Haematopoietic stem cells (HSC) are invaluable, due to their potential to treat malignant and non-malignant diseases. Modern medicine requires a reliable source of human HSCs (hHSCs) for efficient transplantations, which in many cases cannot be obtained from a single donor. Therefore, the ability to amplify donor hHSCs ex vivo would be an ideal alternative. Past attempts to expand hHSCs in vitro, demonstrated that the protocols developed so far have limited success. My research studied the factors which can affect the optimal culture of transplantable HSCs using a 3D culture system that had previously been used to culture HSCs derived from the aorta-gonad-mesonephros (AGM) region of the mouse embryo. This system involved cell culturing at the gas-liquid interface which is particularly sensitive to mechanical disturbances. To overcome this problem, floating Polypropylene support (rings) were designed and tested and I demonstrated that this was able to prolong aggregate culturing for up to 21 days. Further optimisation tests included altering factors such as oxygen levels, and the presence of antioxidants and apoptosis inhibitors in mouse HSCs culture. I have shown that moderate hypoxia (6% O2) did not affect HSCs in culture, while 2% of O2 led to a significant decrease of HSCs activity. Normoxia resulted in higher reactive oxygen species generation, which would likely be detrimental to cells. However, unexpectedly no improvement in repopulation efficiency of cultured HSCs was achieved by the addition of antioxidant. I also found that when the AGM region was dissociated and co-aggregated in the presence of Rho kinase inhibitor a higher level of repopulation was achieved. In addition, troloxpifitrin-a and p38 inhibitor blocked HSC development without affecting progenitor frequency or the total number of live cells. Subclones of mouse stromal cell line (OP9) were used to create a defined haematopoietic niche for hHSC. Functional screening of these lines in co-aggregate culture re- vealed that 3 of the 34 subclones tested were able to maintain hHSC in culture and repopulate immunodeficient mice at a comparable level to uncultured CD34+ cells. The repopulation in engrafted recipients persisted for over 6 months and showed both myeloid and lymphoid potential. These 3 subclones therefore appeared to create a functional niche for hHSCs and were subsequently used to study the impact of a number of factors including SCF, rock inhibitor, TGFb inhibitor, StemRegenin1 (SR), and prolonged culture technique on hHSC expansion. A significant level of fluctuation between experiments was observed and no definitive conclusions could be drawn. I also attempted to establish stromal cell lines from the human AGM region, more specifically from the ventral (AoV) and dorsal (AoD) regions of the dorsal aorta. Despite attempts to immortalise primary stromal cells, all lines went through a growth crisis. Nevertheless, 30 lines were screened for their ability to support haematopoietic cells in co-aggregate culture with results suggesting that lines derived from AoV expanded haematopoietic precursors more efficiently than AoD lines and OP9 control. Many of the tested lines were able to maintain long-term repopulating human HSCs but the level of repopulation was not as high as that achieved from uncultured CD34+ cells. Unfortunately, these human stromal cell lines have an unstable karyotype which may have an impact on their functional characteristics and they may not represent the nature of the primary cells.

O desenvolvimento de novas terapias para o câncer envolve um processo longo e custoso no qual apenas 5% dos candidatos a fármacos em ensaios clínicos são aprovados. Os ensaios celulares são um importante pilar neste processo, no entanto, eles são geralmente feitos em células cultivadas em monocamada, o que apresenta algumas limitações. Assim, o desenvolvimento e aplicação de modelos tridimensionais (3D) para ensaios pré-clínicos tem sido cada vez mais investigado, devido a suas maiores similaridades com tumores in vivo em relação a características físicas, espaciais e bioquímicas. A via PI3K-AKT-mTOR, que está frequentemente desregulada em diversos cânceres, é uma rota metabólica essencial por integrar fatores de crescimento e sinais internos com a expressão de proteínas relacionadas ao crescimento celular. Sendo assim, novos compostos inibitórios dessa via têm sido estudados pelo grupo NEQUIMED como alternativa terapêutica para essas doenças. Dessa forma, este trabalho teve o objetivo de avaliar o potencial citostático e citotóxico de novos inibidores da via da mTOR em culturas celulares bi e tridimensionais de câncer de próstata e hepatocarcinoma. Para isso, o modelo de cultura 3D foi padronizado para ambas linhagens usando duas técnicas diferentes, além da padronização da técnica de redução da resazurina para a determinação da viabilidade celular em 2D e 3D. No ensaio citotóxico, células em monocamada e esferoides foram tratadas com diferentes concentrações dos fármacos de referência e dos novos inibidores e tiveram sua viabilidade determinada pelo ensaio de redução da resazurina. Já para o ensaio citostático, baixas concentrações de células foram plaqueadas em monocamada e tratadas por até 6 dias, tendo sua viabilidade medida a cada 48 h pelo ensaio de MTT. Os esferoides foram tratados por 9 dias com as mesmas substâncias, tendo seu volume medido a cada 3 dias. No geral, os novos compostos não apresentaram efeitos citotóxicos relevantes, contudo, os compostos Neq0438 e seu análogo, Neq0679, tiveram maior efeito citostático que a Rapamicina em culturas 2D e 3D de HepG2. Além disso, os compostos tiveram maior efeito citostático em esferoides do que em células cultivadas em monocamada para as duas linhagens. Isso pode ser justificado pela alteração na expressão de proteínas da via da mTOR em relação ao modelo de cultura, já que foi demonstrado sua maior ativação nos modelos tridimensionais. Futuramente esses compostos poderão ser testados sozinhos, ou em terapia combinada em modelos animais para melhor avaliação de sua segurança e sua evolução no estudo pré-clínico. / The development of new cancer therapies involves a long and expensive process in which only 5% of the clinical trial candidates for new drugs get approval. Cell assays are an essential pillar in this process; however, they are usually carried out in cells grown as a monolayer, which have some limitations. Thus, the development and application of new tridimensional (3D) models for preclinical trials has been deeply investigated, due to their higher similarities with in vivo tumors, regarding physical, spatial and biochemical features. The PI3K-AKT-mTOR pathway, which integrates growth factors and the expressions of proteins for the cellular growth, is often upregulated in several types of cancer. Hence, the NEQUIMED group is studying new pathway inhibitors as a therapeutic alternative for cancer. Here, the cytotoxic and cytostatic effects of the new PI3K-AKT-mTOR inhibitors were assessed in two and three-dimensional cells models of prostate cancer and hepatocarcinoma. To that end, the 3D culture model was standardized for both cell lines using two different techniques. The resazurin reduction assay was also standardized to determine cell viability in 2D and 3D models. For the cytotoxic assay, cells grown in a monolayer and 3D spheroids were treated with a range of concentrations of the reference drugs and new mTOR inhibitors and had their viability assessed by the resazurin assay. For the cytostatic assay, low cell density was plated on 2D and treated for 6 days, having their viability determined every 48 h using the MTT assay. Spheroids were treated for 9 days with the same substances and had their volume measured every 3 days. Overall, the new mTOR inhibitors did not show relevant cytotoxic effects, however, Neq0438 and its analog Neq0679, had a higher cytostatic effect than Rapamycin for both 2D and 3D cultures of HepG2. Besides, all mTOR inhibitors had a higher cytostatic effect on 3D cultures when compared to monolayers, which can be related to the overexpression of the mTOR pathway in this culture system, already reported on the literature. Based on these results, Neq0438 and Neq0679 should now be used alone or in combination using in vivo models to investigate their antitumoral effects.

Development of optogenetically controllable human neural network models can provide an investigative system that is relevant to the human brain. Conventional cultures of neural networks in two-dimensions (2D) have major limitations of scale. For instance, the soma of neurons in 2D is unrealistically flattened and both axon and dendrite outgrowth is restricted. Using a combination of tissue engineering techniques and the inclusion of optogenetically modified human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs), the development of a three-dimensional (3D) human neural culture model within a defined 3D microenvironment is investigated in this study. Light-sensitive neurons were successfully generated by transducing Channelrhodopsin-2 (ChR2) into human iPSC-derived NPCs and neuroblastoma cells (SH-SY5Y) using lentiviral transduction. The use of neuron specific promoters for synapsin-1 (SYN1) and calcium-calmodulin kinase II (CaMKII) in driving the expression of ChR2-Yellow Florescent Protein (YFP) within the mixed neuronal populations from hiPSC-derived neurons (Axol cells) were compared. Viability of the cells at 7 day-post-infection was 80% - 97% in all conditions tested. In line with published literature, transduction efficiency of neurons at day 14 was found to be 3% - 7% for plasmids containing the SYN1 promoter and 2% - 5% for plasmids containing the CaMKII promoter. An increase in promoter driven ChR2-YFP expression was evaluated over 28 days as the neural subpopulations matured. Stably ChR2 expression continued through-out higher passages (≥ P₁₀) and possibly for periods up to several months. Both SYN1 and CaMKII promoters were found to drive the expression of ChR2 in Axol cells targeting inhibitory and excitatory neurons, respectively. 3D culture systems to support cell growth and optogenetic application were developed and characterised. Alginate hydrogel functionalised with short peptide sequence arginine-glycine-aspartate (RGD), and small molecules such as Rho Kinase inhibitor (ROCKi) and ZVAD were incorporated to increase the viability of human pluripotent stem cells (hPSCs). Investigation of cell response reveals that a flow rate of 3 ml/min and an alginate concentration of 1.8% (w/v) are optimal and that stem cell survival is significantly improved through incorporation of RGD and ROCKi. Interestingly, ChR2-YFP expression of Axol and SY5Y cells was detectable when transferred to the 3D culture system. The optogenetically modified neurons were found responsive to light stimulation, showing firing patterns and calcium events typical of early developing neurons (e.g. mixed and burst waves; single and multipeak spikes). Neuronal activities were assessed using calcium imaging. Higher numbers of calcium events were associated with CaMKII driven ChR2-YFP expression than with SYN1 in Axol cells. However, calcium activity in SH-SY5Y cells was most noticeable in neurons expressing ChR2-YFP driven by the SYN1 promoter. In primary rodent neuronal cultures, synchronous calcium firing with repetitive action potentials (APs) resulted from ChR2-YFP expression was driven by both SYN1 and CaMKII promoter upon light stimulation. By combining multi-approaches, we report for the first time on the generation of an in vitro hiPSC-derived neural network model in 3D using functionalised alginate hydrogel and involving optogenetic targeting. Expression of ChR2-YFP was found driven by both SYN1 and CaMKII promoter in the RGD-alginate bead system that cultured with Axol cells.

La maladie d’Alzheimer est une maladie très complexe, aujourd’hui encore mal comprise et cette démence est devenue un réel problème de santé publique. La protéine précurseur de l’amyloïde (APP) et la protéine Tau sont deux acteurs majeurs impliqués dans la maladie. De nombreuses recherches se sont investies dans la compréhension du métabolisme, de l’action et de l’implication de ces deux protéines dans les mécanismes pathologiques de la maladie et d’autres maladies neurodégénératives. Elles sont notamment l’objet de la plupart des approches thérapeutiques passées et actuelles, et étudiées pour le diagnostic biologique de la maladie. Dans ce projet de thèse, notre objectif fut d’explorer le métabolisme des protéines APP et Tau au cours de la différenciation neuronale à l’aide d’outils biochimiques et de systèmes innovants d’immunodétection multiplex très sensibles (MSD®) dans plusieurs modèles de culture cellulaire de la maladie. L’objectif était d’obtenir une vision globale des processus physiopathologiques au travers d’analyses d’échantillons générés au cours de la différenciation neuronale de cellules souches pluripotentes induites (iPSC) de patients Alzheimer comparées aux cellules souches embryonnaires humaines (hESC). Nous avons ainsi généré et caractérisé plusieurs lignées cellulaires d’IPSC d’une personne saine contrôle et de patients atteints des formes sporadiques et familiales de la maladie. Ce projet offre l’opportunité unique de combiner des approches innovantes pour tenter de comprendre comment les fragments et les peptides Ab sont générés, ainsi que les modifications de Tau en conditions normales et pathologiques. / Amyloid precursor protein (APP) and Tau protein are two main molecular actors of the Alzheimer’s disease (AD), which is of prime importance in Human Health. Intensive research is ongoing to understand these proteins’ metabolism, action and implication in the pathological mechanism of these affections. They are the target of most therapeutic approaches and are used for biological diagnosis. In the present PhD project, our objective was to investigate neuronal APP and Tau protein processing and metabolism using biochemical tools and innovative multiplex immunodetection system (MSD®) in diverse cell culture models of AD. The goal was to get a comprehensive view oh physiopathological processes based on the analysis of samples generated in neuronal differentiated human embryonic stem cell and induced pluripotent stem cells derived from AD-patients. We generated several cell lines from an healthy control individual, and AD patients showing sporadic and familial forms of the disease. This project offer the unique opportunity to combine state-to-the-art approaches to understand how the APP fragments and peptides are generated as well as the modifications of the Tau protein in normal and pathological situation.

La mégacaryopoïèse regroupe l’ensemble des processus de différenciation et de maturation des mégacaryocytes (MKs) dans le but de produire des plaquettes capables d’arrêter les saignements. Or ces mécanismes sont mal connus. Afin de mieux les comprendre, nous avons mimé l’environnement médullaire in vitro, en 3D à l’aide d’un hydrogel de rigidité comparable à celle de la moelle osseuse. Dans cette étude nous avons: i) caractérisé le comportement physique de l’hydrogel de méthylcellulose et mis au point la culture de progéniteurs mégacaryocytaires dans ce système, ii) montré la capacité du MK à ressentir les contraintes physiques de son environnement, ainsi que, iii) l’impact de ces contraintes sur la maturation des MKs et la génération des proplaquettes, et enfin, iv) mis en évidence l’existence d’une réponse cellulaire des MKs à la rigidité. Les MKs sont « mécanosensibles », c’est-à-dire capables de ressentir les modifications physiques de leur environnement et de s’y adapter. L’activation de voies de mécanotransduction (dont MKL1) et la réorganisation du cytosquelette en réponse aux contraintes physiques extracellulaires favorisent la maturation des MKs, en termes de ploïdie, d’ultrastructure et in fine de génération de proplaquettes. / Megakaryopoiesis is the process of differentiation and maturation of megakaryocytes (MKs) in the aim to produce platelets able to prevent hemorrhages. These mechanisms are not well known. To better understand the process of platelet formation, we mimicked the medullar microenvironment in vitro, in 3D using hydrogel of stiffness comparable to the bone marrow. In this study we: i) characterized the physical properties of the hydrogel and design the culture of hematopoietic progenitors in this system, ii) showed the MKs ability to feel the physical constraints of their environment, then iii) showed the impact of these constraints on the MK maturation and proplatelet generation, and finally iv) highlighted the MK response to stiffness. MKs are “mecanosensitives”, being able to feel and to adapt to the physicals modifications of the environment. The activation of mechanotransduction pathways (including MKL1) and the cytoskeleton reorganization in response to extracellular physical constraints improves MK maturation, in terms of ploïdy, ultrastructure and ultimately proplatelet generation.

The adult mammary gland is comprised of a bi-layered epithelium of luminal and myoepithelial cells surrounded by an adipocyte-rich fat pad, a highly collagenous extra-cellular matrix (ECM) and a number of other stromal and endothelial cell types. Mammary stem cells (MaSCs) reside within the epithelium and these are capable of repopulating a mammary fat pad that is devoid of epithelium, upon transplantation. It was sought to recapitulate this process of MaSCs repopulating a fat pad using a synthetic fat pad, engineered from a collagen scaffold invested with adipocytes, to provide an in vitro 3D model. Fluorescently tagged murine Axin2-expressing cells were obtained from transgenic mice and seeded into these scaffolds and cultured, mimicking the process of fat pad repopulation. Immunohistochemical analysis demonstrated that Axin2+ myoepithelial cells were rarely capable of forming bi-layered structures that expressed correct myoepithelial localisation and resemblance to a luminal morphology. Breast tumours surrounded by anisotropic (directional) collagen fibres running perpendicular to the tumour boundary are more aggressive and associated with poor patient prognosis. To recapitulate this anisotropic collagen phenotype in vitro, an ice-templating technique was used to modify the structure of the collagen scaffolds producing both non-directional (isotropic) and anisotropic internal architectures. Tumour cells from various breast cancer cell lines were seeded into both isotropic and anisotropic scaffolds to investigate whether this approach could distinguish cell type-specific migratory ability and whether anisotropy affected migration efficiency. Following analysis by confocal microscopy and ImageJ, anisotropic scaffolds were observed to enhance the migratory potential of MDA-MB-231 breast cancer cells. These results highlight the importance of collagen alignment and provide a reproducible method to quantitatively measure cell migration in 3D for cells derived from different breast cancer subtypes. Building on these data, the protocol was adapted to permit the direct investigation of tumour biopsy material. Given the heterogeneity of breast tumours, it was considered important to maintain tumour architecture and stromal components. Thus, murine mammary tumour fragments from two different established mammary cancer models were utilised and cultured in anisotropic collagen scaffolds in the presence or absence of adipocytes to allow an investigation of their influence on tumour cell migration. Further experiments included addition of various therapeutic drugs followed by immunofluorescence microscopy coupled with an optical clearing technique. These data demonstrated the utility of the model in determining both the rate and capacity of tumour cells to migrate through the engineered stroma while shedding light also on the mode of migration. Moreover, the response of different mammary tumour types to chemotherapeutic drugs could be could be readily quantified. To humanize the fat pad for subsequent human tissue analysis, human mesenchymal stem cells (MSC) were obtained from reduction mammoplasties and immortalised, before differentiating them into adipocytes within anisotropic collagen scaffolds. Human breast cancer cells were fluorescently tagged for tracking using lentiviral methods and were seeded into scaffolds invested with differentiated MSCs. Both cell types were successfully co-cultured for 7 days and imaged using multiphoton methods.

Um dos desafios atuais da engenharia de tecidos é o desenvolvimento de biomateriais substitutos e/ou equivalentes que mimetizem o tecido normal. Os estudos empregando cultura celular em monocamada encontram limitações no que concerne às interações bidimensionais entre as células e experimentos utilizando animais, devido à elevada variabilidade, não conseguem predizer os resultados em humanos, comprometendo a sua relevância clínica. À vista disso, a cultura tridimensional de células (3D) utilizando um biomaterial fabricado para promover a proliferação e diferenciação celular tem sido utilizada para recriar a complexidade de um tecido normal, permitindo uma maior e complexa interação celular. Visando mimetizar o ambiente encontrado in vivo, este trabalho investiu no desenvolvimento de uma derme reconstituída (equivalente dérmico) in vitro utilizando como matriz biológica o colágeno, componente mais abundante da derme como suporte para os fibroblastos humanos, assim como na avaliação da fotobiomodulação com luz em 630 nm. Foi preparada uma esponja a partir do colágeno de serosa porcina 1,1% hidrolisado por 96 h. A caracterização do biomaterial foi realizada pela determinação da porosidade, do diâmetro dos poros, da absorção de fluidos e por ensaios de biocompatibilidade, uma vez que estes parâmetros são importantes para a proliferação e diferenciação celular na consequente formação do tecido in vitro. O biomaterial exibiu porosidade de 95,2%, com poros medianos de 44 &#956m estimados por porosimetria de injeção de mercúrio, além de canais com distância média entre as paredes de 78+/-14 &#956m estimado por MEV. Esses valores são considerados como ideais para um biosuporte de crescimento de fibroblastos. A absorção de água e meio de cultura foi de 95% e a esponja não apresentou citotoxicidade para a linhagem celular Vero. Adicionalmente, foi investigado o efeito de irradiação na cultura 3D com luz vermelha (dose 30 J/cm2), que mostrou fotobiomodulação na dose de 30 J/cm2 para cultura de células em monocamada e no início da fase de crescimento celular em cultura tridimensional. Por microscopia confocal, verificou-se que as células cultivadas na presença da esponja (cultura 3D), apresentaram diferenciação e secreção de matriz extracelular. Portanto, os resultados apresentados mostraram que a esponja de colágeno utilizada como biomaterial para suporte celular é eficiente para a produção de uma derme reconstituída (equivalente) in vitro e que a fotobiomodulação em 630 nm na dose de 30 J/cm2 de fato acelera o crescimento celular na matriz. / The development of biomaterials substitutes and/or equivalents to mimic normal tissue is a currently challenge in tissue engineering. Studies using cell monolayer culture presents limitations with respect to two-dimensional interactions between the cells, and experiments using animals cannot predict results in humans, due to the high viability, thus compromising their clinical relevance. In consequence, three-dimensional cell culture (3D) using a biomaterial designed to promote cell proliferation and differentiation has been used to recreate the complexity of a normal tissue, allowing a larger and complex cellular interaction. Aiming to mimic the in vivo environment, the present work refers to create a reconstituted dermis (dermal equivalent) in vitro using collagen, the most abundant component of the dermis, as biological matrix, as support for human fibroblasts, as well evaluate the photobiomodulation with light at 630 nm. First, a sponge was prepared from serous 1.1% porcine collagen hydrolyzed for 96 h. The biomaterial was characterized by determination of its porosity, pore diameter, the fluid absorption and the biocompatibility assays, since these parameters are important to the cell proliferation and differentiation resulting in the in vitro tissue formation. The biomaterial showed porosity of 95.2%, with a median pore of 44 &#956M estimated by mercury porosimetry injection, and channels with an average distance between the walls of 78+/-14 &#956M estimated by SEM. These values are considered as ideal for a biosupport fibroblast growth. The absorption of water and growth medium was 95%, and the sponge showed no cytotoxicity for the Vero cell line. Additionally, it was investigated the effect of irradiation in 3D culture with red light (dose 30 J/cm2), that showed photobiomodulation on the dose 30 J/cm2, for culturing cells in monolayer and in the early-stage of the cell growth in three-dimensional culture. By confocal microscopy, it was verified that the cells cultured in the presence of the sponge (3D culture), allows differentiation and extracellular matrix secretion. Therefore, the results showed that the collagen sponge used as a biomaterial for cell support and the photobiomodulation at 630 nm and dose of 30 J/cm2 are efficient for the production of a reconstructed dermis (equivalent) in vitro.

No description available.

O desenvolvimento de novas terapias para o câncer envolve um processo longo e custoso no qual apenas 5% dos candidatos a fármacos em ensaios clínicos são aprovados. Os ensaios celulares são um importante pilar neste processo, no entanto, eles são geralmente feitos em células cultivadas em monocamada, o que apresenta algumas limitações. Assim, o desenvolvimento e aplicação de modelos tridimensionais (3D) para ensaios pré-clínicos tem sido cada vez mais investigado, devido a suas maiores similaridades com tumores in vivo em relação a características físicas, espaciais e bioquímicas. A via PI3K-AKT-mTOR, que está frequentemente desregulada em diversos cânceres, é uma rota metabólica essencial por integrar fatores de crescimento e sinais internos com a expressão de proteínas relacionadas ao crescimento celular. Sendo assim, novos compostos inibitórios dessa via têm sido estudados pelo grupo NEQUIMED como alternativa terapêutica para essas doenças. Dessa forma, este trabalho teve o objetivo de avaliar o potencial citostático e citotóxico de novos inibidores da via da mTOR em culturas celulares bi e tridimensionais de câncer de próstata e hepatocarcinoma. Para isso, o modelo de cultura 3D foi padronizado para ambas linhagens usando duas técnicas diferentes, além da padronização da técnica de redução da resazurina para a determinação da viabilidade celular em 2D e 3D. No ensaio citotóxico, células em monocamada e esferoides foram tratadas com diferentes concentrações dos fármacos de referência e dos novos inibidores e tiveram sua viabilidade determinada pelo ensaio de redução da resazurina. Já para o ensaio citostático, baixas concentrações de células foram plaqueadas em monocamada e tratadas por até 6 dias, tendo sua viabilidade medida a cada 48 h pelo ensaio de MTT. Os esferoides foram tratados por 9 dias com as mesmas substâncias, tendo seu volume medido a cada 3 dias. No geral, os novos compostos não apresentaram efeitos citotóxicos relevantes, contudo, os compostos Neq0438 e seu análogo, Neq0679, tiveram maior efeito citostático que a Rapamicina em culturas 2D e 3D de HepG2. Além disso, os compostos tiveram maior efeito citostático em esferoides do que em células cultivadas em monocamada para as duas linhagens. Isso pode ser justificado pela alteração na expressão de proteínas da via da mTOR em relação ao modelo de cultura, já que foi demonstrado sua maior ativação nos modelos tridimensionais. Futuramente esses compostos poderão ser testados sozinhos, ou em terapia combinada em modelos animais para melhor avaliação de sua segurança e sua evolução no estudo pré-clínico. / The development of new cancer therapies involves a long and expensive process in which only 5% of the clinical trial candidates for new drugs get approval. Cell assays are an essential pillar in this process; however, they are usually carried out in cells grown as a monolayer, which have some limitations. Thus, the development and application of new tridimensional (3D) models for preclinical trials has been deeply investigated, due to their higher similarities with in vivo tumors, regarding physical, spatial and biochemical features. The PI3K-AKT-mTOR pathway, which integrates growth factors and the expressions of proteins for the cellular growth, is often upregulated in several types of cancer. Hence, the NEQUIMED group is studying new pathway inhibitors as a therapeutic alternative for cancer. Here, the cytotoxic and cytostatic effects of the new PI3K-AKT-mTOR inhibitors were assessed in two and three-dimensional cells models of prostate cancer and hepatocarcinoma. To that end, the 3D culture model was standardized for both cell lines using two different techniques. The resazurin reduction assay was also standardized to determine cell viability in 2D and 3D models. For the cytotoxic assay, cells grown in a monolayer and 3D spheroids were treated with a range of concentrations of the reference drugs and new mTOR inhibitors and had their viability assessed by the resazurin assay. For the cytostatic assay, low cell density was plated on 2D and treated for 6 days, having their viability determined every 48 h using the MTT assay. Spheroids were treated for 9 days with the same substances and had their volume measured every 3 days. Overall, the new mTOR inhibitors did not show relevant cytotoxic effects, however, Neq0438 and its analog Neq0679, had a higher cytostatic effect than Rapamycin for both 2D and 3D cultures of HepG2. Besides, all mTOR inhibitors had a higher cytostatic effect on 3D cultures when compared to monolayers, which can be related to the overexpression of the mTOR pathway in this culture system, already reported on the literature. Based on these results, Neq0438 and Neq0679 should now be used alone or in combination using in vivo models to investigate their antitumoral effects.

La Protéine Prion (PrP) est une glycoprotéine ubiquitaire attachée au feuillet externe de la membrane plasmique par une ancre glycosylphosphatidylinositole (GPI). Cette dernière est l’agent infectieux responsable de la maladie Creutzfeld-Jacob ou « maladie de la vache folle ». Cette protéine existe sous sa forme cellulaire mais également sous sa forme infectieuse, nommée PrPSc (Scrapie). Alors que la fonction de PrPSc est établie au cours de la pathogenèse, la fonction de la protéine cellulaire est beaucoup plus énigmatique notamment chez les mammifères. Il est clairement admis que la forme infectieuse découle d’un changement de conformation de la forme cellulaire. Ainsi afin de mieux appréhender le rôle de la protéine prion dans les cellules saines mais également lors de la pathogenèse il apparaît essentiel d’étudier le trafic de cette protéine. La protéine prion est exprimée dans les cellules neuronales qui sont comme les cellules épithéliales des cellules polarisées. J’ai au cours de ma thèse étudié le trafic de la protéine prion dans les cellules polarisées MDCK. MDCK est la lignée épithéliale sur laquelle nous avons la plus grande connaissance. Dans mon travail j’ai utilisé des cellules MDCK polarisées classiquement en culture bidimensionnelle (2D) mais également en culture tridimensionnelle (3D) où les cellules forment des kystes, structures hautement polarisées, physiologiquement proches de l’épithélium in vivo. Il apparaît que dans les cellules MDCK polarisées sur filtre (en 2D) la localisation de la PrP est controversée. Nous avons trouvé que, contrairement à la majorité des protéines à ancre GPI, la PrP suit la voie de transcytose. La PrP qui se retrouve à la membrane basolatérale est transcytosée vers la membrane apicale. De plus la PrP envoyée à la surface apicale est clivée (clivage alpha) générant deux fragments distincts : le fragment C1, pourvu de l’ancre GPI qui reste associé à la surface apicale et le fragment soluble N1 qui est sécrété dans le milieu de culture des cellules MDCK cultivées en 2D ou dans le lumen des cellules MDCK cultivées en 3D. Mon travail permet de mieux comprendre les études réalisées auparavant mais surtout révèle l’existence d’un mécanisme de transcytose de la protéine prion dans les cellules épithéliales. Cette information est essentielle et nous permet de supposer que ce mécanisme pourrait être également utilisé par les cellules neuronales. / The Prion Protein (PrP) is a ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of “prion diseases” the cellular isoform (PrPC) is an enigmatic protein with unclear function. Prion protein has received considerable attention due to its central role in the development of Transmissible Spongiform Encephalopathies (TSEs) known as “prion diseases”, in animals and humans. Understanding the trafficking, the processing and degradation of PrP is of fundamental importance in order to unravel the mechanism of PrPSc mediated pathogenesis, its spreading and cytotoxicity. The available data regarding PrP trafficking are contradictory. To investigate PrP trafficking and sorting we used polarized MDCK cells (two-dimensional and tree-dimensional cultures) where the intracellular traffic of GPI-anchored proteins (GPI-APs) is well characterized. GPI-APs that are sorted in the Trans Golgi Network follow a direct route from the Golgi apparatus to the apical plasma membrane. The exception to direct apical sorting of native GPI-APs in MDCK cells is represented by the Prion Protein. Of interest, PrP localization in polarized MDCK cells is highly controversial and its mechanism of trafficking is not clear. We found that full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures and that the C1/PrP full-length ratio increases upon MDCK polarization. We revealed that differently from other GPI-APs, PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells and is α-cleaved during its transport to the apical surface. This study not only reconciles and explains the different findings in the previous literature but also provides a better picture of PrP trafficking and processing, which has been shown to have major implications for its role in prion disease.