La leucémie lymphoïde représente environ 30% des cas de cancer chez l’enfant. Elle est souvent causée par des réarrangements chromosomiques impliquant des gènes encodant des facteurs de transcription, qui contrôlent des programmes génétiques complexes. Par exemple, LMO2 (LIM-only 2) est un facteur de transcription oncogénique fréquemment exprimé de façon aberrante dans les leucémies lymphoblastiques aigues des cellules T (T-ALL). Dans l’hématopoïèse normale, LMO2 est essentiel à la génération des cellules souches hématopoïétiques à l’origine de toutes les cellules sanguines. D’ailleurs, certaines cellules leucémiques possèdent des propriétés normalement réservées aux cellules souches hématopoïétiques. Ainsi, l’étude de la fonction de LMO2 dans les cellules souches hématopoïétiques peut être pertinente autant dans le contexte hématopoïétique normal que leucémique. Afin de mettre en évidence de nouvelles fonctions moléculaires pour LMO2, j’ai choisi d’identifier les protéines qui s’y associent. En plus de ses partenaires connus, j’ai identifié plusieurs protéines de transcription/remodelage de la chromatine, en accord avec son rôle transcriptionnel. Plusieurs nouvelles fonctions potentielles ont été révélées, indiquant que cette protéine adaptatrice pourrait faire partie de complexes non transcriptionnels, régulant d’autres processus cellulaires. Les oncogènes comme LMO2 pourraient être des régulateurs à large spectre. Particulièrement, j’ai identifié des interactions entre LMO2 et des protéines de réplication de l’ADN. J’ai montré que LMO2 contrôle la réplication de l’ADN dans les cellules hématopoïétiques, et possiblement durant la leucémogenèse, indépendamment de son rôle transcriptionnel. Ensemble, ces études ont donc permis de révéler de nouvelles fonctions pour LMO2, et pourraient servir de paradigme pour d’autres facteurs de transcription oncogéniques, particulièrement aux autres protéines de la famille LMO, qui sont aussi des oncogènes puissants. / Lymphoid leukemia represents about 30% of childhood cancer cases. It is often caused by chromosomal rearrangements involving genes coding for transcription factors, controlling complex genetic programs. As an example, the oncogenic transcription factor LMO2 (LIM-only 2) is often aberrantly expressed in T cell acute lymphoblastic leukemia (T-ALL). In normal hematopoiesis, LMO2 is essential for the generation of hematopoietic stem cells that give rise to all blood cells. Moreover, some leukemic cells possess properties normally reserved to hematopoietic stem cells. Thus, studying the role of LMO2 in hematopoietic stem cells could be relevant to the contexts of normal hematopoiesis and leukemogenesis. To reveal new molecular functions for LMO2, I chose to identify its associated proteins. In addition to its known protein partners, I identified many proteins involved in transcription/chromatin remodeling, in agreement with its transcriptional role. In addition, several new potential functions have been revealed, indicating that this scaffold protein could be part of non-transcriptional protein complexes, regulating different cell processes. Oncogenes like LMO2 could be master regulators in normal hematopoietic and leukemic cells. Particularly, I identified protein-protein interactions between LMO2 and DNA replication proteins. I demonstrated that LMO2 controls S phase progression in hematopoietic cells, independently of its association in transcriptional complexes. LMO2 overexpression in mice induces T-ALL and affects specifically the cell cycle status of thymocyte progenitors, which are targets of transformation by LMO2. Thus, LMO2 promotes DNA replication in hematopoietic cells, and possibly in leukemogenesis. Together, these studies allowed to reveal new functions for LMO2, and could serve as a paradigm for other oncogenic transcription factors, especially for other LMO proteins which are all potent oncogenes.

Cytochromes P450 are diverse group of heme enzymes found in most species on Earth. In humans they are involved in metabolism of foreign compounds or steroids, bacteria employ cytochromes P450 for utilization of various hydrophobic substrates. General reaction catalyzed by cytochromes P450 is monooxygenation, when one atom of oxygen molecule is introduced into the substrate, while the other is reduced producing water. NADPH:cytochrome P450 oxidoreductase or cytochrome b5 usually serves as an electron donor providing electrons needed for activation of oxygen in eukaryotic organisms, in bacteria small FeS proteins or flavoproteins are these electron donors. It was shown earlier that bacterial electron donor flavodoxin could also interact with human cytochromes P450 in vitro. This thesis employs molecular modeling techniques to support a hypothesis that flavodoxin is responsible for reduction of human (1A2, 2A6, 2A13, 2C9, 2C19, 3A4) and bacterial (101A1 a 176A1) cytochromes P450 heterologously expressed in Escherichia coli. An initial guess of possible mutual orientations of cytochrome P450 and flavodoxin was predicted using information-driven protein-protein docking. The stability of these complexes was examined by directed dissociation method. The most stable orientation for each cytochrome P450 was further...

L’étude des transitions structurales dans les protéines est d’un intérêt crucial car ces transformations sont impliquées dans de nombreux processus biologiques essentiels. De tels phénomènes structuraux peuvent être à l’origine de propriétés remarquables dans les protéines flexibles ou désordonnées, propriétés difficilement accessibles par les techniques structurales usuelles. Le marquage de spin couplé à la spectroscopie de résonance paramagnétique électronique (RPE) est une technique bien adaptée pour l’étude de ces transitions structurales. L’insertion d’un radical nitroxyde sur une cystéine, naturelle ou introduite par mutagenèse dirigée, située à un endroit clé de la protéine permet d’obtenir des informations locales sur les changements structuraux éventuels provoqués par l’ajout d’un partenaire.Cette technique a été appliquée à deux systèmes biologiques comportant un degré de flexibilité différent. La flexibilité de la protéine chaperon NarJ, intervenant dans la biogenèse du complexe Nitrate Réductase de la bactérie Escherichia coli, a été étudiée en présence de son peptide partenaire. Ces études ont permis d’une part de déterminer le site d’interaction et d’autre part, de montrer que l’association des deux partenaires entraîne un verrouillage dans une conformation préférentielle de NarJ. Le deuxième sujet d’étude est la protéine CP12 de Chlamydomonas reinhardtii, intervenant dans la régulation d’un complexe supramoléculaire du cycle de Calvin. La CP12 s’apparente à une protéine intrinsèquement désordonnée, ayant la particularité de posséder des cystéines naturelles et fonctionnelles. Le marquage classique a permis de mettre en évidence un nouveau rôle de son partenaire et de montrer que la CP12 garde un caractère désordonné dans le complexe. Par ailleurs, cette protéine a servi de système d’étude pour développer une nouvelle stratégie de marquage sur Tyrosine et démontrer sa faisabilité. / The study of structural transitions in proteins is of crucial interest because these transformations are involved in many biological processes. Such structural phenomena can be the source of remarkable properties in flexible or disordered proteins, properties hardly accessible by conventional structural techniques. Site-directed spin labeling combined with electron paramagnetic resonance spectroscopy (EPR) is a technique well suited for the study of these structural transitions. The insertion of a nitroxide reagent on a cysteine, natural or introduced by site-directed mutagenesis, located in a key position of a protein provides local information on possible structural changes induced by the addition of a partner. This technique was applied on two biological systems with a different degree of flexibility. The flexibility of NarJ, a chaperon protein involved in the biogenesis of the complex nitrate reductase of Escherichia coli was studied in the presence of its peptide partner. These studies enabled us to determine the interaction site and to show that the association of the two partners induced a locked conformation of NarJ. The second system is the CP12 protein of Chlamydomonas reinhardtii, involved in the regulation of a supramolecular complex of the Calvin cycle. CP12 shares some similarities with the intrinsically disordered protein but having natural and functional cysteines. The conventional labeling allowed us to highlight a new role of its partner and to demonstrate that CP12 remains disordered in the complex. Moreover, this protein was used as a model system to develop a new labeling strategy on tyrosine and to demonstrate its feasibility.

Hydrogen bonds are directional, non-covalent interactions between hydrogen and electronegative atoms. Although generally weak, these interactions are critical to the stability of many biological systems including proteins and DNA. This dissertation explores small molecules in which an intramolecular hydrogen bond is the key determinant of conformation. Chapter 1 introduces the protein Grb2 SH3C, details its role in cancer signalling, and delineates the idea of peptidomimetics—small molecules which are functionalized to mimic the structure of a peptide and disrupt protein-protein interactions. Chapter 2 describes a virtual screen for binders to Grb2 SH3C. From a library of 6.3 million compounds, 34 were tested in vitro and two found to bind to the protein in two orthogonal assays. Chapter 3 describes mimics of the polyproline II helix using a benzoylurea scaffold. A small library of these compounds was synthesized and tested for binding to Grb2 SH3C using SPR, a competition assay, and NMR. Chapter 4 describes attempts to mimic a 310 helix using benzamide-based peptidomimetics. The synthesis and in vitro evaluation of these molecules as ligands of Grb2 SH3C is described. Chapter 5 uses quantum chemical calculations to assess the energies of a series of molecular switches. These calculations benchmark a range of modern density functional theory calculations, and attempt to quantify the accuracy of these methods for a large, flexible system. The role of solvation, entropy, geometry, and torsional angles are assessed in accurately calculating the energies of the critical hydrogen bonds.

Intracellular transport is provided by two major types of molecular motors kinesins and cytoplasmic dynein. Kinesin-1 is a molecular motor that transports molecules and organelles along microtubule tracks anterogradely. Specific protein-protein interactions are required to activate kinesin-1 as the free kinesin exist in an autoinhibited state. The activation of kinesin-1 induces its conformational change, enables microtubule binding and ATP hydrolysis necessary for the directional cargo transport. HDAC6 is a multifunctional protein composed of several domains. It plays an important role in many microtubule dependent processes as HDAC6 is a major tubulin deacetylase. It has been shown that HDAC6 manipulation (inhibition/genetic ablation) affects transport along microtubules but the exact mechanisms are unknown. The effect can be caused either by deacetylation microtubules or direct interaction with molecular motors. This thesis is focused on characterization of interactions between kinesin-1 and HDAC6 that have not been described so far. To this end, we expressed and purified various constructs of kinesin-1 and HDAC6 and tested their interactions by microscale thermophoresis (MST) and hydrogen deuterium exchange (HDX) to determine affinity and interaction sites, respectively. MST data revealed that...

O Transtorno de Déficit de Atenção e Hiperatividade (TDAH) é a doença do neurodesenvolvimento mais comum na infância, afetando cerca de 5,8% de crianças e adolescentes no mundo. Muitos estudos vêm tentando investigar a suscetibilidade genética em TDAH, mas sem muito sucesso. Este estudo teve como objetivo analisar variantes raras e comuns contribuindo para a arquitetura genética do TDAH. Foram gerados os primeiros dados de exoma de TDAH de 30 trios brasileiros em que o filho foi diagnosticado com TDAH esporádico. Foram analisados tanto variações de único nucleotídeo (ou SNVs, single-nucleotide variants) quanto variações de número de cópias (ou CNVs, copy-number variants), tanto nesses trios quanto em outros conjuntos de dados, incluindo uma amostra brasileira de 503 crianças/adolescentes controles, bem como resultados previamente publicados em quatro estudos com variação de número de cópias e uma meta-análise de estudos de associação ao longo do genoma. Tanto os trios quanto os controles fazem parte da Coorte de Escolares de Alto Risco para o desenvolvimento de Psicopatologia e Resiliência na Infância do Instituto Nacional de Psiquiatria do Desenvolvimento (INPD). Os resultados de trios brasileiros mostraram três padrões marcantes: casos com variações herdadas e somente SNVs de novo ou CNVs de novo, e casos somente com variações herdadas. Embora o tamanho amostral seja pequeno, pudemos ver que diferentes comorbidades são mais frequentes em casos somente com variações herdadas. Após explorarmos a composição de variações nos probandos brasileiros, foram selecionados genes recorrentes entre amostras do nosso estudo ou em bancos de dados públicos. Além disso, usando somente genes expressos no cérebro (amostras pós-mortem dos projetos Brain Atlas e Genotype-Tissue Expression), construímos uma rede de interação proteína-proteína \"in silico\" com interações físicas confirmadas por pelo menos duas fontes. Análises topológicas e funcionais dos genes da rede mostraram genes relacionados a sinapse, adesão celular, vias glutamatérgicas e serotonérgicas, o que confirma achados de trabalhos independentes na literatura indicando ainda novos genes e variantes genéticas nessas vias. / Attention-Deficit/Hyperactivity Disorder (ADHD) is the most common neuro-developmental disorder in children, affecting 5.8% of children and adolescents in the world. Many studies have attempted to investigate the genetic susceptibility of ADHD without much success. The present study aimed to analyze rare and common variants contributing to the genetic architecture of ADHD. We generated exome data from 30 Brazilian trios where the children were diagnosed with sporadic ADHD. We analyzed both single-nucleotide variants (SNVs) and copy-number variants (CNVs) in these trios and across multiple datasets, including a Brazilian sample of 503 children/adolescent controls from the High Risk Cohort Study for the Development of Childhood Psychiatric Disorders, and also previously published results of four CNV studies of ADHD involving children/adolescent Caucasian samples. The results from the Brazilian trios showed 3 major patterns: cases with inherited variations and de novo SNVs or de novo CNVs and cases with only inherited variations. Although the sample size is small, we could see that various comorbidities are more frequent in cases with only inherited variants. After exploring the rare variant composition in our 30 cases we selected genes with variations (SNVs or located in CNV regions) in our trio analysis that are recurrent in the families analyzed or in public data sets. Moreover, using only genes expressed in brain (post-mortem samples from Brain Atlas and The Genotype-Tissue Expression project), we constructed an in silico protein-protein interaction (PPI) network, with physical interactions confirmed by at least two sources. Topological and functional analyses of genes in this network uncovered genes related to synapse, cell adhesion, glutamatergic and serotoninergic pathways, both confirming findings of previous studies and capturing new genes and genetic variants in these pathways.

A persistência bacteriana correlacionada à formação de biofilmes bacterianos é, há algum tempo, fonte de grande preocupação médica em virtude de sua ampla associação com a dificuldade de tratamento de infecções crônicas. Por outro lado, as perspectivas de utilização de biofilmes bacterianos em novas aplicações biotecnológicas e até mesmo para fins terapêuticos são promissoras. Há, portanto, grande interesse em compreender os mecanismos que levam as células bacterianas a deixar o estado planctônico, de vida livre, e associarem-se nesses conglomerados celulares altamente complexos. Ao longo das últimas décadas, o segundo mensageiro c-di-GMP – em conjunto com as moléculas que catalisam sua síntese (diguanilato ciclases) e sua degradação (fosfodiesterases) e seus receptores – estabeleceu-se como um elemento central de regulação de uma série de respostas celulares que determinam a formação ou a dispersão de biofilmes. Curiosamente, as proteínas que participam do metabolismo deste segundo mensageiro estão, frequentemente, codificadas múltiplas vezes em um mesmo genoma bacteriano. Em vista dessa observação, estudos mais recentes apontam que, para reger paralelamente uma variedade tão ampla de fenótipos, este sistema opera em modo de alta especificidade de sinalização e que, portanto, o sinal metabolizado por determinados conjuntos de diguanilato ciclases e fosfodiesterases tem alvos celulares específicos. Evidências robustas, porém isoladas até o momento, apontaram que um dos meios pelo qual ocorre a segregação entre sinal produzido e alvo específico é a interação direta entre as proteínas componentes das vias de sinalização. Mais, demonstrou-se que, em algumas vias, a transmissão de sinal ocorre exclusivamente via interação proteica, dispensando a intermediação do sinalizador em si. Para avaliar a validade e relevância global deste mecanismo, propôs-se, neste estudo, a investigação da rede total de interações entre as proteínas tipicamente associadas às vias de sinalização por c-di-GMP em Pseudomonas aeruginosa, utilizando ensaios de duplo-hibrido bacteriano. Para tanto, foram construídas duas bibliotecas de DNA direcionadas e foram feitos testes de interação de forma estratégica para possibilitar o esgotamento e averiguação de todas as possíveis interações entre as proteínas alvo identificadas. O resultado obtido, um mapa inicial, porém abrangente, da rede de interações proteicas em P. aeruginosa, indica uma grande probabilidade de que os mecanismos previamente descritos sejam realmente recorrentes e relevantes para o intermédio da sinalização nesse organismo. Algumas das interações mais robustas encontradas são bastante interessantes e serão, em estudos futuros, mais extensivamente estudadas. / Persister bacteria are correlated to biofilm formation and have been a source of great medical concern due to its close association with the impairment of traditional treatment in combating chronic infections. On the other hand, using bacterial biofilms to create original biotechnological applications or even as a means of therapeutic treatment in medical settings constitutes a promising prospect. There is, therefore, a great interest in understanding the mechanisms that allow bacteria to leave the free-living planktonic lifestyle and associate in these highly complex cellular aggregates. Over the last decades, the second messenger c-di-GMP – and also the molecules involved in its synthesis (diguanylate ciclases) and degradation (phosphodiesterases) along with its receptors – has been established as a key element implicated in regulation of a series of cellular responses that determine biofilm formation or dispersion. Curiously, the proteins that play a part in the metabolism of this second messenger are frequently coded multiple times in single bacterial genomes. Taking this into account, recent studies indicate that, in order to control such a wide range of phenotypes, this system operates via high specificity of signaling – which means that the signal metabolized by a certain set of diguanylate ciclases and phosphodiesterases has specific cellular targets. Robust but yet isolated evidence indicate that a means by which a signal is segregated with its correlated phenotypic response is through direct protein-protein interaction involving the components of these signaling pathways. Even more, there has been strikingly evidence that, in some of these pathways, signal transduction occurs exclusively through protein-protein interaction, entirely dismissing any mediation by the signal molecule. In order to validate and evaluate the global relevance of this type of mechanism, this study proposed the investigation of the entire network of interactions between proteins typically associated with c-di-GMP signaling pathways of Pseudomonas aeruginosa by employing bacterial two-hybrid system assays. To make that possible, two DNA libraries were constructed and interaction essays were performed in a strategic way so that all possibilities of interaction between target proteins were explored. The results obtained from these experiments allowed the construction of a broad map of interactions that, although still primitive, indicates that, chances are, the mechanisms previously described are both recurrent and relevant to signaling regulation in this organism. Some of the interaction partners found are particularly interesting and will be further investigated in future studies.

A proteína Cdc42 (Cell Division Cycle 42) é um membro da família das Rho GTPases, sinalizadores intracelulares conhecidos pelo seu papel na regulação do citoesqueleto. Essa proteína e capaz de ciclar entre um estado ativo (ligado à GTP) e um estado inativo (ligado à GDP) e essa ativação é modulada por diversas proteínas, conhecidas como GEFs (guanine nucleotide-exchange factors), GAPs (GTPase-activating proteins) e GDIs (guanine nucleotide-dissociation inhibitors). Trabalhos recentes têm demonstrado um papel de Cdc42 na apoptose e na senescência, respostas relacionadas e comumente desencadeadas por estresse genotóxico. Neste contexto este trabalho procurou identificar interações de Cdc42 com outras proteínas, que podem ou não estar envolvidas nos mecanismos de resposta ao dano do DNA. Para isso foram utilizadas as linhagens celulares HeLa e MRC-5 submetidas a tratamento com radiação ultravioleta tipo C, a fim de provocar danos no DNA. Foram realizados dois diferentes tratamentos em cada uma das linhagens com diferentes tempos de incubação pós radiação UV, visando a busca de proteínas envolvidas em uma resposta rápida ou tardia ao dano causado. Os lisados celulares desses tratamentos foram submetidos ao pull-down com proteínas recombinantes GST, GST-Cdc42WT (Selvagem) e GST-Cdc42V12 (Mutação constitutivamente ativa). As proteínas purificadas foram digeridas e submetidas à análise por espectrometria de massa e os dados obtidos foram utilizados para a construção de redes de interação proteica. Dentre as proteínas identificadas as que despertaram maior atenção foram: Proibitina-2 (PHB2) encontrada nas amostras incubadas por 48 horas pós irradiação e Cullina-4A (CUL4A) e P53, encontradas em amostra incubada por 5 minutos pós radiação. Essas proteínas possuem papéis em apoptose e reparo de DNA e foram observadas em posições muito próximas de Cdc42 nas redes de interação, fazendo delas interessantes alvos para futuras validações de interação proteica por análises experimentais distintas / The Cdc42 protein (Cell Division Cycle 42) is a member of the Rho family of GTPases, intracellular signalling molecules well known for their role in the cytoskeleton regulation. This protein cycles between an active state (GTP-bound) and an inactive state (GDP-bound) and this regulation is modulated by proteins known as GEFs, GAPs and GDIs. Recent studies demonstrated roles for Cdc42 in apoptosis and senescence, cellular responses commonly triggered by genotoxic stress. This work sought to identify Cdc42 interactions with other proteins that possibly involved in response to DNA damage mechanisms. To reach this aims we used HeLa and MRC-5 cell lines submitted to treatments with ultraviolet C radiation to induce DNA damage. Two experimental conditions were used in each cell line with different times and doses post UV irradiation in order to search for proteins involved in either rapid or delayed response to the installed DNA damage. Cell lysates obtained from these treatments were subjected to pull-down experiments using recombinant proteins GST, GST-Cdc42-WT (Wild type) and GST-Cdc42-V12 (constitutively active mutant). Purified proteins were digested by trypsin, analyzed by mass spectrometry and th obtained data were used for the construction of protein-protein interaction (PPI) networks. Among the identified proteins those that seem more relevant to the aims of this project were: Prohibitin-2 (PHB2), found in samples incubated 48 hours post irradiation; Cullin-4A (CUL4A) and P53, found in samples incubated 5 minutes after radiation. These proteins have roles in apoptosis and DNA repair and were observed in close proximity to Cdc42 in PPI networks, making them interesting targets for future validation by different experimental approaches

Neurodegenerative Erkrankungen (NDs) wie Alzheimer (AD), Parkinson (PD), und amyotrophe lateral Sklerose (ALS) sind Hirnerkrankungen, die durch unlösliche Proteinaggregate in Neuronen oder im Extrazellularraum charakterisiert sind. In dieser Arbeit habe ich für verschiede bekannte und vorhergesagte neurodegenerative Krankheitsproteine (NDPs) Proteininteraktionsnetzwerke erstellt, um mögliche gemeinsame Krankheitsmechanismen genauer zu studieren. Mit Hilfe eines automatisierten Hefe-Zwei-Hybrid-Systems (Y2H) konnte ich 18.663 Protein-Protein-Interaktionen (PPIs) für 449 wildtyp und 22 mutierte Proteine identifizieren. Eine genaue funktionelle Analyse der Interaktionspartner von korrespondierenden wildtyp und mutierten Proteinen ergab deutliche Unterschiede zum einen im Fall von allen untersuchten Proteinen und insbesondere im Fall vom ALS Krankheitsprotein TDP-43. Die identifizierten PPIs wurden außerdem verwendet um krankheitsspezifische Netzwerke zu erstellen und um Proteine zu identifizieren, die mit mehreren NDPs verbunden sind. Ich habe auf diese Weise vier Proteine (APP, IQSEC1, ZNF179 und ZMAT2) gefunden, die mit bekannten NDPs with Huntingtin, TDP-43, Parkin und Ataxin-1 interagieren und so fünf verschiedene NDs miteinander verbinden. Die Reduktion der mRNA Expression von IQSEC1, ZNF179 oder ZMAT2 mit Hilfe von siRNA führte zu einer Verstärkung von pathogenen Mechanismen wie der Aggregation von mutiertem Huntingtin und TDP-43 sowie der Hyperphosphorylierung des Proteins Tau. Außerdem habe ich 22 Proteine entdeckt, die die Aggregation von TDP-43 deutlich verändern und außerdem Mitglieder in sieben vorhergesagten Proteinkomplexen sind. Die Proteinkomplexe habe ich durch Kombination von Interaktionsdaten und Daten eines siRNA Screenings vorhergesagt. Zusätzlich habe ich herausgefunden, dass die Proteine eines vorhergesagten Komplexes, nämlich HDAC1, pRB, HP1, BRG1 und c-MYC, die Aggregation von TDP-43 durch Veränderung von dessen Genexpression beeinflussen. / Neurodegenerative diseases (NDs) such as Alzheimer’s disease (AD), Parkinson’s disease (PD) or amyotrophic lateral sclerosis (ALS) are progressive brain disorders characterized by the accumulation of insoluble protein aggregates in neuronal cells or the extracellular space of patient brains. To elucidate potential common pathological mechanisms in different NDs, I created comprehensive interaction networks for various known and predicted neurodegenerative disease proteins (NDPs). I identified 18,663 protein-protein interactions (PPIs) for 449 bioinformatically selected wild-type target proteins and 22 mutant variants of 11 known NDPs by using an automated yeast two-hybrid (Y2H) system. The functional analysis of the interaction partners of corresponding wild-type and mutant NDPs revealed strong differences in the case of all 11 NDPs and especially for the ALS protein TDP-43. The identified PPIs were used to generate networks for individual NDs such as AD or PD and to identify proteins that are connected to multiple NDPs. For example, I found that five neurodegenerative diseases are connected by four proteins (APP, ZMAT2, ZNF179 and IQSEC1) that link known NDPs such as huntingtin, TDP-43, parkin, ataxin-1 and SOD1. Analysis of publicly available gene expression data suggested that the mRNA expression of the four proteins is abnormally altered in brains of ND patients. Moreover, the knock-down of IQSEC1, ZNF179 or ZMAT2 aggravates pathogenic disease mechanisms such as aggregation of mutant huntingtin or TDP-43 as well as hyperphosphorylation of tau. Additionally, I identified 22 modifiers of TDP-43 aggregation, which are members in 7 protein complexes. These complexes were predicted based on combined data from PPI as well as siRNA screenings. Finally, I found that the proteins HDAC1, pRB, HP1, BRG1 and c-MYC, which form one of the predicted complexes, influence TDP-43 aggregation by altering its mRNA expression.

Proteininteraktionen sind entscheidend für zelluläre Funktion. Interaktionen reflektieren direkte funktionale Beziehungen zwischen Proteinen. Veränderungen in spezifischen Interaktionsmustern tragen zur Entstehung von Krankheiten bei. In dieser Arbeit werden funktionale und pathologische Aspekte von Proteininteraktionen analysiert, um Funktionen für bisher nicht charakterisierte Proteine vorherzusagen und Proteine mit Krankheitsphänotypen zu assoziieren. Verschiedene Methoden wurden in den letzten Jahren entwickelt, die die funktionalen Eigenschaften von Proteinen untersuchen. Dennoch bleibt ein wesentlicher Teil der Proteine, insbesondere menschliche, uncharakterisiert. Wir haben eine Methode zur Vorhersage von Proteinfunktionen entwickelt, die auf Proteininteraktionsnetzwerken verschiedener Spezies beruht. Dieser Ansatz analysiert funktionale Module, die über evolutionär konservierte Prozesse definiert werden. In diesen Modulen werden Proteinfunktionen gemeinsam über Orthologiebeziehungen und Interaktionspartner vorhergesagt. Die Integration verschiedener funktionaler Ähnlichkeiten ermöglicht die Vorhersage neuer Proteinfunktionen mit hoher Genauigkeit und Abdeckung. Die Aufklärung von Krankheitsmechanismen ist wichtig, um ihre Entstehung zu verstehen und diagnostische und therapeutische Ansätze zu entwickeln. Wir stellen einen Ansatz für die Identifizierung krankheitsrelevanter Genprodukte vor, der auf der Kombination von Proteininteraktionen, Proteinfunktionen und Netzwerkzentralitätsanalyse basiert. Gegeben einer Krankheit, werden krankheitsspezifische Netzwerke durch die Integration von direkt und indirekt interagierender Genprodukte und funktionalen Informationen generiert. Proteine in diesen Netzwerken werden anhand ihrer Zentralität sortiert. Das Einbeziehen indirekter Interaktionen verbessert die Identifizierung von Krankheitsgenen deutlich. Die Verwendung von vorhergesagten Proteinfunktionen verbessert das Ranking von krankheitsrelevanten Proteinen. / Protein interactions are essential to many aspects of cellular function. On the one hand, they reflect direct functional relationships. On the other hand, alterations in protein interactions perturb natural cellular processes and contribute to diseases. In this thesis we analyze both the functional and the pathological aspect of protein interactions to infer novel protein function for uncharacterized proteins and to associate yet uncharacterized proteins with disease phenotypes, respectively. Different experimental and computational approaches have been developed in the past to investigate the basic characteristics of proteins systematically. Yet, a substantial fraction of proteins remains uncharacterized, particularly in human. We present a novel approach to predict protein function from protein interaction networks of multiple species. The key to our method is to study proteins within modules defined by evolutionary conserved processes, combining comparative cross-species genomics with functional linkage in interaction networks. We show that integrating different evidence of functional similarity allows to infer novel functions with high precision and a very good coverage. Elucidating the pathological mechanisms is important for understanding the onset of diseases and for developing diagnostic and therapeutic approaches. We introduce a network-based framework for identifying disease-related gene products by combining protein interaction data and protein function with network centrality analysis. Given a disease, we compile a disease-specific network by integrating directly and indirectly linked gene products using protein interaction and functional information. Proteins in this network are ranked based on their network centrality. We demonstrate that using indirect interactions significantly improves disease gene identification. Predicted functions, in turn, enhance the ranking of disease-relevant proteins.