Preterminfants frequently suffer frompulmonary complications due to a physiological and structural lung immaturity resulting in significant morbidity and mortality. Novel in vitro and in vivo models are required to study the underlying mechanisms of late lung maturation and to facilitate the development of new therapeutic strategies. Organoids recapitulate essential aspects of structural organization and possibly organ function, and can be used to model developmental and disease processes. We aimed at generating fetal lung organoids (LOs) and to functionally characterize this in vitro model in comparison to primary lung epithelial cells and lung explants ex vivo. LOs were generated with alveolar and endothelial cells from fetal rat lung tissue, using a Matrigel-gradient and air-liquid-interface culture conditions. Immunocytochemical analysis showed that the LOs consisted of polarized epithelial cell adhesion molecule (EpCAM)-positive cells with the apical membrane compartment facing the organoid lumen. Expression of the alveolar type 2 cell marker, RT2-70, and the Club cell marker, CC-10, were observed. Na+ transporter and surfactant protein mRNA expression were detected in the LOs. First time patch clamp analyses demonstrated the presence of several ion channels with specific electrophysiological properties, comparable to vital lung slices. Furthermore, the responsiveness of LOs to glucocorticoids was demonstrated. Finally, maturation of LOs induced by mesenchymal stem cells confirmed the convenience of the model to test and establish novel therapeutic strategies. The results showed that fetal LOs replicate key biological lung functions essential for lung maturation and therefore constitute a suitable in vitro model system to study lung development and related diseases.

Environmental factors such as diet contribute to the pathogenesis of inflammatory bowel disease (IBD). Epidemiological evidence suggests a robust linkage between IBD and the Western diet, which is often characterized by a high intake of food additives. These additives, including synthetic colourants, are widely used, leading to significant human exposure. Allura Red AC (AR) is one of the most popular synthetic colourants, yet little is known about its impact on human health and the role of AR in the pathogenesis of colitis remains elusive. Serotonin (5-hydroxytryptamine; 5-HT), which regulates various gut physiological processes, has been shown to modulate the gut microbiota and enhance susceptibility to colitis. In this thesis, it was discovered that chronic exposure to AR, at a dose found in commonly consumed dietary products, exacerbated dextran sulfate sodium (DSS)-induced colitis and triggered early onset of disease in the CD4+CD45RBhigh T cell-induced colitis model. AR also induced low grade colonic inflammation in naïve C57BL/6 mice. Exposure to AR was associated with increased colonic 5-HT levels and impaired intestinal barrier function via activation of the myosin light chain kinase (MLCK) pathway. However, AR did not promote colitis in mice lacking tryptophan hydroxylase 1 (Tph1), the rate-limiting enzyme responsible for colonic 5-HT synthesis. Further, AR increased colonic 5-HT levels in germ-free (GF) mice and perturbed the gut microbiota composition in specific pathogen-free (SPF) mice. Transfer of this altered microbiota from the dye-exposed SPF mice to GF mice conferred enhanced susceptibility to DSS-induced colitis. Mechanistically, AR induced reactive oxygen species (ROS) generation and promoted 5-HT secretion via the NF-κB pathway in BON cells. Data in this thesis indicate that the widely used synthetic colourant, AR, promotes colitis via colonic 5-HT in microbiota-dependent and -independent pathways. Collectively, these findings provide important information on enhancing public awareness of its detrimental effects on human health. / Thesis / Candidate in Philosophy / Epidemiological and experimental studies suggest a potential link between inflammatory bowel disease (IBD) and diet. The Western diet, often characterized by a high intake of processed foods, is associated with the growing incidence of IBD. Allura Red AC (AR) is a popular artificial food dye found in highly common processed foods, yet little is known about its impact on human health and disease. Serotonin, a key molecule in the gut, has been implicated in large bowel inflammation. Herein, the potential role of AR in the development of colitis was examined. Across multiple models, AR exposure heightened vulnerability to colitis in mice, an effect attenuated by reduced serotonin production in the gut. The effect of AR in enhancing colitis vulnerability occurred via gut microbiota-dependent and -independent pathways. These studies have identified how AR promotes colitis, findings that may advance public health awareness and impact the health of patients with IBD.

Pancreatic cancer is the seventh leading cause of cancer related mortalities worldwide and incidences are increasing. The prognosis remains poor as the 5-year survival rate is below 10%. This can be partly explained by the silent progression of disease as most patients present with advanced disease at time of diagnosis. In turn, surgical resection, the only potential curative measure, is not possible in nearly 80% of cases due to the occurrence of distant metastasis and/or infiltration of major vessels in close proximity to the pancreas. In patients with localized but advanced disease, resectability can be achieved in some cases by initiation of a neoCTx. However, as neoCTx is commonly conducted by administering multi-drug treatments, severe side effects occur frequently, which require an adaption of drug doses administered. In this study, we revealed the negative impact of these drug dose changes during neoCTx on the patients´ treatment outcome. R0 resections were significantly less frequently observed, and the N-status significantly impacted by the tumor regression grade, which in turn trended towards minor response in the cohort of patients that did not sustain full dose course prior surgery. In turn, treatment of LA PDAC could be improved by increasing the proportion of patients that undergo neoCTx without any changes of the treatment schedule. Patient-derived PDAC organoid could serve as an avatar of patients´ tumor disease on which optimal treatment protocols could be tested. In this study, a large living PDAC PDO biobank successfully has been established from surgical resection specimens as well as EUS guided FNA samples. Subsequently, a new protocol for molecular subtyping of PDAC on organoids was established by assessing the expression level of KRT81 and CFTR, as a replacement for HNF1a, using IF staining. Strikingly, we observed identical PDAC subtypes in PDOs and their respective tissue of origin in nearly all cases. This observation allowed the assumption that PDOs could indeed be used as patient-individual avatars to identify treatment sensitivities and resistances, as they share fundamental molecular properties with the tissue they have been initiated from. Extensive pharmacotyping was performed for many PDO lines by testing the response behavior to the multi-drug regimens FOLFIRINOX and Gem/Pac, as well as their respective single drug compounds. As a result, we observed diverse response patterns for each PDAC PDO line. A poor response to FOLFIRINOX did not necessarily imply a resistance to Gem/Pac. PDO pharmacotyping could guide treatment decision making in the foreseeable future. Moreover, when the non-efficient drug was removed, no changes of overall efficacy of treatment in PDOs was observed, implying that additional therapy improvements could be possible using this ex vivo model. This observation was true for both commonly used chemotherapy protocols, FOLFIRINOX and Gem/Pac and could result in less drug mediated side effects under (neo)adjuvant CTx without impacting treatment efficacy. Yet, the main goal of this study was to assess if PDAC PDOs can be used to predict the neoCTx outcome of PDAC patients. All methods required to address this issue in a prospective clinical trial have been established as a protocol for PDAC PDOs initiation from minimal starting material has been established and subsequently improved resulting in take rates of up to 80%. To support this study, we successfully secured patient enrollment from a second clinical center, which will increase the number of recruited patients in the future. Unfortunately, at the time of writing this thesis, patient numbers were not sufficient to answer the question of the predictive value of PDAC PDOs in regard to the current standard of care.

During the past few decades, the landscape of available therapeutic interventions for cancer treatment has widely expanded, boosted mainly by immunotherapy progress and the precision oncology paradigm. The extensive use of pre-clinical models in cancer research has led to the discovery of new effective treatment options for patients. Despite the notable advancements, some cancer types have found minor benefits from the use of precision-oncology interventions. Characterized by a heterogeneous molecular landscape, bladder cancer is one of the most frequent cancer types in which standard-of- care treatments involve surgical operations accompanied by broad-spectrum chemotherapy. My research stems from the need for precision oncology interventions in bladder cancer and specifically focuses on the development of computational frameworks to guide the discovery of new therapeutic opportunities. This work first introduces the exploration of possible therapeutic interventions in 9p21.3 depleted bladder tumors through the analysis of an in-house large High-Content Drug Screening that tested 2,349 compounds. By combining cell count changes and morphological quantitative features extracted from fluorescence images, we nominate cytarabine as a putative candidate eliciting specific cytotoxic effects in an engineered 9p21.3 depleted bladder cancer model compared to an isogenic wild-type clone. Focusing on the development of computational methodologies to nominate robust context-specific vulnerabilities, I further describe PRODE (PROtein interactions informed Differential Essentiality), an analytical workflow that integrates protein-protein interaction data and Loss of Function screening data. I extensively tested PRODE against the most commonly used and alternative methodologies and demonstrated its superior performance when classifying reference essential and context-essential genes collected from experimental and literature sources. Furthermore, we applied PRODE to a real case scenario, seeking essential genes selectively in the context of HER2+ Breast Cancer tumors. Finally, I report the computational analyses performed on Patient-Derived Organoids (PDOs) established from a bladder cancer cohort. PDOs are demonstrated as informative models when assessing the therapeutic sensitivity of patients to drugs. Overall, this research highlights novel precision-oncology applications by ad-hoc computational analyses that address key open technical and biological challenges in the field of bladder cancer and beyond.

Tuberous sclerosis complex (TSC) is a multisystem tumor-forming disorder caused by biallelic inactivation of TSC1 or TSC2. The primary cause of mortality arises from mesenchymal manifestations in the lung and kidney: pulmonary lymphangioleiomyomatosis (LAM) and renal angiomyolipomas (RAMLs). Despite a well-described monogenic etiology, there remains an incomplete understanding of disease pathogenesis. Consequentially, tractable models which fully recapitulate disease characteristics are lacking. Here, I develop and study novel tissue-engineered models of TSC lung and kidney disease. In my first chapter, I demonstrate that lung-mimetic hydrogel culture of pluripotent stem cell-derived diseased cells more faithfully recapitulates human LAM biology compared to conventional culture on two-dimensional plastic. Leveraging this culture system, I conducted a three-dimensional drug screen using a custom 800-compound library, tracking cytotoxicity and invasion modulation phenotypes at the single cell level. I identified histone deacetylase (HDAC) inhibitors as a group of anti-invasive agents that are also selectively cytotoxic towards TSC2-/- cells. HDAC inhibitor therapeutic effects remained consistent in vivo upon xenotransplantation of LAM cellular models into zebrafish. In my second chapter, I develop a genetically-engineered human renal organoid model which recapitulates pleiotropic features of RAMLs in vitro and upon orthotopic xenotransplantation. I find that loss of TSC1/2 affects multiple developmental processes in the renal epithelial, stromal, and glial compartments. First, loss of TSC1/2 leads to an expanded stroma by favouring stromal cell fate acquisition and alters terminal stromal cell identity. Second, epithelial cells in the TSC1/2-/- organoids exhibit a rapamycin-insensitive epithelial-to-mesenchymal transition. Third, a melanocytic population forms exclusively in TSC1/2-/- organoids, branching from MITF+ Schwann cell precursors of a bona fide neural crest-to-Schwann cell differentiation trajectory. Through these two thesis chapters, I realize the power of tissue-engineered models for the study of TSC. This work offers novel insights into the pathogenesis of RAMLs and identifies a new class of therapeutics suitable for trialing in patients with pulmonary LAM.

Les maladies inflammatoires de l’intestin (MII), incluant la maladie de Crohn et la colite ulcéreuse, sont des maladies chroniques qui résultent d’un dérèglement de la réponse immunitaire aux microorganismes de la lumière intestinale. Des études de séquençages réalisées par le laboratoire du Dr. Rioux, avec ses collègues du International IBD Genetics Consortium ont identifié quatre variants rares, indépendants et causals pour les MII, dans le gène IFIH1. La protéine d’IFIH1 (MDA5) interagit avec certains virus à ARN, afin de déclencher une réponse antivirale de l’immunité innée. Nous avions émis l’hypothèse que ces variants dans IFIH1 diminuaient la réponse antivirale de l’épithélium intestinal, suite à une infection. Nous avons d’abord travaillé avec des lignées cellulaires lymphoblastoïdes (LCLs) obtenues à partir d’individus atteints de MII et qui sont homozygotes ou hétérozygotes composés pour ces variants, ainsi qu’à partir d’individus contrôles (IFIH1 wt). Ces LCLs ont été reprogrammées en cellules souches pluripotentes induites humaines, avant d’être différenciées en cultures épithéliales intestinales. Nos résultats ont d’abord confirmé l’impact des variants sur la structure génique et protéique (IFIH1/MDA5), dans ces modèles cellulaires. Puis, la réponse antivirale a été induite, grâce à la stimulation avec des agents (moléculaire et viral) connus pour stimulés la protéine MDA5. Nous avons démontré que ces variants dans IFIH1 induisaient effectivement une moins grande réponse antivirale, caractérisée par une plus faible expression d’IFNs, comparativement aux contrôles. Finalement, la modulation des IFNs constituerait une avenue potentiellement intéressante pour le traitement des patients atteints des MII et porteurs des variants causals. / Inflammatory Bowel Disease (IBD), including Cronh’s disease and ulcerative colitis, are chronic inflammatory diseases of the gastro-intestinal tract. IBD is associated with a disturbance of the immune response to the microorganisms of the intestinal lumen. Sequencing studies conducted by the laboratory of Dr. John Rioux, in collaboration with his colleagues of the International IBD Genetics Consortium, identified four rare and independent variants in IFIH1, associated to IBD. The protein of IFIH1 (MDA5) interacts with certain RNA viruses to trigger the innate mechanism of antiviral defense. Our hypothesis was that these IFIH1 variants decreased the intestinal epithelial antiviral response, following an infection. We first worked with lymphoblastoid cell lines (LCLs) obtained from IBD patients who are homozygotes or compound heterozygotes for the different variants, as well as from control individuals (IFIH1 wt). These LCLs were reprogrammed into human induced Pluripotent Stem Cells (hiPSCs), before being differentiated into intestinal epithelial cultures. Our results first confirmed the impact the variants on the genetic and protein structure for these models. Then, the antiviral response was triggered by the stimulation of LCLs and intestinal epithelial cells, with agents (molecular and viral) known to stimulate MDA5. We have demonstrated that these IFIH1 variants did indeed induce a lower antiviral response, characterized by lower IFNs expression, compared to control cell lines. Finally, modulation of IFNs could be an interesting avenue for the treatment of IBD patients with the causal variants.

Sertoli cells represent the only somatic cell type within the seminiferous tubules with direct contact to germ cells. Sertoli cells significantly contribute to the development of the testicular niche in a male embryo. Their role during postnatal life is in the regulation and nutrition of germ cells and the formation of the blood-testis barrier to protect these cells. In our laboratory, we have been successful in establishing a cell line of X. tropicalis immature Sertoli cells (XtiSCs) derived from juvenile testes of X. tropicalis. The objective of this thesis was to induce the differentiation process of XtiSCs into mature Sertoli cells. In vitro experiments using several factors or primary culture from adult male X. tropicalis did not show any mature markers in differentiated XtiSCs. Another experiment using cell culture derived from pubertal mice was partially successful in the induction of the differentiation process. These results indicate that XtiSCs do have some differentiation potential into mature Sertoli cells. Part of this work was to test the ability of testicular cells isolated from juvenile males of X. tropicalis to form de novo organoids. In vitro experiments were successful when these cells were cultured in a three-layer matrigel.

Mitochondria are dynamic organelles that function in a complex interconnected network within the cell. Neurons are sensitive and highly energy demanding cells in the brain which require a functioning mitochondrial network that is able to provide ATP and modulate calcium. Mitochondrial networks have yet to be explored which gives rise to the need for specific and efficient molecular tools. In this project, I designed and produced adeno-associated virus vectors carrying a fluorescent reporter gene for imaging mitochondrial networks under human synapsin 1 promoter to target neurons specifically. The design of each vector was conducted with careful consideration of the different components in the plasmid design that are important for optimal expression, which resulted in two constructs; one self-complementary adeno-associated virus vector that marks the mitochondria and one single-stranded that marks mitochondria and the membrane of neurons.  The modularity of viral vectors allows the usage of different serotypes which adapt the vector to the cell type and the model. For this project I chose the serotypes 1 for neurons in vitro and PHP.eB which suits in vivo models since it has better permeability to the blood brain barrier. The production was conducted in human embryonic kidney cells using the triple-plasmid transfection method, followed by extraction and purification. The existence of viral particles was verified through transmission electron microscopy and the DNA titer of the vector through quantitative polymerase chain reaction. The produced adeno-associated virus vectors were delivered into young brain organoids which were not able to express the reporter gene, probably due to not fully developed neurons. The fluorescent protein expression targeting specifically mitochondria and the membrane was however verified in the human embryonic kidney cells during the packaging stages.

<p>Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some <em>in vitro</em> approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different <em>in vitro</em> systems that could help better mimic certain physiological systems that tend to not be mimicked by <em>in vivo</em> models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction.  Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in <em>in vivo</em> models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using <em>in vivo</em> models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.  </p>

Das Protozoon Giardia duodenalis ist eine der Hauptursachen für infektiöse Magen-Darm-Erkrankungen. Die zugrundeliegenden Pathomechanismen sind jedoch nach wie vor unklar. Um die Pathogenität G. duodenalis‘ untersuchen zu können, wird ein Modellsystem benötigt, dass die Komplexität des Darmepithels widerspiegelt. Diese Arbeit zeigt die Etablierung eines Zellkultursystems auf der Basis von organoid-abgeleiteten Epithelien unter Verwendung von filter-basierten Zellkultureinsätzen. Wir haben Protokolle für die Etablierung von organoid-basierten Zellkulturen (ODMs) vier verschiedener Wirte zoonotischer Protozoen unter Verwendung eines einzigen Protokolls erstellt. Die Charakterisierung zeigte, dass das Modellsystem erfolgreich die Polarisierung des Darmepithels nachahmt, aus mehreren Zelltypen besteht und eine Infektion ermöglicht. Der Schwerpunkt der Arbeit lag auf der Analyse der durch G. duodenalis induzierten Barrierestörung in ODMs auf Transkriptions-, Protein- und Funktionsebene. Die Infektion von humanen duodenalen Zellen führte zu einem Verlust der epithelialen Barrierefunktion. Mit Hilfe des transepithelialen elektrischen Widerstandes und Dextran Flux wurde eine Erhöhung der Barrieredurchlässigkeit beobachtet. Die Hemmung von zuvor in immortalisierten Zellmodellen beschriebenen Reaktionswegen konnte die Barrierefunktion nicht wiederherstellen. Stattdessen konnten Veränderungen der Ionenhomöostase sowie den Zusammenbruch der zonula occludens nachgewiesen werden. Der beobachtete Phänotyp konnte auf die Aktivierung des cAMP/PKA/CREB-Signalwegs, als einen von mehreren kausalen Faktoren, zurückgeführt werden. Hier zeigen wir die Etablierung eines aus Organoiden abgeleiteten Modells, das die Untersuchung von G. duodenalis Infektionen in vitro ermöglicht. Mit unserem Modell konnten wir eine neue Reihenfolge von Ereignissen entschlüsseln, die einen der Faktoren während symptomatischer Giardiasis darstellt. / The protozoan Giardia duodenalis is a one of the major causes of gastrointestinal illness. Underlying pathomechanisms remain unclear. An in vitro model system that also mimics the complexity of intestinal epithelium is needed to allow pathogenicity studies. This thesis shows the establishment of a cell culture system based on organoid-derived epithelia using permeable cell culture inserts. We have provided guidelines on the establishment of organoid-derived monolayers (ODMs) of four different hosts of zoonotic protozoa using a single protocol. Characterization showed that the model system successfully mimics intestinal polarization, is composed of multiple cell types and allows for infection with multiple protozoan parasites. As the main focus of the thesis, analysis of G. duodenalis-induced barrier breakdown in ODMs was performed on transcriptional, protein and functional level. Infection of human duodenal, organoid-derived monolayers resulted in a time- and dose-dependent breakdown of epithelial barrier function. Barrier permeability increases were observed ranging from ions to macromolecules as measured by transepithelial electrical resistance and Dextran flux. Inhibition of previously proposed key pathogen-induced pathways observed in immortalized cell models did not rescue barrier dysfunction. We could instead show changes in ion homeostasis, and tight junctional breakdown. While none of the previously proposed effector pathways appeared to be responsible, we could pin-point the observed phenotype to activation of the cAMP/PKA/CREB signaling pathway, as one of the factors of the multifactorial barrier breakdown. The establishment of an organoid-derived infection model is shown, allowing the study of in vitro Giardia duodenalis infections. Using this model, we could decipher a new series of events that may be one of the factors causing the intestinal barrier breakdown observed in symptomatic Giardiasis.