Tesis sobre el tema "Approcci proteomici"

Lo sviluppo di approcci rapidi ed automatizzati come la tecnologia multidimensionale di identificazione proteica (MudPIT) sta rendendo la proteomica uno strumento sempre più efficiente per l’analisi delle proteine in miscele complesse, permettendo l’identificazione di nuovi marcatori biologici che sono di importanza critica per una migliore comprensione della biologia dei tumori e per rendere la sua rilevazione più precoce e meno invasiva. Lo scopo del presente studio era quello di identificare nuove proteine rilasciate dalle cellule di adenocarcinoma duttale del pancreas, usando piccole quantità di campione ed un sistema automatizzato; le evidenze sperimentali così ottenute sarebbero state utilizzate per verificare in vitro ed in vivo, con metodiche più tradizionali quali western blot, RT-PCR ed immunoistochimica, la presenza delle proteine selezionate come possibili marcatori. È stata dunque applicata la tecnologia MudPIT, che incorpora la cromatografia capillare bidimensionale e la spettrometria di massa in tandem, per l’analisi di piccole quantità di surnatanti privi di siero derivanti dalla coltura di cellule Suit-2 non trattate oppure attivate con esteri del forbolo e ionoforo. I potenziali marcatori prescelti sono stati valutati in altre linee cellulari di cancro del pancreas, adenocarcinomi primitivi e xenotrapiantati in topi nu/nu. L’analisi MudPIT effettuata su campioni di 10 μl di surnatanti ha permesso l’identificazione complessiva di 46 proteine tra cellule attivate e non trattate. Di queste proteine, 21 sono classificate come secrete sui database pubblicamente disponibili e 10 non erano state precedentemente associate al carcinoma duttale del pancreas. Questo gruppo comprende le proteine CSPG2/versican, Mac25/angiomodulina, IGFBP-1, HSPG2/perlecan, syndecan 4, FAM3C, APLP2, ciclofilina B, K2 microglobulina, ed ICA69. Le evidenze sperimentali che queste proteine siano rilasciate dalle cellule tumorali in vivo sono state ottenute, per CSPG2/versican e Mac25/angiomodulina, mediante immunoistochimica. L’analisi è stata eseguita tanto su tumori primitivi quanto su di un modello, consistente in cellule della linea Suit-2 incluse in una matrice amorfa (Matrigel®) e trapiantate per una settimana in topi atimici. Si è inoltre dimostrato, mediante il confronto tra cellule non trattate ed attivate con esteri del forbolo, che l’analisi MudPIT può fornire dati semiquantitativi correlati con la quantità relativa di proteina presente nel campione analizzato; anche quest’osservazione è stata convalidata mediante misurazione del diverso livello di espressione di tre proteine rispettivamente inibite (Mac25/angiomodulina), non modificate (CSPG2/versican) ed indotte (MMP-1). Si è poi indagata, su una casistica di 100 pazienti con varie patologie pancreatiche, l’espressione di forme solubili di uPAR (suPAR), il cui ligando uPA era tra le proteine maggiormente indotte in seguito all’attivazione delle cellule in vitro. L’analisi è stata fatta utilizzando una metodica immunoenzimatica (saggio ELISA) su sieri ed urine dei casi disponibili presso la biobanca della clinica chirurgica dell’Università di Verona. E’ stato riscontrato un significativo incremento dei valori di suPAR nei sieri di pazienti affetti da adenocarcinoma duttale del pancreas rispetto alle altre patologie infiammatorie o neoplastiche del pancreas; i dati delle urine, pur se meno netti, indicano comunque una tendenza simile ed incoraggiano ad un incremento del numero di campioni sui quali effettuare ulteriori analisi. Confrontato con altre metodiche, dunque, MudPIT è stato in grado di fornire in modo rapido e riproducibile dati su di una serie di molecole rilasciate da cellule neoplastiche, che hanno quindi caratteristiche di sicuro interesse quali potenziali marcatori di malattia potenzialmente rilevabili nei liquidi biologici. Gli sviluppi futuri di tale approccio comprendono, oltre all’ampliamento dell’analisi MudPIT su un numero maggiore di linee cellulari, lo sviluppo di nuovi reagenti per l’identificazione quelle molecole per le quali essi non sono attualmente disponibili.
Non disponibile

Doutoramento em BioQuímica
A cárie dentária é uma doença complexa que afecta uma grande parte da população mundial independentemente do sexo, idade ou etnia. Este processo é dependente de factores biológicos que se encontram presentes na saliva e placa dentária. Em seguimento do referido, amostras de saliva foram colectadas de indivíduos caracterizados em função dos índices DMFT e DMFS. A avaliação dos convencionais parâmetros clínicos como por exemplo fluxo salivar, capacidade tampão, pH usados na avaliação do risco para a cárie dentária em combinação com dieta, hábitos de higiene e tabagismo foram realizados para todos os indivíduos participantes do qual se observou a ausência de uma positiva correlação com o índice DMFT. Uma vez que os factores biológicos presentes na saliva influenciam o processo da cárie dentária, o objectivo deste trabalho consistiu na investigação de uma possível correlação entre as proteínas e peptídeos da saliva e o processo da cárie dentária. A caracterização das proteínas e peptídeos da saliva foi alcançada utilizando electroforese bidimensional (2-DE), cromatografia líquida de alta resolução (HPLC) combinada com a espectrometria de massa (MS), do qual resultou a identificação de 38 proteínas das quais 12 foram identificadas pela primeira vez por 2-DE e 22 peptídeos por HPLC-MS também identificados pela primeira vez. Ensaios realizados para o estudo da composição da película dentária seguiram a mesma metodologia descrita para a caracterização das proteínas e peptídeos da saliva sendo realizados inicialmente in vitro e confi rmados posteriormente por ensaios in vivo. A adsorção dos componentes salivares à hidroxiapatite é um processo selectivo com predominância de componentes salivares de baixo peso molecular. Contudo, amilase, lactoferrina, IgA salivar e anidrase carbónica VI foram também identificadas. A extracção sequencial usando guanidina e ácido trifluoroacético das proteínas/peptídeos adsorvidas à hidroxiapatite permitiu uma avaliação da força das ligações estabelecidas. Destes ensaios verificou-se que proteínas ricas em prolina (PRP-1/3), cistatina S, statherina e histatina 1 estabeleciam interacções fortes com a hidroxiapatite permanecendo adsorvidas após extracção com guanidina. As proteínas caracterizadas da saliva e da película dentária foram correlacionadas com o índice DMFT apresentando uma predominância de elevadas quantidades de cistatinas, PRP -1/3, statherina e histatina 1 no grupo de indivíduos sem cárie. O reduzido número de fragmentos em associação com as elevadas quantidades de cistatinas podem sugerir um controle mais eficiente da actividade proteólitica evitando desta maneira a degradação de importantes proteínas salivares no grupo de indivíduos sem cárie. A composição da película dentária é afectada pela composição proteica da saliva encontrando-se as referidas proteínas em maior quantidade. Os dados obtidos sugerem uma eficiente protecção por parte das proteínas da saliva contra a cárie dentária em particular a PRP-1/3, statherina e histatina 1, provavelmente devido à sua participação nos processos de remineralização na superfície do dente, e das cistatinas na diminuição da actividade proteólitica.
Dental caries is a complex disease process that affects a large proportion of the world population, regardless of gender, age and ethnicity. This process is dependent upon biological factors that are present within saliva and dental plaque. Following this, whole saliva was collected from selected individuals characterised according its DMFT and DMFS scores. Evaluation of the conventional clinical parameters such as flow rate, buffering capacity, pH used for caries risk assessment in combination with diet, hygiene and smoke habits was performed for all participating subjects showing absence of a statistic positive correlation with DMFT index. Since biological factors present on saliva influence dental caries process, the aim of this study was to investigate how salivary proteins and peptides are correlated with this pathology. Characterisation of salivary proteins and peptides was achieved using twodimensional gel electrophoresis (2-DE) and high performance liquid chromatography (HPLC) in combination with mass spectrometry (MS) resulting in the identification of 38 proteins, being 12 proteins identified by 2-DE and 22 peptides by HPLC-MS were identified for the first time. Experiments to study enamel pellicle composition were performed following the same methodology described for salivary proteins and peptides, initially in vitro being supported with in vivo assays. Adsorption of salivary components to hydroxyapatite showed to be a selective process with a predominance of low molecular weight salivary components. However, amylase, lactoferrin, S-IgA, carbonic anhydrase VI were also identified. A sequential extraction, using of guanidine and trifluoroacetic acid, of the adsorbed proteins/peptides to hydroxyapatite allowed to evaluate the strength of the establish interactions. From this experiments, proline-rich proteins (PRP -1/3), cystatin S, statherin, histatin 1 exhibited a strong interaction with hydroxyapatite remaining adsorbed after guanidine extraction. Characterised salivary proteins from whole saliva and enamel pellicle were correlated with DMFT index showing a predominance of higher amounts of cystatins, PRP-1/3, statherin and histatin 1 in caries free group. Decreased number of fragments in association with higher amounts of cystatins may suggest a more effective control in proteolytic activity which avoid the degradation of important salivary proteins from caries free group. Acquired pellicle composition is affected by whole saliva protein composition being the above referred proteins present in higher amounts. Obtained data suggest an effective protective role of several salivary proteins to dental caries in particular of PRP-1/3, statherin and histatin 1, possibly due to their participation on remineralization processes at the tooth surface, and of cystatins probably by decreasing proteolytic activity.

2004/2005
In questo lavoro si è cercato di fornire gli strumenti per l'analisi del proteoma della membrana plasmatica con particolare interesse nei confronti delle glicoproteine e delle eventuali modificazioni della loro componente oligosaccaridica, nell'ambito deii'HCC, con lo scopo di individuare nuovi marker glicoproteici da utilizzare in diagnostica e terapia. La componente oligosaccaridica delle glicoproteine di membrana viene coinvolta e continuamente rimaneggiata in diversi processi biologici, che vanno dalla regolazione del sistema immunitario alla comunicazione cellulare, dallo sviluppo embrionale alla capacità patogenetica degli agenti infettivi, dal ripiegamento della catena lineare dei polipeptidi fino allo sviluppo dei tumori e di altre importanti patologie[1J. La limitata disponibilità di dati sperimentali di riferimento per quanto riguarda un approccio di proteomica della membrana plasmatica, ha reso ardua l'interpretazione di molti dei risultati ottenuti riportati in questo lavoro di Tesi. In via preliminare si è reso necessario mettere a punto la maggior parte dei protocolli sperimentali atti ad ottenere il maggior grado di informazioni possibile in merito all'espressione differenziale delle glicoproteine di membrana. Questa fase propedeutica ma indispensabile ha impegnato gran parte del tempo richiesto per lo sviluppo di questo progetto di ricerca. L'approccio sperimentale ha previsto l'utilizzo di due modelli di linea epatocitaria. La linea CHANG, derivante da tessuto di fegato normale, mostra una notevole somiglianza con le cellule normali di fegato ed è citata spesso in letteratura come modello di epatocita in condizione fisiologica[2J. Le cellule HepG2 sono una linea cellulare stabilizzata in coltura derivata da cellule di un epatocarcinoma umano. In primo luogo è stato necessario mettere a punto un metodo di estrazione, confrontando e modificando alcune delle metodofogie già esistenti, al fine di sviluppare una strategia che permettesse di ottenere i risultati migliori in termini di purezza e arricchimento del campione proteico4 Più precisamente, tra queUe disponibili, due sono state messe a confronto e svituppate a seconda detre nostre esigenze . Analizzando i campioni di proteine estratte secondo la strategia differenziate proposta da MoUoyf31 dopo separazione etettroforeticai si è osservato un potenziale arricchimento in proteine dl membrana,. ma la contaminazione da parte della componente dtopfasmatica o proveniente dalle membrane degli organe Ui è. risultata essere ancora troppo a.fta .. Al metodo appena lndicato si è prefertto· queflo che prevede fa marcatura con un derivato della biotina e Ja successiva purificazione su colonna funzionalizzata con avidina[4l: si .è dimostrato, infatti, che attraverso questo metodo estrattivo si possono ottenere proteine che presentano un peso molecolare elevato e che per la maggior parte appartengono alla classe deHe glicoproteine, essendoci una buona corrispondenza tra n profiJo proteico rivelato in colorazione argentica e quello rivelato con un metodo di colorazione specifico per le glicoproteine (ProQ Emerald 300). Inoltre, tramite analisi di immunocitochimica, in fase pre-estrattiva, e di western blot si è verificato che tutte le proteine estratte sono biotinilate; infine, dai gel bidimensionali ottenuti sono evidenziabili le caratteristiche tipiche delle glicoproteine, che si presentano come trenini di spot costituiti delle diverse glicoforme esistenti, differenti tra loro sia per pi che per massa relativa. l'osservazione di questi risultati ci ha fatto ragionevolmente supporre che il metodo di estrazione e purificazione prescelto portasse, effettivamente, ad un arricchimento in proteine di membrana. Successivamente l'analisi comparativa eseguita sulle mappe prote;che relative alta linea· cellulare· CHANG ed HepG2 ha messo in luce numerose differenze, dì tipo proteìco, esistenti a livello della membrana cellulare, ma ha evidenziato anche aJcune somigJianze degne di nota. s; è sceJto dì cominciare l'identificazione delle proteine da quelle che risultavano comuni ad entrambe le linee cellulari e che, ad una prima osservazione dei gel, si presentavano come treni di spot associabili a diverse glicoforme di una glicoproteina. Le analisi di spettrometria di massa hanno fornito risultati interessanti·; anche se inaspettati.. Di particolare importanza è il ritrovamento di segnali attribuibili a proteine con funzioni di Chaperoninei4J .. Tra queste sono state identificate, costantemente:: GR.P78/Bip, HSP60, MTHSP75, HSP90, gp96/GRP94 per entrambe le linee cellulari., mentre POI è stata identificata nelle HepG2. Ed è stata proprio " l' inusualità " di .questo dato che ci ha stimolato a proseguire su una nuova linea interpretativa e a verificare fa possibilità che effettivamente queste proteine fossero presenti su una membrana plasmatica dei modelli cellulari studiati1 da un lato per vatidare le metodologie sviluppate, dall'altro per sfruttare il potenziale informativo fornito da un dato che, seppure anomalo, rimane comunque estremamente interessante. La particolarità di questo risultato risiede nella "anomala" localizzazione topografica di questa dasse di proteine che, normalmente, hanno una tipica.. ma non esclusiva.. localizzazione citoplasmatica o collocazione a livello di reticolo endoplasmatico. Per molte di queste chaperonine si è cercato di dare un interpretazione all'inconsueta localizzazione. In questo lavoro sono state analizzate in maniera più dettagllatate proteine che, tra quelle identificate, presentavano aspetti interessanti sia da.t punto di v.ista funzionale (HSP90 e GRP78) che glicobiologico (gp96). Caratteristica di· tutte- le- proteine- con localizzazione· a llveflo· def- RE, come- GRP94 e GRP78, è la presenza, nella porzione C-terminale, di una particolare sequenza amminoacidica KDEL {lys-Asp-Giu-Leu) che ne garantirebbe la· permanenza a- livello- del· REr51.. Nonostante questa peculiarità, esistono diversi riscontri sperimentali che dimostrano la localizzazione dì GRP78 e gp96 anche a livello della membrana plasmatìca dove sì. assocerebbero con altre proteine in alcuni casi non ancora identificate, per formare complessi di diverse dimensioni.. I meccanismi molecolari chiamati in causa per spiegare la "fuga" di proteine KDEL dal RE alla superficie della membrana plasmatica sono diversi. Ad esempio alcuni dati sembrerebbero attribuire questo evento ad una saturazione dei recettori per KDEL con conseguente perdita di alcune proteine che sarebbero in grado di migrare verso la membrana plasmatica. In altri casi il difetto nel sistema di ritenzione potrebbe essere dovuto alla presenza di .forme tronche delle proteine o difettive del dominio di riconoscimento. Un'altra ipotesi prevede che l'associazione delle proteine KDEL con proteine che sono destinate ad essere esportate verso la membrana plasmatica possa bloccare stericamente H dominio KDEL, impedendone l'interazione con il· rispettivo recettore e comportando la comigrazione verso la membrana plasmatica. Queste ossetvazioni, per quanto interessanti, rappresentano comunque solo interpretazioni finalistiche di un comportamento- che, alla fuce dei risultati riportati in questo favoro e di· quelli in letteratura, potrebbe essere molto più importante e di maggior significato biologico: non è un caso che tutti i dati più significativi e, al momento,. più. c.ompJeti. riguardano forme ceJJuJari associate a. trasformazioni neoplastiche .. Su HSP90, in letteratura, sono state fatte le considerazioni più interessanti. Dati recenti attestano la sua localizzazione sulla superficie cellulare in particolare sulla -membrana -dei -neuroni nelle fasi .precoci delt.o sviluppo de.l sistema nervoso: si ipotizza che questa chaperonina sia coinvolta nella migrazione cellulare[6J. Inoltre, è stato proposto che, sulla superficie cellulare, .HSP90 svolgesse un .ruolo attivo, in questo caso ln senso migratorio, partecipando a qualche meccanismo che· porta la cellula a· staccarsi dalla matrice extracellulare e dalle cellule vicine. Questo dipenderebbe dalla stretta relazione che esiste tra HSP90 e MMP2, enzima. coinvolto nel rimodellamento-della-matrice extracellulare[7l. Dal punto dì vista dì un approccio glicomico alla trasformazione neoplastìca e facendo salvo il concetto ormai accettato e dimostrato della stretta associazione tra. Ja trasformazione neo.pJastica e la: modificazione dei. pattem di glicosilazione appare piuttosto interessante l'osservazione secondo la quale alcune di queste proteine vengano attivate ad alti livelli in presenza di inibitori della glicosilazione. Le alterazioni della glicosilazione potrebbero essere, entro certi limiti, assimilate agli effetti prodotti dal trattamento con inibitori della glicosilazione. Non bisogna dimenticare, inoltre, che questi chaperone molecolari sono deputati al controllo e alla successiva eliminazione di proteine non correttamente ripiegate e/o glicosilate: una loro alterata funzionalità potrebbe risolversi in una mancata eliminazione o sequestramento detta proteina non funzionale con conseguente trasporto della stessa al compartimento di competenza. La presenza, quindi, di proteine non correttamente gticosilate sulla membrana plasmatica potrebbe essere· dovuta a meccanismi di· eliminazione alterati a livello del RE- e del· Golgi. Certamente questa è semplice considerazione ipotetica che, in ogni caso, potrebbe costituire una buona base di partenza per ulteriori e più a-pprofonditi. studi. In questo lavoro si è cercato non solo di ottenere gli strumenti per facilitare la comprensione del proteoma di membrana ma anche. porre. te basi per lo studio e ·la caratterizzazione degli N-glicani associati a questo compartimento. Quest'ultimo aspetto sperimentale è piuttosto rilevante: la possibilità di sviluppare una gUcoproteomica in senso stretto- si è sempre scontrata con .ta sostanziale incompatibilità dei metodi disponibili in letteratura, che comportavano o la perdita della componente saccaridica o n· sacrificio di quella proteicarsJ. Fintanto che l'approccio glicoproteomico era rivolto esclusivamente all'identi.ficazione de.l complessQ delle proteine espresse da una cellula; ciò· non· ha mai costituito un problema; quando· invece si rende necessaria un'analisi di un compartimento esclusivo come quello della membrana plasmatica, dove la componente glicoproteica è poco rappresentata, il discorso è diverso. In taf senso, f'ottimìzzazìone degfi approcci sperimentali di 2-DE che consentono la simultanea caratterizzazione della porzione oligosaccaridica e di quella proteica è auspicabile se non indispensabile... Proprio in quest'ottica risiede l'importanza dei risultati ottenuti in questo ·lavoro, ossia nell'aver messo a punto un efficiente metodo di degli cosilazione in gelr9J in associazione alla separazione 20-E, che permettesse di mantenere integra ed analizzabile sia la componente oligosaccaridica che quella proteica, per lo sviluppo di una completa glicomica della membrana plasmatica.
XVIII Ciclo
1974
Versione digitalizzata della tesi di dottorato cartacea.

Dissertation presented to obtain the PhD degree in Biochemistry
Most echinoderm species share an outstanding capacity for regeneration that is maintained throughout the adult animal lifespan. Regeneration allows these deuterostomes to recover from predation injuries or selfinduced arm autotomy, which are known to occur frequently in nature. Although echinoderms are extremely interesting in terms of their phylogenetic proximity to chordates, most areas of echinoderm research have been neglected in recent years. These wonderful animals quickly shifted from being the preferred animal models in the 19th-20th centuries of the pioneer regenerationists to scientific oblivion. Other species, for which the possibility of conducting genetic studies became available, are now favored. After the sequencing of an echinoderm species genome, the sea urchin Strongylocentrotus purpuratus in 2006, several scientific reports of interesting molecular studies were published.(...)
Fundação para a Ciência e a Tecnologia

In this study, my aim was to investigate oligodendrocyte signalling using a non-candidate, unbiased, proteomic approach. First, in collaboration with my industrial sponsors Merck Sharpe and Dohme, the potential of using 2-dimensional differential in gel electrophoresis (2D DIGE) to study oligodendrocyte signalling was evaluated. Although differentially expressed proteins between the 2 proteomes under analysis could be identified, it was concluded that 2D DIGE would be an unsuitable method to study oligodendrocyte biology. An alternative approach was therefore pursued in which a specific subset of the proteome was analysed. Previously, α6β1 integrin has been shown to be involved in oligodendrocyte survival and morphological differentiation. A protocol was developed whereby protein complexes co-immunoprecipitated with α6β1 integrin could be identified by mass spectroscopy analysis. Multiple proteins were identified including β1 integrin, contactin, plectin and heterogeneous ribonuclear protein K (hnRNP K). Characterisation and function of contactin in oligodendrocyte α6β1 integrin signalling was then investigated. Contactin expression throughout oligodendrocyte in vitro differentiation was confirmed by both Western blotting and Immunocytochemistry. The interaction of contactin and α6β1 was also verified by traditional co-immunoprecipitation. siRNA knockdown of contactin had no effect on in vitro oligodendrocyte morphological differentiation. However, contactin siRNA knockdown did block oligodendrocyte enhanced survival of the α6β1 integrin substrate, laminin-2, in response to platelet derived growth factor (PDGF). Therefore, by taking a proteomic approach, I have identified a novel interaction between α6β1 integrin and contactin, and a potential role of contactin in α6β1 integrin mediated survival signalling.

Intermediate filament proteins (IFPs) form the main structural elements of a wool fibre. The IFPs of wool are comprised of two families; the acidic type I family and the neutral-basic type II family. During follicle development, one type I and one type II IFP develop into an obligate heteropolymer, which, through a series of associations with other heteropolymers, forms an intermediate filament. Two-dimensional polyacrylamide gel electrophoresis (20-PAGE) methods have been used to provide high-resolution separation of wool IFPs. Improvements in the method for maintaining reducing conditions and chaotrope constitution, combined with low % T polyacrylamide gels, allowed the high-resolution separation of the two keratin IFP families and their individual family members. The IFPs were separated to produce a clearly defined spot pattern, with numerous discrete minor spots not previously observed. Genomic studies have reported that there are eight genes which produce eight abundant IFPs in wool. It was hypothesised that the large number of additional spots seen on a 20-PAGE gel was due to post-translational modification (PTM) of the protein. Several common PTMs of proteins produce charge heterogeneity, including phosphorylation and glycosylation. However, analysis of wool IFPs by 20- PAGE techniques and mass spectrometry revealed no evidence of phosphorylation or glycosylation modifications. Conformational equilibria as a cause of protein charge heterogeneity has recently been reported. Investigations with both the type I and type II IFPs have shown that when single protein spots from a 20-PAGE separation are eluted, re-focused and re-electrophoresed, several spots are formed on both the acidic and basic side of the original spot. The cause of this heterogeneity is thought to be a conformational equilibrium between several different forms of the same protein in the rehydration solution used for the first dimension. This technique allowed the accurate assignment of IFPs resolved by 20-PAGE to protein families. Fractionation methods to separate the IFPs and intermediate filament associated proteins (IFAPs) were successfully developed. Further fractionation into the type I and type II IFPs was achieved along with partial success at isolating individual spots. In vitro assembly experiments with the different IFP families gives important information about the strength of different protein pairings. To date there are no reproducible, efficient, in vitro assembly conditions for keratinised wool IFPs. A comprehensive study to investigate assembly conditions for keratinised wool IFPs was undertaken. Assembly of filaments from IFPs was achieved after a partial digestion with chymotrypsin. Filaments were formed that varied in diameter from 10 to 40 nm, showing that higher ordered structures were being formed. This demonstrates that IFPs can be successfully assembled in vitro to form filamentous structures that may be able to be manipulated for biomaterial uses.

Mestrado em Bioquímica - Bioquímica Clínica
A hipertensão arterial pulmonar é uma doença com mau prognóstico que coloca em risco a vida dos pacientes, e cuja severidade e sintomas estão fortemente relacionados com a função do ventrículo direito. A caquexia cardíaca é uma complicação da insuficiência cardíaca crónica que resulta de um desequilíbrio entre as vias catabólica e anabólica. Este desequilíbrio é consequência de uma série de processos imunológicos, metabólicos e neurohormonais. Apesar de vários biomarcadores terem sido propostos no contexto da insuficiência cardíaca, a maioria é usada apenas para indicação de prognóstico. A aplicação da proteómica à análise de fluidos biológicos para estudo da insuficiência cardíaca e caquexia associada poderá ser útil na identificação de um painel complementar de biomarcadores com melhor desempenho e poder de diagnóstico. De modo a caracterizar o efeito da hipertensão arterial pulmonar nos perfis proteico e proteolítico da urina dos pacientes, recorreu-se a tecnologias como SDS-PAGE nanoLC-MS/MS e zimografia. Os dados da análise por nanoLC-MS/MS foram submetidos à base de dados UniProt, sendo depois analisados com base em ferramentas bioinformáticas. Foi identificado um total de 277 proteínas, sendo que 51 delas eram comuns a todos os indivíduos. Várias proteínas exclusivas foram identificadas tanto nos pacientes com hipertensão arterial pulmonar, como nos indivíduos saudáveis. A WNK4 foi a única proteína comum aos 2 pacientes. Em suma, os resultados evidenciam uma elevada actividade proteolítica na urina de pacientes com hipertensão arterial pulmonar, enfatizando a inflamação e caquexia associadas à doença, e permitiram também sugerir a WNK4 como um novo potencial biomarcador para o diagnóstico da hipertensão arterial pulmonar.
Pulmonary arterial hypertension is a life-threatening disease associated with poor prognosis, whose severity of symptoms and survival are strongly related with right ventricular function. Cardiac cachexia is a serious complication of chronic heart failure that results from an imbalance of catabolic and anabolic pathways. This imbalance is caused by a series of immunological, metabolic and neurohormonal processes. Although several biomarkers have been proposed in the context of heart failure, most of them present only potential prognostic value. The application of biofluids’ proteomics to the study of heart failure and related cachexia may help to identify a panel of complementary biomarkers with better performance and diagnostic power. In order to characterize the effect of pulmonary arterial hypertension on the urinary protein and proteolytic profiles, SDS-PAGE nanoLC-MS/MS and zymography were performed. Data from nanoLC-MS/MS was submitted to UniProt database and then were analyzed using bioinformatics tools. A total of 277 proteins were identified and 51 of them are common between all the individuals. Several exclusive proteins were identified in both pulmonary arterial hypertension patients and healthy subjects and WNK4 was the only common protein between pulmonary arterial hypertension patients. Taken together, data highlight a high proteolytic activity in the urine of pulmonary arterial hypertension patients, emphasizing the disease-related inflammation and cachexia and also allow to suggest WNK4 as a new potential biomarker for the diagnosis of pulmonary arterial hypertension.

I risultati riportati nella tesi riguardano lo studio di caratterizzazione di due ecotipi autoctoni molisani di lenticchia (Lens culinaris M.) e l'utilizzo di un nuovo approccio per la genotipizzazione delle varietà locali. In particolare il lavoro si è concentrato su tre argomenti principali: 1) l'uso della proteomica per studiare la variabilità genetica esistente tra diverse popolazioni di lenticchia, 2) la risposta di varietà autoctone di lenticchia allo stress salino e 3) l'uso del loop-mediated isothermal DNA amplification (LAMP) per l'amplificazione dei microsatelliti (SSR). Per raggiungere il primo obiettivo, lo studio si è concentrato sull'analisi proteomica del seme maturo di lenticchia. L'utilizzo dell'elettroforesi bidimensionale ha permesso l'individuazione di 135 spot ben risolti, che rappresentano la prima mappa del proteoma del seme maturo di lenticchia. Inoltre l'analisi statistica multivariata, effettuata utilizzando i dati quantitativi degli spot risultati differenzialmente espressi, ha portato all'individuazione delle proteine che risultano indispensabili per la differenziazione delle diverse popolazioni di lenticchia analizzate, come riportato in Scippa et al., 2010. La diversità delle due varietà locali di lenticchia (Capracotta e Conca Casale) del Molise è stata studiata utilizzando un approccio integrato costituito da analisi a livello morfologico, genetico e proteomico, come dimostrano i risultati pubblicati in Viscosi et al., 2010. Inoltre, un ulteriore studio è stato eseguito al fine di approfondire le informazioni sulla caratterizzazione proteomica delle lenticchie di Capracotta e Conca Casale. I dati ottenuti sono stati elaborati mediante un'analisi statistica uni- e multivariata mediante cui sono state individuate le proteine, risultanti principalmente coinvolte nella risposta agli stress abiotici e biotici, che caratterizzano i due ecotipi autoctoni. Successivamente, il lavoro ha studiato a livello fisiologico e biochimico, la risposta delle due varietà molisane di lenticchia e di cinque varietà commerciali allo stress salino. In particolare è stato analizzato come lo stress salino influenza la germinazione e causa modificazioni nell'espressione di alcune proteine. Inoltre, l'analisi statistica multivariata è stata effettuata per identificare le proteine importanti coinvolte nella risposta a questo tipo di stress. Infine la tesi di dottorato si è focalizzata sull'individuazione di un nuovo approccio molecolare da utilizzare ai fini della genotipizzazione delle varietà locali. Il lavoro è stato svolto incorporando un microsatellite (SSR) del riso all'interno di un LAMP assay e ha dimostrato che i risultati sono coerenti con l'analisi eseguita mediate amplificazione del microsatellite attraverso la tradizionale PCR. Inoltre, il LAMP assay è caratterizzato da un' elevata sensibilità, dato che risulta in grado di amplificare il DNA target anche quando questo è presente in poche copie. Il lavoro riportato nella tesi di dottorato rappresenta una novità nel campo del proteoma del seme di lenticchia e dimostra che la proteomica è un potente strumento per l'analisi della biodiversità in ecotipi appartenenti ad una stessa specie. Inoltre, il lavoro conferma che gli studi di proteomica possono notevolmente contribuire a comprendere i complessi meccanismi coinvolti nella risposta della pianta allo stress salino. L'originalità della tesi di dottorato è rappresentata anche dal nuovo uso del metodo LAMP per amplificare SSR.
The results reported in the thesis are related with the study of characterization of two autochthonous lentil (Lens culinaris M.) landraces of Molise region and of a new approach for genotyping of local varieties. In particular the work has been focused on three major issues: 1)use of proteomics to investigate natural variation within and between lentil populations, 2) response of lentil to salt stress and 3) use of the loop-mediated isothermal DNA amplification (LAMP) method to amplify SSR. To accomplish the first objective investigations have been focused on the proteomic analysis of mature seed of lentil. The use of 2DE couple to the MS/MS allowed the identification of 135 well resolved spots which represent the first lentil seed proteome reference map. In addition the multivariate statistical analyses carried out on spots, resulted differentially expressed between different lentil populations, led to detection of the proteins which were essential for population discrimination, as shown in the paper Scippa et al., 2010. The diversity of two lentil landraces (Capracotta and Conca Casale) from Molise was studied using a combination of morphological, genetic and proteomic analyses, as shown by results published in the paper by Viscosi et al., 2010.Moreover, a further study has been carried out to deepen the knowledge about proteomic characterization of Capracotta and Conca Casale. The data obtained were elaborated by uni-and multivariate statistical analysis to identify the proteins that characterize the lentil ecotypes, mainly involved in abiotic and biotic stress responses. On the base of aforementioned results, the PhD project proceeded in analyzing the physiological and proteomic response of two lentil landraces from Molise and five commercial varieties to salt stress. In particular it has been investigated how salt stress affected seed germination and caused changes in expression of proteins. Furthermore, the multivariate statistical analysis was performed using quantitative data of spots in order to identify important proteins involved in saline stress response. The third aim of the PhD thesis was the detection of a new molecular approach for genotyping of local varieties. Work has been carried out by incorporation rice simple sequence repeat (SSR) motif within a LAMP assay. It has been demonstrated that results were consistent with analysis performed by PCR. In addition, LAMP assay was characterized by high sensitive being able to amplify from near single copy of target. The work reported in the PhD thesis represents a novelty in the field of proteome of lentil seed and it surely shows that proteomics is a powerful tool for analysis of biodiversity in ecotypes of a single plant species. Furthermore, the work confirms that proteomics studies can significantly contribute to understand the complex mechanisms involved in the plant response to salt stress. The originality of the PhD thesis is also represented by novel use of the loop-mediated isothermal DNA amplification method to amplify SSR.

The concept of endothelial progenitor cells (EPCs) has attracted considerable interest in cardiovascular research, but despite a decade of research there are still no specific markers for EPCs and results from clinical trials remain controversial. To identify membrane proteins, which could serve as potential markers for EPCs, an alternative proteomic method was adopted to obtain sufficient membrane material for proteomic analysis. Microparticles (MPs), the intact vesicles formed from the plasma membrane, were harvested from the conditioned medium of EPC cultures and their protein composition was analysed by liquid chromatography-tandem mass spectrometry. Surprisingly, the platelet-specific integrin alpha lib emerged as the most abundant integrin in EPC cultures. Subsequent experiments confirmed that the conventional methods for isolating peripheral blood-derived mononuclear cells (PBMNCs) lead to a substantial contamination with platelets. Notably, platelets readily disintegrate into platelet MPs when activated during PBMNCs isolation conditions. These platelet MPs are taken up by the PBMNCs, which acquire "endothelial" characteristics (CD31, von Willebrand factor [VWF], lectin-binding), and angiogenic properties. In a large population-based study (n =526), platelets emerged as a positive predictor for the number of colony-forming units and early outgrowth EPCs. This study provides the first evidence that the cell type consistent with current definitions of an EPC phenotype may arise from an uptake of platelet MPs by mononuclear cells resulting in a gross misinterpretation of their cellular progeny. In addition, the release of platelet MPs at sites of vascular injury may play a role in orchestrating tissue repair and contribute to the activation of an angiogenic programme in monocytes by inducing the expression of non-coding regulatory RNA, known as microRNAs, which act as translational repressers. The uptake of platelet MPs by mononuclear cells induced the release of the CXCL7 chemokine which in turn guided a de novo induction of miR-885-5p in mononuclear cells. The increased expression of miR-885-5p resulted in the targeted reduction of the actin-bundling protein LCP-1, facilitating the adhesive and migratory ability of mononuclear cells. These findings demonstrate the advantage of using an unbiased proteomic approach to assess cellular phenotypes and advise caution in attributing the benefits inclinical trials using unselected bone marrow mononuclear cells (BMCs) to stem cell-mediated repair.

Mastocytosis is a myeloproliferative neoplasm characterized by infiltration of clonally derived mast cells in different tissues. According to mast cells localization, it is possible to discriminate cutaneous mastocytosis (CM) from systemic mastocytosis (SM), the latter involving at least an extracutaneous organ, like bone marrow, liver, spleen and gastrointestinal tract. Some of the SM patient can develop also cutaneous lesions (SM+C). Oral cavity is commonly involved in the symptomatology. Disease classification is often tricky. In the first part of this thesis, in order to highlight possible qualitative/quantitative modifications of the salivary proteome associated to the different forms of the disease, we investigated salivary samples collected from 6 CM, 35 SM patients, among which 8 with only systemic symptoms (SM-C) and 27 with both systemic and cutaneous symptoms (SM+C), and 48 healthy controls by a top-down proteomic approach. Low-resolution HPLC-ESI-MS analysis of the acid soluble fractions of saliva highlighted different proteomic profiles in the three patients’ groups, showing that the salivary samples of the patients were characterized by a down-regulation of peptides and proteins involved in the homeostasis and defense of the oral cavity, and in the innate immunity and in inflammation not only in the oral cavity but at systemic level, such as aPRPs, statherin, histatins and cystatins. Only two proteins with regulatory roles in the innate immunity and inflammation, S100A8 and antileukoproteinase, resulted up-regulated in patients differently to all the other salivary proteins analyzed, suggesting the establishment of a response by the organism to the injuries caused by the disease. Interestingly, some differences have been found among the patients in the concentration of α-defensins 1, thymosin β-4, and the truncated forms of cystatin D-R26 variant, and some truncated forms of P-B and statherin. Correlation between the protein/peptide levels and tryptase concentration evidenced that acidic PRPs, statherin and P-B fragments, and cystatin D-R26 des1-5 correlated positively just in SM-C group, while thymosin β-4 correlated negatively. Since the interesting data on cystatin D, in the second part of the thesis I focused on the characterization of the salivary protein complex aggregating to the cystatin D-C26 variant (named by us SIC-D). Indeed, the C-26 variant is usually undetectable in acid soluble fraction of saliva but measurable in whole saliva. Pools of whole saliva from 4 CM, 3 SM-C, 14 SM+C, and 20 sex/age matched healthy controls, were submitted to immunoprecipitation with cystatin D-C26 antibody followed by SDS-PAGE/western-blot under reducing and non-reducing conditions. Since the low volume of CM samples, the tryptic digestion, and the nano-HPLC-high-resolution-MS/MS analysis were performed only in SM-C, SM+C and control samples. The quantitative comparison was performed with Proteome Discoverer 2.2 software. SIC-D included 44 proteins, among which IgA, IgG, PIgR, annexins, α-defensin 1/2, S100A8, carbonic anhydrase 6, prolactin-inducible protein, lysozyme C and dermicidin. Several qualitative/quantitative differences were highlighted with respect to controls and between the two patient groups. The most relevant were: all the patients exhibited lower levels of IgA, PIgR, DMBT-1 and S100A8 than controls, but higher levels of IgG, α-defensins 1/2 and carbonic anhydrase 6. The highest level of cystatin D-C26 was found in SM+C patients, which were different from SM-C for annexin A2. Both SM-C and SM+C showed the presence of antileukoproteinase and S100A14. The results on the acid-soluble fraction of saliva and the preliminary results on the SIC-D complex are promising in order to find candidate markers able to discriminate the different forms of mastocytosis.

Der SET-Bereich befindet sich unter den verschiedenen Proteinsequenzbereichen, die mit epigentischer Regulation hauptsächlich durch die Präsenz von Trihtorax (trxG) und Polycomb (PcG) Gruppen von Chromatinmodifikatoren in Zusammenhang gebracht werden. Nach der Entdeckung des ersten SET-Bereichs vor einigen Jahren, welcher die Histon-Lysin-Methyltransferase (Su(var)39) enthält, wurde den Proteinen mit SET-Bereich sehr viel Aufmerksamkeit geschenkt. Obwohl die Histon-Lysin-Methylierung schon länger als 30 Jahre bekannt ist, war ihre Funktion vor diesem überragenden Ergebnis größten Teils unbekannt. In meiner Arbeit beschreibe ich die kombinatorische und funktionale Charakterisierung von 3 Hefe Proteinkomplexen durch die Anwendung von proteomischer SEAM (Sequential Epitope Tagging and Mass Spectrometry). Zwei dieser Komplex enthalten einen SET-Bereich und die Dritte ist der Rad6 Komplex aus S. pombe (Sp_Rad6C). Der Set1 Komplex (Set1C) beinhaltet 8 Bausteine, methylisiert Lysin 4 in Histon H3 und ist die erste, entdeckte Histone H3 Lysin 4 Methyltransferase. Es beinhaltet ein Ash2 Homolog (Bre2), einen bekannten Baustein von trxG. Kürzlich wurden Rad6 beinhaltende Komplexe gezeigt, die in engem Bezug zu der H3-K4 und H3-K79 Methylation durch ubiquitinierung von Histon H2B und die Etablierung von trans-histonen Signalwegen stehen. Unsere Analysen von Sp-Rad6C führten zu mehreren interessanten Ansichten. Der Set3 Komplex (Set3C) hat keine feststellbare Aktivität einer Methyltransferase, enthält jedoch zwei Histon deacetylasen (HDACs) ? eine klassische HDAC (Hos2) und eine NAD-abhängige HDAC (Hst1). Unsere funktionelle Analyse von Set3C zeigt, dass Set3C bei der Regulierung des meiotischen Genexpressionsprogramms in knospenden Hefen (S. cerevisiae) beteiligt ist. Evolutionbiologisch betrachtet, ist die Spalt-Hefe (S. pombe) sehr weit von S. cerevisiae entfernt und wird meist als ein besserer Modellorganismus fur höhere Eukaryoten angesehen. In einem Versuch, unser Wissen uber andere Organismen zu vergrößern, haben wir ähnliche Untersuchungen in S. pombe unternommen und haben herausgefunden, dass Set1C in beiden Hefen sehr stark konserviert ist. Darüberhinaus waren die Set1-Ash2 Verbindungen konserviert und wir nehmen an, dass auch in höheren Eukaryoten Set1-ahnliche Methyltransferasen Ash2-ahnliche Proteinen angehören. Dies wurde später durch mehrere Studien von anderen Gruppen bestätigt, die an Säugetieren arbeiten. Was Set3C anbelangt, wurden unsere weiteren Analysen nur durch vergleichende Proteomik beschränkt. Wir zeigen, dass der proteomische Kern von Set3C in Spalt-Hefe konserviert wird. Im Gegensatz zu Set3C in S. cerevisiae, beinhaltet diese in S. pombe nur eine HDAC, die zur Hos2 Familie gehört,. Die präsentierte Arbeit hat auch viele Auswirkungen auf die übergreifende Organisation von Proteomen. Wir beschreiben verschiedene Beispiele von gemeinsamen Komponenten zwischen unterschiedlichen Komplexen und prägen den Begriff "proteomischer Hyperlink". Wir waren in der Lage zu zeigen, dass proteomische Kerne sogar für unwesentliche Proteinkomplexe hoch konserviert sind. Die generelle proteomische Schaltung über proteomische Hyperlinks scheint jedoch verworrener und unvorhersehbar zu sein. Wir schlussfolgern, dass die Erschaffung von zuverlässigen, detailierten, proteomischen Abbildungen, welche auf dem Wissen von niederen Organismen fundieren, zur Zeit nicht möglich ist.

Breast cancer represents a heterogeneous collection of different diseases characterized by different pathological and biological features, clinical presentation, clinical behaviour, response to treatment and outcome. In current practice, pathological diagnosis and classification of breast cancer is based mainly on well-established traditional morphologic features. However, morphological features alone do not adequately reveal the molecular heterogeneity and complexity of breast cancer. Still, there are relatively few biomarkers widely used in prognostication in invasive breast cancer and in predicting response to targeted therapies, and even fewer of value in the clinical management of the pre-invasive disease of ductal carcinoma in situ (DCIS). There is therefore an unmet need for biomarkers for better classification, better prediction of prognosis and of prediction of response to therapy for both invasive breast carcinoma and DCIS. The co-expression of a HER2/HER3 combination results in more aggressive tumour growth and is associated with endocrine and chemotherapy resistance, driven not simply by receptor expression but also by signalling via the receptors dimers. Therefore, methods which directly query signalling pathway activation in breast cancer specimens are anticipated to provide important insights into the molecular ‘‘logic’’ that distinguishes cancer from normal tissues and potentially to have an important impact on personalized intervention strategies. The aim of this thesis has been directed at evaluating candidate biomarkers in breast cancer. This has been targeted at examining attributes associated with known functional properties of candidate drivers of disease or resistance to treatment rather than those traditionally based on altered expression of these biomarkers. Specifically, the work was directed at the HER1-3 members of the EGFR family of growth factor receptor in breast cancer. In this project I have in part developed, tested and evaluated two methods, which have the ability to detect protein-protein complexes at a single molecule level and thus allow the study of signalling pathways in situ. The first method is an in-house coincidence detection technology created from two recombinant fusion proteins and the second is a commercially available proximity ligation assay (PLA) method. Both approaches were able to detect the target proteins with high sensitivity and specificity, however the proximity ligation assay was subsequently used here to assess the protein complexes and activation status of the EGFR family in breast cancer patient’s samples. The patient study cohort is derived from a consecutive series of approximately 293 cases of primary operable invasive breast cancers obtained from the Guy’s and St Thomas (King’s Health Partner’s) Breast Cancer Biobank archive presenting between 1990 and 1992. In these cases, 9 different biomarkers were studied for HER family expression level, dimer expression and activation status using proximity ligation assays (PLAs). The relationship between HER activation status, dimer expression and relapse free survival (RFS) was investigated and stratified multivariate regression analysis identified factors influencing patient prognosis. In conclusion, PLA successfully and reproducibly detected HER protein complexes and phosphorylation in vivo. A significant association was identified between high levels of phosphorylated HER2 and reduced recurrence-free survival (RFS) in invasive lobular carcinoma (p = 0.04, HR 0.99, 95% CI: 0.997-1.002). High levels of HER1/HER3 dimers were associated with reduced RFS in T1 (<2cm) breast cancer patients, (p = 0.02, HR 1.84, 95% CI: 1.08-3.13). Similarly, high levels of HER1/HER3 and HER2/HER3 dimers were associated with reduced RFS in breast cancer patients with N1 nodal status (p <0.0001, HR 1.84, 95% CI: 0.58-1.93) and (p <0.0001, HR 0.64, 95% CI: 0.45-0.90) respectively). Work in this thesis demonstrates that in situ detection of HER protein complexes and activation status can be monitored robustly and with specificity in clinical specimens, providing novel prognostic information. This technique was also applied successfully to assess the HER family in a smaller number of DCIS cases. This novel technique and approach could potentially be applied for patient stratification and assist in the selection of more individualized treatment options according to tumour molecular characteristics.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen associated with severe nosocomial infections in immunocompromised hosts and patients with cystic fibrosis (CF). During infection the bacteria secrete proteins that are essential to the infection process. Several of these virulence-associated proteins have been identified using genetic methods. The aim of this research, using a proteomic approach, was to identify novel extracellular proteins that are secreted by P. aeruginosa during infection of a CF patient. Extracellular proteins from P. aeruginosa strain PAO1 grown in vitro were separated by two-dimensional gel electrophoresis (2-DE). The humoral response of chronically infected CF patients to the separated proteins was elucidated using western blotting. Growth phase, cell population and iron limitation were identified as important regulators of the extracellular proteome. The number of extracellular proteins significantly increased upon entry into stationary phase, as did the number of proteins detected by CF patient sera. The detection of several known quorum-controlled proteins by patient sera indicated the importance of this regulatory mechanism during infection. In iron-limiting medium, the proportion of proteins detected by CF patient sera significantly increased compared to extracellular proteins from cells grown in iron-replete conditions. Proteomic analysis of a PAO1 pvdS mutant strain showed that PvdS (an iron-regulated alternative sigma factor) directs production of many extracellular proteins made during infection of a CF patient. Examination of extracellular proteins from a second strain, PA4, indicated it had a shared set of extracellular proteins. The identities of selected proteins were determined and these included well-characterised extracellular virulence factors such as elastase (LasB). Also identified were proteins with a potential virulence role such as azurin (a copper containing redox protein), PA2939 (a likely aminopeptidase) and proteins with unknown functions. This study provides the first evidence for the production of these proteins during infection. In summary, the proteomics methodology developed here facilitated the rapid identification and enumeration of proteins secreted by P. aeruginosa during infection.

Tamoxifen is an effective and well-tolerated treatment for early disease and/or pre-menopausal patients with breast cancer (BC); although many women go on to develop resistance. Currently the five-year survival rate following Tamoxifen resistance (TR) is < 20%; hence the mechanisms need to be better understood. Recent research has focussed on specific pathways, however additional mechanisms are involved and we investigated these using cell line models of BC (MCF7) and TR using a variety of proteomic approaches. Differential expression and phosphorylation of proteins between the MCF7 and TR cell lines were detected by antibody arrays; which detected changes in Mitogen Activated Protein Kinases and Receptor Tyrosine Kinases family members, and in apoptosis related proteins. There were 21 novel proteins found to be altered in TR. 262 quantifiable proteins were found using SILAC; 29% over expressed in resistance and 25% down regulated. 5 were subsequently picked for validation by Western blot and 2 of these (IQGAP1 and cortactin) were chosen for further investigation with siRNA and functional assays. IQGAP1 was found to play a role in TR; as decreasing expression of IQGAP1 using SiRNA decreased the proliferation of TR cells and significantly modulated the TR cells ability to invade matrigel.

Der SET-Bereich befindet sich unter den verschiedenen Proteinsequenzbereichen, die mit epigentischer Regulation hauptsächlich durch die Präsenz von Trihtorax (trxG) und Polycomb (PcG) Gruppen von Chromatinmodifikatoren in Zusammenhang gebracht werden. Nach der Entdeckung des ersten SET-Bereichs vor einigen Jahren, welcher die Histon-Lysin-Methyltransferase (Su(var)39) enthält, wurde den Proteinen mit SET-Bereich sehr viel Aufmerksamkeit geschenkt. Obwohl die Histon-Lysin-Methylierung schon länger als 30 Jahre bekannt ist, war ihre Funktion vor diesem überragenden Ergebnis größten Teils unbekannt. In meiner Arbeit beschreibe ich die kombinatorische und funktionale Charakterisierung von 3 Hefe Proteinkomplexen durch die Anwendung von proteomischer SEAM (Sequential Epitope Tagging and Mass Spectrometry). Zwei dieser Komplex enthalten einen SET-Bereich und die Dritte ist der Rad6 Komplex aus S. pombe (Sp_Rad6C). Der Set1 Komplex (Set1C) beinhaltet 8 Bausteine, methylisiert Lysin 4 in Histon H3 und ist die erste, entdeckte Histone H3 Lysin 4 Methyltransferase. Es beinhaltet ein Ash2 Homolog (Bre2), einen bekannten Baustein von trxG. Kürzlich wurden Rad6 beinhaltende Komplexe gezeigt, die in engem Bezug zu der H3-K4 und H3-K79 Methylation durch ubiquitinierung von Histon H2B und die Etablierung von trans-histonen Signalwegen stehen. Unsere Analysen von Sp-Rad6C führten zu mehreren interessanten Ansichten. Der Set3 Komplex (Set3C) hat keine feststellbare Aktivität einer Methyltransferase, enthält jedoch zwei Histon deacetylasen (HDACs) ? eine klassische HDAC (Hos2) und eine NAD-abhängige HDAC (Hst1). Unsere funktionelle Analyse von Set3C zeigt, dass Set3C bei der Regulierung des meiotischen Genexpressionsprogramms in knospenden Hefen (S. cerevisiae) beteiligt ist. Evolutionbiologisch betrachtet, ist die Spalt-Hefe (S. pombe) sehr weit von S. cerevisiae entfernt und wird meist als ein besserer Modellorganismus fur höhere Eukaryoten angesehen. In einem Versuch, unser Wissen uber andere Organismen zu vergrößern, haben wir ähnliche Untersuchungen in S. pombe unternommen und haben herausgefunden, dass Set1C in beiden Hefen sehr stark konserviert ist. Darüberhinaus waren die Set1-Ash2 Verbindungen konserviert und wir nehmen an, dass auch in höheren Eukaryoten Set1-ahnliche Methyltransferasen Ash2-ahnliche Proteinen angehören. Dies wurde später durch mehrere Studien von anderen Gruppen bestätigt, die an Säugetieren arbeiten. Was Set3C anbelangt, wurden unsere weiteren Analysen nur durch vergleichende Proteomik beschränkt. Wir zeigen, dass der proteomische Kern von Set3C in Spalt-Hefe konserviert wird. Im Gegensatz zu Set3C in S. cerevisiae, beinhaltet diese in S. pombe nur eine HDAC, die zur Hos2 Familie gehört,. Die präsentierte Arbeit hat auch viele Auswirkungen auf die übergreifende Organisation von Proteomen. Wir beschreiben verschiedene Beispiele von gemeinsamen Komponenten zwischen unterschiedlichen Komplexen und prägen den Begriff &quot;proteomischer Hyperlink&quot;. Wir waren in der Lage zu zeigen, dass proteomische Kerne sogar für unwesentliche Proteinkomplexe hoch konserviert sind. Die generelle proteomische Schaltung über proteomische Hyperlinks scheint jedoch verworrener und unvorhersehbar zu sein. Wir schlussfolgern, dass die Erschaffung von zuverlässigen, detailierten, proteomischen Abbildungen, welche auf dem Wissen von niederen Organismen fundieren, zur Zeit nicht möglich ist.

As the complexity of our society and computational resources increases, so does the complexity of the problems that we approach using evolutionary search techniques. There are recent approaches to deal with the problem of scaling evolutionary methods to cope with highly complex difficult problems. Many of these approaches are biologically inspired and share an underlying principle: a problem representation based on basic representational building blocks that interact and self-organize into complex functions or designs. The observation from the central dogma of molecular biology that proteins are the basic building blocks of life and the recent advances in proteomics on analysis of structure, function and interaction of entire protein complements, lead us to propose a unifying framework of thought for these approaches: the proteomics approach. This thesis propose to investigate whether the self-organization of protein analogous structures at the representation level can increase the degree of complexity and ``novelty'' of solutions obtainable using evolutionary search techniques. In order to do so, we identify two fundamental aspects of this transition: (1) proteins interact in a three dimensional medium analogous to a multiset; and (2) proteins are functional structures. The first aspect is foundational for understanding of the second. This thesis analyzes the first aspect. It investigates the effects of using a genome to proteome mapping on evolutionary computation. This analysis is based on a genetic algorithm (GA) with a string to multiset mapping that we call the proportional genetic algorithm (PGA), and it focuses on the feasibility and effectiveness of this mapping. This mapping leads to a fundamental departure from typical EC methods: using a multiset of proteins as an intermediate mapping results in a \emph{completely location independent} problem representation where the location of the genes in a genome has no effect on the fitness of the solutions. Completely location independent representations, by definition, do not suffer from traditional EC hurdles associated with the location of the genes or positional effect in a genome. Such representations have the ability to self-organize into a genomic structure that appears to favor positive correlations between form and quality of represented solutions. Completely location independent representations also introduce new problems of their own such as the need for large alphabets of symbols and the theoretical need for larger representation spaces than traditional approaches. Overall, these representations perform as well or better than traditional representations and they appear to be particularly good for the class of problems involving proportions or multisets. This thesis concludes that the use of protein analogous structures as an intermediate representation in evolutionary computation is not only feasible but in some cases advantageous. In addition, it lays the groundwork for further research on proteins as functional self-organizing structures capable of building increasingly complex functionality, and as basic units of problem representation for evolutionary computation.
Ph.D.
School of Computer Science
Engineering and Computer Science
Computer Science

Proteomics is the large scale study of proteins in cells or organisms. The purpose of this study was to characterize the proteomic alterations occurring in a prostate cancer (LNCaP) cell line after treatment with delta-tocotrienol (a form of vitamin E not very prevalent from most dietary sources). We found that both gamma- and delta-tocotrienols induced time and concentration dependent growth inhibition and programmed cell dead (apoptosis) in LNCaP cells. Secondly, we used two-dimensional gel electrophoresis (2-DE) to characterize changes in protein expression levels associated with this treatment. Our results show that a specific set of proteins are regulated at both early and late times following treatment with delta-tocotrienol and these proteins have been characterized by their apparent molecular weights and isoelectric points. The alteration observed at early time points are particularly interesting because these changes are likely to reflect the underlying molecular mechanisms for triggering cancer cell death.

Allergic contact dermatitis (ACD) is a prevalent skin condition caused by chemical haptens, which enter the epidermis, and by modifying self-proteins, render them immunogenic via the activation of hapten-specific T-cells. It is currently not known which specific protein modifications are responsible for sensitization. This work investigates the extreme sensitizer DNCB, which confers a dinitrophenyl (DNP) protein modification. Immortalised keratinocytes (HaCaT), incubated with DNCB were compared to ex vivo human skin using immunofluorescence and western blot detection of DNP-proteins, showing widespread protein modification and similarities between tissue and cells when using a clinical dose. Proliferation assays using lymphocytes from DNCB-sensitive donors showed responses to DNP proteins isolated from DNCB-treated HaCaT cells and primary keratinocytes. While western blot analysis of pH gradient separated fractions identified a number of DNP-proteins in the DNP-HaCaT cell lysates, these were not immunogenic in lymphocyte assays. The model protein human serum albumin (HSA) was used to investigate the modification kinetics of DNCB by identifying which amino acid residues were changed more readily using a range of DNCB concentrations and incubation times. Cysteine residues, including those in disulphide bonds and particularly cysteine 34 are more readily modified than lysine in HSA, suggesting that DNCB is able to alter the structure of proteins. A novel process of hapten-reversal by a process termed ‘thiolysis’ was found to remove DNP groups from the cysteine residues of synthetic peptides derived from the sequence of HSA containing cysteine 34 using the reducing agent dithiothreitol. Identical peptides with C34>K34 showed no such hapten-reversal. This corresponds to the unexpected immunogenicity of the cysteinyl peptide and also the DNP modified tripeptide glutathione. Anti-DNP western blots show that the DNP group is transferred to other proteins during incubation with human monocytes in culture. This suggests a cellular process of removing DNP groups and GILT, a thiol reductase present in the endosomes is presented as a candidate for this process. This work demonstrates that DNCB can generate a wide variety of DNP protein adducts and that cysteinyl moieties are able to stimulate lymphocyte proliferation by way of hapten transfer. This highlights a potentially novel process involved in the mechanism of contact allergy.

Mestrado em Bioquímica Clínica
Currently, diastolic heart failure (DHF) is recognized an important cause of cardiovascular mortality and morbidity reaching approximately 50% of HF cases. The growing incidence of cardiovascular risk factors, such as obesity and overweight are associated with a worse prognosis in patients with cardiovascular disease, prompting the onset of diastolic dysfunction. In fact, currently the adipose tissue is considered an important modulator of cardiac function. Adipokines, pro-inflammatory cytokines and other important substances are released by this tissue and seem to play an important role in the induction of the cardiac dysfunction. Thus, considering the high prevalence of obesity in patients with diastolic dysfunction and lack of information about this relation, the aim of this work is to characterize the profile of substances released by adipose tissue under conditions of DHF, using a proteomic approach. The visceral (VAT) and epicardial (EAT) adipose tissue from obese animals with diastolic HF (n=3, obese ZSF1) with their respective lean controls without diastolic HF (n=3, lean ZSF1) were analysed. Firstly, the optimization of methodology for analysis of tissues proteome was performed, testing three different extraction protocols. The selection of protocol was made considering the larger number of extracted proteins, since it there were no significant differences in performed evaluations. After execution of the extracted protocol elected, it was performed the separation of proteins and peptides by one-dimension gel electrophoresis and high-performance liquid chromatography and tryptic digestion used to further mass spectrometry identification. In VAT, the results showed in obesity a decrease of catalase protein and increase of superoxide dismutase, peroxiredoxin-1 and A1 and A2-annexin proteins leading to believe that there are inflammation, oxidative stress and, at the same time, a metabolism protection in order to prevent the increase of reactive oxygen species and progression of inflammation. In addition, it was also was evidenced a decrease of the aldehyde dehydrogenase, mitochondrial suggesting greater susceptibility to oxidative stress. In EAT of the obese ZSF1, the results presents a decrease of 3-ketoacyl-CoA thiolase protein enzyme as a compensatory mechanism in order to inhibit fatty acid oxidation and an increase of lumican and collagen-alpha-1(I) proteins suggesting a link between inflammation caused by obesity and increases of adipose tissue extracellular matrix. Curiously, both tissues demonstrated the increase of cardiac contractile proteins in obese adipose tissue, which are consistent with several reported myofilamentary changes in DHF. Finally, it was analysed differences between the VAT and EAT, which suggest that both tissues present a different proteome. This work is a general approach to the protein composition of the epicardial and visceral adipose tissue of the animal model ZSF1, providing importants differences in adipose tissue proteome between obese animals with DHF and respective controls and helping the future development researches more focused on the functional role of proteins changes in DHF.
Atualmente é reconhecido que a insuficiência cardíaca (IC) diastólica (ICD) constitui uma importante causa de morbilidade e mortalidade cardiovascular atingindo cerca de 50% dos casos de IC. O aumento da incidência de comorbilidades, tal como a obesidade são fatores de risco que se associam a um pior prognóstico em pacientes com ICD. De facto, atualmente ao tecido adiposo é atribuído um importante papel de modulação da função cardíaca. As adipocinas, citocinas pró-inflamatórias assim como outras substâncias libertadas por este tecido aparentam desempenhar um papel predominante na indução da disfunção cardíaca. Assim, considerando a elevada prevalência de obesidade em pacientes com disfunção diastólica e a falta de informação sobre esta relação, o objetivo deste trabalho é caracterizar o perfil de substâncias libertadas pelo tecido adiposo em condições de ICD, recorrendo a uma abordagem proteómica. Neste sentido utilizou-se o tecido adiposo visceral (TAV) e epicárdico (TAE) de animais obesos com ICD (n=3 , ZSF1 obesos) e os respetivos controlos magros sem ICD (n=3, ZSF1 magros). Primeiro procedeu-se à otimização da metodologia para análise do proteoma dos tecidos onde foram testados 3 protocolos distintos de extração de proteínas. A seleção do protocolo foi feita considerando o número de proteínas extraídas, uma vez que não existiram diferenças significativas nas avaliações realizadas. Após a execução do protocolo de extração elegido, efetuou-se eletroforese de primeira dimensão e cromatografia líquida de alta resolução para a separação de proteínas e péptidos e procedeu-se à identificação dos fragmentos por espectrometria de massa. No TAV, os resultados demonstraram, na obesidade, uma diminuição das proteína catalase e um aumento das proteínas superóxido dismutase, peroxirredoxina-1 e anexina-A1 e A2 levando a crer que existe inflamação, stress oxidativo mas, simultaneamente, uma resposta compensatória do tecido adiposo para prevenir o aumento de produção de espécies reativas de oxigénio e a progressão da inflamação. Observou-se ainda uma diminuição da proteína aldeído desidrogenase mitocondrial, o que sugere maior suscetibilidade ao stress oxidativo. No TAE dos ZSF1 obesos, os resultados demonstraram uma redução da enzima 3-cetoacil-CoA tiolase sugerindo um mecanismo compensatório a fim de inibir a oxidação dos ácidos gordos, e ainda um aumento das proteínas colagénio-alfa1(I) e lumican, sugerindo uma ligação entre a inflamação causada pela obesidade e alterações da matriz extracelular do tecido adiposo. Curiosamente, os dois tecidos demonstraram o aumento de proteínas contrácteis cardíacas nos obesos ZSF1, as quais são consistentes com várias alterações miofilamentares apresentadas na ICD. Por fim, a análise das diferenças entre o TAV e o TAE revela claras diferenças no proteoma de cada um destes tecidos. Este trabalho apresenta uma abordagem generalista da composição proteica dos tecidos adiposos visceral e epicárdico do modelo animal ZSF1, indicando importantes diferenças no proteoma do tecido adiposo entre animais obesos com ICD e os respetivos controlos e direcionando o desenvolvimento futuro de investigações mais específicas focadas no papel funcional das proteínas mais alteradas na ICD.

Liquid chromatography-mass spectrometry (LC-MS) based proteomic methods have provided archaeologists with a powerful tool for the discovery and identification of proteins within artifacts. Traditionally, discovery-based methods have utilized a non-targeted full mass scan method in an attempt to identify all proteins present within a given sample. However, increased sensitivity is often needed to target specific proteins in order to test hypotheses. Proteins present within archaeological materials present a unique challenge, as they are often subjected to a variety of chemical transformations both before and after burial. Any preserved proteins will be present within a complex mixture of compounds, and full mass scans often fail to detect less abundant proteins of interest. Consistent and reliable targeted methods are needed to detect protein biomarkers. Taphonomic experimentation was employed as a means to identify the effect of particular processes and conditions on the preservation of mare's milk proteins. In addition, three LC-MS methods were evaluated for their efficiency in identifying mare's milk-specific peptide biomarkers from experimental pottery samples. The ability to reliably detect the presence of these species-specific peptides can help provide evidence about past cultural groups, including the origins of dairying and animal domestication.

Chez les vertébrés, le foie est un des organes majeurs du métabolisme en étant le siège de la régulation de différentes voies du métabolisme qui contrôlent l’homéostasie du glucose et des lipides. En se basant sur des travaux de recherche récents suggérant que le système de conjugaison de l'ubiquitine est engagé en réponse à différents signaux métaboliques, nous avons réalisé une analyse protéomique globale dans le but d’identifier des protéines ubiquitylées dans le foie de souris soumises á un protocole de jeûne – re-nourrissage. Parmi les 117 protéines différemment ubiquitylé sur le jeûne ou le re-nourrissage, nous avons identifié complément 3 (C3) dans le foie de souris réalimentées. Nous avons observé qu'un produit d'activation de C3, C3a, est ubiquitylé dans les hépatocytes primaires traités avec les médias riches en nutriments. Ainsi, nous proposons que l'ubiquitination de C3 joue un rôle dans la régulation des fonctions inflammatoires ou métaboliques de C3 dans le foie
In vertebrates, the liver has developed to be a major metabolic organ able to control glucose and lipid homeostasis. It activates or inhibits specific pathways in a regulated manner, depending on the metabolic state of our organism. Based on the emerging experimental evidence suggesting that the ubiquitin conjugation system is engaged in response to different metabolic cues, we conducted a global proteomic analysis to identify metabolic pathways modified by ubiquitylation. To this end, we used livers of mice subjected to a fasting – refeeding protocol. Amongst the 117 proteins differentially ubiquitylated upon fasting or refeeding conditions, we identified complement 3 (C3) to be ubiquitinated in livers of refed mice. We observed that an activation product of C3, C3a, is ubiquitylated in primary hepatocytes treated with nutrient-rich media. Thus, we suggest that the ubiquitylation of C3 plays a role in the regulation of inflammatory or metabolic functions of C3 in the liver

The development of new biomarkers for cancer patients would be advantageous in population screening for the early detection of cancers, pathological diagnosis, assessment of prognosis, tailoring treatment to individuals, and assessment of treatment response. With this in mind different proteomic approaches were used to identify biomarkers which could potentially aid prognosis and predict response in gastrointestinal and ovarian cancer. Raf Kinase Inhibitor Protein (RKIP) was originally purified from bovine brain extracts and named phosphatidylethanolamine-binding protein (PEBP). It has subsequently been shown to be a widely expressed and highly conserved protein. Several recent studies have suggested that RKIP may suppress metastasis in melanoma, prostate, and breast cancer, as reduction or loss of RKIP expression was observed in metastatic cell lines and metastatic tissue. In this part of the project RKIP expression was assessed by immunohistochemistry in tissue microarrays (TMA) from patients with colorectal and ovarian cancer. The results confirmed the findings of earlier studies and suggest that the level of RKIP expression is significantly and inversely associated with metastatic disease and can predict the risk of metastatic relapse in patients with no evidence of metastases at presentation. The level of RKIP expression as a prognostic factor was independent of sex, age, tumour site, mitotic index, lymphovascular invasion and tumour stage. Cytokeratin 18 (CK18) is an epithelial-specific cytokeratin that undergoes cleavage by caspases during apoptosis. Measurement of caspase-cleaved (CK18-NE) or total cytokeratin 18 (CK18) from epithelial-derived tumours could be a simple, non-invasive way to monitor or predict responses to treatment. Soluble plasma CK18-NE and CK18 were measured by ELISA from 73 patients with advanced gastrointestinal adenocarcinomas before treatment and during chemotherapy, as well as 100 healthy volunteers. Both CK18-NE and total CK18 plasma levels were significantly higher in patients compared to the healthy volunteers (p=0.015, p<0.001). The total CK18 baseline plasma levels prior to treatment were significantly higher (p=0.009) in patients who develop progressive disease than those who achieve partial response or stable disease and this correlation was confirmed in an independent validation set. The peak plasma levels of CK18 occurring in any cycle following treatment were also found to be associated with tumour response, but peak levels of CK18-NE did not reach significance (p=0.01, and p=0.07, respectively). A surface-enhanced laser desorption-ionisation mass spectrometry (SELDI-MS) pilot study on serum from 8 oesophageal cancer patients and 8 healthy volunteers revealed a novel biomarker, ~4kDa, downregulated in patients (p=0.012). An expanded 30 tumour/normal study was performed for validation which confirmed the down-regulation of this potential biomarker (p<0.0001). Attempts to identify tentatively suggested that the peptide may be inter-alpha-trypsin inhibitor heavy chain H4 precursor, which was interesting as a cleavage fragment of inter-alpha -trypsin inhibitor heavy chain H4 had been previously found to be up-regulated in patients with ovarian cancer, and down-regulated in patients with breast cancer. However, it was not possible to confidently confirm this identification. In a further part of this study, haptoglobin was found to be significantly more abundant in the serum from patients with oesophageal cancer compared to healthy volunteers. It was straightforward to isolate and identify and would be amenable to immunoassay as there are good antibodies available for confirmation. In conclusion, with the current lack of effective markers of metastatic relapse in colorectal cancer, a straightforward test like RKIP expression in the primary tumour may be a very cost-effective way to identify which patients may derive greater benefit from adjuvant treatment and closer post-operative surveillance, and in patients with advanced gastrointestinal malignancy levels of plasma CK18 are a potential marker of tumour response.

Mestrado em Medicina e Oncologia Molecular
Master Degree Course in Molecular and Oncology Medicine
CLASPs são proteínas altamente conservadas que participam na segregação dos cromossomas através do seu papel fundamental na interface cinetocoro-microtúbulo durante a mitose. Em levedura, Drosophila, e Xenopus, um único ortólogo da CLASP está presente, o qual é necessário para a formação do fuso mitótico através da regulação da dinâmica dos microtúbulos a nível do cinetocoro. Em mamíferos, no entanto, somente a CLASP1 tem sido implicada na divisão celular, apesar da existência de um segundo parólogo, CLASP2. Neste estudo descrevemos a localização mitótica da CLASP2 humana em células HeLa e mostramos que a sua localização nos cinetocoros, centrossomas, e fuso durante toda a mitose é notavelmente semelhante à CLASP1. A análise de fibroblastos embrionários de ratinho KO para a Clasp2 revelou que a localização da CLASP1 no cinetocoro e a resposta do checkpoint da Mad2 não estão comprometidos pela ausência de CLASP2. Para melhor compreender as funções das CLASPs em mitose, nomeadamente para determinar potenciais funções redundantes, nós realizamos a depleção de cada CLASP por RNAi. Notavelmente, a depleção isolada de cada CLASP não induziu qualquer erro significativo na mitose; a redução dos níveis de ambas as CLASPs por RNAi causou severos defeitos mitóticos a nível do fuso (principalmente células com fusos multipolares), e conteúdo anormal de DNA (aneuploidia). No geral, estes resultados sugerem que CLASP1 e CLASP2 possuem papéis sobreponíveis durante a mitose. A fim de compreender os mecanismos moleculares subjacentes à função das CLASPs humanas durante a mitose, nós realizamos um estudo proteómico para a identificação das proteínas interactoras da CLASP1 durante a mitose através de espectrometria de massa. Os nossos resultados confirmaram as interações entre CLASP1 - CLIP-170 e LL5ß, ambos descritas em interfase. Além disso, novas interacções foram encontradas como CENP-E, Astrin, GCC185, CENP-J/CPAP, MARK2 e a nova proteína KIAA0802. Em células interfásicas, as CLASPs acumulam-se no aparelho de Golgi e esta acumulação está relacionada com a presença de microtúbulos estabilizados. No entanto, há pouca informação a respeito da função de Golgi durante a mitose. A análise da distribuição mitótica da GCC185 mostrou que em estadios precoces a GCC185 localiza-se em torno dos centrossomas, e dispersa-se em metafase e anafase. Em telofase, a GCC185 relocaliza-se na região perinuclear e nos centrossomas. De notar que nós encontramos um co-localização extensiva entre a GCC185 e a CLASP1 (especialmente em profase, prometafase e telofase). A interacção entre a GCC185 e a CLASP1 foi também confirmada em extractos celulares interfásicos. Finalmente, muitas +TIPs como o APC, CLIP-170/Restin e EB1 têm sido implicadas em processos de aneuploidia e tumorigénese, formando uma complexa rede proteica com as CLASPs. Para determinar as implicações da CLASP1 nos mecanismos de tumorigénese, realizamos uma pesquisa mutacional numa linha celular humana derivada do carcinoma do cérvix (células HeLa). Na nossa análise mutacional encontramos três deleções: duas correspondem a isoformas da CLASP1 resultantes de splicing alternativo (737 1538 e 1125 1164), e a terceira deriva da perda parcial do exão 21 (673 679). Estas mutações podem estar relacionadas à instabilidade dos cromossomas que conduz a mutações aleatórias, ou podem reflectir que o gene CLASP1 é um alvo principal para mutações. Estes resultados incentivam a um exame maior para mutações da CLASP noutras linhas celulares tumorais e tumores primários. No geral, nós encontramos que a CLASP1 e CLASP2 possuem papéis redundantes durante a mitose, cuja ausência pode originar diversos defeitos mitóticos, conduzindo em último efeito à aneuploidia. Adicionalmente, descobrimos novas interacções moleculares com importantes proteínas mitóticas e fornecemos a ligação molecular entre a função da CLASP e o papel desconhecido do aparelho de Golgi durante a mitose.
CLASPs are well-conserved microtubule plus-end-tracking proteins that participate in chromosome segregation through their key role at the kinetochore-microtubule interface. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2. Here we describe the mitotic localization of human CLASP2 in HeLa cells and show that its localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1. Analysis of Clasp2 KO mouse embryonic fibroblasts revealed that CLASP1 kinetochore localization and Mad2 checkpoint response is not compromised in the absence of CLASP2. To further understand CLASP roles in mitosis, namely to rule out potential redundant functions, we performed single CLASP depletion by RNAi. Remarkably, single CLASP depletion caused no significant impairment of mitosis, while reducing the levels of both CLASPs by RNAi caused severe mitotic spindle defects (mainly cells with multipolar spindles), and abnormal DNA content (aneuploidy). Overall, these results suggest that CLASP1 and CLASP2 play overlapping roles during mitosis. In order to understand the molecular mechanisms underlying the function of human CLASPs during mitosis, next we performed a proteomic study for the identification of CLASP1 interacting proteins during mitosis by mass-spectrometry. Our results confirmed the interactions between CLASP1 CLIP-170 and LL5ß, both described in interphase. Moreover, new interactors were found such as CENP-E, Astrin, GCC185, CENP-J/CPAP, MARK2 and the novel protein KIAA0802. In interphase cells, CLASPs accumulate at the Golgi apparatus and this accumulation is related to the presence of stabilized microtubules. However, there is little information concerning Golgi function during mitosis. Analysis of GCC185 mitotic distribution showed that in early stages GCC185 localizes around centrosomes, and disperses in metaphase and anaphase. In telophase, GCC185 re-localizes to the perinuclear region and centrosomes. Noteworthy, we found an extensive co-localization between GCC185 and CLASP1 (especially in prophase, prometaphase and telophase). The interaction between GCC185 and CLASP1 was also confirmed in interphase cell extracts. Finally, many +TIPs like APC, CLIP-170/Restin and EB1 have previously been implicated in aneuploidy and tumourigenesis, forming a complex protein network with CLASPs. To determine the implications of CLASP1 for the mechanisms of tumourigenesis, we performed a mutational screening in a human cell line derived from cervix carcinoma (HeLa cells). In our mutational analysis we found three deletions: two of them correspond to CLASP1 alternative spliced isoforms (737 1538 and 1125 1164), and the third derives from the partial loss of exon 21 (673 679). These mutations could be related to chromosomal instability leading to random mutations, or may reflect that CLASP1 gene is a main target for mutations. These results encourage a larger survey for CLASP mutations in other tumour cell lines and primary tumours. Overall, we found that CLASP1 and CLASP2 play redundant roles during mitosis, whose absence can originate several mitotic defects, and ultimately lead to aneuploidy. Additionally, we uncovered new molecular interactions with important and novel mitotic proteins and provided the molecular linkage between CLASP function and the still mysterious role of the Golgi apparatus during mitosis.

Titanium is among the most commonly used metals as a standard treatment for bone defects. Despite its excellent mechanical and biocompatibility properties, Ti is relatively bioinert and surface modifications are necessary to improve its bioactivity. The sol-gel process allows this modification through the development of coatings with controlled release of bioactive compounds. On the other hand, the growing demand for biomaterials with better performances created the need to develop new in vitro methods more predictive of in vivo results. Considering this, the application of proteomics represents an interesting alternative to be explored. Herewith, the present doctoral thesis aimed to develop sol-gel coatings doped with different bioactive compounds and its characterization employing proteomics to determine predictive biomarkers of possible in vivo results.
Programa de Doctorat en Ciències Biomèdiques i Salut

Mestrado em Medicina e Oncologia Molecular
Master Degree Course in Molecular and Oncology Medicine
CLASPs são proteínas altamente conservadas que participam na segregação dos cromossomas através do seu papel fundamental na interface cinetocoro-microtúbulo durante a mitose. Em levedura, Drosophila, e Xenopus, um único ortólogo da CLASP está presente, o qual é necessário para a formação do fuso mitótico através da regulação da dinâmica dos microtúbulos a nível do cinetocoro. Em mamíferos, no entanto, somente a CLASP1 tem sido implicada na divisão celular, apesar da existência de um segundo parólogo, CLASP2. Neste estudo descrevemos a localização mitótica da CLASP2 humana em células HeLa e mostramos que a sua localização nos cinetocoros, centrossomas, e fuso durante toda a mitose é notavelmente semelhante à CLASP1. A análise de fibroblastos embrionários de ratinho KO para a Clasp2 revelou que a localização da CLASP1 no cinetocoro e a resposta do checkpoint da Mad2 não estão comprometidos pela ausência de CLASP2. Para melhor compreender as funções das CLASPs em mitose, nomeadamente para determinar potenciais funções redundantes, nós realizamos a depleção de cada CLASP por RNAi. Notavelmente, a depleção isolada de cada CLASP não induziu qualquer erro significativo na mitose; a redução dos níveis de ambas as CLASPs por RNAi causou severos defeitos mitóticos a nível do fuso (principalmente células com fusos multipolares), e conteúdo anormal de DNA (aneuploidia). No geral, estes resultados sugerem que CLASP1 e CLASP2 possuem papéis sobreponíveis durante a mitose. A fim de compreender os mecanismos moleculares subjacentes à função das CLASPs humanas durante a mitose, nós realizamos um estudo proteómico para a identificação das proteínas interactoras da CLASP1 durante a mitose através de espectrometria de massa. Os nossos resultados confirmaram as interações entre CLASP1 - CLIP-170 e LL5ß, ambos descritas em interfase. Além disso, novas interacções foram encontradas como CENP-E, Astrin, GCC185, CENP-J/CPAP, MARK2 e a nova proteína KIAA0802. Em células interfásicas, as CLASPs acumulam-se no aparelho de Golgi e esta acumulação está relacionada com a presença de microtúbulos estabilizados. No entanto, há pouca informação a respeito da função de Golgi durante a mitose. A análise da distribuição mitótica da GCC185 mostrou que em estadios precoces a GCC185 localiza-se em torno dos centrossomas, e dispersa-se em metafase e anafase. Em telofase, a GCC185 relocaliza-se na região perinuclear e nos centrossomas. De notar que nós encontramos um co-localização extensiva entre a GCC185 e a CLASP1 (especialmente em profase, prometafase e telofase). A interacção entre a GCC185 e a CLASP1 foi também confirmada em extractos celulares interfásicos. Finalmente, muitas +TIPs como o APC, CLIP-170/Restin e EB1 têm sido implicadas em processos de aneuploidia e tumorigénese, formando uma complexa rede proteica com as CLASPs. Para determinar as implicações da CLASP1 nos mecanismos de tumorigénese, realizamos uma pesquisa mutacional numa linha celular humana derivada do carcinoma do cérvix (células HeLa). Na nossa análise mutacional encontramos três deleções: duas correspondem a isoformas da CLASP1 resultantes de splicing alternativo (737 1538 e 1125 1164), e a terceira deriva da perda parcial do exão 21 (673 679). Estas mutações podem estar relacionadas à instabilidade dos cromossomas que conduz a mutações aleatórias, ou podem reflectir que o gene CLASP1 é um alvo principal para mutações. Estes resultados incentivam a um exame maior para mutações da CLASP noutras linhas celulares tumorais e tumores primários. No geral, nós encontramos que a CLASP1 e CLASP2 possuem papéis redundantes durante a mitose, cuja ausência pode originar diversos defeitos mitóticos, conduzindo em último efeito à aneuploidia. Adicionalmente, descobrimos novas interacções moleculares com importantes proteínas mitóticas e fornecemos a ligação molecular entre a função da CLASP e o papel desconhecido do aparelho de Golgi durante a mitose.
CLASPs are well-conserved microtubule plus-end-tracking proteins that participate in chromosome segregation through their key role at the kinetochore-microtubule interface. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2. Here we describe the mitotic localization of human CLASP2 in HeLa cells and show that its localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1. Analysis of Clasp2 KO mouse embryonic fibroblasts revealed that CLASP1 kinetochore localization and Mad2 checkpoint response is not compromised in the absence of CLASP2. To further understand CLASP roles in mitosis, namely to rule out potential redundant functions, we performed single CLASP depletion by RNAi. Remarkably, single CLASP depletion caused no significant impairment of mitosis, while reducing the levels of both CLASPs by RNAi caused severe mitotic spindle defects (mainly cells with multipolar spindles), and abnormal DNA content (aneuploidy). Overall, these results suggest that CLASP1 and CLASP2 play overlapping roles during mitosis. In order to understand the molecular mechanisms underlying the function of human CLASPs during mitosis, next we performed a proteomic study for the identification of CLASP1 interacting proteins during mitosis by mass-spectrometry. Our results confirmed the interactions between CLASP1 CLIP-170 and LL5ß, both described in interphase. Moreover, new interactors were found such as CENP-E, Astrin, GCC185, CENP-J/CPAP, MARK2 and the novel protein KIAA0802. In interphase cells, CLASPs accumulate at the Golgi apparatus and this accumulation is related to the presence of stabilized microtubules. However, there is little information concerning Golgi function during mitosis. Analysis of GCC185 mitotic distribution showed that in early stages GCC185 localizes around centrosomes, and disperses in metaphase and anaphase. In telophase, GCC185 re-localizes to the perinuclear region and centrosomes. Noteworthy, we found an extensive co-localization between GCC185 and CLASP1 (especially in prophase, prometaphase and telophase). The interaction between GCC185 and CLASP1 was also confirmed in interphase cell extracts. Finally, many +TIPs like APC, CLIP-170/Restin and EB1 have previously been implicated in aneuploidy and tumourigenesis, forming a complex protein network with CLASPs. To determine the implications of CLASP1 for the mechanisms of tumourigenesis, we performed a mutational screening in a human cell line derived from cervix carcinoma (HeLa cells). In our mutational analysis we found three deletions: two of them correspond to CLASP1 alternative spliced isoforms (737 1538 and 1125 1164), and the third derives from the partial loss of exon 21 (673 679). These mutations could be related to chromosomal instability leading to random mutations, or may reflect that CLASP1 gene is a main target for mutations. These results encourage a larger survey for CLASP mutations in other tumour cell lines and primary tumours. Overall, we found that CLASP1 and CLASP2 play redundant roles during mitosis, whose absence can originate several mitotic defects, and ultimately lead to aneuploidy. Additionally, we uncovered new molecular interactions with important and novel mitotic proteins and provided the molecular linkage between CLASP function and the still mysterious role of the Golgi apparatus during mitosis.

Substantia nigra is the most affected brain region in Parkinson's disease. Substantia nigra is characterized by strong pigmentation derived by the presence of neuromelanin (NM), a particular melanic pigment that accumulates during aging in neurons in typical membrane-bounded organelles with high levels of lipids (dolichols). The occurrence of an elevated number of NM organelles in the most vulnerable brain region during Parkinson’s disease is of special interest. It is not clear if NM organelles may interfere with the vulnerability of neurons during aging. Nevertheless, many evidences support the correlation between aging and neurodegeneration, and the accumulation of intracellular indigestible materials (damaged proteins, inclusion bodies and lipofuscins). The accumulation of indigestible materials inside the cell may increase cellular stress and neurodegeneration. In this study, samples of NM organelles (whole and without membranes) and NM pigment were isolated from human post-mortem tissue (substantia nigra) of healthy subjects. Samples were analysed in duplicate to obtaining the proteomic profile of the NM organelle using MudPIT proteomic technology. A total of 1142 proteins were identified, of which 48 were observed with high confidence as representative of samples. Comparing results from different samples, some proteins were assigned to different regions of the NM organelle (NM pigment, membrane, soluble portion). From the proteomic profile emerges the lysosomal nature of the NM organelle: among the 48 representative proteins, more than 20 are lysosomal proteins. In addition, other groups of proteins were identified: proteins involved in protein folding (alpha-crystallin) and degradation (ubiquitin), proteins important to iron homeostasis (ferritin L), antioxidant proteins and proteins involved in intracellular aggregates formation. Furthermore, typical biomarkers of lysosomal storage disease are observed (mitochondrial subunit c, saposins). On the other hand, any enzyme potentially involved in the melanogenic process and any lipids biosynthetic enzyme were identified. This study demonstrates that the NM organelle has the features of both a lysosomal/autophagic organelle and an aged and damaged lysosome accumulating indigestible material. These data are in agreement with recent hypothesis suggesting that the NM organelle formation originate by sequestration of NM and other potentially toxic molecules as damaged proteins, damaged lipids and metals. Our data suggest that this protective mechanism, during aging, may induce accumulation of indigestible material inside the NM organelles and, as consequence, a decreased degradative function of the cell. In summary, e suggest that NM organelle could confer protection to neurons and, at the same time, could increase cellular vulnerability in aging conditions.

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.

Dissertação apresentada para a obtenção do grau de doutor em Bioquímica pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia
Arsenic is a metalloid that occurs naturally in soils and is toxic to living organisms at high concentrations. The arsenic poisoning can occur indirectly (food chain) or directly through drinking water. In humans the chronic exposure to arsenic is linked to cancer, vascular diseases and skin lesions. In countries like Bangladesh the problems related to arsenic poisoning are very dramatic and has become a public health problem. Selenium is an essential micro-nutrient to humans and it is known for its anti-cancer and anti-oxidant properties. This element is present in nature at small amounts and enters the food chain through the plants that uptake it from the soils. Although there are some references to diseases related with selenium poisoning, they are rarely documented and for this reason the effects of selenium toxicity in humans remains unknown. Throughout its evolution, many organisms have developed strategies and mechanisms to excrete heavy metals preventing their adverse effects. In the late 90’s a study with small mammals showed that an enriched diet in selenium had decreased the arsenic toxic effects on mammals exposed to high concentrations of this metalloid. Afterwards the bio-formation of a metabolite that contained in its composition arsenic and selenium was identified. This metabolite was easily excreted by the organism which suggesting the presence of a biological mechanism for the detoxication of metals in mammals. The present work studies the possibility to use selenium as an ecological solution to avoid/diminish the toxicity of arsenic in drinking waters, using food supplements as a selenium source. Techniques were developed with the goal of determining the total amount and speciation of selenium in biological samples and food supplements by HPLC and ET-AAS. For solid-liquid selenium extraction it was used an enzymatic digestion accelerated with ultrasonic energy. This methodology, that has reduced the extraction time from hours to minutes, was firstly reported in 2004 in the Analytical Chemistry journal and since then it has been extensively used by the scientific community. A bibliographic review has been developed in order to establish the state of the art and to enhance the divulgation of this methodology between the scientific community. To study the antagonistic effects of selenium and arsenic in biological systems, freshwater clams (C. fluminea) were exposed during 21 days to different concentrations of these elements. The determination of arsenic and selenium on the clam’s soft tissue (digestive gland and remains body) was performed by ET-AAS. For the identification of proteins by peptide mass fingerprint, a new and fast ultrasonication assisted enzymatic digestion with immobilized trypsin (on magnetic particles and glass beads) method was developed
Fundação para a Ciência e Tecnologia,financial support

Protein prenylation is an important post-translational modification that occurs in all eukaryotes; defects in the prenylation machinery can lead to toxicity or pathogenesis. Prenylation is the modification of a protein with a farnesyl or geranylgeranyl isoprenoid, and it facilitates protein-membrane and protein-protein interactions. Proteins of the Ras superfamily of small GTPases are almost all prenylated and of these the Rab family of proteins forms the largest group. Rab proteins are geranylgeranylated with up to two geranylgeranyl groups by the enzyme Rab geranylgeranyltransferase (RGGT). Prenylation of Rabs allows them to locate to the correct intracellular membranes and carry out their roles in vesicle trafficking. Traditional methods for probing prenylation involve the use of tritiated geranylgeranyl pyrophosphate which is hazardous, has lengthy detection times, and is insufficiently sensitive. The work described in this thesis developed systems for labelling Rabs and other geranylgeranylated proteins using a technique known as tagging-by-substrate, enabling rapid analysis of defective Rab prenylation in cells and tissues. An azide analogue of the geranylgeranyl pyrophosphate substrate of RGGT (AzGGpp) was applied for in vitro prenylation of Rabs by recombinant enzyme. Alternatively, geranylgeranylated proteins (including Rabs) were labelled with AzGG via metabolic labelling of live cells with AzGGOH. Once Rabs were tagged with an azide moiety they could be labelled via a bioorthogonal ligation reaction with a trifunctional alkyne probe. The probe contained a fluorophore for in-gel fluorescence analysis and a biotin affinity label for affinity purification of labelled proteins. The conditions for protein tagging, labelling, affinity purification and LC-MS/MS analysis were optimised significantly during this work. Affinity purified proteins were identified and in some cases quantified using LC-MS/MS techniques, with iTRAQ labelling for quantification. Rab prenylation was probed in cell culture, in cells treated with the drug Mevastatin and in tissue from mouse models with defects in the prenylation machinery.

The molecular mechanism that leads to ischemic preconditioning and hence to ischemic tolerance, are not completely understood although it is clear that multiple effectors and pathways contribute to the instauration of this neuroprotective profile. To study the mechanism/pathway involved in the ischemic tolerance, brain proteins, plasma proteins and plasma metabolites were analyzed in preconditioning stimulus, in severe stroke and in preconditioned mouse model. A conventional 2-DE approach was used to study technical replicates of pooled brain proteins revealing an involvement of energy metabolism, mitochondrial electron transport, synaptic vesicle transport and antioxidant processes; moreover network analysis suggested an involvement of the androgen receptor that was validated on technical replicates of pooled brain proteins by western blot analysis revealing an increased expression in preconditioned stimulus animals. Plasma proteins were analyzed using a i-DE Le-MS/MS approach on technical replicates of pooled plasma proteins revealing decreased levels of epidermal growth factor receptor (EGFR) and increased levels of insuline like growth factor acid labile subunit (IGFALS), which expression was paralleled by increased insulin like growth factor 1 (IGFi) plasma concentration, as validated by ELlSA on biological replicates, in preconditioning stimulus animals. Finally an untarget metabolomics analysis was applied to technical replicates of pooled plasma proteins revealing fatty acid oxidation and branched-chain aminoacid metabolism as the main biological processes modulated in ischemic tolerance and highlighted an involvement of the aminoacid leucine, carnitine esters and adenosine. The results described in this thesis represents the first application of both' proteomic and metabolomic approaches in cerebral ischemic sets, highlighting the androgen receptor as an important mediator between proteins and metabolites and adding new evidence to the current knowledge on ischemic preconditioning that may represent a starting point for future experiments on investigating candidate pathways that relate to the androgen receptor.

Lo studio della componente proteica in alimenti di origine animale, nasce dall’esigenza di dover comprendere come tali componenti, modifichino la propria conformazione chimico-fisica e biochimica, attraverso i processi tecnologici, l’azione enzimatica e le condizioni di stoccaggio della materia prima e del prodotto finito. I salumi e i prodotti lattiero caseari, possono vantarsi di essere prodotti prestigiosi della produzione alimentare agricola Italiana. L’approccio proteomico studiato sui prodotti a base di carne cotti ha evidenziato notevoli differenze sia quantitative che qualitative indotte dal processo tecnologico, dalla cottura, se sottoposto a taglio o sminuzzamento e salatura. I risultati dimostrano che nei prosciutti cotti e nei prodotti emulsionati, le aggregazioni proteiche riflettono le condizioni di trasformazione, che per i prodotti cotti sono caratterizzate principalmente da ponti disolfuro e nei prodotti emulsionati da legami inter-proteina di natura covalente. Accade di frequente che le industrie lattiero-casearie del nostro Paese si approvvigionino di latte in polvere, latte a lunga conservazione, latte e cagliate congelate nazionali ed estere, ed altri prodotti che spesso risultano essere di bassa qualità. Materie prime conservate e semilavorati sono utilizzati dall’industria lattiero-casearia per il loro basso costo e per far fronte ad una concorrenza sempre più agguerrita della grande distribuzione e delle grandi industrie. Pertanto, lo studio proteomico, permette di allestire procedure analitiche che consentono di tutelare il marchio di qualità e il “Made in Italy”. Le tecniche elettroforetiche adottate su mozzarelle di bufala Campane DOP, hanno rivelato la presenza di diverse condizioni di proteolisi, indotte dall’idrolisi della β-CN ad opera della plasmina, e frammenti dell’αs1-CN, per conto della chimosina, riscontrate anche mediante spettrometria di massa, indicando l’uso di latte congelato o refrigerato, cagliate congelate o semi-prodotti di basa qualità e non idonei alla produzione della mozzarella di bufala Campana con denominazione di origine, in quanto il Regolamento CE n°1107, prevede esclusivamente l’utilizzo di latte di bufala fresco e proveniente da bufale allevate nella stessa zona di produzione. Lo studio ha portato anche alla determinazione di un “indice di freschezza”, monitorando il livello di proteolisi di 75 campioni di mozzarella di bufala Campana DOP e tre campioni controllo. In questa analisi sono stati rilevati i valori quantitativi della β-CN e i frammenti della γ-CN e, confrontando le intensità delle bande, è stato possibile calcolare questo indice chiamato Φ. Più il valore di Φ calcolato è stato elevato maggiore era la freschezza della mozzarella. Inoltre, lo studio è stato condotto monitorando l’effetto che il congelamento ha sul latte di bufala, quantificando la β-CN mediante tecniche immunochimiche, quale Test Elisa indiretto, che riconosce l’antigene mediante un anticorpo specifico ed il complesso così formatosi è rivelato con un anticorpo secondario aspecifico a cui è legato un gruppo cromogeno che si colora dopo l’azione enzimatica. I risultati di questa prima indagine hanno mostrato che la plasmina può agire sui campioni anche durante il congelamento.
The study of animal food proteins arises from the necessity to understand how such components modify their chemical-physical and biochemical behaviour through technological processes, enzymatic actions and the storage conditions of the raw material to reach at the finished product. The meat and dairy products can boast of being prestigious products of Italian food production. The proteomic approach developed on cooked meat products showed significant quantitative and qualitative differences induced by the technological process: cooking, cutting or crushing and salting. The results demonstrate that in cooked hams and emulsified products, protein aggregations reflect the processing conditions, which for cooked products are characterized primarily by disulfide bridges and in emulsified products from inter-protein bonds of covalent nature. It often happens that the dairy industries of our country use powdered milk, UHT milk, frozen milk and curds, national or foreign, and other products that often turn out to be of low quality. The dairy industry use frozen raw materials and semi-preserved for their low cost and to cope with an increasingly competition of large retailers and major industries. Therefore, the proteomic study allows to set up the analytical techniques which allow to protect the quality mark and the "Made in Italy". The electrophoretic techniques adopted on buffalo mozzarella PDO revealed the presence of several conditions of proteolysis, induced by the hydrolysis of β-CN by plasmin activity and fragments dell'αs1-CN by chymosin activity, also confirmed by mass spectrometry. These results indicated the use of frozen or refrigerated milk, frozen curds or low quality semi-preserved products and so on, not suitable for the production of buffalo mozzarella PDO with designation of origin, as the EC Regulation n ° 1107 provides only the ‘use of buffalo fresh milk from buffalo reared in the same production area’. This study also led to the determination of an "index of freshness" monitoring the level of proteolysis of 75 samples of buffalo mozzarella PDO, plus three control samples. In this analysis there were detected quantitative values of β-CN and fragments of γ-CN and comparing the intensity of the bands, it was possible to calculate this index called Φ. The higher was Φ value the higher was the freshness. In addition, the study was conducted by monitoring the freezing effect on buffalo milk, quantifying the β-CN through immunochemical technique, indirect test Elisa, which recognizes the antigen by a specific antibody and the complex is revealed with a non-specific secondary antibody, which carries a chromogenic group, which is colored after enzymatic action. The results of this first investigation showed that the plasmin can act during freezing on the samples.

Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory conditions of the gastrointestinal tract including Crohn’s disease (CD) and ulcerative colitis (UC). Pathogenic mechanisms of IBDs, etiology and behavior, are not fully understood. They are characterized by a great extent of heterogeneity, in terms of phenotypic presentation and response to different therapies. These aspects lead to a great variability of the efficacy of different therapeutic strategies inducing patient to suffer and imply enormous costs for healthcare systems. In severe IBD and in corticosteroid-dependent or –resistant cases, the use of biological drugs, targeted towards TNF (infliximab, adalimumab) or α4β7-mediated lymphocyte adhesion (vedolizumab) is indicated. However, 20-40% of patients do not respond to biological agents, leading to an increase of direct and indirect costs and unnecessary exposure of patients to possible adverse events. Nowadays, the diagnostic and prognostic tools for IBD and the outcome of therapy are largely based on evaluation of clinical symptoms in combination with endoscopy, histology, radiology and non-specific biomarkers from serum or stools. There are no reliable clinical or molecular predictors of response to anti-TNF or anti-leukocyte adhesion drugs. The aim of the project is to promote personalized medicine in IBD, using serum proteomic profiling, to identify potential molecular markers that may predict the behavior of the disease and the response vs. failure of anti-TNF or anti-leukocyte adhesion treatment strategies in IBD patients. After obtaining written informed consent, we prospectively enrolled all the consecutive IBD patients afferent to Gastroenterology and Digestive Endoscopy Unit of IRCCS Policlinico San Donato. All diagnoses must have been previously confirmed by clinical, endoscopic and histologic criteria. Age and sex matched healthy controls were also be enrolled. Clinical data, such as, disease, medication and family medical history were collected; disease location, extension and behavior were classified according to Montreal classification, whereas clinical activity was evaluated using clinical scores, i.e. Harvey-Bradshaw Index (HBI) as appropriate. Patients underwent blood collection for serum. Successively, we obtained Protein Matrix Assisted Laser Desorption Ionization (MALDI) profiling from the collected sera. A total of 40 sera from healthy control and 32 sera from male adult patients affected by CD were analyzed. Before MALDI analysis, the samples underwent immunodeplection in order to eliminate the high abundant protein fractions from the serum. From MALDI analysis, we obtain best separating peaks between different conditions, which represent characteristic serum profiles. The best separating peaks were compared along different groups. Healthy controls versus responder and non responder were compared first, to identify the best peaks able to define control samples and disease samples. To identify the best peaks able to define differences between responders and non responders, these two groups were compared at I infusion and at II infusion time. Finally, total MALDI spectra from controls, responders and non responders were compared together at I infusion and at II second infusion time. This comparison showed one particular peak (corresponding to 3155, 98 m/z) that was changed in all samples and normalized at control level after treatment. This finding could indicate that this peak is typical of the disease. In conclusion specific protein profiles appear to be associated with the absence of response to anti-TNF in CD patients and one single peak is differentially expressed in controls, CD responder to anti-TNF and non-responder; thus, further investigations are required in order to identify the protein that the peak corresponds to.

The main work presented in this thesis describes proteomics strategies applied to study the trafficking and turnover of glutathione reductase (Glr1) isoforms in the cytosol and mitochondria of Saccharomyces cerevisiae. Additional work was performed in order to understand mass spectrometric response factors and how they can affect peptides representation in the mass spectra. The opportunity to study two sub proteomes involved in biofilm formation of Pseudomonas aeruginosa PAO1 arose during my PhD and their analysis is also presented. Glr1 is a low abundant protein involved in the defence mechanisms against reactive oxygen species, which are sources of many diseases. Because of its biological relevance, considerable effort has been made in order to understand its functional role in the cell. This protein has been studied using biochemical strategies. In yeast, the cytosolic and mitochondrial forms of glutathione reductase are expressed by the same gene, GLR1, using alternative start codons. Translation from the first AUG codon generates the mitochondrial form incorporating a transit peptide necessary for import into the mitochondria. If the translation starts at the second AUG codon, the cytosolic counterpart is generated. Biochemical approaches show that the first AUG codon is not in favourable context and it has been suggested that leaky scanning accounts for the abundance of the cytosolic protein. The analysis of Glr1 forms by mass spectrometry was demanded because only the N-terminal region is informative about similarities and differences between cytosolic and mitochondrial forms. The protein is also of low abundance in both cytosol and mitochondrial compartments. A genetically modified strain, over-expressing this protein was, therefore, used throughout this work in order to analyse glutathione reductase in the mitochondria. This was not possible with the wild-type strain. Because the first AUG codon is now in context, the over-producing strain (MORF) yields both cytosolic and full length mitochondrial isoforms in the cytosol. Analysis of the mitochondrial protein shows that the cleavage of the pre-sequence is not specific. Three different forms of the mitochondrial N-terminal peptide were detected. Some attention was also devoted to glutathione reductase turnover in both cytosol and mitochondrial compartments using the genetically modified strain. Both N-terminal peptides generated from translation starting in the first and second AUG codon as well as mid-chain peptides from the cytosol fraction and one mid-chain peptide from the mitochondrial fraction, were used to calculate relative turnover measurements. My results illustrate that the mitochondrial protein is in faster turnover than the cytosolic counterpart. Moreover, the long and short forms observed in the cytosol also show slightly different turnover rates, the long form presenting faster turnover than the short form. Rapid turnover therefore maintains the level of glutathione reductase in the mitochondria. Despite the exquisite sensitivity of mass spectrometry, its restricted dynamic range compared with the dynamic range of the entire proteome is limiting for such studies. Peptides have different responses in the mass spectrometry and factors such as hydrophobicity and gas-phase basicity, can also contribute to low detectability of some peptides. To maximise the mass spectrometric response of peptides especially the ones derived from low abundant proteins, is extremely important. My thesis therefore includes a study of mass spectrometric response of peptides generated by different enzymes. Applying the kinetic method, the importance of the position of basic residues on gas-phase basicity and thus on the mass spectrometric response was demonstrated. In addition, the opportunity to carry out a related study on the proteome analysis of membrane vesicles and matrix within biofilms of Pseudomonas aeruginosa PAO1 has arisen and results of this study were presented in the final results chapter. It is the first time that two-dimensional chromatography was applied to analyse these sub-proteomes. Moreover, previous studies were mostly limited to the planktonic population; here the proteomes of membrane vesicles and extracellular matrix with the biofilm were addressed.

La thématique des maladies infectieuses représente un réel enjeu politique, économique et de santé publique. Les objectifs de mes travaux de thèse étaient de développer des approches protéomiques pour identifier, détecter, caractériser et/ou quantifier des protéines et de les appliquer à l’étude de maladies infectieuses pour lesquelles des données de protéomique pourraient ouvrir à de nouvelles approches thérapeutiques et/ou diagnostiques. Les stratégies d’identification et de détection des protéines développées pour l’étude de la maladie de Lyme ont permis de prouver la faisabilité du diagnostic par spectrométrie de masse et de proposer des protéines candidates-vaccin. Les stratégies de quantification mises en place pour l’étude de la toxoplasmose ont permis d’identifier et de quantifier un complexe protéique clef. Les stratégies de caractérisation du N-terminome pour l’étude du paludisme ont permis d’apporter des preuves expérimentales des processus de maturation et d’adressage des protéines
Infectious diseases represent a real challenge in terms of politic, economic and public health. The purposes of my thesis works were to develop proteomic approaches able to identify, to detect, to characterize and/or to quantify proteins and to apply these approaches to the study of infectious diseases for which proteomic data cou Id open to new therapeutic and/or diagnostic strategies. Identification and specific detection strategies developed in the study of Lyme's disease allowed to prove the feasibility of diagnosis by mass spectrometry and to propose new vaccine-candidate proteins. Quantification strategies developed in the study of toxoplasmosis allowed us to identify and to quantify a key protein complex of the parasite. N-terminome characterization strategy developed in the study of malaria allowed us to bring experimental proofs of proteins processing and trafficking

Thesis (Ph. D.)--University of California, San Diego, 2010.
Title from first page of PDF file (viewed May 6, 2010). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (leaves 97-109).

MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level. Studies in mouse embryonic stem cells (mESCs) where DGCR8; a major protein involved in their biogenesis has been knocked out, have shown that the global loss of canonical miRNAs results in cell cycle defects, delayed and reduced expression of markers of differentiation, and an inability to downregulate pluripotency markers upon differentiation. By conducting a 2D-DIGE study, comparing protein expression in wild type and DGCR8-/- mESCs, the aim was to study the effects that the loss of DGCR8 has on the proteome of mESCs when grown under proliferative conditions. The loss of DGCR8 in mESCs resulted in the deregulation of proteins with a chaperone function and those involved in glucose metabolism. Notably enzymes involved in glycolysis were reduced, whereas those involved in the TCA cycle were upregulated compared to wild type cells. mESCs are known to be highly glycolytic and the form of glucose metabolism used by cells has been linked to their capacity to differentiate. A second DIGE study was carried out on DGCR8-/- mESCs individually transfected with Embryonic Stem Cell Cycle specific (ESCC) miRNAs, to establish novel targets of these miRNAs and study their effect on the proteome. The study revealed that the ESCC miRNAs influence the expression of glucose metabolism proteins, notably Aldolase A, a key enzyme for glycolysis was identified in both studies as being an indirect target of the ESCC miRNAs. High resolution nuclear magnetic resonance spectroscopy further revealed differences in metabolism between the DGCR8-/- mESCs transfected with the ESCC miRNAs and those transfected with a control miRNA, indicative of a switch from predominantly glucose metabolism in the wild type mESCs to glutaminolysis for energy generation in the DGCR8-/-. Therefore the same miRNAs that control the embryonic stem cell cycle, also play a major role in the metabolic status of these cells, which may in turn play a role in the controlling the balance between pluripotency and differentiation. At the time of writing this is the first study using proteomic techniques to compare DGCR8-/- and wild type mESCs, and to explore the effect of the ESCC miRNAs.