culture minimum standard. L'insensibilité de hid3Δ au brefeldin A, mais sa sensibilité au golgicide A démontrent que Hid3 fonctionne dans le transport antérograde à travers l'appareil de Golgi. Afin d'explorer des rapports indiquant que le renouvellement des protéines pourrait être modifié dans hid3Δ, j'ai entrepris une étude de protéomique à la quantification label-free. La régulation positive de la voie de signalisation MAPK de tension a démontré que les cellules étaient dans un état de tension dans des conditions normales de croissance. De plus, des composants de protéine dans plusieurs voies de signalisation étaient modifiés, pouvant affecter une large gamme de processus cellulaires
Schizosaccharomyces pombe has become an important model system to study physiological, biochemical and genetic processes in humans. This work is part of a continuing project to study how altered Golgi function contributes to diseases, such as cancer, or causes of genetic abnormalities. The HID-1 protein of C. elegans and humans are peripheral membrane proteins of the Golgi apparatus and are part of the DYMECLIN superfamily of proteins. Animals have both a HID1 and a DYM gene. In C. elegans, HID-1 maintains normal cellular growth and in humans reduced expression of HID1 has been implicated in tumour proliferation. Loss of DYM in humans leads to skeletal deformation and potentially mental retardation. S. pombe has three HID-1 orthologues, but no DYM. In contrast, many unicellular and multicellular eukaryotes have only DYM. S. pombe mutants lacking Hid1 and Hid3 were sensitive to oxidative stress and growth of hid3Δ was stopped in standard minimal media. Insensitivity of hid3Δ to brefeldin A but sensitivity to golgicide A demonstrated that Hid3 operates in anterograde protein transport through the Golgi. In order to investigate reports that protein turnover might be altered in hid3Δ, I undertook a proteomics study using label-free protein quantification. Up-regulation of the MAPK stress signalling pathway demonstrated that cells were under a state of stress under standard growth conditions. In addition, protein components of Ras signalling, microtubule dynamics and chromatin remodelling were altered potentially affecting a wide variety of processes from cell cycle regulation to metabolism

2014 - 2015
It is known that prenatal exposure to pollutants and particularly heavy metals can have long term damaging consequences on infants, due to their accumulation in-body. Since the 1990s, ten million tonnes of waste have been illegally dumped in the area around Caserta and Naples. Thus, direct exposure to waste and heavy metals during the last two decades was very frequent in the so-called “Lands of fires”. The number of children suffering from cancer and of malformed fetuses in Italy's "Land of Fires", an area where toxic waste has been dumped by the mafia, is reported significantly higher than elsewhere in the country. In this thesis we examined the proteome of the umbilical cords from malformed fetuses obtained by therapeutic abortions, after mothers' being exposed to the pollution on “land of fire” during early pregnancy, and analyzed the differences between umbilical cords from malformed fetuses to healthy ones. The main goals were to understand the impact of the contamination by heavy metals on the fetus development, and to identify new putative biomarkers of exposure to metal contaminants. All umbilical cords were obtained in Campania region (Naples and Caserta, mainly in the “land of fires”). The collection of the biological samples was carried out in collaboration with the Caserta Hospital “Sant’Anna e San Sebastiano” and with the Avellino Hospital “San Giuseppe Moscati”. A proteomic approach based on Filter-Aided Sample Preparation (FASP) method was set up and performed. This bio-analytical strategy combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics, greatly reduces the time required for sample preparation and enables more flexibility in sample processing. Protein identification and quantification were performed by matching mass spectrometry data in on-line protein database, using the MaxQuant 1.5.2.8 software. Statistical analyses were employed to identify proteins whose levels were sensibly different in the umbilical cords from malformed fetuses. Gene Ontology (GO) classification was used in order to obtain functional information of the differentially expressed proteins and to correlate them to the embryonic development. Finally, Matrix Metalloproteinases (MMPs) have been shown to play significant roles in a number of physiological processes, including embryogenesis and angiogenesis, but they also contribute to the development of pathological processes. Thus, gelatin zymography technique was performed to detect MMPs enzymatic activity in the umbilical cords. Our results support a significant role of MMPs in the fetus development. [edited by author]
XIV n.s.

There is increasing evidence that the aberrant expression of cancer-testis (CT) antigens - a family of ca. 150 proteins that are both autoimmunogenic and mainly restricted to tumours in various types of human cancers - makes them attractive immunotherapy targets, as well as possible cancer diagnostic markers. We carried out a retrospective serological study of primary and secondary autoimmune responses of various cohorts of cancer patients prior to and/or following a variety of distinct treatments (chemotherapy, radiotherapy and immunotherapy), using a large number of archived human serum samples. Our goals were to develop and validate a novel cancer-testis and -associated antigen microarray platform and to then explore its utility and general applicability in the cancer immunology field. In addition, we sought to cross-correlate our protein microarray data from specific cohorts with in vitro T-cell re-stimulation assays for a selected subset of patients. Furthermore, as a means of determining the biological significance of our protein microarray data, we also collected clinical patient data where possible. The underlying hypothesis of our study was that there were measurable differences in autoantibody repertoires towards tumour-specific and -associated antigens between pre- and post-treated cancer patient samples (using various trial therapies), potentially augmented by prior chemo- or radiotherapy, which would correlate with likelihood of response of individual patients to a given therapeutic treatment - including those treatments that aim to generate T-cell responses - and which would also correlate with the nature and extent of individual patient responses to treatment.

Thesis (M.S. in environmental science)--Washington State University, August 2009.
Title from PDF title page (viewed on Aug. 7, 2009). "School of Earth and Environmental Sciences." Includes bibliographical references (p. 85-94).

Alzheimer’s disease (AD) is a neurodegenerative disease and the major cause of dementia, affecting more than 50 million people worldwide. Chronic pain is long-lasting, persistent pain that affects more than 1.5 billion of the world population. Overlapping and heterogenous symptoms of AD and chronic pain conditions complicate their diagnosis, emphasizing the need for more specific biomarkers to improve the diagnosis and understand the disease mechanisms. To characterize disease pathology of AD, we measured the protein changes in the temporal neocortex region of the brain of AD subjects using mass spectrometry (MS). We found proteins involved in exo-endocytic and extracellular vesicle functions displaying altered levels in the AD brain, potentially resulting in neuronal dysfunction and cell death in AD. To detect novel biomarkers for AD, we used MS to analyze cerebrospinal fluid (CSF) of AD patients and found decreased levels of eight proteins compared to controls, potentially indicating abnormal activity of complement system in AD. By integrating new proteomics markers with absolute levels of Aβ42, total tau (t-tau) and p-tau in CSF, we improved the prediction accuracy from 83% to 92% of early diagnosis of AD. We found increased levels of chitinase-3-like protein 1 (CH3L1) and decreased levels of neurosecretory protein VGF (VGF) in AD compared to controls. By exploring the CSF proteome of neuropathic pain patients before and after successful spinal cord stimulation (SCS) treatment, we found altered levels of twelve proteins, involved in neuroprotection, synaptic plasticity, nociceptive signaling and immune regulation. To detect biomarkers for diagnosing a chronic pain state known as fibromyalgia (FM), we analyzed the CSF of FM patients using MS. We found altered levels of four proteins, representing novel biomarkers for diagnosing FM. These proteins are involved in inflammatory mechanisms, energy metabolism and neuropeptide signaling. Finally, to facilitate fast and robust large-scale omics data handling, we developed an e-infrastructure. We demonstrated that the e-infrastructure provides high scalability, flexibility and it can be applied in virtually any fields including proteomics. This thesis demonstrates that proteomics is a promising approach for gaining deeper insight into mechanisms of nervous system disorders and find biomarkers for diagnosis of such diseases.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2007.
Submitted by Luis Felipe Souza (luis_felas@globo.com) on 2008-11-12T17:50:40Z No. of bitstreams: 1 Dissertacao_2007_AlexandreRosa.pdf: 9082399 bytes, checksum: 9fe59d69d2f47fa7ddf981b510013376 (MD5)
Approved for entry into archive by Georgia Fernandes(georgia@bce.unb.br) on 2008-12-08T18:26:42Z (GMT) No. of bitstreams: 1 Dissertacao_2007_AlexandreRosa.pdf: 9082399 bytes, checksum: 9fe59d69d2f47fa7ddf981b510013376 (MD5)
Made available in DSpace on 2008-12-08T18:26:42Z (GMT). No. of bitstreams: 1 Dissertacao_2007_AlexandreRosa.pdf: 9082399 bytes, checksum: 9fe59d69d2f47fa7ddf981b510013376 (MD5)
Biomedical research commonly starts by raising a hypothesis to solve a problem. In this context, scientists select the most appropriate method(s) to answer the question and solve a common dilemma. Over the past decade, we have witnessed a revolution of new technologies in molecular biology – the Omics Science. Highscale technologies such as metabolomics, cytomics, genomics and proteomics are changing the way we study complex biological systems. Current approaches to understanding the functional diversity of an organism preferentially strive for a systems biology approach whereby first the phenotypic classification of a specific cytome is achieved prior to an attempt to perform proteomic analysis. In this context, to better understand the features that involve neutrophil activation and programmed cell death in the pathological and healthy states, this study proposes the integration of cell biology approaches such as flow cytometry with a very robust proteomics platform in an attempt to integrate data at the molecular level with phenotypic data of neutrophils. The application of subcellular fractionation method using digitonin detergent extraction to enrich cytosolic proteins from neutrophils was found reproducible, simple to perform, and inexpensive. ________________________________________________________________________________________ ABSTRACT
Pesquisas biomédica comumente começa com a elaboração de uma hipotese para resolver um problema. Nesse contexto, cientistas selecionam o(s) metodo(s) mais apropriado(s) para responder a questão e solucionar um dilema. Nos últimas anos, nós temos testemunhado uma revolução de novas tecnologias em biologia molecular – a ciência -Omica. Tecnologias de alta-escala tais como metabolômica, citômica, genômica e proteômica estão mudando o modo que estudamos sistemas biológicos complexos. Métodos contemporâneos para o entendimento da diversidade funcional em um dado organismo preferencialmente abordam uma visão de biologia de sistemas onde primeiro a classificação fenótipa de um citoma é alcançado antes de uma tentativa de caracterizar o proteoma de tal célula. Dentro desse contexto, e para proporcionar um melhor entendimento dos componentes envolvidos na ativação e morte celular programada dos neutrófilos nos estados patológicos e sano, esse estudo propõe a integração de métodos em biologia celular tal como citometria de fluxo com uma robusta plataforma proteômica em uma tentativa de integrar dados a nível molecular com dados fenótipicos de neutrófilos. Fracionamento subcelular usando um método de extração e enriquecimento de proteínas citosólicas com o detergente digitonina foi otimizado nesse trabalho, e encontrado ser altamente reprodutível, fácil de realizar, e de baixo custo.

Rice (Oryza sativa L.) is without doubt one of the major crops worldwide, as its consumption is continuously increasing, especially in low- and lower-middle-income countries where it is the most important staple. This cereal has been domesticated for a long time, result of which several species and varieties are now available. It was key during the green revolution when its production increased in more than 2-fold times, and in addition has a deep cultural background in all the regions where it is grown. Hence, it is of uttermost importance for researchers and breeders to broaden and expand the knowledge on this cereal on all the study and research areas, especially now that we are living in the 21st century, which is marked by climate change. This phenomenon is one of the most menacing as it will reduce the quantity and quality of arable land due to salinization of soil as well as water scarcity which is the single and most important factor that determines global crop yields. In this sense, this thesis addresses to three important topics on rice that will help researchers and breeders for the improvement of rice varieties in the forthcoming future: (i) Analysis of salinity tolerant rice plants subjected to high salt concentration through a combined approach of shotgun proteomics and physiological characterization for the identification of new key proteins involved in the tolerance to this stress. In this study we provide a huge database for future studies of the salinity-stress response in rice varieties harboring the Saltol region, as we detected more than 2000 proteins involved during the early stages of salinity stress in the shoots and roots of the FL478 rice line. Moreover, this study highlights the importance of examining both the shoots and roots because their salinity tolerance traits are different and respond to different requirements of the rice plant. Finally, the information presented here expands our knowledge on adaptive processes under high salinity in rice plants and, notably, in rice roots. (ii) Phytohormones analysis in dwarf varieties through a broad phytohormone profiling method developed during this thesis for the characterization of phytohormone levels during the development of plants with contrasting heights that will allow to have more information for developing new dwarf mutant varieties. In this work, we reported that GA19 seems to have a crucial role in gibberellin availability in rice as its levels were much higher than all the other gibberellins in all tissues. In addition, it has been demonstrated that the GA20ox-2 mutation is not the only factor affecting height in rice, as a mutated variety had an increased growth during the half of its development period. Finally, we established for the first time a simple and broad phytohormone extraction and detection protocol that allows to identify 13 gibberellins and ABA, JA and IAA in several tissues at different phenological stages. (iii) Improvement and enhancement of anther culture protocols in rice for obtaining higher rates of stabilized green double haploid plants using different cold-pretreatments and additives in the growing media such as hormones and antimitotics in the growing media for their subsequent commercialization. It was evidenced that the best cold-pretreatment was 10ºC for 9 days, and that colchicine addition greatly enhances the production of green double haploid plantlets. Moreover, we have introduced for the first time a post-anther culture treatment with good prospects. Finally, several of the rice lines produced were assayed in rice paddy fields where they displayed good agronomical behavior.
El arroz (Oryza sativa L.) es sin duda uno de los principales cultivos del mundo, especialmente en países en vías de desarrollo donde es el alimento básico, teniendo además un importante componente cultural. Fue clave en la revolución verde cuando su producción aumentó más del doble. En el siglo 21 marcado por el cambio climático, se verá reducida la cantidad y calidad de las tierras cultivables debido a la salinización del suelo y la escasez de agua. Por tanto, es de gran importancia para los investigadores y mejoradores ampliar el conocimiento que se tiene sobre este cereal en todas las áreas de estudio. En este sentido, esta tesis abordó tres temas importantes referentes a este cereal: (i) Un estudio del proteoma, de la parte aérea y radicular, de plantas de arroz tolerantes a la salinidad sometidas a una alta concentración de sal con el objetivo de identificar de nuevas proteínas clave involucradas en la tolerancia a este estrés. Proporcionamos una gran base de datos, más de 200 proteínas involucradas en este estrés y destacamos la importancia de las raíces para la tolerancia a este estrés. (ii) Análisis de fitohormonas en variedades enanas a través de un amplio y rápido método desarrollado durante esta tesis que permite, por primera vez en arroz, la extracción y detección de fitohormonas que posibilida la identificación de 13 giberelinas y ABA, JA e IAA en varios tejidos en diferentes etapas fenológicas. Se observó que la GA19 parece tener un papel crucial en la disponibilidad de giberelinas en el arroz, ya que sus niveles son los más elevados en todos los tejidos. La mutación GA20ox-2 no es el único factor que afecta la altura ya que la variedad mutada alcanza la altura de variedades wild-type. (iii) Por último, se reportan dos métodos de aumento de la eficiencia del protocolo de cultivo de anteras, técnica muy útil para la obtención de plantas de arroz dihaploides estables. El aumento del rendimiento se debió a nuevos pretratamientos de frío, así como la modificación de los medios de cultivo mediante diferentes concentraciones de hormonas y colchicina. Se introdujo, por primera vez, un tratamiento de diploidización para plantas haploides.

Thesis (Ph. D.)--University of Maryland, College Park, 2005.
Thesis research directed by: Chemistry. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

People are, in their daily lives, exposed to a number of airborne foreign compounds that do not normally affect the body. However, depending on the nature of these compounds, dose and duration of exposure, various airway symptoms may arise. Early symptoms are often manifested as upper airway mucosal inflammation which generates changes in protein composition in the airway lining fluid. This thesis aims at identifying, understanding mechanisms and characterizing protein alterations in the upper airway mucosa that can be used as potential new biomarkers for inflammation in the mucosa. The protein composition in the mucosa was studied by sampling of nasal lavage fluid that was further analyzed with a proteomic approach using twodimensional gel electrophoresis and mass spectrometry. Additionally, by studying factors on site through environmental examination, health questionnaires and biological analyses, we have tried to understand the background to these protein alterations and their impact on health. Respiratory symptoms from the upper airways are common among people who are exposed to irritative and microbial agents. This thesis have focused on personnel in swimming pool facilities exposed to trichloramine, metal industry workers exposed to metalworking fluids, employees working in damp and moldy buildings and infants diagnosed with respiratory syncytial virus infection. The common denominator in these four studies is that the subjects experience upper airway mucosal inflammation, which is manifested as cough, rhinitis, phlegm etc. In the three occupational studies, the symptoms were work related. Notably, a high prevalence of perceived mucosal symptoms was shown despite the relatively low levels of airborne irritants revealed by the environmental examination. Protein profiling verified an ongoing inflammatory response by identification of several proteins that displayed altered levels. Interestingly, innate immune proteins dominated and four protein alterations occurred in most of the studies; SPLUNC1, protein S100A8 and S100A9 and alpha-1-antitrypsin. Similarly, these proteins were also found in nasal fluid from children with virus infection and in addition a truncated form of SPLUNC1 and two other S100 proteins (S100A7-like 2 and S100A16), not previously found in nasal secretion, were identified. Altogether, the results indicate the potential use of a proteomic approach for identifying new biomarkers for the upper respiratory tract at an early stage in the disease process after exposure to irritant and microbial agents. The results indicate an effect on the innate immunity system and the proteins; SPLUNC1, protein S100A8 and S100A9 and alpha-1-antitrypsin are especially promising new biomarkers. Moreover, further studies of these proteins may help us to understand the molecular mechanisms involved in irritant-induced airway inflammation.

Proteomics to evaluate the effect of cryopreservation on post-tahw stallion sperm OBJECTIVES AND METHODS The success of cryopreservation in stallion sperm is lower than bull (1). Stallions are commonly selected on the basis of athletic performance records, pedigree and conformation. “Freezability” of semen is a term used to indicate survival rates of sperm populations after a freezing-thaw cycle on the basis of motility parameters. About 20-50% of stallions have sperm with unacceptable freezability (2) and a a lot of variations inter- ad intra stallion also exist, altoughth many different extenders were tested to improve quality of frozen semen. The aim of this study was to compare by proteomic analysis sperm protein expression profile from 6 stallions as studs. They were selected in groups of “high” or “low” freezability quality, according to WBFSH (World Breeding Federation for Sport Horses) guideline ( 35% minimum of PMS, Progressive Motile Spermatozoa). Whole sperm proteins were extracted by 0,5 mL commercial paillettes of frozen semen. After centrifugation cycles to remove extender (INRA82 with abundant skim milk powder declared) protein extracts by 2 commercial paillettes for each stallion were loaded on 7 cm immobilized pH gradient (4-8) IPG strip and separated by IEF (Iso Electro Focusing) as the first dimension, and then with upright SDS-PAGE (polyacrylamide gel electrophoresis) as the second. Every analisys were repeated in double. A usefull method to eliminate extender’s caseine was carried out to avoid interference with semen protein focalisation around pI 4. RESULTS Comparative proteomics analysys of all gels by imagine analysis software showed that expression of 10 protein spots (fig. 1) is related to high or low freezability parameters. Spots 25,27,18 are similar to sp32 (proacrosin binding protein, 28-29kDa, pI 4.8-5.2, teorical 61kDa, 5.9 pI), implicated in acrosomial reaction. In gels appears as a triplette of spots at the same pI, maybe to indicate post-translational modifications. Spot 12 is similar to CRISP3 (Cysteine-rich secretory protein) seminal plasma protein specific to stallion sperm and positively related to fertility (3). Spots 17 and 23 are similar to kallicrein-1E2 (KLK2), protein isoforms, negatively related to prengnancy rate and positively related to ejaculate volume (3). Spots 31 and 32 are highly expressed in “good” freezer stallions, and are compatible with aSFP (acidic protein bovine seminal plasma, 11-12 kDa pI 4.9), a protein of boar seminal plasma spermadhesine, and may play a role against ROS (reactive oxygene species). Spot 68 and 30 can be similar to bovine BSP A1/A2 (16 kDa, pI 4.7-5.5) also called PDC-109, a bull heparin-binding proteins of the seminal plasma positively correlated with good freezeability in this species (4). CONCLUSION Comparative proteomics analysis i this study showed that the expression levels of several proteins are related to high or low semen freezability. Some of that proteins are seminal plasma proteins, and we could hypotithesize that they are involved in protection sperm during cryoconservation and in preservation against oxidative damage. This study also confirme that mere knowledge of the gene sequences is insufficent to elucidate biochemical changes in sperm function, expecially related to post-traslational protein modifications. This study also demonstrate that 2-DE is a crucial step in the workflow of a proteomic approach, and the importance of a correct method to extraction proteins from semen frozen with addicted extender, instead of usual methods based on standard tissues protocols. Further large-scale proteomic studies will lead to the development of novel biomarkers of good semen freezabilty, essential to abtain higher and easier reproductive efficiency and to ansure lower cost and time-loss by breeders.
INTRODUZIONE – Con il miglioramento delle biotecnologie inerenti l’inseminazione artificiale (IA), la conservazione e il trasporto del materiale seminale, il potenziale riproduttivo maschile ha assunto un’importanza spiccata in termini zootecnici e commerciali. Nel cavallo la selezione degli stalloni impiegati come riproduttori si basa soprattutto alla valutazione delle performance sportive e delle caratteristiche morfofunzionali dell’animale. Attualmente il valore riproduttivo di uno stallone è basato essenzialmente su parametri seminali classici (concentrazione, motilità progressiva e totale, morfologia, numero totale di spermatozoi), benché sia ormai noto che tale valore è il risultato di complesse interazioni di tipo genetico, ambientale e biologico. Il valore riproduttivo degli stalloni non è al momento quantificabile, come accade invece nel bovino, mediante l’assegnazione di un Indice genetico Quantitativo basato su test di progenie, e i dati sull’Indice di Fertilizzazione (percentuale di concepimento per ciclo ovulatorio) sono sporadici, soprattutto in seguito all’utilizzo di seme congelato. E’ noto infatti che la crioconservazione attiva reazioni ossidative e riduce il tempo di sopravvivenza degli spermatozoi post-scongelamento. Rispetto alla specie bovina inoltre la qualità del seme congelato equino è molto inferiore, e solo il 20-50% degli stalloni hanno parametri seminali accettabili dopo lo scongelamento (1). Esistono poi forti variazioni inter- e intra- individuali nella congelabilità del seme degli stalloni non ancora chiarite con le convenzionali analisi del seme (2). Si è iniziato a parlare di proteomica dello sperma nel 1997, e da allora alcuni studi sono stati condotti sull’uomo (3,4,5) e su varie specie animali (6,7,8,9). Il vantaggio offerto dalla proteomica è quello di avere in una sola separazione elettroforetica bidimensionale un quadro completo delle proteine espresse in un preciso momento da un dato comparto cellulare di un individuo. L’obiettivo di questo lavoro è l’analisi proteomica di campioni di seme congelato di stalloni con caratteristiche seminali differenti post-scongelamento. MATERIALI E METODI - Il seme congelato di 4 stalloni raccolto presso un Centro di Riproduzione Equina riconosciuto (Intermizoo spa, Vigonza, PD), è stato sottoposto ad analisi elettroforetiche bidimensionali. 4 paillettes commerciali sono state analizzate per ogni stallone, di cui si conoscono i valori relativi alla concentrazione, motilità totale (MOT) e spermatozoi progressivamente motili (PMS) post-scongelamento, calcolate mediante dispositivo computerizzato CASA (Computerized Assisted Sperm Analyzer) (Hamilton Thorne®, Biosciences). In accordo con le linee guida della WBFSH (world breeding federation for sport horses), che stabilisce come standard minimo di utilizzo del seme congelato un valore di 35% di PMS, i soggetti analizzati sono stati inquadrati come“good” (campione 6 e 7) e “bad” freezer (campione 3 e 5). Il pellet cellulare ottenuto dopo 5 cicli di centrifugazione (2000G per 20’) è stato risospeso in 800 µL di tampone di estrazione (7 M urea, 2 M Tiourea, 4% Chaps, 1% DTT) e poi sonicato (5 cicli, in ghiaccio). Dopo centrifugazione (2000G per 5’) al fine di eliminare i residui cellulari, le proteine di ciascun campione sono state quantificate mediante 2D QuantiKit® (BioRad). E’ stata poi eseguita l’isoelettrofocalizzazione su strip non lineari a gradiente di pH immobilizzato (range 4-8) da 7 cm (10), mediante IPGphor II® (GE Healtcare) fino al raggiungimento di 70KV/h totali. Dopo equilibrazione, le strip sono state trasferite su gel (SDS-PAGE 12,5% acrilamide) per la separazione in seconda dimensione su Mini Protean 2D cell® (Biorad). I gels ottenuti sono stati poi colorati utilizzando un protocollo standard di colorazione con Coomassie colloidale (11), e digitalizzati per l’analisi d’immagine degli spot con software dedicati. RISULTATI - Sono stati identificati 124 spot. L’analisi comparativa ha evidenziato numerose differenze inter-individuali. Alcuni spot differentemente espressi tra i diversi campioni sono stati identificati mediante ricerca bibliografica di mappe proteomiche esistenti in altre specie (4,6,7,8,9,12), in attesa di ulteriori analisi mediante spettrofotometria di massa (Fig.1). DISCUSSIONE - Gli spot 18, 25 e 27 potrebbero corrispondere alla sp32 (proacrosin binding protein, 28-29kDa, pI 4.8-5.2) proteina implicata nella capacitazione e ben conservata tra le specie. Nei gels tale proteina appare sottoforma di una catena di tre spot con punti isolettrici lievemente dissimili, ad indicare probabilmente modificazioni post-translazionali (12) quali la fosforilazione che avviene alla capacitazione. Lo spot 12 sembrerebbe corrispondere alla CRISP3 (cysteine-rich secretory protein), 25 kDa, pI 7,54), proteina specie specifica del plasma seminale equino e correlata positivamente alla fertilità (8). Gli spot 17 e 23 corrisponderebbero alle isoforme della kallicreina (27 kDa pI 5.51), correlate negativamente alla fertilità nello stallone (8). Gli spots 31 e 32 sembrano essere simili alla aSFP (acidic protein bovin seminal plasma, 11-12 kDa pI 4.9), appartenente alla famiglia delle spermadesine (6), avente ruolo di protezione della membrana spermatica dalla perossidazione lipidica indotta dai ROS (reactive oxygen species) e risultata associabile ad una migliore congelabilità nel seme di toro (7). Gli spot 30 e 68, potrebbero corrispondere alla BSP A1/A2 bovina (16 kDa, pI 4.7-5.5) detta anche PDC109, proteina del plasma seminale che si lega specificatamente ai fosfolipidi di membrana degli spermatozoi. La sua abbondanza nel plasma seminale dei tori con buona congelabilità, induce a ipotizzare che abbia un ruolo determinante nella protezione della membrana spermatica durante le procedure di crioconservazione (7). Questi dati confermano che la proteomica può essere di notevole ausilio in andrologia, in quanto gli spermatozoi sono cellule altamente specializzate, con una membrana molto complessa e ricca in proteine, e subiscono forti modificazioni del corredo proteico nel corso della maturazione. Inoltre, a causa del complesso riarrangiamento del DNA e dell’estrusione di gran parte del citoplasma, sono cellule dalle limitate difese contro i ROS (reactive oxygen species). E’ noto che la manipolazione e i metodi di crioconservazione amplificano gli effetti deleteri dei ROS sul seme. Un’accurata indagine proteomica potrebbe svelare modificazioni a carico del corredo proteico indotte da alti livelli di ROS nel seme. L’utilizzo di questa tecnica elettroforetica potrebbe inoltre permettere lo studio delle modificazioni dei parametri seminali allo scongelamento utilizzando protocolli diversi di crioconservazione. Infine l’identificazione di ulteriori spot con diversa espressione e la correlazione con parametri seminali e dati sulla fertilità potrebbero consentire l’individuazione di biomarkers predittivi del potenziale riproduttivo degli stalloni e la messa a punto di test di screening.

Muscular dystrophies are a group of diseases that are often caused by loss-of-function mutations affecting the dystrophin glycoprotein complex (DGC). The common feature of the diseases is muscle degeneration, which is often associated with mental retardation and various retinal defects, including ones of synaptic transmission. However, the mechanisms of the disease remain largely unknown, especially those in the central nervous system. I have focused on dystroglycan (DG), the transmembrane protein in the DGC that links the cytoskeleton to the extracellular matrix and is essential for muscle survival and brain development. I have used proteomics and Drosophila genetics to study DG function in the brain and retina.
Using proteomics I found that beta-DG is directly associated with the GTPase dynamin 1 in the retina and in the brain together with alpha-DG and Grb2, and immunohistochemically beta-DG was colocalized with dynamin 1 in the outer plexiform layer where photoreceptor terminals are localized. Moreover, loss of DG in differentiated DG-null embryonic stem cells significantly increases dynamin-mediated transferrin-uptake and re-expression of DG in null cells by infection with an adenovirus containing DG reduced transferrin uptake to levels seen in wild-type cells. This result implies that one of mechanisms in muscular dystrophy might be the altered synaptic vesicle endocytosis, especially in the retina where synaptic transmission defect has been known for decades.
Muscular dystrophies show not only impaired retinal synaptic transmission and several DG-related congenital muscular dystrophies also display retinal structural defects. To further understand the roles of DG in the retina, I used Drosophila eye as a model and demonstrated for the first time that DG is required cell-autonomously for photoreceptor morphogenesis in the developing visual system. Deficiency of DG in the eye causes severe disruption of retinal structure, aberrant lens formation and abolition of electroretinogram in the adult fly eye. These adult defects appear derived from autonomous photoreceptor cell (PRC) defects in the early pupa including size arrest, loss of polarity and progressive degeneration. All defects in the eye, however, can be reversed by re-expression of wild type DG in DG-deficient PRCs, suggesting DG functions cell-autonomously in PRCs and non-autonomously for lens. In the 3rd instar larvae DG is present in the apical tips and the basal membranes of PRCs, two polarized locations opposing the extracellular matrix. At the pupal stage it continues to mainly distribute at the apical rhabdomere and basal membrane of PRCs. Over-expression of DG leads to larger ommatidia but the PRC number remains unchanged, suggesting that DG is both necessary for and sufficient to promote PRC expansion. By rescue experiments, I demonstrated that the extracellular DG alone could not rescue DG-deficient eye defects, whereas the intracellular DG can substantially ameliorate PRC degeneration and structural defects while some PRCs remain disorganized, a sign of disrupted PRC planar polarity in absence of the extracellular DG. Therefore, our data suggest that the degeneration and planar polarity disruption in DG-deficient PRCs are two independent processes that appear to require the respective function of intracellular and extracellular DG. In summary, our experiments demonstrated several novel findings and provided the basis for future investigations on DG function and the molecular mechanisms of nervous system defects in muscular dystrophies.

The WHO classification of brain tumors is based on histological features and the aggressiveness of the tumor is classified from grade I to IV, where grade IV is the most aggressive. Today, the correlation between prognosis and tumor grade is the most important component in tumor classification. High grade gliomas, glioblastomas, are associated with poor prognosis and a median survival of 14 months including all available treatments. Low grade meningiomas, usually benign grade I tumors, are in most cases cured by surgical resection. However despite their benign appearance grade I meningiomas can, without any histopathological signs, in some cases develop bone invasive growth and become lethal. Thus, it is necessary to improve conventional treatment modalities, develop new treatment strategies and improve the knowledge regarding the basic pathophysiology in the classification and treatment of brain tumors. In this thesis, both proteomics and metabolomics have been applied in the search for biomarkers or biomarker patterns in two different types of brain tumors, gliomas and meningiomas. Proteomic studies were carried out mainly by surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS). In one of the studies, isobaric tags for relative and absolute quantitation (iTRAQ) labeling in combination with high-performance liquid chromatography (HPLC) was used for protein detection and identification. For metabolomics, gas-chromatography time-of-flight mass spectrometry (GC-TOF-MS) has been the main platform used throughout this work for generation of robust global metabolite profiles in tissue, blood and cell cultures. To deal with the complexity of the generated data, and to be able to extract relevant biomarker patters or latent biomarkers, for interpretation, prediction and prognosis, bioinformatic strategies based on chemometrics were applied throughout the studies of the thesis. In summary, we detected differentiating protein profiles between invasive and non-invasive meningiomas, in both fibrous and meningothelial tumors. Furthermore, in a different study we discovered treatment induce protein pattern changes in a rat glioma model treated with an angiogenesis inhibitor. We identified a cluster of proteins linked to angiogenesis. One of those proteins, HSP90, was found elevated in relation to treatment in tumors, following ELISA validation. An interesting observation in a separate study was that it was possible to detect metabolite pattern changes in the serum metabolome, as an effect of treatment with radiotherapy, and that these pattern changes differed between different patients, highlighting a possibility for monitoring individual treatment response.  In the fourth study of this work, we investigated tissue and serum from glioma patients that revealed differences in the metabolome between glioblastoma and oligodendroglioma, as well as between oligodendroglioma grade II and grade III. In addition, we discovered metabolite patterns associated to survival in both glioblastoma and oligodendroglioma. In our final work, we identified metabolite pattern differences between cell lines from a subgroup of glioblastomas lacking argininosuccinate synthetase (ASS1) expression, (ASS1 negative glioblastomas), making them auxotrophic for arginine, a metabolite required for tumor growth and proliferation, as compared to glioblastomas with normal ASS1 expression (ASS1 positive). From the identified metabolite pattern differences we could verify the hypothesized alterations in the arginine biosynthetic pathway. We also identified additional interesting metabolites that may provide clues for future diagnostics and treatments. Finally, we were able to verify the specific treatment effect of ASS1 negative cells by means of arginine deprivation on a metabolic level.

Upon ageing, a progressive disruption of protein homeostasis often leads to extensive protein aggregation and neurodegeneration. It is therefore important to study at the proteome level the origins and consequences of such disruption, which so far have remained elusive. Addressing this problem has recently become possible by major advances in mass spectrometry-based (MS) proteomics, which allows the identifications and quantification of thousands of proteins in a variety of biological samples. In the first part of this thesis, I analyse proteome-wide MS data for the nematode worm C. elegans upon ageing, in wild type (WT), long-lived and short-lived mutant strains. By comparing the total abundance and the soluble abundance for nearly 4000 proteins, I provide extensive evidence that proteins are expressed in adult worms at levels close to their solubility limits. With the use of sequence-based prediction tools, I then identify specific physico-chemical properties associated with this age-related protein homeostasis impairment. The results that I obtained reveal that the total intracellular protein content remains constant, in spite of the fact that the proteome undergoes wide remodeling upon ageing, resulting into severe protein homeostasis disruption and widespread protein aggregation. These results suggest a protein-dependent decrease in solubility associated with the protein homeostasis failure. In the second part of the thesis, I determine and classify potential interactions of misfolded protein oligomers with other proteins. This phenomenon is widely believed to give rise to cytotoxicity, although the mechanisms by which this happens are not fully understood. To address this question, I process and analyse MS data from structurally different oligomers (toxic type A and nontoxic type B) of the protein HypF-N, incubated in vitro with proteins extracted from murine cell cultures. I find that more than 2500 proteins are pulled down with the misfolded oligomers. These results indicate that the two types of oligomers interact with the same pool of proteins and differ only in the degree of binding. Functional annotation analysis on the groups reveals a preference of the oligomers to bind proteins in specific biological pathways and categories, including in particular mitochondrial membrane proteins, RNA-binding proteins and molecular chaperones. Overall, in this study I complement the powerful and high-throughput experimental approach of MS proteomics with bioinformatics analyses and prediction algorithms to define the physical, chemical and biological features of protein homeostasis disruption upon ageing and the interactome of misfolded oligomers.

Myotonic Dystrophy type 2 (DM2) is caused by a DNA microsatellite expansion within the ZNF9 gene leading to an abnormal splicing pattern largely responsible for the pathological condition. To better define the functional changes occurring in human DM2 myotubes, we performed a quantitative proteome comparison between myotubes of DM2 and control patients using two-dimensional gel electrophoresis followed by mass spectrometry. Our results indicate that the proteins, altered in DM2 cultures, belong to two major functional categories: i) mitochondrial components, with a reduction of Elongation factor Tu (EFTu), Heat Shock Protein 60 (HSP60), Glucose Regulated Protein 75 (GRP75) and Dienoyl-CoA-Isomerase, an enzyme involved in fatty acids degradation; ii) the ubiquitin proteasome system, with increase of the 26S proteasome regulatory subunit 13 and a reduction of Proteasome subunit Apha 6 and of Rad23B homolog. Altered ubiquitin-proteasomal activity is supported by a global reduction of cytosolic ubiquitinated proteins, nonetheless the accumulation of ubiquitin-protein conjugates after proteasomal inhibitor MG-132 treatment is maintained in DM2 and control cells, suggesting a higher degradation rate for the proteasome in myoblasts from patients affected by the disease. Although future work is required to clarify how these changes affect the protein degradation machinery and mitochondrial function and to evaluate if these changes also occur in the biopsies of DM2 patients, these results identify the mitochondrial proteins and the ubiquitin-proteasomal system as candidates potentially relevant to DM2 pathogenesis. Further analysis performed in Human skin fibroblasts primary cultures, obtained from patients biopsies, revealed an Hyperpolaryzation of the mitochondrial membrane potential involving DM2 cells, indicating a putative functional issue for mitochondria. As the evaluation of the Cytocrome c release following hydrogen peroxide treatment showed a differential response to this stress inducing compound, we pointed out a functional involvement of the DM2 mitochondrial-mediated cellular response to oxidative stress.

Poverty stricken areas of the world are affected by Neglected Tropical Diseases, with an estimated 1 billion sufferers. As well as inadequate living conditions and healthcare, there has been very little pharmaceutical incentive to tackle these diseases. As a result, the diseases are still spreading. Drugs available on the market suffer from poor efficacy, high toxicity, increasing resistance and inappropriate dosing for rural treatment. The nature of many NTDs prevents the use of vaccinations. Therefore, more efficacious and safe treatments are sought after. The folate pathway has been extensively studied in a number of organisms, with its essentiality exploited in a number of drugs and drug targets. The same cannot be said for the kinetoplastids. Drug discovery programmes have focused on targeting enzymes of the folate metabolism with very little clinical success. Despite showing significant inhibition of the parasitic enzymes, potency is seen to decrease in cellular and animal models. Understanding how the folate pathway operates in these organisms could provide insight into where and how anti-folate compounds bind. This information could then be used to facilitate better drug treatments for the kinetoplastids. This thesis describes a number of approaches undertaken to better understand folate metabolism in kinetoplastids. Clinical and literature anti-folate compounds were immobilized onto resins, followed by chemical proteomics, utilizing novel techniques (iTRAQ), to allow for target identification. Using competition studies, specific and non-specific targets were identified in parasitic lysate (T. brucei and L. major) for each anti-folate compound. This method was further exploited by creating a folate resin (Folate beads). The resin had the potential to pull down 9 proteins from the “folate-ome”. In future studies, the resin can be used to enrich for the folate proteins in kinetoplastids and related organisms. Alongside the studies of the folate proteins, it was also desired to study proteins involved in the essential pterin pathway. This pathway has not been extensively studied in kintoplastids, with the exception of PTR1 (by-pass protein for DHFR). The failure to synthesise pterin derivatives for bead coupling led to a fragment screening campaign being carried out on QDPR in leishmania major. Working through a triage workflow, two moderately potent fragments were identified, showing inhibition against LmQDPR. Through structure-free optimization strategies, greater than 100 optimized fragments were synthesised in a bid to understand SAR. Although this work remains incomplete, LmQDPR has been successfully crystalized with 23 hit fragments, which are awaiting further biophysical analysis to understand binding.

The present research work concerns the development of new analytical methods aimed to the study of the proteome and the metallome of placental tissue and the evaluation of its possible modifications and/or alterations due to gestational diabetes. Proteomics analysis was performed by different experimental approaches based on mass spectrometry (ESI-MS and MALDI-MS), and on one- and two-dimensional electrophoresis. These analytical techniques are presently the most suitable experimental approaches to be used in omics applications thanks to their high sensitivity and specificity. Results showed the presence of protein species differently expressed in the case of gestational diabetes; in particular, it was measured an increment of the chorionic somatomammotrophin level and a decrease in the concentration of alpha, beta and gamma chains of fibrinogen and tubulointerstitial nephritis antigen-like in the case of placental tissues impaired by GDM. Metallomics analysis was carried out by ICP-MS, an analytical technique characterized by high sensitivity and robustness. The study reported an increased concentration of Se and a decrease of Cd level in pathological placental tissues compared to healthy ones. Furthermore, this approach allowed the evaluation of the effectiveness of the sampling procedure, the influence of the blood on the obtained results and showed the importance of other parameters, such as the lifestyle of the patients, in the statistical evaluation of the experimental data. In conclusion, the obtained data might be useful for the study and the explanation of the biochemical processes that characterize the disease and for the determination of new biomarkers aimed to the development of innovative diagnostic tests for gestational diabetes.
Il lavoro di ricerca presentato concerne lo sviluppo e il perfezionamento di metodi analitici atti allo studio del proteoma e del metalloma del tessuto placentare e alla valutazione di eventuali sue modificazioni e/o alterazioni in presenza di diabete gestazionale. Le analisi di proteomica sono state effettuate mediante metodologie analitiche basate sulla spettrometria di massa (in particolare sono state utilizzate le tecniche di ionizzazione MALDI e ESI) e sull’elettroforesi mono e bi-dimensionale. Allo stato attuale dell’arte queste tecniche analitiche risultano essere gli approcci sperimentali più adatti a condurre indagini di questo tipo grazie all’elevata sensibilità e specificità che le caratterizza. I risultati analitici hanno evidenziato la presenza di specie proteiche diversamente espresse nel caso di diabete gestazionale; in particolare è stato notato un incremento dei livelli di somatomammotropina corionica e una diminuzione della concentrazione delle catene alfa, beta e gamma del fibrinogeno e di Tubulointerstitial nephritis antigen-like nelle placente complicate da GDM. Le analisi di metallomica sono state condotte mediante ICP-MS, a sua volta tecnica analitica caratterizzata da elevata sensibilità e robustezza. Le analisi hanno rivelato un aumento della concentrazione di Se e una diminuzione dei livelli di Cd nelle placente patologiche rispetto a quelle sane. L’approccio seguito ha permesso di valutare l’efficacia del campionamento e delle procedure pre-analitiche, l’influenza del tessuto ematico sui risultati ottenuti e ha mostrato l’importanza di altri parametri, come ad esempio lo stile di vita della gestante, nella valutazione statistica dei dati sperimentali. In conclusione, si può affermare che le informazioni ottenute possono essere utili per lo studio e la delucidazione dei processi biochimici che caratterizzano la patologia e per la determinazione di nuovi bio-marcatori che permettano lo sviluppo di test diagnostici innovativi per il diabete gestazionale.

Helicoverpa armigera is an insect, causes important economical losses in crops. To reduce this loss, chemical insecticides such as pyrethroids have been commonly used against H. armigera in farming areas all over the world. However, excess and continuous usages of them cause resistance development in H. armigera. Insects develop resistance against applied insecticides by following three main mechanisms
by reducing the amount of insecticide entering into the insect body, developing insensitivity of the insecticide effective site and increasing detoxification metabolism of insecticides such as increased metabolism of them in midgut tissue of H. armigera. Therefore, changes in differentially expressed midgut proteins were analysed at protein level with two-dimensional gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) together with examine biochemical activity changes of certain detoxification enzymes such as esterases (EST) and glutathione S-transferases (GST). Moreover, transcriptional level analysis of certain genes from EST and GST systems together with cytochrome P450 monooxygenases (CYP450) system were done with quantitative real-time PCR method, too. According to the comparative proteome analysis, it was found that H. armigera field samples overcome pyrethroid stress mainly by increasing energy metabolism related proteins expressions such as ATP synthase, Vacuolar ATPase A and B and arginine kinase proteins. Furthermore, certain detoxification enzymes such as thioredoxin peroxidase and NADPH cytochrome P450 reductase were up-regulated in Mardin population, suggesting that they were actively participating in response to pyrethroid stress. NADPH cytochrome P450 reductase could play a role in detoxification of toxic pyrethroid metabolites such as 3-phenoxybenzaldehyde. However, while glutathione S-transferases (GSTs) were not found up-regulated in the comparative proteome analysis, biochemical assays (GST-CDNB, GST-DCNB and GST-PNBC) showed significant increases in enzyme activities in the Adana and in the Mardin field population, as compared to the susceptible strain. Furthermore, GST-DCNB and GST-PNBC activities showed significant increase in Ç
anakkale population. As overcoming energy crisis may lead to an increase in oxidative stress, detoxification enzymes (GSTs and thioredoxin peroxidase) might be involved in pathways for eliminating toxic reactive oxygen species such as H2O2. Similarly, although esterases (EST) were not found as differentially expressed, biochemical assays for ESTs showed significant increases in enzymatic activities in the Adana and the Mardin field populations. Thus, ESTs are also proposed to be involved in developing resistance as an initiator of pyrethroid metabolism in H. armigera from Turkey. Quantitative real-time PCR results showed that while CYP9A14 gene expression was up-regulated in all analyzed field populations, CYP9A12 gene expression was up-regulated in both Ç
anakkale and Mardin populations. CYP4S1 gene expression was also up-regulated only in Mardin field population. However, while CYP6B7 gene expression together with CYP9A12 and CYP4S1 genes expressions were down-regulated in Adana population, CYP6B7 gene expression was not significantly changed in both Ç
anakkale and Mardin populations. In addition, GST, GSTX01 and ESTX018 gene expressions were not significantly changed in all field populations in comparison to susceptible population. Therefore, CYP9A14, CYP9A12 and CYP4S1 genes proposed to be involved in detoxification of toxic pyrethroid metabolites possibly through regulation of NADPH cytochrome P450 reductase. In conclusion, it is suggested that one of the main mechanisms of resistance development is increased energy metabolism in the midgut tissue of H. armigera which may be a general prerequisite for compensating the costs of energy-consuming detoxification processes.

The declining reserves of fossil fuel twined with an increasing concern about the environmental consequences of burning these fuels and rising carbon dioxide levels, means that a more sustainable replacement is required. Lignocellulosic biomass is an attractive candidate that has been shown to be the best sustainable alternative source to produce bioethanol for liquid transportation fuels. It has enormous availability, is renewable and cost-effective. As an agricultural residue, it does not compete with food production. However, lignocellulosic biomass of plant cell walls is composed mainly of cellulose, hemicellulose and lignin, which are extremely resistant to digestion. Converting this biomass to useful products of fermentable sugars for bioethanol production has met with little success as harsh pretreatment and costly enzyme applications are required. An arsenal of enzymes and a synergistic mechanism are required to deconstruct recalcitrant lignocellulosic biomass for an efficient production of lignocellulosic bioethanol. To achieve this goal, this study used transcriptomic and proteomic approaches with the objective of identifying new genes and enzymes involved in lignocellulose degradation. This revealed that the only one AA10 of Cellulomonas fimi was among the highest enzymes identified during the degradation of cellulose. Another other 20 hypothetical proteins co-expressed with CAZymes have been identified including a potentially exclusively new C. fimi β-glucosidase (PKDP1) that contains a PKD-domain and oxidoreductase predicted function of PQQ-domain. A naturally mutagenized C. fimi population also was screened from an adaptive evolution experiment involving exposure to a wheat straw environment. One of the strains in the adaptive population (Strain-6) showed a higher association with wheat straw biomass, which may be an indication of the strategy that being used by the adapted strain to tackle obstinate substrates to sustain growth. These results show many new enzymes would be revealed from the C. fimi repertoire in order to have a better enzymatic cocktails for lignocellulose breakdown. For the future, this encourages a deeper understanding of lignocellulose deconstruction mechanisms by an orchestra of multiple enzymes in a bacterial system.

Thesis (Ph. D.)--Joint Program in Chemical Oceanography (Massachusetts Institute of Technology, Dept. of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2011.
Cataloged from PDF version of thesis.
Includes bibliographical references.
A combination of uptake field studies on natural phytoplankton assemblages and laboratory proteomic and physiological experiments on cyanobacterial isolates were conducted investigating the interactions of cadmium (Cd), zinc (Zn), and phosphorus (P) in marine Synechococcus. Enriched stable isotope field uptake studies of ¹¹⁰CD in the Costa Rica Upwelling dome, a Synechococcus feature, showed that uptake of Cd occurs in waters shallower than 40 m, correlates positively with chlorophyll a concentrations and is roughly equivalent to the calculated upwelling flux of cadmium inside the dome. In laboratory experiments, Synechococcus WH5701 cells exposed to low picomolar quantities of free Cd under Zn deficiency show similar growth rates to no added Cd treatments during exponential growth phase, but show differences in relative abundances of many proteins involved in carbon and sulfur metabolism suggesting a great metabolic impact. During stationary phase, chronic Cd exposure in this coastal isolate causes an increase in relative chlorophyll a fluorescence and faster mortality rates. The interactions of acute Cd exposure at low picomolar levels with Zn and phosphate (PO4³-) were investigated in Synechococcus WH8102, an open ocean isolate. The presence of Zn appears vital to the response of the organism to different PO4 ³- cocentrations. Comparisons with literature transcriptome analyses of PO4 ³- stress show similar increases in relative abundance of PO4 ³- stress response proteins including a PO4 ³- binding protein and a Zn-requiring alkaline phosphatase. A bacterial metallothionein, a Zn-associated protein, appears to be correlated with proteins present under low PO4 conditions. Together, these experiments suggest that the interactions of Cd and Zn can affect Synechococcus and play a role in the acquisition of PO4 ³-.
by Alysia Danielle Cox.
Ph.D.

Aim of the PhD work was the development and validation of methods for the extraction of quantitative biomarkers from in vivo molecular imaging (from PET/CT oncological studies) to be correlated with ex vivo molecular imaging indexes (from hystological and proteomic data).

ABSTRACT Renal cell carcinoma (RCC) is among the 10 leading causes of cancer-related deaths worldwide and its incidence is increasing steadily. At the time of diagnosis, 30% of patients have metastases and another third will develop metastatic disease within 10 years. Moreover metastatic RCC is radio- and chemotherapy resistant. Diabetic nephropathy (DN) is a common diabetic complication that causes a renal massive functional deficit making dialysis or transplantation essential for the survival of patients and it is associated with alterations in the expression of several renal proteins. Therefore, it is important to identify differentially expressed proteins to be used as potential diagnostic and/or prognostic markers in RCC and DN. A promising strategy is comparative subcellular proteomics of urinary exosomes, membranous vesicles released from cells and membrane microdomains, lipid arfts and caveolae. About 30 ml of urines were collected from 29 clear cell RCC patients and 28 healthy matched controls and stored at -80°C, after antiproteases addition. Diabetes was induced in 10 Sprague Dawley rats by streptozotocin injection. Control rats (n=4) underwent only buffer injection. Out of 10 diabetic rats, 4 were chronically treated with insulin. After 3 months, 24 hours urines were collected. In both cases (RCC patients and diabetic rats with their healthy controls respectively) exosomes were then isolated from total urines by ultracentrifugation after cells and debris clearing. As regards RCC esults show that the protein profile of urinary exosomes is peculiar: the most abundant urinary proteins of plasmatic origin (i.e. albumin) are markedly reduced, while the Tamm Horsfall protein (THP) is highly enriched and affected by a significant biological variability. Moreover, protein profiles of exosomal fractions isolated from urine of ccRCC and matched controls show some difference. Mass spectrometry analysis of two pools of exosomes isolated from CTRL and RCC patients urines permits the identification of 261 proteins in CTRL and 187 proteins in RCC. Some of these protein Aquaporin-1, Matrix Metallo-protease 9 and Carbonic Anhydrase 9, display differential amount in ccRCC patient urine exosomes by EF/WB. Moreover subcellular fractions were prepared by differential centrifugation from surgical samples of RCC and adjacent normal kidney (ANK). MD were isolated from plasma-membrane-enriched fractions after treatment with Triton X-100 and sucrose density gradient ultracentrifugation. MD derived from RCC and ANK tissues of 7 patients were pooled, and proteins separated by 4-12% and 12% gel electrophoresis. After Coomassie Blue staining, bands were excised and analyzed by LC-MS/MS after trypsin digestion. We identified 83 proteins from microdomains isolated from RCC tissue, and 95 proteins from ANK. About 60% of the identified proteins are membrane-associated and about half of these resulted microdomain-associated. GRAVY scores assignment shows that most identified proteins (about 70%) are in the hydrophobic range. From a functional point of view, we found proteins involved in signal transduction (Ras related proteins), channels (Aquaporin-1), carriers (P-glycoprotein) and cytoskeleton structural constituents (Spectrin). We chose some promising proteins such as Renal dipeptidase (identified only in ANK MD) or Emmprin (overexpressed in RCC tissue) and investigated their differential expression of by WB About DN study, protein profiles of exosomes obtained from the three groups of rat (healthy controls CTRL, diabetic D and diabetic treated with insulin DI) show differences, mainly at low molecular weights. Some differential bands were excised and analyzed by LC-MS/MS after digestion by tripsin. In particular we identified the Major Urinary Proteins, known as MUPs from CTRL and DI gels, while these proteins are not present in the same bands excised from D gels. MUPs regulate glucose and lipid metabolism suggesting an involvement in hyperglycemia, insulin resistance and/or glucose intolerance in diabetes.