Rozprawy doktorskie na temat „Proteomics approaches”

Absolute quantitative proteomics typically relies on the use of stable isotope labelled internal standards introduced in a known amount. Comparative signal intensities of the labelled and unlabelled peptides allow the inference of protein concentration.

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

Le trafic rétrograde permet le transport des protéines de la membrane plasmique vers le réticulum endoplasmique, via l'appareil de Golgi, évité la dégradation des lysosomes. Des études antérieures ont montré que le transport rétrograde est crucial pour les fonctions cellulaires telles que la signalisation, l'homéostasie ionique, et l'établissement de gradients du morphogène Wnt. Par ailleurs, le transport rétrograde joue un rôle essentiel dans l'internalisation cellulaire des facteurs pathogènes comme les toxines protéiques et les protéines de virus. Toutefois, la liste actuelle des protéines cargos est limitée, et il est probable que de nombreuses fonctions cellulaires du transport rétrograde restent encore à découvrir. De toute évidence, un fort besoin existe pour une caractérisation plus poussée de cette voie de transport. Dans cette étude, quatre différentes approches protéomiques ont été développées visant à identifier les protéines membranaires prenant la route du transport rétrograde: SNAP-tag, sulfatation, la FKBP, et la streptavidine. Parmi ceux-ci, l'approche SNAP-tag s'est avéré être la stratégie la plus efficace pour identifier les candidats du fret de la voie rétrograde. Cette stratégie est basée sur la modification covalente du protéome de la membrane plasmique avec un benzylguanine (BG) dérivés. Seules les protéines membranaires BG-taggées qui sont ensuite transportés par voie rétrograde peuvent coupler covalentement à une protéine de fusion de SNAP-tag localisée dans la TGN. L'approche a été validée, étape par étape, en utilisant une protéine cargo rétrograde bien étudiée, toxine Shiga sous-unité B (STxB). Nous avons pu montrer que les STxB peuvent être capturés et identifiées par l'approche SNAP-tag. Par ailleurs, couplé à la LC-MS, les candidats des nouvelle protéines de la voie rétrograde ont été identifiés. Les trois autres approches ont guidé notre choix vers la stratégie la plus efficace en protéomique, en apportant des possibilités d'expérimentation et de défauts dans les domaines de la chimie organique et en biologie cellulaire. Généralement, ce travail de thèse a permis de développer une nouvelle stratégie protéomique qui pourraient être appliquée pour identifier les nouvelles candidats de la route rétrograde. Nous avons mis au point une approche dynamique en protéomique qui complète la protéomique traditionnelle. Par ailleurs, le concept d'utiliser des outils chimiques pour étudier le transport rétrograde peut également être appliqué à d'autres voies d'endocytose.

Recent technological developments in analytical chemistry spurred the analysis of historical, archaeological and paleontological objects. The Identification of proteins and small molecules from cultural heritage objects is crucial to characterize the materials used by the artists and it can provide invaluable information for designing restoration interventions. All most the developed analytical procedures require at least a micro sampling from the object. However, non-invasive techniques are always preferred for the analysis of precious and unique objects. A part of this PhD research focused on the development and application of new non-invasive methods for the analysis of cultural heritage. A new method for the non-invasive analysis of proteins and small molecules with mass spectrometry from cultural heritage objects was discussed; the results obtained using a non-invasive imaging instrument on ancient Egyptian mural paintings were also presented; the development and application of non-invasive methods that use portable infrared spectroscopy instrumentation were shown. The recent revolution in mass spectrometry technology with the introduction of high throughput instruments and techniques has led to the widespread expansion of advanced analytical methods in health science. But today, the main target of modern mass spectrometry analysis in biomedical research can be summarize as the development of effective and reliable approaches able of discriminating diseased conditions at their earliest stage, in a non or minimally-invasive manner. The aim of the second part of this PhD research was the development and application of non-invasive methods for the analysis of biological materials. A new method for the non-invasive analysis and characterization of adenoma in colon rectal cancer was presented and a combined bi and mono-dimensional gas chromatography mass spectrometry approach for the identification of new biomarkers for prostate cancer in serum was discussed.

In this work, tandem mass spectrometry (MS/MS)-based quantitative protocols were developed to facilitate differential protein expression analysis and biomarker discovery via a two-step sample interrogation strategy: (a) global protein profiling and differential expression analysis by spectral counting; and, (b) biomarker candidate validation by targeted screening, i.e., multiple reaction monitoring (MRM). Preliminary experiments were performed to evaluate the performance of the spectral counting method. The method proved to be applicable for proteins with spectral counts⠥2, and a close-to-linear relationship between protein concentration and spectral count data was achievable at protein concentration levels <0.1 μM. The detection limit was 40-800 fmol. A protein/peptide library containing ~10,000 peptide entries that facilitates the development of future MRM experiments, was developed. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum, the peptide retention time, and the top 10 most intense product ions that correspond to a given parent peptide. Only proteins identified by at least two spectral counts are listed. An MRM experiment was performed to demonstrate the successful applicability of this peptide library for the identification of putative biomarkers in proteomic samples.
Master of Science

The field of subcellular proteomics aims to describe and analyze all the proteins present in a precise subcellular compartment at a given time. In contrast to whole-cell or whole-organism proteomics, the analysis of individual organelles has provided simpler proteomes from which relevant biological information could be more easily derived. To date, the protein complement of several subcellular structures, including the mitochondria, lysosome, peroxysome, phagosome and nucleus has been described. The powerful and rapidly evolving instrumentation as well as the development of biochemical and bioinformatics tools now allow scientists to derive whole organelle models based on the proteomic data generated. This field however still faces numerous challenges. Among those is the analysis of membrane-associated proteins, whose large and hydrophobic character complicate their extraction, subsequent separation and analysis by mass spectrometry. Another emerging limitation in subcellular proteomic resides in the difficulty in collecting enough material of a very pure preparation of the organelle of interest, which still depend on lengthy and labor-intensive density-based centrifugation as the method of choice for subcellular fractionation. The work presented in this thesis describes the development of new methods for subcellular proteomics that address the above-mentioned limitations, and their application to relevant biological models. In chapter 2, we present the design of a non-discriminatory investigative approach to study membrane proteins. Relying on detergent-free and gel-free procedures, this strategy allowed identification of hundreds of cell surface-exposed proteins from freshly ejaculated bovine spermatozoa, as presented in chapter 3. Diverging from traditional cell fractionation protocols, we have refined in chapter 4 a fluorescence-assisted organelle sorting method and have employed it to acquire the proteome of corticotropes-derived secretory granules. Our proc
Le domaine de la protéomique subcellulaire vise à décrire et analyser toutes les protéines présentes dans un compartiment subcellulaire précis à un temps donné. Contrastant avec la protéomique des cellules ou d'organismes complets, l'analyse d'organelles individuelles a généré des protéomes plus simples desquels l'information biologique pertinente peut être plus facilement extraite. À ce jour, le complément de protéines de plusieurs structures subcellulaires, incluant la mitochondrie, le lysosome, le peroxysome, le phagosome et le noyau a été décrit. L'évolution rapide et la puissance de l'instrumentation disponible couplées au développement d'outils biochimiques et bioinformatiques permettent maintenant aux scientifiques de générer des modèles d'organelles complets basés sur les données générées par la protéomique. Ce domaine, cependant, fait toujours face à plusieurs défis. Parmi ceux-ci, on doit mentionner l'analyse des protéines associées à la membrane dont la taille et l'hydrophobicité compliquent l'extraction, la séparation subséquente et l'analyse par spectrométrie de masse. Une autre limitation émergeante en protéomique subcellulaire est l'obtention d'une préparation très pure d'organelles d'intérêt et ce, en quantité suffisante, qui dépend toujours de longues et laborieuses centrifugations basées sur la densité comme méthode de choix pour la fractionnement subcellulaire. Le travail présenté dans cette thèse décrit le développement de nouvelles méthodes en protéomique subcellulaire qui s'adressent aux défis mentionnés précédemment, et leur application à des modèles biologiques pertinents. Dans le chapitre 2, nous présentons l'élaboration et la mise au point d'une approche investigatrice non discriminatoire pour étudier les protéines de membrane. Basée essentiellement sur des procédures sans détergent et sans gel de séparation, cette stratégie a permis l'identification de centaines$

DOTTORATO DI RICERCA IN CHIMICA DEL FARMACO (XXV CICLO) Proteomic approaches in Drugs and Biomarkers Discovery Dott. Andrea Pancotti Tutor: Prof. Marina Carini Co-tutor: Prof. Sergio Romeo Abstract My thesis project is focused on development and application of proteomic approaches in two fundamental research areas: the drug discovery process and the discovery of disease biomarkers. In particular, new and reliable strategies by mass spectrometry for the fast identification and validation of drug targets, lead compounds and disease biomarkers have been considered. Proteomic Approaches in Drug Discovery In Prof. Romeo’s group new compounds have been identified with high in vitro efficacy against the intraerythrocytic stages of Plasmodium falciparum (P.f.) (IC50 <10 nM),1 but the molecular target is unknown and preliminary results indicate that these compounds may exerts their activity against P.f. through multiple targets. Proteomics offers a unique tool for target identification2 and several proteomic approaches are available, one of the most interesting is the so called chemical proteomics, which couple affinity purification methods with mass spectrometry and therefore permits to increase selectivity and sensitivity.3 In order to facilitate the mass spectrometric analysis, it would be necessary to know the target localization into P.f. of our compounds. Therefore last year, fluorescence compounds, as analytical tools for preliminary target identification, were designed. These compounds have been tested in the Prof. Taramelli’s laboratory against D10 and W2 strains, but only few compounds resulted to be active, and activities were elicited only in a micromolar range. These activities were not sufficient to conduct fluorescent studies, therefore further optimizations were undertaken. Considering the recent SAR obtained in our laboratory, compound 1 has been synthesized characterized by the presence of coumarin and a propylendiamine linker between leucine and the fluorescent group. This compound was active against P.f (IC50 D10 and W2= 13 nM) and fluorescent studies are being performed. In the meanwhile we are focusing our attention on the target identification. The adopted chemical proteomics approach requires the preparation of an alkyne derivate of the lead compound that will be incubated in a Pf lysate. Then trough click chemistry the protein bound alkyne derivative will be reacted with an azide containing a cleavable linker and a terminal biotin unit (compound 3). The lysate will be purified by affinity based chromatography using streptavidin beads. Then the cleavable linker will be cleaved and the protein-compound complex analyzed using HPLC-MS analysis. Thus propargylic derivative 2 was synthesized. It was tested in the Professor Taramelli’s laboratory and resulted to be very active against Pf. (IC50 D10= 8nM/ IC50 W2= 12nM). Compound 3 has been synthesized and it is characterized by a biotin unit, an acylhydrazone cleavable linker, a poliethylenglicol spacer and a terminal azide. In order to verify if compound 2 and 3 were useful tools to perform the procedure that we aimed to follow, the optimized click chemistry reaction was performed between the two compounds obtaining compound 4. In order to test the whole proteomics procedure, compound 4 was immobilized on agarose streptavidin beads and after the selective cleavage of the acylhydrazone cleavable linker and reduction of intermediate, compound 5 was obtained and characterized through direct infusion ESI-MS spectrum. After this successful result the final proteomics analysis is being conducted in collaboration with Professor Taramelli’s laboratory. Proteomic Approaches in Biomarkers Discovery One of the most exciting areas of proteomic research is the identification and validation of disease biomarkers which can be used as measurements within clinical studies and for the purpose of predictive diagnosis. The study of proteomics is considered to be a key for the characterization of human diseases and disease states, and mass spectrometry technology plays a crucial role in this disease research. We will focus in particular on detection and quantization of post-translational modifications of proteins caused by oxidative and carbonyl stress. While the development of specific antibodies against modified proteins has made it possible to confirm the occurrence of oxidative stress in vivo and its involvement in several physio-pathological conditions, the resultant chemical modifications of proteins has not yet been explored. Hence we need proteomic tools, which can sensitively detect oxidative damage on peptides and proteins: these tools would be helpful to understand exactly when, how, and where the damage occurs, and to gain a deeper insights into the mechanism of onset, progression, and/or complication of the diseases. Proteomics approaches have led to the identification of the protein/s showing high sensitivity to oxidation/carbonylation, and among them human serum albumin (HSA) was found to be the main target in the circulation.4-6 During the first year we demonstrated covalent modification of HSA-Cys34 by acrolein in patients undergoing hepatectomy surgery by using a mass spectrometric approach based on a triple quadruple mass spectrometer in precursor ion scan mode, able to identify unknown modifications of Cys34 by reactive carbonyl species (RCS) generated by lipid peroxidation. However, parallel studies performed in our laboratories demonstrated that in some pathologic conditions (i.e. uremic subjects), Cys34 undergoes disulfide formation by cysteinylation, thus limiting its availability for reaction with RCS. Hence, Cys 34-adducts couldn’t always be useful as carbonylation biomarkers. Therefore, I focused my attention on another HSA nucleophilic site (His-146), previously recognized as a potential oxidation/carbonylation target7. Digesting HSA with tripsin or tripsin+chimotripsin, two different peptides containing His-146 have been obtained: H*PYFYAPELLFFAK and H*PY, respectively. Anyway, analysis of tripsin or tripsin+chimotripsin digested HNE-HSA adduct did not lead to identification of any covalent modifications on His 146. Using an high-resolution, high mass accuracy mass spectrometer (LTQ-Orbitrap), performing a full scan analysis in data-dependent mode followed by data analysis with the SEQUEST algorithm it was possible to explain that the absence of any signals relative to RCS-H*PYFYAPELLFFAK adducts was due to different missed cleavages. The analyses relative to the H*PY peptide indicated the formation of one adduct only, H(HNE)PYF, but no significant fragmentation of the corresponding [M+H]+ was observed in MS/MS experiments, even working at high collision energy (40eV). Maybe the reason could be that the peptide is too short for an optimal fragmentation. Taking into account these observations, we have considered as alternative approach the application of a new HSA digestion strategy, based on the use of chimotripsin only. In silico studies indicated the EIARRH*PY peptide as a new tag containing the target residue His 146 in the middle, exactly like Cys-34 in the LQQC*PF peptide, previously analyzed with satisfactory results. This new peptide has also the appropriate length to be analyzed by HPLC-MS/MS. Hence, the EIARRH*PY peptide seemed to possess the optimal characteristics to start a new MS strategy involving His-146 as a carbonyl adduction site on HSA. Thus, HSA was isolated from human plasma by affinity chromatography, reduced with NaBH4 and digested with chimotripsin only in order to obtain the expected peptide. The peptide mixture was analyzed by LTQ-Orbitrap in data dependent scan mode and afterwards by the SEQUEST algorithm, comparing the obtained data with the primary sequence of the protein. The results of the analysis confirmed the formation of the expected peptide. In this way we created the bases for the future studies on the EIARRH*PY peptide, in order to identify any covalent modifications of His146 induced by RCS. References 1. Vaiana, N.; Marzahn, M.; Parapini, S.; Liu, P.; DelAgli, M.; Pancotti, A.; Sangiovanni, E.; Basilico, N.; Bosisio, E.; Dunn, B. M.; Taramelli, D.; Romeo, S. Bioorganic & Medicinal Chemistry Letters 2012, 22, 5915. 2. Brown, D.; Superti-Furga, G. Drug Discov Today 2003, 8, 1067. 3. Godl, K.; Wissing, J.; Kurtenbach, A.; Habenberger, P.; Blencke, S.; Gutbrod, H.; Salassidis, K.; Stein-gerlach, M.; Missio, A.; Cotten, M.; Daub, H. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 15434. 4. Mera, K.; Anraku, M.; Kitamura, K.; Nakajou, K.; Maruyama, T.; Otagiri, M. Biochem. Biophys. Res. Commun. 2005, 334, 1322. 5. Anraku, M.; Kitamura, K.; Shinohara, A.; Adachi, M.; Suenaga, A.; Maruyama, T.; Miyanaka, K.; Miyoshi, T.; Shiraishi, N.; Nonoguchi, H.; Otagiri, M.; Tomita, K. Kidney Int. 2004, 66, 841. 6. Aldini, G.; Vistoli, G.; Regazzoni, L.; Gamberoni, L.; Facino, R. M.; Yamaguchi, S.; Uchida, K.; Carini, M. Chem. Res. Toxicol. 2008, 21, 824. 7. Aldini, G.; Gamberoni, L.; Orioli, M.; Beretta, G.; Regazzoni, L.; Facino, R. M.; Carini, M. J. Mass Spectrom. 2006, 41, 1149.

A diverse range of high-dimensional datasets has recently become available to help elucidate the functioning of biological systems and defects within those systems leading to disease. All of these new technologies come with the challenges of determining how the raw data should be efficiently processed or normalised and, subsequently, how can the data best be summarised for more complex downstream analysis. There are many approaches to summarising and normalising omics data, with new methods frequently being developed. To date, there has not been a comprehensive evaluation of existing methods for many omics data types. This thesis focusses on systematically evaluating existing methods for three different types of omics data and, having identified limitations in the current methods, also proposes new approaches to improve their quality. Firstly, CAGE-seq data are considered. A two-stage method based on a novel region-finding algorithm followed by a classifier that integrates sequence patterns surrounding the identified regions is shown to possess superior performance to two existing methods. Similarly, a novel data summarisation approach to gene expression data, which integrates changes in location and scale into a unified metric, demonstrates benefits in two-class classification problems. The error rates are found to be competitive with existing methods, and the feature selection has higher stability and increased biological relevance. Finally, in the proteomics setting, there are many choices for how to summarise peptides to proteins, as well as issues relating to batch effects and whether internal controls are necessary. By developing a broad variety of performance metrics, and an accompanying web-based framework, novel recommendations about peptide to protein summaries and batch correction algorithms are made, and a surprising result regarding the necessity of internal standards is revealed.

L’objectiu general d’aquesta tesi doctoral va ser desenvolupar, analitzar i validar noves eines bioinformàtiques que converteixin les dades crues de metabolòmica (adquirides per espectrometria de masses) en coneixement biològic amb la finalitat d’estudiar les alteracions en el proteoma i metaboloma de cèl·lules humanes del pigment retinal exposades a condicions de hiperglucèmia i/o hipòxia. Per assolir aquest objectiu general, aquesta tesi s'ha estructurat en dos blocs principals: ⁻ Objectius metodològics: (i) Hem analitzat les bases de dades espectrals de masses de metabolòmica basades en LC / MS. (ii) Finalment, hem generat i millorat la caracterització de les dades de metabolómica de LC / MS centrant-nos en l'anotació de MS1 i MS2. ⁻ Objectius biològics: (iii) Hem proposat un nou mètode que detecta i analitza els canvis en les xarxes d'interacció proteïna-proteïna (PPI) per condicions d’hiperglucèmia i / o hipòxia. (iv) Hem presentat un flux de treball nou que és capaç de predir i validar les alteracions metabòliques degudes a condicions d’hiperglucèmia i / o hipòxia integrant a la vegada les dades d'expressió de proteïnes en les xarxes metabòliques.
El objetivo general de esta tesis doctoral fue desarrollar, analizar y validar nuevas herramientas bioinformáticas que conviertan los datos crudos de metabolómica (adquiridos por espectrometría de masas) en conocimiento biológico con el fin de estudiar las alteraciones en el proteoma y metaboloma de células humanas del pigmento retinal expuestas a condiciones de hiperglucemia y / o hipoxia. ⁻ Objetivos metodológicos: (i) Hemos analizado las bases de datos espectrales de masas de metabolómica basada en LC / MS. (ii) Finalmente, hemos generado y mejorado la caracterización de los datos de metabolómica de LC / MS centrándonos en la anotación de MS1 y MS2. ⁻ Objetivos biológicos: (iii) Hemos propuesto un nuevo método que detecta y analiza los cambios en las redes de interacción proteína-proteína (PPI) por condiciones hiperglucemia y / o hipoxia. (iv) Hemos presentado un flujo de trabajo novedoso que es capaz de predecir y validar las alteraciones metabólicas debidas a condiciones hiperglucemia y / o hipoxia integrando a la vez los datos de expresión de proteínas en las redes metabólicas.
The general objective of this doctoral thesis was to develop, analyse and validate new bioinformatic tools for converting raw MS-based metabolomics data into biological knowledge, in order to study alterations in the proteome and metabolome of human retinal pigment epithelium cells exposed to hyperglycemic and/or hypoxic conditions. To reach this general objective, this thesis has been structured in two main blocks: ⁻ Methodological aims: (i) We have analysed mass spectral databases for LC/MS-based untargeted metabolomics. (ii) Finally, we have generated and improved the characterization of LC/MS metabolomics data focusing on MS1 and MS2 annotation. ⁻ Biological aims: (iii) We have proposed a novel method that detects and analyses changes in protein-protein interaction (PPI) networks by hyperglycemic and/or hypoxic conditions. (iv) We have presented a novel workflow which is able to predict and validate metabolite alterations due to hyperglycemic and/or hypoxic conditions integrating protein expression data in metabolic networks.

Specialized metabolism (secondary metabolism) in glandular trichomes of sweet basil (Ocimum basilicum L.) and accumulation of specialized metabolites (secondary metabolites) in rhizomes of turmeric (Curcuma longa L.) was investigated using proteomic and metabolomic approaches, respectively. In an effort to further clarify the regulation of metabolism in the glandular trichomes of sweet basil, we utilized a proteomics-based approach that applied MudPIT (multidimensional protein identification technology) and GeLC-MS/MS (gel enhanced LC-MS/MS) to protein samples from isolated trichomes of four different basil lines: MC, SW, SD, and EMX-1. Phosphorylation, ubiquitination and methylation of proteins in these samples were detected using X!tandem. Significant differences in distribution of the 755 non-redundant protein entries demonstrated that the proteomes of the glandular trichomes of the four basil lines were quite distinct. Correspondence between proteomic, EST, and metabolic profiling data demonstrated that both transcriptional regulation and post-transcriptional regulation contribute to the chemical diversity. One very interesting finding was that precursors for different classes of terpenoids, including mono- and sesquiterpenoids, appear to be almost exclusively supplied by the MEP (2-C-methyl-D-erythritol 4- phosphate) pathway, but not the mevolonate pathway, in basil glandular trichomes. Our results suggest that carbon flow can be readily redirected between the phenylpropanoid and terpenoid pathways in this specific cell type. To investigate the impact of genetic, developmental and environmental factors on the accumulation of phytochemicals in rhizomes of turmeric, we performed metabolomic analysis in a 2x2x4 full factorial design experiment using GC-MS, LC-MS, and LC-PDA. Our results showed that growth stage had the largest effect on levels of the three major curcuminoids. Co-regulated metabolite modules were detected, which provided valuable information for identification of phytochemicals and investigation of their biosynthesis. Based on LC-MS/MS data, 4 new diarylheptanoids were tentatively identified in turmeric rhizomes using Tandem-MSASC, a home-made software tool that automatically recognizes spectra of unknown compounds using three approaches. Based on our metabolomic results, we proposed two new strategies, “metabolomics-guided discovery” and “correlation bioassay”, to identify bioactive constituents from plant extracts based on information provided by metabolomic investigation.

Sperm cryopreservation is currently the most suitable technique to store boar sperm samples for a long period of time. However, not all boar ejaculations present the same freezability, this allows the classification of boars and their ejaculations into good (GFE) and poor (PFE) freezability boars/ejaculates. In this context, the present Thesis aims to gain new insights into the evaluation and prediction of sperm freezability, thereby preventing PFE cryopreservation. Through Western blot it was determined whether small heat shock protein 10 (ODF1/HSPB10) and voltage-dependent anion channel 2 (VDAC2) were reliable freezability markers. Furthermore the sperm and seminal plasma proteome of GFE and PFE were compared through two-dimensional difference gel electrophoresis (2D-DIGE), seeking potential markers for boar sperm freezability. The main conclusion of this Dissertation is that VDAC2, ACRBP and TPI in fresh sperm and FN1 in fresh seminal plasma may be used to predict the sperm resilience to withstand cryopreservation procedures
La criopreservació espermàtica és actualment la tècnica més eficient per emmagatzemar les mostres d’esperma de porc durant llargs períodes de temps. Però, no totes les ejaculacions presenten la mateixa congelabilitat, permetent classificar tant els porcs com les seves ejaculacions entre els/les de bona (GFE) i mala (PFE) congelabilitat. Així, aquesta Tesi pretén adquirir noves aproximacions en l’avaluació i predicció de la congelabilitat espermàtica, per evitar la criopreservació de les PFE. Mitjançant Western Blot, es va determinar si dues proteïnes (ODF1/HSPB10 i VDAC2) eren marcadores de congelabilitat. També, el proteoma espermàtic i el del plasma seminal de GFE i PFE va ser comparat mitjançant gels 2D-DIGE, per tal de cercar marcadors potencials de la congelabilitat espermática. Les conclusions són que la VDAC2, la ACRBP i la TPI en l’esperma fresc i la FN1 en el plasma seminal poden ser emprades per predir la resistència de les ejaculacions porcines a la criopreservació

The consideration of possible future clean-fuel (H2), biodegradable plastic and potential anticancer drug has made the fresh-water unicellular cyanobacterium, Synechocystis sp. pee 6803 one of the most attractive cyanobacteria strains to commercial and industrial markets. To fully exploit the potential of these natural products, it is essential to understand the cellular response of this organism toward environmental stimulations. In this study, Synechocystis sp. was subjected to qualitative 'shotgun'-based proteomic analysis, and a total of 776 unique proteins, covered 24% of the entire proteome, were successfully identified. The use of Synechocystis sp. as a model of organism also revealed the potential of gel-based prefractionation over more classical chromatography approaches. The reliability of the iTRAQ-mediated quantitative proteomic approach was also assessed using Synechocystis sp., Sulfolobus solfataricus (an Archaea) and Saccharomyces cerevisiae (an Eukaryotes). Based on multiple replicate analyses, biological variation was found to have the greatest impact on results, and thus biological replicates were recommended for inclusion in all future iTRAQ experiments. This investigation also revealed at least ±50% variation is necessary for significant protein expression to have occurred. The functional proteomic approaches in this thesis covered two main fundamental ideas: firstly to investigate the basic cellular response of this organism toward photoperiod (Le. light-dark cycle); secondly, to examine the cellular stress response as a consequence of macronutrient deficiency, specifically phosphate starvation. The cellular response as a result of circadian influence was also studied via combining results from both transcriptomic and proteomic approaches; and possible interactions (e.g. post-translational modification) between mRNA and protein were also discussed.

The innate immune system is of central importance to the early containment of infection. When receptors of innate immunity recognize molecular patterns on pathogens, they initiate an immediate immune response by inducing the expression of cytokines and other host defense genes. Altered expression or function of the receptors, the molecules that mediate the signal transduction cascade, or the cytokines themselves can predispose individuals to infectious or autoimmune diseases. Here we used genomic approaches to uncover novel components underlying the innate immune response to cytosolic DNA and to characterize variation in the innate immune responses of human dendritic cells to bacterial and viral ligands. In order to identify novel genes involved in the cytosolic DNA sensing pathway, we first identified candidate proteins that interact with known signaling molecules or with dsDNA in the cytoplasm. We then knocked down 809 proteomic, genomic, or domain-based candidates in a high-throughput siRNA screen and measured cytokine production after DNA stimulation. We identified ABCF1 as a critical protein that associates with DNA and the known DNA-sensing components, HMGB2 and IFI16. We also found that CDC37 regulates stability of the signaling molecule, TBK1, and that chemical inhibition of CDC37 as well as of several other pathway regulators (HSP90, PPP6C, PTPN1, and TBK1) potently modulates the innate immune response to DNA and to retroviral infection. These proteins represent potential therapeutics targets for infectious and autoimmune diseases that are associated with the cytosolic DNA response. We also developed a high-throughput functional assay to assess variation in responses of human monocyte-derived dendritic cellsto LPS (receptor: TLR4) or influenza (receptors: RIG-I and TLR3), with the goal to ultimately map genetic variants that influence expression levels of pathogen-responsive genes. We compared the variation in expression between the dendritic cells of 30 different individuals, and within paired samples from 9 of these individuals collected several months later. We found genes that have significant inter- vs. intra-individual ariation in response to the stimuli, suggesting that there is a substantial genetic component underlying variation in these responses. Such variants may help to explain differences between individuals’ risk for infectious, autoimmune, or other inflammatory diseases.

When treating patients with cancer, the ability to predict a patient’s response to treatment is an important tool to allow therapy to be tailored for best outcome. Therefore, the need exists for a test to forecast a patient’s response using a sample that is readily accessible, and provides an accurate reflection of a patient’s response to a disease or treatment. Profiling biological fluids, such as plasma or urine, has gained considerable interest in recent years. This is because these fluids are readily available and are expected to provide an accurate representation of a patient’s response to treatment. As such, much effort has been put into finding biomarkers or prognostic indicators. Abnormal protease activity has been linked to the progression of cancer due to their involvement in several processes vital to the survival and proliferation of the disease. These include metastasis, resistance to apoptosis and angiogenesis, amongst others. In addition, dysregulated protease activity has been linked to poor response to chemotherapy as well as tumour regrowth following radiotherapy. Therefore, an increased understanding regarding the activity of a patient’s proteases may provide the clinician with more information as to how best to treat the patient. Therefore, monitoring protease activity has been suggested as a potential marker to predict a patient’s response to cancer treatment. Most enzyme activity assays are currently performed by fluorescence spectroscopy. However, these workflows suffer from limited sensitivity and linear range. Therefore, an alternative, more sensitive assay is required. Mass spectrometry (MS) is a highly sensitive analytical technique routinely used to quantify changes in biological systems. As such, MS has the potential to be used in enzyme activity assays. This study illustrates the development of a novel MS based method to monitor the activity of target enzymes in plasma; specifically asparaginyl endopeptidase (AEP) and caspase-3 using mass spectrometry. These enzymes have been linked to poor chemotherapeutic response in childhood leukaemia and tumour regrowth post-radiotherapy respectively. This project will describe the development and optimisation of each stage of a five step sample preparation and analysis method. This includes the design of enzyme substrates designed to be cleaved by the target enzyme, whilst reducing the effect of other enzymes acting on this substrate, how best to enrich samples for these target peptides, as well as determining the best MS technique to monitor these peptides. In addition, this project describes a comparison between this assay and an existing fluorescence assay when monitoring AEP activity in biological samples such as plasma and whole cell lysates. The application of this method in quantifying caspase-3 activity in plasma samples is also examined.

Sphingolipids (SLs) are one of the major classes of lipids in eukaryotes. In addition to being essential structural components of cell membranes, SLs also play capital roles as signalling molecules. Ceramides (Cer) are a family of bioactive SLs consisting of a long chain base (LCB), known as the sphingoid base, linked to a fatty acid (FA) of variable chain length via an amide bond. Due to their metabolic inter-relations with other SL species, Cer are considered key intermediates in the SL pathway. Cer are important second messengers in several cellular processes including apoptosis, autophagy, cell differentiation and sensescence. Ceramide synthases (CerS) are a group of enzymes, primarily localised at the endoplasmic reticulum, that catalyse the N–acylation of sphingoid bases such as sphingosine (So) and dihydrosphingosine (dhSo), using acyl CoA thioesters of variable chain lengths, to afford Cer and dihydroceramides (dhCer), respectively. Six isoforms of CerS (CerS1–6) have been identified in mammals. Each CerS isoform utilizes a small subset of FA-CoAs of defined chain lengths and, thus, each of them produces specific Cer populations. In the recent years, it has become apparent that Cer with different acyl chains vary in their biophysical properties and in the signalling pathways they participate. Furthermore, Cer with defined acyl chain lengths have been found to be implicated in the onset of a variety of human diseases, including cancer, type-2 diabetes mellitus, Alzheimer’s disease, multiple sclerosis and cardiomyopathy. In this context, the development of appropriate tools to study the activity of CerS enzymes, which is crucial to decipher the molecular mechanisms by which Cer elicit their effects, was the ultimate goal of the present doctoral thesis. The first part of this thesis was devoted to the development of a new CerS activity assay based on the Förster Resonance Energy Transfer (FRET) phenomenon. To that end, we designed and synthesized a series of fluorescently labelled (or labelable) 1-deoxy sphingoid probes derived from spisulosine, a small library of clickable FA analogues of different chain lengths, and a collection of bicyclo[6.1.0]nonyne (BCN) or 1,2,4,5-tetrazine (Tz) based fluorescent reagents. The absorption and fluorescence emission properties of these compounds was thoroughly studied in various solvents by means of cuvette-based experiments. Based on these studies, we anticipated that a highly efficient FRET process would take place between the donor-acceptor fluorophore pairs that had been selected, namely MCC/NBD and NBD/NR. Next, the metabolic incorporation of the different spisulosin-based probes and the FA analogues was evaluated in various biological contexts. Mass spectrometry analysis evidenced an extensive metabolization of the synthetic LCB probes and the FA analogues by CerS enzymes to form the corresponding Cer metabolites. Unfortunately, the FA analogues were also incorporated into other lipidic metabolic pathways, resulting in the generation of a strong fluorescence background after the fluorescent labelling reactions. Our different attempts to solve this issue were unfruitful and, thus, the development of the FRET based fluorescence assay to determine the CerS activity could not be achieved. The second part of this thesis was aimed at the development of new click-formed proteolysis targeting chimeras (CLIPTACs) targeting the ubiquitination and proteasomal degradation of CerS, as an alternative to small molecule inhibitors for the modulation of the CerS activity. To this end, we designed and synthesized four BCN derivatives containing known ligands for recruiting different E3 ubiquitin ligases. These BCN-tagged E3 ligase recruiters will be used in future studies in combination with an azido-functionalized analogue of the CerS substrate Jaspine B to obtain the desired CLIPTACs.
Los esfingolípidos (SLs) son una de las principales categorías de lípidos presentes en los organismos eucariotas. Los SL no sólo son componentes estructurales esenciales de las membranas celulares, sino que también actúan como moléculas señalizadoras. Las ceramidas (Cer) son una tipología de SL que están formadas por una base esfingoide y una cadena de ácido graso de longitud variable unidos a través de un enlace amida. Las Cer participan como segundos mensajeros en procesos celulares como la apoptosis, la autofagia, la diferenciación celular y la senescencia. Las ceramida sintasas (CerS) son un grupo de enzimas del retículo endoplasmático que catalizan la N-acilación de bases esfingoides, como la esfingosina, utilizando acil-CoAs de distintas longitudes, para dar Cer. En los mamíferos se han descrito seis isoformas de la CerS y cada una de ellas tiene preferencia por un pequeño grupo de ácidos grasos de longitud de cadena definida, por lo que cada una produce perfiles de Cer característicos. En los últimos años se ha visto que la longitud de la cadena acilo de las Cer influye en sus propiedades biofísicas y en las cascadas de señalización en las que participan. Además, se sabe que ciertas Cer están involucradas en el desarrollo de distintas enfermedades como el cáncer, la diabetes, el Alzheimer o la esclerosis múltiple. En este sentido, el desarrollo de herramientas adecuadas para el estudio de la actividad de CerS es fundamental para descifrar los mecanismos moleculares a través de los cuáles actúan las Cer, siendo este el objetivo principal que se persiguió en la presente tesis doctoral. La primera parte de la tesis se centró en el desarrollo de un nuevo ensayo para determinar la actividad CerS por medio del fenómeno de FRET. Para ello, se diseñaron y sintetizaron una serie de sondas esfingoides derivadas de la espisulosina, una pequeña quimioteca de análogos de ácidos grasos “clicables” de distintas longitudes de cadena y una colección de reactivos fluorescentes marcados con un grupo biciclo[6.1.0]nonino (BCN) o 1,2,4,5-tetrazina (Tz). Las propiedades de absorción y emisión de fluorescencia de estos compuestos fueron estudiadas en varios disolventes a través de experimentos “en cubeta”. En base a estos experimentos anticipamos que las parejas de fluoróforos seleccionadas eran adecuadas para su uso en experimentos de FRET. A continuación, se evaluó la incorporación metabólica de las distintas sondas esfingoides y de los varios análogos de ácidos grasos en medios biológicos. Los estudios de lipidómica mostraron que tanto las sondas como los análogos de ácidos grasos eran procesados por las CerS para dar las Cer correspondientes. Sin embargo, los análogos de ácidos grasos también entraron en otras rutas metabólicas de los lípidos dando lugar a un elevado ruido de fondo tras la reacción de marcaje de fluorescencia. Nuestros intentos para solventar este problema fueron en vano y, por tanto, finalmente no fue posible la implementación del ensayo de fluorescencia para medir la actividad CerS. En la segunda parte de la tesis se propuso el desarrollo de nuevos CLIPTACs dirigidos a la degradación de CerS, como alternativa a los inhibidores clásicos para la modulación de la actividad CerS. Para ello se diseñaron y sintetizaron cuatro derivados de BCN que contuvieran un ligando para reclutar distintos enzimas E3 ligasas de ubiquitina. Estos reclutadores de E3 ligasa serán utilizados en un futuro en combinación con un derivado de la Jaspina B, un análogo del sustrato de las CerS, para obtener los CLIPTACs deseados.

Protein post-translational modifications (PTMs) play a key role in the field of functional proteomic because they regulate activity, localization, and interaction with other cellular molecules, such as proteins, nucleic acids, lipids and cofactors. One of these modifications is named lysine acetylation and, although has been known from 50 years, it is still not completely understood, given its low stoichiometry and technical challenges in identifying modified sites. This PTM involves the transfer of an acetyl group from the donor acetyl-CoA to a protein and it is capable of affecting protein functions, regulating enzymatic activity and protein subcellular localization. The cornerstone of this thesis is the investigation of post-translational lysine acetylation, in proteins involved in lipotoxicity in type 2 diabetes mellitus and in proteins involved in fibroblast activation in the context of breast cancer. The main research project involves the investigation of acetylation in pancreatic β cells in the context of type 2 diabetes mellitus (T2DM) and experiments have been carried out in the Department of Pharmacy of the University of Pisa, under the guide of Prof. Lucacchini. A second project, carried out at the Beatson Institute for Cancer Research in Glasgow under the supervision of Dr Zanivan, examines acetylation in normal fibroblasts and cancer-associated fibroblasts in the context of breast cancer. Type 2 diabetes mellitus is a complex metabolic disorder, characterized above all by insulin resistance in muscle, liver and adipose tissue. One typical predisposition factor is obesity and our aim in this project is to investigate the effect of a diet rich in saturated fatty acids on pancreatic islets in type 2 diabetes. Palmitate is the most common saturated free fatty acid in human plasma and has been used for in vitro and in vivo studies on lipotoxicity. We used palmitate to study the mechanisms of lipotoxicity in rat β cell lines first and in human pancreatic islets then. Palmitate has been found to cause impairment of insulin secretion through mitochondrial alterations. However, the relationship between type 2 diabetes and mitochondrial dysfunctions is still under investigation and requires further studies. On this basis, by two dimensional gel electrophoresis, western blot and liquid chromatography-mass spectrometry we evaluated the effects of palmitate on mitochondrial proteins from INS-1E cells and on proteins from human pancreatic islets (HPI) after 24h palmitate exposure. The second project aimed to set up a quantitative proteomic approach to study lysine acetylation in normal (NFs) and cancer associated fibroblasts (CAFs), derived from human mammary tissues. Extensive comparative proteomic studies on human mammary fibroblasts, carried out in the Proteomic group of Dr Zanivan, revealed that the levels of some metabolic proteins and enzymes are altered in CAFs upon activation. In particular, it has been found that such proteins are involved in the generation of acetyl-CoA, resulting in higher acetyl-CoA levels in CAFs than in normal fibroblasts. Acetyl-CoA is the building block for protein acetylation reactions as well as for fatty acid and cholesterol synthesis. To investigate whether acetyl-CoA can be used to acetylate proteins and in this way regulating cellular functions in a differential manner between NFs and CAFs, we approached to study the acetylome of these cells using a bottom-up proteomics approach. Stable isotope labelling by amino acids in cell culture (SILAC) has been used to accurately quantify the acetylome of NFs and CAFs and an antibody-based strategy allowed the enrichment of acetylated peptides. Findings linking lysine acetylation to fibroblast activation, and acquisition of a proinvasive phenotype, could allow us to better understand mechanisms underlying this activation and discover novel therapeutic targets in cancer.

Proteomics is a rapidly growing discipline dealing with structure, molecular interactions, conformational dynamics, modifications and the functions of proteins. Mass spectrometry (MS) and (nuclear magnetic resonance) (NMR) have been used comprehensively to study protein interactions. Acyl carrier protein (ACP) interacts with more than 30 partner proteins during either fatty acid or polyketide biosynthesis. In order to be fully activated ACP gains a 4'-phosphopantetheinyl (4'-PP) group from coenzyme A using acyl carrier protein synthase (AcpS) via posttranslational modification. Protein-protein interactions of the ACP from the actinorhodin (act) polyketide synthase (PKS) complex and AcpS were investigated using oxidative footprinting with hydroxyl radicals generated from the Fenton reaction. Chemical modification of acidic residues was also used to investigate the interaction between these proteins using l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) this acted as a zero length cross linker to induce modification with Me-Glycine. MS was used to identify the modified residues and peptides and the extent of modification. Several residues were found to be protected in the complex between the two proteins and these may participate in the interaction interface between ACP and ACPS. Isotopically labelled ACP was expressed and purified and multidimensional NMR experiments were recorded to investigate this interaction interface identified using oxidative footprinting techniques. Chemical shift perturbations for ACP residues were calculated, and these revealed that many residues were affected by oxidation of ACP. Oxidation of methionine to methionine sulfoxide was confirmed. Metabolomics is a discipline which deals with metabolites in a biological system. It provides a wealth information for disease diagnosis, drug discovery, toxicology and genetic modification. Attempts have been made in this thesis to utilize metabolomics in biometrics. Mice were used as a model to attempt to determine individuals' age by their scent. In this part of the project chemometric methods were used to discriminate mice using a gas chromatography-mass spectrometry dataset of volatile organic compounds obtained from their urine. Principal component regression (PCR), partial least squares regression (PLSR) and support vector regression (SVR) were used to determine mouse age. Mice could be discriminated by their age using SVR without overfitting the data.

Les coquilles de mollusques ont très souvent été employées par les populations préhistoriques, comme source de matière première, à la confection d'ornements vestimentaires ou de parures. Ces objets, largement transformés, constituent un témoignage inestimable pour comprendre les cultures passées et la façon dont des populations disparues ont exploité leur environnement naturel. Abondamment retrouvés dans les sites archéologiques, les restes coquilliers sont le plus souvent minuscules, modifiés ou bien retrouvés à l'état de fragments, à un point tel qu'il est impossible de leur assigner une appartenance taxonomique.Depuis quelques années, les approches protéomiques, basées sur la sensibilité des techniques de spectrométrie de masse, ont été utilisées pour l'étude de restes bio-archéologiques, ouvrant ainsi un champ disciplinaire nouveau, la paléoprotéomique. Alors que la protéomique est classiquement employée sur des coquilles modernes pour en comprendre les mécanismes moléculaires de biominéralisation, la paléoprotéomique de fragments de coquilles archéologiques n'a jamais été réalisée jusqu'à présent, pour plusieurs raisons: faibles quantités d'échantillons (et, par conséquent, de protéines), données de séquences très parcellaires sur les protéines coquillières de mollusques.Ce travail de thèse représente donc la première tentative d'analyse des protéines coquillières d'échantillons archéologiques par une approche protéomique, telle que réalisée dans le domaine de la biominéralisation. Trois concepts-clés sont mis en exergue: les protéines de coquille contiennent de l'information taxonomique; elles peuvent être préservées à des échelles de temps archéologiques; elles peuvent être utilisées comme "code-barre" moléculaire pour identifier les taxons dont sont issus les restes archéologiques.En premier lieu, l'approche protéomique (HPLC-MS/MS) a été testée sur la coquille d'un bivalve-modèle, Spondylus gaederopus, pour en caractériser son contenu protéique. Nos données indiquent que le répertoire protéique coquillier de cette espèce diffère de celui de formes voisines et comprend des séquences "taxon-spécifiques". En second lieu, des "empreintes de masse peptidique" (PMF en anglais) par MALDI-TOF ont été réalisées sur les coquilles récentes de 34 espèces de mollusques, couvrant un large spectre de taxons trouvés sur des sites archéologiques. Les spectres PMF obtenus ont une signature particulière, contiennent une information taxonomique, un "code-barre", applicable à des échantillons archéologiques. En troisième lieu, des expériences de vieillissement accéléré ont simulé la diagenèse de coquilles de Spondylus. La stabilité des protéines coquillières a été étudiée en combinant immunochimie et protéomique quantitative. Les données démontrent que Spondylus est un bon modèle, favorable à la préservation des protéines de coquilles. Certains processus chimiques de clivage peptidique ont pu être analysés finement. Les patrons de dégradation des protéines sont cependant complexes, une complexité non prédite par les modèles théoriques. Enfin, des ornements archéologiques, provenant de divers sites européens mésolithiques et néolithiques ont été étudiés par protéomique couplée à des analyses microstructurales et géochimiques. La réalisation d'empreintes de masse peptidique (PMF) n'a pas permis de lever le voile sur l'identité taxonomique des échantillons, sans doute du fait des très faibles quantités de protéines restantes, ces échantillons requérant des analyses plus complètes par HPLC-MS/MS. Par contre, les données obtenues sur de minuscules boutons-doubles ont clairement montré que ces derniers ont été fabriqués à partir de nacre de moules d'eau douce (unionidés) largement réparties en Europe
Archaeological mollusc shell artifacts and ecofacts are valuable sources to study past cultures and provide insights on how people exploited their environments. Mollusc shells were often used as raw materials to make personal ornaments and are abundantly found in archaeological sites. However, minute, heavily worked and/or fragmented shell ornaments are rarely identified at different taxonomic levels, due to limited availability of analytical approaches to determine taxon-specific (diagnostic) features. In recent years, proteomics, which exploits the high sensitivity of modern mass spectrometry techniques, has been successfully applied to the study of a variety of bioarchaeological remains, opening a new research field referred to as ‘palaeoproteomics’. While modern mollusc shells represent key study models in biomineralization research to investigate the molecular mechanisms of biocalcification and mineral deposition, palaeoproteomics on archaeological shells has never been carried out before. The challenges are mainly due to the intrinsic physico-chemical features of their substrates (including low protein content), and the paucity of protein sequences for reference.Therefore, this work presents a comprehensive proteomic-based investigation of archaeological mollusc shells, grounded in biomineralization research, and the first application of palaeoproteomics to shells. The main objective of this PhD was to investigate three key concepts: whether shell proteins carry taxonomic information, could be preserved over archaeological times scales and could be used as molecular barcodes for the taxonomic determination of ancient shell artifacts.High performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was used for an in-depth proteomics characterisation of a selected model, Spondylus gaederopus, demonstrating that its shell-associated proteins are very different from other models and likely represent lineage-specific sequences. Peptide mass fingerprinting (PMF) by MALDI-TOF mass spectrometry was employed to obtain sequence information from thirty-four different molluscan taxa. Intracrystalline shell proteins displayed clearly distinct PMFs indicating that they may encode taxonomic information and could be used as molecular barcodes to identify archaeological mollusc shells. Accelerated aging experiments were performed to mimic the diagenesis of Spondylus shell and the stability of intracrystalline shell proteins was investigated by a combination of immunochemistry and quantitative TMT proteomics. We concluded that Spondylus represents a favourable system for protein preservation, as evidenced by the thermal stability of shell peptide sequences. However, the observed degradation patterns pinpointed the complexity of the whole diagenesis process, which does not entirely follow expected trajectories from theoretical models.Finally, a set of archaeological shell ornaments, recovered from different Mesolithic and Neolithic European prehistoric sites, were studied by proteomics coupled with structural, biomolecular and geochemical analyses. The studies showed that PMF by MALDI-TOF approach was not able to characterise these archaeological samples, which are very degraded or and/or have low protein contents, and the analysis of ancient shell proteins requires higher sensitivity, as offered by HPLC-MS/MS mass spectrometry. Indeed, palaeoproteomic analysis by HPLC-MS/MS of small “double-button” ornaments identified that they were made of freshwater mother-of-pearl shells, resolving the long-standing debate over their biological origin and provenance. The research carried out in this thesis highlights the immense potential of proteomics-based approaches to study archaeological and palaeontological shell remains

Neurodegenerative diseases are associated with extensive physical and mental debilitation, significant costs to the healthcare system, as well as great emotional and financial burden to the patients, their families and care providers. Despite progress in our understanding of the mechanisms behind neurodegenerative diseases, the vast majority are still currently untreatable. Synapses are important pathological targets in a range of disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and lysosomal storage disorders, such as Batten disease. Loss of synaptic connections and impairments in synaptic function are present in the initial stages of neurodegenerative conditions and throughout the course of disease progression. Therefore, synaptoprotective strategies are regarded as a potentially key factor in the development of effective therapies aimed at preventing or halting neurodegeneration. Despite the continuously growing body of research elucidating the molecular mechanisms that modulate synaptic function and vulnerability, the contribution of these pathways to neurodegenerative diseases is far from fully characterized. In addition, there are frequent issues regarding the applicability of the research performed using in vitro and small animal models of disease to develop therapeutic strategies for use in human patients. In the work described in this thesis, we initially validated the involvement of a selection of key synaptic targets, previously identified as regulators of synaptic degeneration in lower animal models, including mice and Drosophila, in a large animal model of neurodegenerative disease: CLN5 Batten sheep. Subsequently, we explored two of these individual synaptic protein targets in more detail (calretinin and α-synuclein), to further investigate their contribution to synaptic function and stability. Calretinin is a poorly characterized protein, primarily known for its calcium buffering capacities and high levels of expression in a subpopulation of interneurons. In this work, we show calretinin is expressed in previously unreported cell populations, including motor axons and synapses from the peripheral nervous system, and that it is enriched in synapses in vitro. Furthermore, we show calretinin responds dynamically to synaptic activity and is directly involved in neurodegenerative pathways, as demonstrated by its ability to influence the course of Wallerian degeneration and apoptotic cell death. α-synuclein plays a central role in the pathophysiology of Parkinson’s disease and contributes to the maintenance of synaptic transmission and mitochondrial function. However, questions still remain about how to effectively manipulate α- synuclein to obtain therapeutic benefits. Therefore, we sought to explore downstream targets of α-synuclein in order to uncover new pathways through which this protein may influence synaptic stability. Using proteomics on mice lacking α-synuclein and in vitro cell systems we identified sideroflexin 3 (sfxn3). We show sfxn3 is localized at the inner mitochondrial membrane and that it functions outside the main canonical pathways of mitochondria energy production. In addition, overexpression of sfxn3 in Drosophila led to a significant loss of synaptic boutons at the level of the neuromuscular junction, suggesting regulated levels of sfxn3 are important for the maintenance of synaptic connections. Altogether, the work developed in this thesis provides novel insights into pathways regulating synaptic stability and function. We not only provide evidence that the molecular targets studied are affected in a large animal model of neurodegenerative disease, and are therefore likely to be relevant to studies in human conditions, but we also uncover two new molecular targets capable of independently regulating synaptic form and function.

Fresh fruits and vegetables (FFVs) are the most frequently wasted foods because of their perishability and handling requirements. However, there is a lack of information on how much each step of the supply chain impacts FFVs quality, particularly on tomatoes, and what measures need to be taken for an immediate and effective impact on waste reduction. There is also no information on how the supply chain affects the proteome of the tomato and what proteins are differentially regulated by the most impactful steps of the supply chain. The objectives of the work presented on this thesis were to evaluate the decline in the overall quality and quantify tomato waste at each step of the supply chain, from the farm to consumer; and to determine what proteins are impacted by the decline in quality that is associated with temperature abuse. To determine overall quality and tomato waste, light-red tomatoes were exposed to an optimum temperature (13 °C) and eighteen different time-temperature scenarios, normally encountered during supply chain, and sensory and physicochemical attributes measured at each step. To determine the impact of chilling and non-chilling temperatures normally encountered during tomato supply chain, on the proteome, light-red tomatoes were exposed to an optimum temperature (13 °C) and to two time and temperature supply chain scenarios (2 °C and 25 °C) that showed the most negative impact on tomato overall quality, and physicochemical and proteomic attributes were measured at each step. For the first tomato harvest, the steps with the highest impact on quality and waste were shipping to distribution center (DC; 20°C), cooling at the grower (25°C) and storing at the consumer (4°C). For the second tomato harvest, shipping to the store (2°C), cooling at the farm (10°C) and displaying at the store (20°C) negatively impacted quality. High temperatures during cooling, shipping and store display impacted sensory quality and resulted in increased weight loss, and decreased sugar, carotenoids, and ascorbic acid contents. Although low temperatures during shipping, cooling and consumer did not impact tomato sensory quality, they contributed to a decline in sugar, carotenoids and ascorbic acid contents. Overall, the most impactful steps on tomato quality and waste, regardless of the temperature, were shipping to DC, cooling, shipping to stores, displaying at the store, and consumer storage. Analysis of the differentially expressed proteins in the tomato showed that metabolic proteins were greatly impacted by temperature abuses such as phosphomannomutase, heme oxygenase 1, and MAP kinase; and that proteins regulating cellular membrane integrity such as vacuolar protein sorting-associated protein were also impacted.

The vascular endothelium is one of the major targets of oxidative stress, playing an important role in the development of vascular-related disorders, such as atherosclerotic disease1. Oxidative stress increases the formation of reactive species (ROS), such as peroxides and hydroxyl radicals. ROS are signalling molecules and are able to induce leukocyte adhesion and promote the vascular endothelial permeability, that leads to the dysfunction of endothelium- related signal transduction pathways as well as redox-regulated transcription factors2. ROS cause reorganization of the actin filament, formation of an intracellular gap and changes in the cell shape. The endothelium contains adherents and tight junctions that are responsible for maintaining the restrictive barrier. Figure A1 shows the subcellular endothelial sites regulated by oxidative stress. In the normal structure of vascular endothelium, the lateral cell border contains occluding protein in association with zonula occludens-1 (ZO-1); VE-cadherin in association with a-, b-, and g-catenins; other proteins such as platelet endothelial cell adhesion molecule 1 (PECAM-1). Cortical actin filaments are directly linked with ZO-1, vinculin and α-actinin, that connect catenin to VE-cadherin in actin filament network. Intercellular adhesion molecule-1 (ICAM-1) expressed on the endothelial luminal surface might also involve in cortical actin filaments. ECs continuously express surface adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1), ICAM-1, ICAM-2 and P-selectin (stored in Weible-Palade bodies). However, oxidative stress causes phosphorylation and reorganization of occludin and PECAM-1, reduces levels of vascular endothelium catenins and actin-binding proteins. In the actin filament network, oxidant stress causes the increased formation of stress fibers and decreased cortical actin band, resulting in disruption of the tight and adherens junctions. ROS promote cell contraction and stress fiber formation due to the increased phosphorylation of myosin light chain (MLC). Additionally, oxidative stress functionally upregulates adhesion molecules

Data reported in the present thesis were obtained in two different projects. Accordingly, the thesis is divided into two chapters. The first chapter, which refers to papers I and II, reports a study aimed at unravelling the physiologic role of the cellular prion protein (PrPC) using an in vivo model of skeletal muscle regeneration. The second chapter refers to paper III in which the two dimensional electrophoresis (2DE) approach in combination with tandem mass spectrometry was used to identify potential biological markers of the illegal treatment of bulls with growth promoting agents (GPA). By focusing on the relationship between PrPC and skeletal muscle regeneration in a live model, in the first research line (Chapter I) we investigated if and how the protein influences the proliferation and the differentiation of muscle precursor cells. PrPC is a cell surface glycoprotein involved in the onset of rare and fatal neurodegenerative disorders, known as transmissible spongiphorm encephalopathies (TSE) or prion diseases. TSE occur when PrPC converts into a conformationally modified isoform that originates the prion, a novel infectious and neuro-pathogenic agent. Although much information is now available on the different routes of prion infection, both the mechanisms underlying prion neurotoxicity and the physiologic role of PrPC remain unclear. Nonetheless, use of different animal and cell models has suggested a number of putative functions for the protein, ranging from cell protection against oxidative and apoptotic challenge, to cell adhesion, proliferation and differentiation. Skeletal muscles express significant amounts of PrPC, and have been related to PrPC pathophysiology by several findings. Therefore, in order to clarify the physiologic role of PrPC, we employed a degeneration/regeneration protocol to the tibialis anterior muscle, which allowed us to compare the regeneration in mice expressing, or not, PrPC. The analyzed histological and biochemical parameters provided proof for the physiologic relevance of PrPC commitment in signalling events involved in muscle regeneration. Indeed, we observed that the absence of PrPC significantly delayed the regenerative process compared to WT muscles. In particular, we found that the lack of PrPC caused attenuation of the signalling pathway triggered by TNF-, which in turn decreased the activation of the p38 kinase pathway, and – consequently – later exit from the cell cycle, and differentiation, of myogenic precursor cells. Importantly, restoring PrPC expression completely rescued the PrP-KO muscle phenotype, highlighting that regulation of signalling pathways by PrPC has clear physiologic importance in an extraneural tissue. The second research line, described in Chapter II, was aimed at setting up a proteomic-based strategy to identify illicit drug treatments in bulls. Classical assays for detecting this kind of illegal practice are not suited to detect compounds either of unknown chemical structure, or present at levels below the quantification threshold of the presently used analytical techniques. The successful application of histological analyses of target organs, which are indirectly modified following these treatments, has suggested that approaches based on the biological effects of the molecules under consideration, rather than the direct detection of their residues, could be potentially valuable in the field. The most relevant advantage of this methodology is that cellular or tissue modifications by drugs remain evident long time after the end of illicit treatments, when chemical residues are no longer, or hardly detectable. On the other hand, this approach is significantly limited by subjective experience and evaluation skill of technicians. Thus new strategies are needed for detecting indirect biomarkers in animal fluids or tissues. These biomarkers can be naturally occurring molecules, such as proteins that are modified in structure, or in concentration, following variations of the normal condition of the animal. To identify possible biologic markers of illicit drug treatments of beef cattle, we adopted a proteomic approach, including 2D differential in gel electrophoresis (DIGE) and mass spectrometry analysis, to compare the protein expression pattern of muscle specimen from experimentally treated bulls and control animals. To this aim, bulls belonging to the treated cohort were subjected to three different pharmacological protocols, including use of growth promoting agents (GPA). Two of these treatments showed a remarkable anabolic effect compared to untreated animals, resulting in an altered skeletal muscle proteome. 2DE protein maps from treatment and control groups were compared using the DeCyder software for 2D-DIGE maps analysis. We then set out to identify, using a MALDI-tandem mass spectrometry (MS/MS) approach, all proteins showing a significant alteration in their expression levels following administration of GPA. Among differentially expressed 169 proteins, 29 were identified, most of which were found to be involved in muscle contraction and energy metabolism. These results corroborate previous findings on the mechanism of action of GPA, and may be useful to design new strategies for the discovery of illicit pharmacological treatments in bulls.
I dati riportati nella presente tesi sono stati ottenuti in due diversi progetti. Pertanto, la tesi è divisa in due distinti capitoli. Il primo capitolo, che si riferisce agli articoli I e II, riporta uno studio volto a chiarire il ruolo fisiologico della proteina prionica cellulare (PrPC) utilizzando un modello in vivo di rigenerazione del muscolo scheletrico. Il secondo capitolo si riferisce all'articolo III, in cui si è cercato di individuare possibili marcatori biologici di trattamento illecito di vitelloni con agenti promotori della crescita (GPA), utilizzando un approccio di elettroforesi bidimensionale (2DE), in combinazione con spettrometria di massa. Focalizzando l'attenzione sul rapporto tra PrPC e la rigenerazione del muscolo scheletrico in un modello in vivo, nella prima linea di ricerca (Capitolo I) abbiamo indagato se e come la proteina influenza la proliferazione e la differenziazione delle cellule precursori del muscolo. PrPC è una glicoproteina ancorata alla membrana esterna delle cellule coinvolta nella comparsa di malattie neurodegenerative rare e mortali, conosciute con il nome di encefalopatie spongiformi trasmissibili (EST) o malattie da prioni. L'evento alla base delle EST è la conversione della PrPC in una isoforma con una modificata conformazione che dà origine al prione, un agente infettivo neurotossico. Anche se ora sono disponibili molte informazioni sulle diverse vie di infezione da parte del prione, sia i meccanismi alla base della neurotossicità, sia il ruolo fisiologico della PrPC rimangono poco chiari. Tuttavia, l'uso di diversi modelli animali e cellulari ha suggerito molteplici funzioni putative per la PrPC, che vanno dalla protezione cellulare contro lo stress ossidativo e stimoli apoptotici, all'adesione, proliferazione e differenziazione cellulare. Il muscolo scheletrico esprime quantità significative di PrPC, e molti studi l'hanno correlato alla fisiopatologia della proteina. Pertanto, al fine di chiarire il ruolo fisiologico della PrPC in questo tessuto, abbiamo impiegato una protocollo di degenerazione/rigenerazione del muscolo tibiale anteriore, che ci ha permesso di confrontare il processo rigenerativo in topi che esprimono, o meno, PrPC. I parametri istologici e biochimici analizzati hanno fornito prove della rilevanza fisiologica della PrPC e del suo coinvolgimento negli eventi di segnalazione coinvolti nella rigenerazione muscolare. Infatti, è stato osservato che l'assenza della PrPC ritarda significativamente il processo di rigenerazione rispetto ai muscoli WT. In particolare, abbiamo trovato che la mancanza di PrPC causa un'attenuazione della via di segnalazione attivata dal TNF-alpha, che porta ad una ridotta attivazione della chinasi p38, e - conseguentemente - ritarda l'uscita dal ciclo cellulare e la differenziazione dei precursori miogenici. È importante sottolineare che il ripristino dell'espressione della PrPC abolisce completamente il fenotipo osservato nei muscoli di topi PrP-KO, sottolineando che la regolazione delle vie di segnalazione da parte PrPC ha una chiara importanza fisiologica anche in tessuti extraneuronali. La seconda linea di ricerca, descritta nel capitolo II, è stata volta a creare una strategia basata su tecniche di proteomica per l'identificazione di trattamenti farmacologici illeciti in vitelloni. L'approccio classico per la rilevazione di questa pratica illegale non è adatto ad individuare composti sia di struttura chimica sconosciuta, sia di farmaci presenti a livelli inferiori alla soglia di quantificazione delle tecniche analitiche attualmente impiegate. Il successo delle analisi istologiche di organi bersaglio, che vengono indirettamente modificati a seguito di questi trattamenti, ha suggerito che gli approcci basati sulla ricerca degli effetti biologici delle molecole in esame, piuttosto che sulla rilevazione diretta dei loro residui, potrebbero essere molto utili. Il vantaggio più rilevante di questa metodologia è che le modificazioni del tessuto indotte da un trattamento farmacologico rimangono evidenti molto tempo dopo la fine dei trattamenti illeciti, quando i residui chimici non sono più, o quasi, rilevabili. D'altra parte, questo approccio è notevolmente limitato dalla capacità di valutazione dei tecnici e l'analisi e influenzata dalla soggettività. Per questo, sono necessarie nuove strategie per il rilevamento dei biomarcatori indiretti presenti nei fluidi animali o nei tessuti. Questi biomarcatori possono essere molecole naturalmente presenti, come ad esempio proteine che abbiano subito modifiche nella struttura, o nella concentrazione, a seguito di variazioni della condizione fisiologica dell'animale. Per identificare tali marcatori biologici di trattamenti farmacologici illeciti nei bovini da carne, abbiamo adottato un approccio proteomico, mediante elettroforesi differenziale su gel in due dimensioni (2D-DIGE) e analisi in spettrometria di massa, al fine di confrontare i pattern di espressione proteica di muscolo scheletrico tra animali trattati farmacologicamente e di controllo. A questo scopo, i vitelloni appartenenti al gruppo di trattamento sono stati sottoposti a tre differenti protocolli farmacologici, mediante l'impiego di agenti promotori della crescita. Due di questi trattamenti hanno portato ad un notevole effetto anabolico rispetto agli animali non trattati, mostrando di conseguenza un'alterazione del proteoma del muscolo scheletrico. Le mappe proteiche dei campioni appartenenti ai gruppi di trattamento e di controllo sono state confrontate utilizzando il software DeCyder per analisi di dati derivanti da 2D-DIGE. Si è poi cercato di identificare, con un approccio di spettrometria di massa (MALDI) in tandem (MS/MS), tutte le proteine che mostrano una significativa alterazione nei loro livelli di espressione in seguito a somministrazione di agenti promotori della crescita. Tra le 169 proteine che cambiano in espressione in seguito al trattamento farmacologico, sono state identificate 29 proteine diverse, la maggior parte delle quali è coinvolta nella contrazione muscolare e nel metabolismo energetico. Questi risultati confermano i precedenti risultati sul meccanismo d'azione degli agenti promotori della crescita, e possono essere utili per sviluppare nuove strategie per l'identificazione di trattamenti farmacologici illeciti nei bovini da carne.

Les gènes et les protéines n’agissent pas de manière isolée dans les cellules, mais interagissent plutôt pour faire leurs fonctions dans les processus biologiques. Ces interactions peuvent être représentées sous forme de grands réseaux dans lesquels les nœuds sont des gènes ou des protéines et les arêtes représentent leurs interactions. Diverses approches basées sur la théorie des graphes ont été développées pour extraire la connaissance fonctionnelle contenue dans ces réseaux. Néanmoins, ces méthodes ont été principalement appliquées à des réseaux individuels, en ignorant la diversité des interactions biologiques. Nous déclarons que ces différents types d’interactions peuvent être représentés sous la forme de réseaux multiplexes, c’est-à-dire des ensembles de réseaux partageant les mêmes nœuds, ce qui permet une description plus précise des systèmes biologiques. Cette thèse est focalisée sur le développement de nouveaux algorithmes étendant aux réseaux multiplexes certaines méthodes populaires de la théorie des graphes en biologie computationnelle, ainsi que sur leur application à l’étude des maladies humaines. Du côté des applications, nous nous concentrons sur les maladies liées au vieillissement prématuré, un groupe de maladies génétiques ressemblant à certains aspects du vieillissement physiologique à un âge précoce. Nous avons appliqué nos algorithmes pour détecter les modules associés à plus de 70 syndromes annotés avec un phénotype lié au vieillissement prématuré. Les résultats ont révélé le paysage des processus moléculaires perturbés dans ces maladies, qui peuvent être mis en parallèle avec les caractéristiques du vieillissement physiologique
Genes and proteins do not act isolated in cells but rather interact to perform their functions in signaling pathways, molecular complexes, or, more generally, biological processes. These interactions can be represented as large networks in which nodes are genes or proteins and edges represent their interactions. Various graph-theory based approaches have been developed to extract the functional knowledge contained in biological networks. Nevertheless, these methods have been mainly applied to individual networks, ignoring the diversity of biological interactions. We state here that these different types of interactions can be represented as multiplex networks, i.e. collections of networks sharing the same nodes, leading to a more accurate description of biological systems. This thesis focuses on the extension from individual to multiplex networks of some of the state-of-the-art guilt-by-association methods in computational biology, and on their application to the study of human diseases. On the application side, we concentrate on premature aging diseases, a group of rare genetic disorders that resemble some aspects of physiological aging at an early age. In this framework, we applied our algorithms to detect the modules associated to more than 70 disorders annotated with at least one premature aging related phenotype. The results revealed the landscape of perturbed molecular processes in premature aging diseases, which can be paralleled with the hallmarks of physiological aging to help identifying common and specific features

CKD is diagnosed when there’s a decreased kidney function shown by a GFR less than 60 ml / min (established for a reference man with 1.73 m² body surface area), or markers of kidney damage, or both, of at least 3 months duration. The severity of complications increases in parallel with the GFR decline. We focused on three comorbidities extremely common in CKD patients: oxidative stress and inflammation; anemia and dysbiosis. We investigated these CKD comorbidities both with in vitro and in vivo approaches. More in detail, regarding in vivo studies, we measured oxidative stress biomarkers in a population of ESRD patients before and after the hemodialysis treatment, comparing the results with a population of healthy subjects; we evaluated oxidative stress biomarkers in the plasma of HD patients before, during and after two type of iron treatments (intravenous and sucrosomial iron). Regarding in vitro experiments, we focused on two uremic toxins, urea and indoxyl sulphate, and we evaluated their effects on a human endothelial cell line (Human Microvascular Endothelial Cells 1, HMEC-1).

Type 2 diabetes is characterized by increased levels of glucose in the blood originating from insulin resistance in insulin sensitive tissues and from reduced pancreatic insulin production. Around 400 million people in the world are diagnosed with type 2 diabetes and the correlation with obesity is strong. In addition to life style induction of obesity and type 2 diabetes, there are indications of genetic and epigenetic influences. This thesis has focused on the characterization of primary human adipocytes, who play a crucial role in the development of type 2 diabetes. Histones are important proteins in chromatin dynamics and may be one of the factors behind epigenetic inheritance. In paper I, we characterized histone variants and posttranslational modifications in human adipocytes. Several of the specific posttranslational histone modifications we identified have been characterized in other cell types, but the majority was not previously known. Moreover, we identified a variant of histone H4 on protein level for the first time. In paper II, we studied specific histone H3 methylations in the adipocytes. We found that overweight is correlated with a reduction of H3K4me2 while type 2 diabetes is associated with an increase of H3K4me3. This shows a genome-wide difference in important chromatin modifications that could help explain the epidemiologically shown association between epigenetics and metabolic health. Caveolae is a plasma membrane structure involved in the initial and important steps of insulin signaling. In paper III we characterized the IQGAP1 interactome in human adipocytes and suggest that IQGAP1 is a link between caveolae and the cytoskeleton. Moreover, the amount of IQGAP1 is drastically lower in adipocytes from type 2 diabetic subjects compared with controls implying a potential role for IQGAP1 in insulin resistance. In conclusion, this thesis provides new insights into the insulin signaling frameworks and the histone variants and modifications of human adipocytes.

Proteomics have come a long way since the mid 1990s, with many life-scientists adopting proteomic approaches to understand gene function. Whilst genomic information itself provides an excellent foundation for biomedical research, the complexity of a human cannot be explained by the genomics approach alone. Proteins are the ultimate effector of cell function, thus, studying the dynamic proteome complement of a cell at any given time is a better reflection of the immediate cell environment and its subsequent disease states. Proteins exert their roles in a living organism by interacting with other proteins, so it is believed that a protein's interactions and its functions are related. The study of protein interactions for biological discovery is now routinely carried out in high-throughput formats. Protein microarrays, in particular protein-protein interaction arrays, are valuable tools in protein functional studies, facilitating the unbiased systematic screening for potential interactors. This hypothesis- generating approach is essential in this post-genomic era to bridge the gap between genomics and proteomics, increasing the efficiency of biological research. This PhD describes the functional characterisation of Raf Kinase Inhibitor Protein (RKIP) using protein microarrays for the first time. Whilst the exact physiological role of RKIP remains unclear, RKIP has been associated with an increasing number of diseases, particularly in cancer. This microarray study provided an insight into RKIP’s role in the cell, with the identification of a broad spectrum of interactors not previously associated with its current known function. Findings from this study supported RKIP’s role as a metastatic suppressor, and its function as a scaffold protein. Whilst the advantages of protein microarrays for functional studies are clear, a number of limitations remain. The limitations associated with the current detection strategies in protein microarrays were addressed, with the application of a novel quantum dot-based detection strategy for the detection of protein interactions on microarrays. The performance of quantum dots was comparable to current Alexa-based detection strategies. Biological validation of the novel interactors are routinely carried out by immunoprecipitations (IP) and western blots, which introduces a bottle neck in the high throughput workflow. This thesis reports the development of an integrated co-IP / SILAC (stable isotope labelling by amino acids in culture) approach to compliment the rate of discovery made possible by microarrays.

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

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.