The Self-Limiting Dynamics of TGF-β Signaling In Silico and In Vitro, with Negative Feedback through PPM1A Upregulation

The TGF-β/Smad signaling system decreases its activity through strong negative regulation. Several molecular mechanisms of negative regulation have been published, but the relative impact of each mechanism on the overall system is unknown. In this work, we used computational and experimental methods to assess multiple negative regulatory effects on Smad signaling in HaCaT cells. Previously reported negative regulatory effects were classified by time-scale: degradation of phosphorylated R-Smad and I-Smad-induced receptor degradation were slow-mode effects, and dephosphorylation of R-Smad was a fast-mode effect. We modeled combinations of these effects, but found no combination capable of explaining the observed dynamics of TGF-β/Smad signaling. We then proposed a negative feedback loop with upregulation of the phosphatase PPM1A. The resulting model was able to explain the dynamics of Smad signaling, under both short and long exposures to TGF-β. Consistent with this model, immuno-blots showed PPM1A levels to be significantly increased within 30 min after TGF-β stimulation. Lastly, our model was able to resolve an apparent contradiction in the published literature, concerning the dynamics of phosphorylated R-Smad degradation. We conclude that the dynamics of Smad negative regulation cannot be explained by the negative regulatory effects that had previously been modeled, and we provide evidence for a new negative feedback loop through PPM1A upregulation. This work shows that tight coupling of computational and experiments approaches can yield improved understanding of complex pathways.Singapore-MIT AllianceMechanobiology Institute, SingaporeInstitute of Bioengineering and Nanotechnology (Singapore)National University of Singapore (NUS Graduate School for Integrative Sciences and Engineering scholar)Singapore-MIT Alliance for Research and Technolog

SYSTEMATIC ANALYSIS OF TGF-ß SIGNALING AND ACTIVATION IN LIVER FIBROSIS

Ph.DDOCTOR OF PHILOSOPH

Computational modeling of TGF- signaling

Tese de mestrado integrado em Engenharia Biomédica e Biofísica (Engenharia Clínica e Instrumentação Médica), Universidade de Lisboa, Faculdade de Ciências, 2020O fator de transformação do crescimento beta ou TGF-β é uma citocina que consta nos 33 membros da superfamília do TGF-β, envolvida em processos fundamentais da célula, nomeadamente na proliferação, migração, diferenciação das células e apoptose celular, assim como na homeostasia dos tecidos. Consequentemente, o mau funcionamento da via de sinalização do TGF-β está associado a diferentes patologias. Tendo em conta este facto e a grande diversidade de respostas biológicas por si originadas, esta cascata de sinalização é um grande alvo de pesquisas, da parte de diversos autores, através da implementação de diversos modelos computacionais. A morfologia da célula não é tida em conta na implementação da maioria dos modelos correntes da via de sinalização canónica do TGF-β. Porém, esta característica celular é normalmente vista como uma medida de grande relevância para ditar o modo como a célula responde a estímulos mecânicos externos. A resposta das células ao ambiente extracelular, caracterizada pela conversão de estímulos mecânicos em sinas bioquímicos, tem a designação de mecanotrasdução. As integrinas, que são uma família de receptores de sinais e de adesão células, convertem estímulos mecânicos em sinais bioquímicos, constituindo um meio de comunição entre as células e o ambiente extracelular. Deste modo, as interacções entre a via de sinalização do TGF-β e as integrinas constituem uma forma de mecanotransdução. Um entendimento mais profundo da dinâmica da via de sinalização do TGF-β através de um modo dependente da morfologia da célula e um entendimento do papel que o crosstalk entre as integrinas e a via de sinalização canónica do TGF-β tem sobre a expressão genética permitem, consequentemente, perceber melhor a conexão entre esta via e o comportamento celular perante estímulos mecânicos. Tal conhecimento detém o potencial para prever interações entre as células e biomateriais e poderá, possivelmente, ser aplicado a diferentes terapias, tais como terapias associadas ao controlo do desenvolvimento de tumores. Devido às razões previamente referidas, neste trabalho é apresentado um novo modelo computacional para a via de sinalização canónica do TGF-β, contendo um módulo de crosstalk com as integrinas. O modelo construído é espacial, sendo implementado através de equações diferenciais parciais. As equações utilizadas são do tipo reação-difusão. Os principais objectivos do presente trabalho são avaliar o impacto que a morfologia da célula detém sobre a sinalização downstream e perceber qual é a influência da crosstalk entre a via de sinalização canónica do TGF-β e as integrinas sobre a expressão genética. Deste modo, o modelo implementado neste trabalho divide-se em dois módulos – Módulo I e Módulo II. O modelo standard, que consiste no modelo da via de sinalização canónica do TGF-β, corresponde ao Módulo I. O Módulo I do modelo foi construído com base em dois artigos: Claus et al., 2013 e Nicklas & Saiz, 2013. As equações que envolvem as diferentes espécies da via de sinalização canónica do TGF-β, assim como as condições de fronteira aplicáveis às membranas citoplasmática e nuclear, foram adaptadas de Claus et al., 2013, e a secção do modelo respeitante ao trafficking de receptores foi adaptado de Nicklas e & Saiz, 2013. As equações de reação-difusão implementadas no Módulo I traduzem os passos gerais da cascata de sinalização do TGF-β referidos por diversos autores: os recetores do TGF-β são ativados pelo ligando TGF-β, recrutando e fosforilando o Smad2/3 junto à membrana citoplasmática. O Smad2/3 fosforilado, pSmad2/3, pode associar-se ao Smad mediador, o Smad4, formando um complexo, o pSmad2/3 + Smad4. Este complexo migra para o núcleo, onde se associa ao DNA, induzindo expressão genética. Quando o pSmad2/3 não se associa ao Smad4, pode migrar diretamente para o núcleo, tal como moléculas de Smad2/3 e moléculas de Smad4 podem deslocar-se até ao núcleo sem estarem associadas a outras espécies. O processo de formação do complexo pSmad2/3 + Smad4 também pode ocorrer no núcleo da célula. O Módulo II do modelo diz respeito à implementação de diferentes interações de crosstalk entre as integrinas e a via de sinalização do TGF-β. As interações de crosstalk incluídas neste módulo são: 1) A upregulation dos recetores do TGF-β despoletada pelas integrinas; 2) O aumento da constante catalítica da reacção de fosforilação do Smad2/3 mediada pelo complexo formado pelo TGF-β e os seus recetores, complexo C, sendo este aumento despoletado pelas integrinas; 3) A estabilização dos recetores do TGF-β despoletada pelas integrinas, resultando na diminuição da taxa de degradação do complexo C; 4) A formação de um complexo composto pelas integrinas e pelo complexo C, complexo IC, que se liga ao Smad2/3, provocando um aumento da constante catalítica da reação de fosforilação do Smad2/3. Foram realizadas diferentes experiências in silico para atingir os objetivos previamente referidos. Relativamente ao Módulo I, de modo a avaliar o impacto da morfologia celular sobre a expressão genética induzida pelo TGF-β, foram feitas simulações para diferentes geometrias da célula, nomeadamente para elipses e rectângulos com diferentes rácios de largura e altura. Foram ainda realizadas simulações para diferentes áreas da célula, num intervalo de valores que se estende desde a área estabelecida no modelo standard até 10 vezes a área do modelo standard. A avaliação do impacto da área da célula a nível downstream é realizada para as geometrias acima referidas. Adicionalmente, os parâmetros do modelo foram submetidos a uma análise de sensibilidade. No que diz respeito ao Módulo II, realizaram-se diferentes simulações para diferentes tipos de crosstalk que se estabelecem entre as integrinas e a via de sinalização do TGF-β para perceber a influência que um aumento dos parâmetros associados a cada tipo de crosstalk possui sobre a concentração do complexo pSmad2/3 + Smad4 no núcleo. Esta incrementação dos valores atribuídos a cada parâmetro é feita de modo independente ou para dois ou três parâmetros em simultâneo. A análise das diferentes simulações realizadas, com o intuito de avaliar apenas o impacto da geometria da célula, indica que a espécie participante desta via de sinalização que mais sofre discrepâncias na sua concentração nuclear por rácio largura/altura, devido a mudanças na geometria da célula, é o Smad2/3. As restantes espécies, nomeadamente o pSmad2/3, o complexo pSmad2/3 + Smad4 e o Smad4, sofrem discrepâncias idênticas por unidade de rácio largura/altura. Constatou-se que existe uma tendência de decréscimo na concentração nuclear do Smad2, pSmad2/3 e complexo pSmad2/3 + Smad4 com o aumento do rácio largura/altura da célula. Por outro lado, constatou-se um aumento da concentração nuclear do Smad4 por unidade de rácio largura/altura. As simulações efectuadas para avaliar o efeito que as dimensões da célula possuem sobre a cascata do TGF-β, a nível downstream, indicam que com o aumento do tamanho da célula, existe diminuição da concentração do complexo pSmad2/3 + Smad4 no núcleo da célula, havendo um declínio na expressão genética. Esta conclusão é aplicável a todas as geometrias da célula consideradas. Concretamente, considerando a passagem da área standard da célula para uma área 10 vezes superior, constata-se uma diminuição de cerca de 50% na concentração nuclear do complexo pSmad2/3 + Smad4 para todos os formatos da célula. Ainda relativamente a resultados do Módulo I, os diversos parâmetros do modelo foram submetidos a uma análise de sensibilidade que revelou que a concentração do complexo pSmad2/3 + Smad4 no núcleo é mais sensível às reacções do modelo do que a processos de difusão. No entanto, um decréscimo de 100% do valor dos parâmetros de difusão revela uma sensibilidade considerável a nível downstream da via de sinalização, o que sugere que ainda assim os parâmetros de difusão devem ser incluídos em modelos que visem estudar a dinâmica da cascata do TGF-β. As simulações realizadas para estudar o efeito da crosstalk entre as integrinas e a via de sinalização do TGF-β sugerem que de todos os tipos de interação implementados no modelo construído, o tipo de interação que causa maior upregulation da concentração nuclear do complexo pSmad2/3 + Smad4 é o decréscimo da taxa de degradação dos receptores devido à estabilização dos receptores do TGF-β por parte das integrinas. Por outro lado, o tipo de interacção de crosstalk que menos contribui para a expressão genética é o aumento da constante catalítica de fosforilação do Smad2/3 mediada pelo complexo IC. Em adição, a expressão genética sofreu mais upregulation, em geral, quando houve uma intensificação simultânea dos efeitos de dois tipos de crosstalk com as integrinas, comparativamente com uma forma de crosstalk ou três tipos de interação em conjunto. Os próximos passos associados ao trabalho apresentado neste documento são o estabelecimento de um conjunto de experiência in vitro para analisar o efeito da morfologia da célula e da mecanotransdu-ção, com o intuito de validar os resultados e conclusões obtidos através do conjunto de experiências in silico. Adicionalmente, outras formas de crosstalk entre as integrinas e a cascata do TGF-β deverão ser incluídas no modelo, tais como a interação da via de sinalização com proteínas reguladas pela actina-G e actina-F do citoesqueleto da célula. Estas proteínas são libertadas quando existe remodelação do cito-esqueleto e um desequilíbrio nos seus níveis resulta na expressão de receptores de ligandos da família TGF-β.The TGF-β is a cytokine involved in fundamental cell processes, such as cell migration, proliferation and apoptosis. A deeper insight into the dynamics of the TGF-β pathway through a cell morphology-dependent manner and into the role that the crosstalk with the integrins has upon downstream signaling of this pathway allows a better understanding of the link between the TGF-β pathway and mechanotransduction cues, therefore holding the potential to uncover interactions between biomaterials and cells and apply this knowledge to different therapies. In this document, a spatial computational model for the canonical TGF-β pathway is presented, including a module for the crosstalk with the integrins. The equations used to model this system are partial differential equations of the reaction-diffusion type. The model was created to evaluate the impact of cell shape and size, as well as crosstalk with the integrins, on genetic expression. The results of the experiments suggest that a bigger width/height ratio of the cell induces less con-centration of the nuclear Smad2/3, nuclear phosphorylated Smad2/3 and nuclear phosphorylated Smad2/3 + Smad4 complex, for elliptical and rectangular cell shapes. Conversely, the concentration of the nuclear Smad4 increases as the width/height ratio of the cell increases, for elliptical and rectangular cell shapes. The nuclear species of the TGF-β pathway that suffers more variation in its nuclear concentration across cell shapes is the Smad2/3. Considering a range of values that go up to 10 times the original area of the cell for different cell shapes, as the area of the cell increases, the concentration of the nuclear phosphorylated Smad2/3 + Smad4 complex decreases by approximately 50%. Sensitivity analysis of the model parameters indicate that genetic expression elicited by the TGF-β when considering diffusive processes differs considerably from results without considering this phenomenon. It is suggested that the way of crosstalk which more heavily increases genetic expression is the decrease of the degradation rate of the receptors due to stabilization from the integrins. Experimental procedures also suggest that the concentration of the nuclear phosphorylated Smad2/3 + Smad4 complex undergoes more upregulation, in general, by the joint behavior of two ways of crosstalk interaction than by the effects of one way of crosstalk independently or the combination of three ways of crosstalk out of the considered interactions

The role of the miR-200 family in the epithelial-to-mesenchymal transition (EMT)

Over the last decade, microRNAs (miRNAs) have earned a lot of attention due to their critical roles in several biological processes and human diseases. However, progress in this field has been limited by the difficulty of discovering miRNA target genes, as each miRNA can potentially bind to hundreds of different mRNAs.(...

La protéine à 7TM GPR50 : un nouveau régulateur de la voie de signalisation TGFβ

During the last years, it became more and more accepted, that orphan G Protein coupled receptors (GPCRs) with a transmembrane spanning heptahelical core (7TM) can have ligand-independent functions. One of those 100 orphan GPCRs is GPR50, a 7TM protein with a long cytosolic domain. Recently, studies revealed ligand-independent functions for GPR50, where it has the capacity to modulate the activity of other proteins upon complex formation. By applying a tandem affinity purification approach we sought to identify further putative interacting partners of GPR50. One of the identified binding partners is the transforming growth factor β (TGFβ) receptor type I (TβRI).The TGFβ-dependent signal transduction pathway of serine/threonine kinases is a pathway with direct signal flow from ligand over the receptor to its substrates, the Smads which translocate into nucleus where they bind DNA and regulate gene expression. An important question concerns the generation of specificity and fine-tuning of TGFβ-dependent signaling. Throughout the years, an important number of proteins which regulate the activity of the TGFβ signal transduction pathway in a positive or negative manner have been identified. Most of them act in a cell-context-dependent manner, allowing the regulation of TGFβ signaling adapted to the particular circumstances.We report here the complex formation of GPR50 and TβRI on the plasma membrane. The consequence of this interaction is the GPR50-mediated induction of a constitutive activation of the TβRI and its downstream signaling in a TGFβ ligand-independent manner. This has been monitored by Smad2/3 phosphorylation, Smad2/3-Smad4 complex formation and their subsequent translocation into the nucleus, where they activate Smad-dependent gene expression. In order to decipher the molecular mechanism that allows this activation, we showed that GPR50 competes with the negative regulator, that prevents leaky TGFβ signaling, the gatekeeping molecule FKBP12, for binding to the TβRI. We identified a motif in FKBP12 involved in the interaction with TβRI with similarities to a motif in GPR50, providing a molecular basis for the replacement of FKBP12 by GPR50 in the TβRI complex. We showed that GPR50 is capable of activating the TβRI even in the absence of the TβRII, which normally is required for activating the TβRI by phosphorylation. This reveals a previously unknown mode of activation of the TβRI in absence of the TGFβ ligand and TβRII. In order to identify the functional consequences of this crosstalk, we studied migration and growth of MDA-MB-231 breast cancer cells stably overexpressing GPR50. In these cells, TGFβ-like pro-migratory and anti-proliferative effects have been observed.Future research will help to identify tissues and biological circumstances, where this crosstalk could take place for putting this novel mode of regulation of TGFβ signaling pathway into a context-dependent-manner. Additionally our work established another ligand-independent task for the orphan 7TM protein GPR50, consolidating its function as binding partner and activity modulator.La protéine GPR50, qui fait partie de la famille des récepteurs de la mélatonine, est classée, avec une centaine d’autres protéines à sept domaines transmembranaires (7TM), dans la catégorie des récepteurs couplés aux protéines G hétérotrimériques (RCPG) orphelins, c’est-à-dire pour lesquels aucun ligand n’a pu être identifié. De plus en plus d’études montrent que les 7TM peuvent avoir des fonctions indépendantes d’un ligand. C’est le cas de GPR50 qui inhibe les fonctions du récepteur de la mélatonine MT1 en interagissant directement avec lui. Nous avons cherché à identifier d’autres partenaires associés à GPR50 en appliquant la technique de purification par affinité en tandem et avons mis en évidence son interaction avec un récepteur du facteur de croissance Transforming Growth Factor ß (TGFβ), le récepteur de type I (TβRI).Nous décrivons ici la formation d’un complexe entre GPR50 et le récepteur TβRI au niveau de la membrane plasmique, avec pour conséquence l’induction d’une activité constitutive du récepteur et des voies de signalisation en aval en l’absence de TGFβ, mais également en l’absence du récepteur TßRII qui est habituellement indispensable pour l’activation de TβRI par phosphorylation. Cette activité constitutive se traduit par la phosphorylation des protéines Smad2 et Smad3, leur intégration dans un complexe avec Smad4, la translocation du complexe dans le noyau et finalement l’activation de la transcription de leurs gènes-cibles. Nous avons décrypté les mécanismes moléculaires de cette activation constitutive en montrant que GPR50 entre en compétition, pour l’interaction avec TβRI, avec le régulateur négatif FKBP12, une protéine inhibitrice de l’activité basale du récepteur en l’absence de ligand. Nous avons identifié dans la queue intracytoplasmique de GPR50 un motif répétitif similaire à la séquence de FKBP12 impliquée dans son interaction avec TβRI , motif qui constitue la base moléculaire de cette compétition.Nous avons étudié les conséquences fonctionnelles de cette activation en surexprimant GPR50 de manière stable dans la lignée cellulaire MDA-MB-231, dérivée d’un cancer de sein. Nous avons observé dans ces cellules des effets pro-migratoires et anti-prolifératifs similaires à ceux causés par l’administration de TGFβ.En conclusion, ce travail décrit un nouveau mode d’activation du récepteur TβRI en l’absence de ligand, mais identifie également une nouvelle fonction indépendante d’un ligand pour le RCPG orphelin GPR50. En perspective de ce travail, nous allons essayer d’identifier des conditions biologiques où cette interaction pourrait prendre place afin de confirmer ces résultats dans un contexte plus physiologique

Identification of distinct markers to differentiate natural and induced T regulatory cells in cancer.

Regulatory T cells (Tregs) are a distinct subset of CD4+ T cells that play a vital role in maintaining immune homeostasis and peripheral tolerance, thereby preventing autoimmunity. Tregs are generally categorised into two main subsets; natural and induced Tregs. In cancer, Tregs are found extremely enriched in the tumour microenvironment and contribute to the inhibition of anti-tumour response and tumour progression. The origin of tumour-infiltrating Tregs (whether it is nTregs or iTregs) is still enigmatic, since there are no distinct biomarkers which can differentiate between the two subsets. Therefore, the aim of this study is to identify cell surface biomarkers that can differentiate phenotypic features of iTregs from nTregs in the context of cancer. With this aim, an in vitro murine model was successfully developed to generate CD4+CD25++Foxp3+ iTregs from purely sorted naïve CD4+CD25-Foxp3- T cells in the presence of TGF-β1. The induction of iTregs was assessed using flow cytometry. Methylation status of Foxp3-TSDR and Foxp3 stability was assessed. Naïve CD4+CD25-Foxp3- T cells and CD4+CD25+Foxp3+ nTregs were purely sorted using cell sorting. Five biologically different subsets of CD4+ cells including naïve CD4+CD25-Foxp3- T cells, activated CD4+CD25-Foxp3- T cells, naïve CD4+CD25+Foxp3+ nTregs, activated CD4+CD25+Foxp3+ nTregs and CD4+CD25++Foxp3+ iTregs were subjected to quantitative proteomic profiling using SWATH-MS. Subcellular fractionation methods were employed to isolate membrane and cytoplasmic proteins from each of the subsets. Quantitative proteomic data were analysed using artificial neural networks. The results revealed that 4 distinct membrane biomarkers (PLP2, ITIH4, HEM6 and MAVS) were differentially upregulated in iTregs compared to other subsets. EPHX1 (HYEP) was identified upregulated only in naïve nTregs and downregulated in iTregs and other subsets. The biomarkers were further tested. Pathway enrichment analysis of iTregs showed a distinct metabolic pathway enrichment in iTregs indicating a mechanistic insight into the iTreg development. Once validated in humans these proteins could be used as a biomarker for iTreg or as a drug target for the selective depletion for better immunotherapeutic outcome in cancer patients

Modulation des réactions alloimmunitaires par les cytokines maîtresses IFN-γ et TGF-β

L’injection de cellules immunologiquement compétentes à un hôte histo-incompatible amène une réaction qui peut se traduire par la maladie du greffon-contre-l’hôte (GVHD). La GVHD demeure une barrière importante à une utilisation plus répandue de la greffe allogénique de cellules hématopoïétiques (AHCT), pourtant un traitement efficace pour traiter de nombreuses maladies. Une meilleure compréhension des mécanismes qui sous-tendent cette pathologie pourrait en faciliter le traitement et la prévention. L’Interféron-gamma (IFN-γ) et le Transforming Growth Factor-béta (TGF-β) sont deux cytokines maîtresses de l’immunité impliquées dans la fonction et l’homéostasie des cellules greffées. Nous démontrons chez la souris que l’IFN-γ limite la reconstitution lympho-hématopoïétique de façon dose-dépendante en mobilisant des mécanismes d’apoptose et en inhibant la prolifération cellulaire. Le TGF-β est quant à lui généralement connu comme un immunosuppresseur qui contrôle l’immunité en utilisant plusieurs voies de signalisation. Le rôle relatif de ces voies en AHCT est inconnu. Nous avons étudié une de ces voies en greffant des cellules provenant de donneurs déficients pour le gène SMAD3 (SMAD3-KO), un médiateur central de la voie canonique du TGF-β, à des souris histo-incompatibles. Bien que l’absence de SMAD3 ne cause aucune maladie chez nos souris donneuses, l’injection de cellules SMAD3-KO amène une GVHD du colon sévère chez le receveur. Cette atteinte est caractérisée par une différenciation Th1 et une infiltration massive de granulocytes témoignant d’un rôle central de SMAD3 dans la physiologie des lymphocytes T CD4 et des cellules myéloïdes. Nous avons focalisé ensuite nos efforts sur le rôle de SMAD3 chez les lymphocytes T CD4 en sachant que SMAD3 était actif chez les lymphocytes T CD4 tolérants. Nous avons découvert que SMAD3 était rapidement inactivé après une activation des cellules T, suggérant que l’inactivation de SMAD3 était fonctionnellement importante pour briser l’état de tolérance. Des études de micro-puces d’ADNc nous ont montré que SMAD3 contrôlait en effet l’expression de nombreux transcrits de gènes connus comme étant reliés à la tolérance et/ou à des processus biologiques dont les rôles dans le maintien de la tolérance sont plausibles.The injection of immuno-competent cells into a histo-incompatible host can result in the development of Graft-versus-Host disease (GVHD). GVHD is the most significant barrier to a more widespread use of allogeneic hematopoietic cell transplantation (AHCT), a potent treatment for several diseases. A better understanding of the pathophysiological underpinnings of GVHD would facilitate the design of rational approaches to treat and prevent this complication of AHCT. Gamma-interferon (IFN-γ) and Transforming Growth Factor-beta (TGF-β) are master cytokines of immunity and have a role in the function and homeostasis of transplanted cells. Using a murine model, we show that IFN-γ curtails lympho-hamatopoitic reconstitution in a dose-dependent fashion by increasing apoptosis and by limiting donor cell proliferation. TGF-β is an immunosuppressive cytokine that controls immune cells through multiple signaling pathways. The relative contribution of these pathways in AHCT is unknown. We specifically studied the role of one of these pathways by transplanting SMAD3 deficient cells (SMAD3-KO) in histo-incompatible hosts. SMAD3 is a key mediator of the so-called canonical TGF-β signaling pathway. Although SMAD3-KO donor mice are healthy, the injection of SMAD3-KO cells leads to severe GVHD in the hosts, characterized by intestinal involvement associated with Th1 skewing and massive granulocyte infiltration. These findings hint at a crucial role for SMAD3 in CD4 T-cell and myeloid cell biology. We then focalized on the role of SMAD3 in CD4 T cells knowing that SMAD3 is active in tolerant, resting CD4 T cells. We found that SMAD3 was rapidly inactivated upon T cell activation, suggesting that SMAD3 inactivation was functionally important to break the state of tolerance. Our cDNA microarray experiments show that indeed, SMAD3 regulates the transcript levels of multiple genes known to be involved in T cell tolerance and in biological processes plausibly related to immune tolerance

Non-coding RNA and transcriptional regulation in CD4 T cell lineages

CD4 T cell lineage choice epitomises the ability of the immune system to become tailored to a specific threat and provides a framework for understanding the mechanisms behind cell specification. The differentiation of T effectors from naïve cells gives rise to pro-inflammatory lineages including T helper 1 (Th1) and Th2 and anti-inflammatory regulatory T cells (Treg). An additional lineage of Treg also exits the thymus in parallel to naïve T cells and together these Treg are required for prevention of autoimmunity. These T cell lineages are distinct in terms of their cytokine production and functional effects but also through their differences in gene expression and its regulation, which are orchestrated by the presence of lineage-specifying transcription factors specific for each lineage. In addition, post-translational modification of histones also provide insights into this transcriptional regulation and more recently the pervasive and tissue-specific transcription of multiple classes of RNA species without protein coding capacity, non-coding RNA (ncRNA), has been found to play a role in cell differentiation and function. In this thesis I identify several ncRNAs with differential expression different T cell lineages. This includes ncRNAs upregulated Treg compared to T responders. The characterisation of these, including their expression in the autoimmune context of systemic lupus erythematosus (SLE), is presented and their possible biological functions are examined. The relevance of histone modifications for influencing Treg identity in SLE is also investigated. An additional class of ncRNAs that originate from gene enhancer regions, eRNA, is also investigated in the context of Th1 versus Th2 lineage choice. This enhancer transcription is increased genome-wide in Th1 cells at enhancers with high density T-bet binding in, termed ‘super-enhancers’. The functional relevance of these eRNAs, including at the super-enhancer upstream of the Th1 signature cytokine gene, IFNG, is also investigated in knockdown experiments

Biomarker discovery for disease progression and metastasis in prostate cancer: a multi-omic approach

Prostate cancer (PCa) is the most common cancer in men and the third most common cause of cancer-related deaths in Europe, which is primarily due to the development of metastasis, which decreases the 5-year survival rate to 30 %. The development of metastasis is the major cause of death in cancer patients and the process highly implicated in the ability of cancer cells to spread is called epithelial to mesenchymal transition (EMT). The aim of this study was to use inducible in vitro EMT models for the discovery of novel disease associated biomarkers through the use of multi-omics datasets. For this, two PCa cell lines were stimulated with transforming growth factor β (TGF-β), resulting in apparent morphological changes indicating a cellular change in the direction of an increased mesenchymal morphology. Induction of EMT was confirmed using quantitative real-time PCR, immunofluorescence staining and western blot analysis. To improve the understanding of underlying changes and for the discovery of novel biomarkers, proteomic and transcriptomic profiles of both models in their induced and non-induced states were generated. Their subsequent integration highlighted 13 potential biomarkers indicative for the process of EMT in PCa and metastasis development. Out of the 13 core markers, four of these were taken forward and further validated using tissue microarrays and the in silico analysis of publicly available datasets. The generated results have supported the association of all 4 markers with EMT and disease progression, however two markers were identified to be of particular interest (DPYL3 and SDPR). These two markers have shown significant differences between primary PCa and castration-resistant prostate cancer (CRPC) and Gleason scoring. Furthermore, both of them were shown to be predictive for disease-recurrence. Overall, the generated results have highlighted the successful application of an integrated omics approach for the discovery of novel disease-associated biomarkers for PCa progression