Saliva of Ixodid ticks contains a whole array of pharmacologically active molecules with vasodilatory, antihemostatic, and immunomodulatory activities. This thesis focuses on two types of salivary proteins, serpins and cystatins, and their role in immunomodulation. These protease inhibitors are known to affect many biological functions. To better understand their role in tick saliva we examined their effect on dendritic cells and their ability to modulate the immune response after pathogen infection. As model pathogens, Borrelia spirochetes and tick-borne encephalitis virus were used.

Proper orientation of the mitotic spindle is essential during animal development for the generation of cell diversity and organogenesis. To understand the molecular mechanisms regulating this process, genetic studies have implicated evolutionarily conserved proteins that function in diverse cell types to align the spindle along an intrinsic cellular polarity axis. This activity is achieved through physical contacts between astral microtubules of the spindle and a distinct domain of force generating proteins on the cell cortex. In this work, I shed light on how these proteins form distinct cortical domains, how their activity is coupled to their subcellular localization, and how they provide cytoskeletal and motor protein connections that are required to generate the forces necessary to position the mitotic spindle. I first discuss the mechanisms by which Mushroom body defect (Mud; NuMA in mammals), provides spindle orientation cues from various subcellular locations. Aside from its known role at the cortex as an adapter for the Dynein motor, I reveal novel isoform-dependent Mud functions at the spindle poles during assembly of the mitotic spindle and astral microtubules, thus implicating Mud in spindle orientation pathways away from the cell cortex. Moreover, through collaborative efforts with former lab members, I describe molecular regulation and assembly of two ‘accessory’ pathways that activate cortical Mud-Dynein, one through the tumor suppressor protein Discs large (Dlg), and another through the signaling protein Dishevelled (Dsh). I demonstrate that the Dlg pathway is spatially regulated by the polarity kinase atypical Protein Kinase C (aPKC) through direct phosphorylation of Dlg. This signal relieves Dlg autoinhibition to promote cortical recruitment of the Dlg-ligand Gukholder (Gukh), a novel microtubule-binding protein that provides an additional connection between astral microtubules and the cortex that is essential for activity of the Dlg pathway. Lastly, I determine that the Dsh accessory pathway provides an alternative cytoskeletal cue by recruiting Diaphanous (Dia), an actin nucleating protein. By demonstrating interchangeability between the two accessory pathways, we conclude that Mud-Dynein is activated by various cytoskeletal cues and that the mode of activation is cell-context dependent. This dissertation includes unpublished and previously published co-authored material. / 10000-01-01

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Orientadores: Eduardo Galembeck, Carmen Verissima Ferreira Halder / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T23:42:26Z (GMT). No. of bitstreams: 1 Milani_Renato_D.pdf: 7268876 bytes, checksum: dfdb9d30aafa024539f758590409a4ca (MD5) Previous issue date: 2014 / Resumo: A leucemia mieloide crônica, caracterizada principalmente pelo gene de fusão BCR-ABL, ainda necessita de novos tratamentos aos pacientes, como ocorre com outros tipos de câncer. A resistência a quimioterápicos é um dos principais obstáculos a serem superados para o sucesso em seu tratamento. Assim, a identificação dos mecanismos moleculares que promovem e mantêm o fenótipo resistente a múltiplas drogas (MDR) é de extrema importância para a evolução dos protocolos terapêuticos. Contudo, ainda pouco se sabe sobre as vias de sinalização envolvidas nestes eventos. O mapeamento das vias de sinalização nas células resistentes pode gerar informações para o entendimento da resistência, bem como apontar alvos para a intervenção farmacológica. Neste trabalho, apresentamos uma análise comparativa do proteoma e fosfoproteoma das células K562 (célula leucêmica não resistente) e Lucena-1 (célula leucêmica resistente a múltiplas drogas). Diversas ferramentas biocomputacionais foram criadas para auxiliar na análise dos dados, com destaque para uma ferramenta, batizada de PhosphoActivity, capaz de enriquecer conjuntos de dados obtidos a partir de fosfoproteomas com os sítios responsáveis pela ativação e pela inibição das proteínas associadas a cada fragmento fosforilado. Estas ferramentas foram empregadas para reduzir o conjunto de 2209 proteínas e 4257 peptídios fosforilados correspondentes a 2053 fosfoproteínas identificadas por espectrometria de massas. Através da combinação de dados experimentais com predições baseadas em aprendizado de máquina, foram selecionadas 145 proteínas e fosfoproteínas para validação. A seleção inclui fatores de transcrição e proteínas estruturais, como ?-catenina, HDAC6 e o filamento intermediário vimentina. As proteínas e fosfoproteínas identificadas e validadas através de métodos computacionais e experimentais apontaram o envolvimento de vias como a reorganização do citoesqueleto, a proliferação celular e o metabolismo de carboidratos na quimiorresistência de Lucena-1. Além disso, a identificação da proteína tirosina fosfatase LMW-PTP como tendo um papel central na resistência em Lucena-1 aponta a natureza complexa e multifatorial deste processo / Abstract: Chronic myeloid leukemia, characterized by the BCR-ABL fusion gene, still poses challenges to patient treatment. One of them is chemoresistance, a major barrier for successful therapy approaches. Still, the molecular mechanisms responsible for promoting and keeping the multiple drug resistance (MDR) phenotype are largely unknown. The mapping of phosphorylation events in resistant cells may improve disease understanding at the cellular level and suggest new targets for pharmacological intervention. Here we present a comparative analysis of the proteome and phosphoproteome in K562, a chronic myeloid leukemia cell line, and Lucena-1, a K562-derived chemoresistant line. We developed several bioinformatics tools to help analyze the data, such as a phosphoproteomics dataset enrichment tool, titled PhosphoActivity, that is able to retrieve documented sites responsible for the activation or inhibition of the proteins related to each phosphorylated fragment. These tools were employed to sift through 2290 proteins and 4257 phosphorylated peptides corresponding to 2053 phosphoproteins previously identified by mass spectrometry. Combining experimental data with support vector machine-based predictions, we selected 145 proteins and phosphoproteins for validation. The selection includes transcription regulators and structural proteins, such as ?-catenin, HDAC6 and the intermediary filament vimentin. Proteins and phosphoproteins identified and validated through computational and epxerimental methods suggest the involvement of pathways such as cytoskeleton rearrangement, cell lproliferation and carbohydrate metabolism in the chemoresistance of Lucena-1. Furthermore, the identification of LMW-PTP, a protein tyrosine phosphatase, as having a pivotal role in the resistance process in Lucena-1, suggests it as a complex and multifactorial process / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Orientador: Carmen Veríssima Ferreira Halder / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T16:08:32Z (GMT). No. of bitstreams: 1 Abrantes_JuliaLauraFernandes_D.pdf: 4562084 bytes, checksum: fba9dbca16cd31c006b311ff23a0b41b (MD5) Previous issue date: 2013 / Resumo: O melanoma é o tipo mais agressivo de câncer de pele. Seu tratamento permanece como um grande desafio, já que em estágio avançado torna-se extremamente refratário aos tratamentos convencionais. miR-34a é um microRNA supressor de tumor com expressão normalmente reduzida em células cancerosas. A fim de investigar o papel de miR-34a como supressor do melanoma, o principal objetivo deste estudo foi identificar alvos moleculares modulados pela expressão ectópica de miR-34a na linhagem celular de melanoma metastático humano SK-mel-103. miR-34a reduziu significativamente a viabilidade das células de melanoma, o que deve estar relacionado, pelo menos em parte, com o aumento na expressão da proteína pró-apoptótica Bax, ativação da caspase-3 e clivagem da PARP-1. Estes dados sugerem que miR-34a foi capaz de induzir apoptose nas células de melanoma. Além disso, houve redução na expressão de CDK4, CDK6, E2F3 e pRb, proteínas relacionadas com a progressão do ciclo celular. Aumento na expressão de p21, um inibidor de CDKs, também foi observado nessas células. Algumas moléculaschave envolvidas com os processos de proliferação celular e apoptose, como proteínas oncogênicas (Axl, AKT, ERK 1/2, ?-catenina e c-myc) e proteínas supressoras de tumor (p53 e PTEN), foram "down- e upreguladas" por miR-34a, respectivamente. Interessantemente, o fluxo autofágico foi aumentado por miR-34a, efeito que não foi correlacionado com alterações adicionais na viabilidade das células de melanoma. O aumento no fluxo autofágico ocorreu, provavelmente, como uma resposta celular ao estresse de retículo e a agregação de proteínas induzidos por miR-34a, fenômenos que também podem ter contribuído para a indução de apoptose nesse contexto. Os dados obtidos neste estudo trouxeram novos aspectos moleculares da ação de miR-34a como supressor tumoral, e permitem apontar este microRNA como um potencial alvo terapêutico contra o melanoma metastático humano / Abstract: Melanoma is the most aggressive form of skin cancer. Its treatment remains a big challenge, since in advanced stage it is extremely refractory to conventional treatments. miR-34a has emerged as an important tumor suppressor, and its expression is normally reduced in cancer cells. To provide more information about the role of miR-34a as a melanoma suppressor, the main goal of this study was to identify key molecular players modulated by ectopic expression of this microRNA in the metastatic melanoma cell line SK-mel-103. miR-34a caused a reduction of melanoma cells viability, what may be related, at least in part, with the increased expression of pro-apoptotic marker, Bax, activation of caspase 3 and PARP-1 cleavage, which indicates that miR-34a triggered apoptosis in melanoma cells. In addition, the expression of CDK4, CDK6, E2F3 and pRb, proteins related to the cell cycle progression, was reduced. An increase in p21 expression, a CDK inhibitor, was also detected in these cells. Some key molecules involved with proliferation and apoptosis processes, such as oncogenic proteins (Axl, AKT, ERK 1/2 kinases, ?- catenin and c-myc) and tumor suppressor proteins (p53 and PTEN), were down- and upregulated by miR-34a, respectively. Interestingly, the autophagic flux was stimulated by miR-34a, but this effect was not correlated with further alterations in cell viability. The increased autophagy occurred probably as a cellular response against the reticulum stress and the protein aggregation induced by miR-34a in melanoma cells, which can also be contributing to the cell death by apoptosis in this context. Our findings brought up novel molecular aspects about the role of miR-34a as melanoma suppressor. The broad action of this microRNA on key molecular players of melanoma aggressiveness was crucial for reprogramming these cells in favor of apoptosis. Altogether, this study pointed out miR-34a as a potential therapeutic agent against advanced melanoma / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Themis est une protéine de signalisation des récepteurs des lymphocytes T (TCR) essentielle pour la sélection positive des cellules T. La fonction moléculaire de Themis a été controversée mais de récentes études suggèrent qu'il est un régulateur positif des voies de signalisation des TCR. Nous avons montré dans une étude préliminaire que Themis interagit avec des déubiquitinases et qu'il est ubiquitiné dans les thymocytes. L'objectif de ma thèse était de caractériser les mécanismes moléculaires qui régulent l'ubiquitination de Themis et de déterminer si ces processus affectent la fonction de Themis durant le développement des lymphocytes T. Nous avons montré que si l'expression des ARNm codant pour Themis diminue dans les stades précoces de la sélection positive, son expression protéique est parallèlement augmentée, suggérant une stabilisation de Themis par des modifications post-traductionnelles durant cette étape. Nous avons montré que la déubiquitinase USP9X déubiquitine Themis pour stabiliser son expression durant la stimulation des TCR. L'ensemble de nos résultats proposent qu'USP9X soit activé durant la stimulation des TCR grâce à son recrutement dans les complexes proximaux des TCR par l'intermédiaire de l'adaptateur Grb2 et Themis, entrainant la stabilisation de l'expression de Themis. Nous pensons que ce mécanisme est important pour maintenir l'expression de Themis durant la sélection positive afin de favoriser l'induction d'un signal des TCR soutenu, requis pour l'efficacité de ce processus. / The protein Themis is a new actor of the T cell receptor (TCR) signaling essential for the positive selection of T cells. The molecular function of Themis has been controversial but recent findings suggest that it acts as positive regulator of TCR signaling. We demonstrated in an initial research that Themis interacts with deubiquitylases and is covalently associated to ubiquitin chains in thymocytes. The aim of my PhD project was to characterize the molecular process that regulates the ubiquitination of Themis and to investigate how these post-translational modifications affect Themis function during T cell development. We demonstrated that Themis mRNA expression is progressively decreased after positive selection whereas Themis protein expression is enhanced at the early stages of positive selection, suggesting that Themis is stabilized by post-translational modifications during positive selection. We demonstrated that USP9X allows the deubiquitination of Themis and its stabilization following TCR engagement. Ours results suggest that USP9X is activated during TCR engagement following its recruitment to proximal signaling complexes through Grb2 and Themis, leading to the deubiquitination and stabilization of Themis expression. We believe that this mechanism is important to sustain Themis expression during positive selection and to promote durable TCR signals required for the efficiency of this process.

Lors d’une réponse immune, les lymphocytes T et les cellules présentatrices d’antigènes (CPA) interagissent entre elles. La synapse immunologique (SI), interface de contact entre les deux cellules, est le site où une cascade de signalisation se met en place. Les lymphocytes T subissent alors un profond réarrangement au niveau de la membrane plasmique et du cytoplasme : les protéines impliquées dans cette signalisation sont alors recrutées à la synapse immunologique. Nous nous intéressons à une nouvelle structure appelée “l’antisynapse” qui se localise au pôle opposé à celui de la synapse immunologique. L’objectif de notre étude est de déterminer la composition de cette nouvelle structure et sa cinétique d’apparition et de disparition. Afin d’étudier cette structure, nous faisons des conjugués in vitro entre des CPA et des lymphocytes T et nous observons la formation de ces contacts sur cellules vivantes ou cellules fixés. L’antisynapse est composée de molécules de signalisations que l’on retrouve classiquement à la synapse immunologique, tels que LAT, CD3, lck ou la PI3K. Grâce à la sonde fluorescente ROZA récemment développée au laboratoire1, nous avons montré que la kinase ZAP-70 est activée à l’antisynapse. Ces observations sont cohérentes avec le fait que nous avons déjà observé la présence de protéines avec des tyrosines phosphorylées à ce pôle. Cette structure précoce et transitoire s’observe fréquemment et apparaît très souvent avant que la synapse ne puisse être détectée. Son apparition est indépendante de la signalisation en aval du TCR et peut être déclenchée par un signal d’adhésion. D’autre part, le cytosquelette de microtubules semble jouer un rôle majeur dans sa disparition. Le rôle de l’antisynapse est toujours en cours d’étude mais nous avons déjà pu montrer qu’elle constituait un point de stockage pour les protéines destinées à former la synapse immunologique au moment de sa formation. Grâce à cette structure nous essayons de mieux comprendre comment le signalosome s’assemble dans la cellule T. Nous voulons également comprendre comment une telle structure peut apparaître aussi rapidement et quelles sont les voies de signalisation mises en jeu dans sa formation. / During the immune response, T lymphocytes and antigen presenting cells (APC) are known to develop strong interactions. The immunological synapse (IS), structure established at the interface between the two cells, is the site where a cascade of signaling events is initiated and may lead to a physiological response. T lymphocyte undergoes a profound rearrangement in the plasma membrane and in the cytoplasm: proteins which are involved in the signaling are recruited to the immunological synapse. We have recently described a new structure that we have called antisynapse (ASI), located at the cell pole opposite to the synapse1. The purpose of this work is to characterize the components of this new structure and their kinetic of appearance and disappearance. To study this structure, we made in vitro contact between APC and T lymphocytes and we observed these conjugates either in live or fixed conditions. Surprisingly, the antisynapse contains most of the signaling molecules classically reported as components of the immunological synapse such as LAT, CD3, Lck or PI3K. By using the fluorescent probe ROZA that we recently developed1, we have shown that ZAP-70 is activated at the antisynapse. This observation is consistent with the fact that we have also observed the presence of tyrosine-phosphorylated proteins at the ASI. Interestingly, we have observed that LFA-1, a protein involved in the adherence, is also found at the ASI. Our results indicate that this transient structure develops frequently and appears rapidly after the contact between the T cell (around one minute) and the APC. Surprisingly, antisynapse formation is independent on TCR signaling but is triggered by adhesion. Furthermore, it disappears using the microtubule network. The role of the antisynapse is currently under investigation but we have shown that it constitutes a stock of proteins ready to go to the forming immune synapse. We currently try to take advantage of this structure to better understand how the T cell signalosome may assemble and to find out if, functionally, the T cell takes advantage of this structure. We also try to understand how this paradoxical structure can appear so rapidly and what are the signaling pathways involved in its establishment.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Anna Zolkiewska / ADAM12 (A Disintegrin and Metalloprotease 12) is a cell surface protease, which is deregulated in many human diseases. High expression of ADAM12 in triple-negative breast cancers (lacking estrogen receptor, progesterone receptor, and HER2 expression) is associated with poor patient prognosis. My dissertation focused on the understanding of the biological functions of ADAM12 in triple-negative breast cancers. I found that ADAM12 is significantly upregulated in the claudin-low molecular subtype of breast cancer. Claudin-low tumors are typically triple-negative and are enriched in cancer stem cells. Here, I demonstrated that the loss of ADAM12 expression not only decreased the number of cancer stem-like cells in vitro but also significantly compromised the tumor-initiating capabilities of breast cancer cells in vivo. This is the first evidence showing that ADAM12 might regulate the cancer stem cell-like phenotype in triple-negative breast cancers. I also discovered a novel mechanism of ADAM12-regulated signaling by transforming growth factor β (TGFβ) through modulation of TGFBR1 mRNA expression in breast cancer cells. Lastly, I characterized the effects of six different somatic mutations in the ADAM12 gene found in human breast cancers on the intracellular trafficking, post-translational processing, and function of ADAM12 protein. Collectively, the findings of this study support the notion that ADAM12 with catalytically active metalloprotease domain is required for the progression of triple-negative breast cancers.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death in the United States. Aldo-keto reductase 1 B10 (AKR1B10) is highly expressed in colon and small intestine of normal humans, but its expression is lost or markedly down-regulated in tissues of patients with ulcerative colitis (UC) and CRC. AKR1B10 is a monomeric cytosolic enzyme with strong enzymatic activity to α, β-unsaturated carbonyl compounds, protecting cells from carbonyl lesions; AKR1B10 also mediates de novo synthesis of long chain fatty acids and membrane lipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2). To study the etiopathogenic role of AKR1B10 in UC and CRC, our lab generated AKR1B8 deficient (AKR1B8 -/-) mice. AKR1 B8 is the orthologue in mice of human AKR1B10,

The major human pathogen Streptococcus pneumoniae is a unique model for the study of eukaryotic-type serine/threonine protein kinases and its cognate phosphatases in bacteria, since it encodes only a single signaling pair composed of the StkP protein kinase and PhpP phosphatase. This signaling pair plays a role in several cellular processes, mainly in cell wall biosynthesis and cell division. StkP and PhpP proteins with a pleiotropic effect appear to regulate a complex signaling cascade by phosphorylation of many substrates. However, only a few have been characterized so far. Using MS analysis, we have identified about 90 phosphopeptides that are potential substrates for the StkP kinase and PhpP phosphatase. This diploma thesis is focused on the characterization of the new substrate Spr0929 and its role in pneumococcal physiology. One of the objectives was to investigate cell morphology of strains carrying deletion of the spr0929 gene in different genetic backgrounds. It turned out that the role of Spr0929 in cell morphology is strain specific. The growth curves of strains with this deletion were compared to that of the wild type in various physiological conditions as well. As Spr0929 contains a nucleoid-associated domain called NdpA, determination of its cell localization was an important...