Tumour dissemination is a major reason for failure of therapy for many tumour types therefore there is a requirement for novel targets & therapies. The αIIbβ3 and αvβ3 integrins have been demonstrated to have significant involvement at many stages of the tumour dissemination process including, tumour cell adhesion, migration, metastasis and angiogenesis, and thus the β3 integrins are a potential target for therapeutic antagonism with small molecules. Because of the clear interaction between the different integrin types, targeting integrins as a therapeutic strategy requires targeting more than one integrin type. Consequently, the ICT is developing a group of novel new αIIbβ3 and αvβ3 integrin dual antagonists. One of the main challenges is having a relevant, validated experimental model that expresses these integrins. The aim of the work presented here is to develop and validate an in vitro αIIbβ3 and αvβ3 integrin expressing assay of tumour cell invasion. The spheroid invasion assay has the advantage over standard monolayer transwell chamber invasion assays of being a 3-dimensional assay, and thus mimics better the cell-cell interactions and architecture that are present in a tumour compared to the monolayer-based assay. A panel of human cancer cell lines known to express one of the molecular targets of interest, αvβ3 integrin was evaluated for the ability to form spheroids and to invade through collagen matrices. One glioma cell line, U87-MG, demonstrated consistent spheroid formation and invasion and was thus selected for further studies. Optimum conditions were established for use of U87-MG in the invasion assay, and the assay was validated using a known inhibitor of invasion, LiCl and known β3 antagonist, cRGDfV. Subsequently a group of novel small molecule β3 antagonists were evaluated at nontoxic concentrations using the assay. Both LiCl and cRGDfV inhibited spheroid invasion through the gel in a dose-dependent manner, thus validating the assay. Furthermore, when the novel small molecule β3 antagonists were evaluated using the model, a dose and time dependent reduction in U87-MG spheroids invasion in collagen was observed. In further work initial steps were taken to construct a cell line which expresses both αIIbβ3 and αvβ3 integrin to use in the model to assess for dual integrin antagonism. In conclusion, this work has established a validated assay which has been utilised for some compounds to evaluate a group of novel small molecule β3 integrin antagonists with encouraging results.

Cells in multicellular organisms are surrounded by a complex three-dimensional macromolecular extracellular matrix (ECM). This matrix, traditionally thought to uniquely serve a structural function providing support and strength to cells within tissues, is increasingly being recognized to have pleiotropic effects in neurogenesis and regeneration processes such as neocortex folding, stem cell niche maintenance, peripheral nerve regeneration, axonal growth, and many more. ECM mediates these processes via cell-ECM interactions which provide the cells with a wealth of signals including biophysical and mechanical cues in a spatiotemporal manner. Owing to the importance of the surrounding microenvironment, modern neural tissue engineering strategies have focused on the development of engineered biomaterials capable of finely instructing the neuronal response according to their physicochemical characteristics. Neurons and neural stem cells are in fact sensitive to their mechanical and topographical environment, and cell–substrate binding contributes to this sensitivity by activating specific signaling pathways for basic cell function. In addition, the advances in nanotechnology have opened the possibility of introducing decorative nano-motifs that interact with cells at the molecular level. Successful strategies in tissue engineering are driven by not only advances in the synthesis of highly instructive biomaterials but also greatly depend on the right selection of cell sources. As a matter of fact, advances in neural tissue engineering have been strongly hampered by the poor availability of cell sources, considering that primary neurons are the only type of cells that do not proliferate. The discovery of induced pluripotent stem cells (iPSCs) has addressed many of the cell-related limitations in neural tissue engineering, offering the possibility to consistently produce a wide range of neural cell lines. Advances in cell biology have led to the development of iPSCs-derived brain spheroid, which surely represent the most promising tools for several neural tissue engineering applications ranging from in vitro modelling of neurodegenerative diseases (i.e., Parkinson's, Huntington's and Alzheimer's), biomaterials testing and drug screening platforms. The overarching goal of my doctoral work was to engineer biomaterials with instructive physicochemical properties to elicit beneficial cellular responses that are suitable for different neural tissue engineering applications such as nerve regeneration and 3D in vitro modelling. In the first study (Chapter 2), I evaluated the compounded effects of surface stiffness and micro-topography on dorsal root ganglion and human bone-marrow mesenchymal stem cells behavior. To this end, arrays of parallel microchannels of different geometries were introduced on the surface of chitosan films by electrophoretic replica deposition. In addition, a novel chemical crosslinking with citric acid was performed to both enhance the long-term stability of the chitosan films and fine-tune the surface stiffness for the investigation of its role in cell behavior. In the second study (Chapter 3), I developed a novel nanocomposite consisting of a collagen hydrogel decorated with glycine-derived carbon nanodots (Gly-CNDs). After a comprehensive physicochemical characterization of the resulting nanocomposite, I evaluated the effects exerted on neuronal differentiation and electrophysiological maturation of mouse iPSCs-derived brain spheroid. In the third study (Chapter 4), I optimized an alignable collagen-based hydrogel characterized by anisotropically oriented fibers with potential applications in both peripheral and central nervous system repair. I established a protocol that encompasses the introduction in the collagen solution of biodegradable laminin-functionalized magnetic microbeads and the time-controlled application of an external magnetic field. The regenerative potential of the hydrogel was unveiled using mouse iPSCs-derived neural stem cells.

Academic: Ovarian cancer is the leading cause of death among female gynecologic cancers. Current treatments include surgical debulking, and chemotherapy. However, better interventions are needed to reduce the mortality rate of metastatic disease. Ovarian cancer cells have displayed the ability to aggregate and form 3D homogeneous and heterogeneous spheroids, which can function as micrometastases. Ovarian cancer spheroids survive independently prior to adhering to an endothelial tissue. Since aggregation has been shown to provide a survival advantage to the spheroids and increased their aggressive phenotype, this study aimed to investigate how the metabolism of ovarian cancer cells change in 3-dimensional (3D) culture. Examining metabolic pathways and identifying markers of metabolic change could provide the scientific base for new, targeted interventions for this disease. Spheroids of both homogeneous and heterogeneous composition demonstrated overall lower metabolic capacity than their adherent counterparts. Spheroids had a lower basal energetic demand than adherent cells, paralleled by lower maximal respiration capacity, glycolytic capacity, and spare respiratory capacity. We conclude that the lower energetic demand of spheroids may be a mechanism to prolong death by reserving energy and metabolic cellular processes; this may render anti-metabolic drug treatment with AICAR or metformin ineffective against disseminating ovarian cancer aggregates. General: Ovarian cancer is currently the leading cause of death among female gynecologic cancers. While treatments exist, better interventions are needed to reduce the mortality rate in this form of cancer. Ovarian cancer cells have displayed the ability to aggregate and form 3D homogeneous and heterogeneous spheroids, which can function as micrometastases. Ovarian cancer spheroids survive independently prior to adhering to an endothelial tissue. Since aggregation has been shown to provide a survival advantage to the spheroids and increased their aggressive phenotype, this study aims to investigate how the metabolism of ovarian cancer cells change in 3-dimensional (3D) culture. Examining metabolic pathways and identifying markers of metabolic change could provide the scientific base for new, targeted interventions for this disease. / Master of Science

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological malignancy due to the insufficient accurate screening programs for the early detection of EOC. To improve the accuracy of the early detection, there is a need to deeply understand the mechanism of EOC progression and the interaction between cancer cells with their unique microenvironment. Therefore, this work investigated the metabolic shift in the mouse model for progressive ovarian cancer, and evaluated the effects of hypoxic environment, spheroid formation as well as stromal vascular fractions (SVF) on the metabolic shift, proliferation rate, drug resistance and protein markers in functional categories. The results demonstrated an increasingly glycolytic nature of MOSE cells as they progress from a tumorigenic (MOSE-L) to a highly aggressive phenotype (MOSE-FFL), and also showed changes in metabolism during ovarian cancer spheroid formation with SVF under different oxygen levels. More specifically, the hypoxic environment enhanced glycolytic shift by upregulating the glucose uptake and lactate secretion, and the spheroid formation affected the cellular metabolism by increasing the lactate secretion to acidify local environments, modulating the expression of cell adhesion molecules to enhance cell motility and spheroids disaggregation, and up-regulating invasiveness markers and stemness makers to promote ovarian cancer aggressive potential. Hypoxia and spheroid formation decreased ovarian cancer cells growth but increased the chemoresistance, which leads to the promotion of aggressiveness and metastasis potential of ovarian cancer. SVF co-cultured spheroids further increased the glycolytic shift of the heterogeneous of ovarian cancer spheroids, induced the aggressive phenotype by elevating the corresponding protein markers. Decreasing the glycolytic shift and suppression of the proteins/pathways may be used to inhibit aggressiveness or metastatic potential of ovarian cancer heterogeneous of ovarian cancer spheroids, induced the aggressive phenotype by elevating the corresponding protein markers. Decreasing the glycolytic shift and suppression of the proteins/pathways may be used to inhibit aggressiveness or metastatic potential of ovarian cancer. / Master of Science

The majority of this work is concerned with the local-linear convective instability analysis of the incompressible boundary-layer flows over prolate spheroids and oblate spheroids rotating in otherwise still fluid. The laminar boundary layer and the perturbation equations have been formulated by introducing two distinct orthogonal coordinate systems. A cross-sectional eccentricity parameter e is introduced to identify each spheroid within its family. Both systems of equations reduce exactly to those already established for the rotating sphere boundary layer. The effects of viscosity and streamline-curvature are included in each analysis. We predict that for prolate spheroids at low to moderate latitudes, increasing eccentricity has a strong stabilizing effect. However, at high latitudes of ϴ ≥ 60, increasing eccentricity is seen to have a destabilizing effect. For oblate spheroids, increasing eccentricity has a stabilizing effect at all latitudes. Near the pole of both types of spheroids, the critical Reynolds numbers approach that for the rotating disk boundary layer. However, in prolate spheroid case near the pole for very large values of e, the critical Reynolds numbers exceed that for the rotating disk. We show that high curvature near the pole of prolate spheroids is responsible for the increase in critical Reynolds number with increasing eccentricity. For both types of spheroids at moderate eccentricity, we predict that the most amplified modes travel at approximately 76% of the surface speed at all latitudes. This is consistent with the existing studies of boundary-layer flows over the related rotating-disk, -sphere and -cone geometries. However, for large values of eccentricity, the traveling speed of the most amplified modes increases up to approximately 90% of the surface speed of oblate spheroids and up to 100% in the prolate spheroid case.

Although researchers have been able to print small, simple, and avascular tissues, they have been unsuccessful in creating large, complex and vascularized organs. Printing large and complex three-dimensional tissues or organs involves utilizing a large quantity of cellular spheroids and layer-by-layer addition of spheroids. In this study, an in-house cell spheroid fabrication system was developed to produce cell spheroids with human liver cells (hepG2), human endothelial cells (hEC), human neural stem cells (hNSC), and induced pluripotent stem cells (iPSC). It offers the ability of fabricating uniform-sized spheroids repeatedly, which is essential when large and complex structures need to be produced. In order to test the spheroids’ ability to fuse, hEC spheroids were placed in line with one another and revealed successful fusion. Overall, the results indicate the in- house developed cell spheroid fabrication system can play a major role in bioprinting by providing researchers with uniform-sized spheroids in large quantities, consistently.

Le cancer de l'ovaire représente la première cause de décès par cancer gynécologique. La survie globale des patientes à 5 ans est inférieure à 30%. Ce sombre pronostic s'explique à la fois par la découverte tardive de la maladie et par le développement d'une chimiorésistance. L'ascite est un fluide exsudatif qui est fréquemment accumulé dans la cavité péritonéale au cours de la progression des cancers de l'ovaire. Ce « microenvironnement tumoral » particulier contribue à la dissémination des cellules cancéreuses et à leurs implantations péritonéales.L'objectif global du travail de thèse a été, d'une part d'évaluer l'influence de l'ascite sur le comportement des cellules cancéreuses ovariennes et d'autre part, d'étudier les mécanismes de résistance à la perte d'ancrage des cellules cancéreuses ovariennes.Nous avons ainsi démontré que l'ascite induit une transition épithélio-mésenchymateuse partielle et que les modifications des comportements cellulaires observées sont dépendantes des intégrines alpha-v.Deux ligands de ces intégrines, la vitronectine et la fibronectine, ont été purifiés selon un protocole original permettant la caractérisation des deux protéines à partir d'une même ascite. Ces protéines ascitiques ont des propriétés différentes selon leur origine, donc selon les patientes dont elles sont issues, et influencent le comportement adhésif des cellules avec un degré variable. L'importance de la signalisation dépendante des intégrines alpha-v et des voies MAP Kinases a également été démontrée dans l'établissement d'une résistance des sphéroïdes tumoraux à l'anoïkis.En perspective, approfondir les connaissances des processus cellulaires et moléculaires conduisant à la dissémination intrapéritonéale et à l'émergence de chimiorésistance ainsi que déterminer le rôle potentiel de protéines ascitiques dans ces processus pourraient permettre la découverte de nouvelles cibles thérapeutiques. / Ovarian cancer is the most lethal gynaecological malignant disease, mainly due to late diagnosis and to acquired chemoresistance. An exudative fluid named ascites is frequently accumulates within the abdominal cavity during ovarian cancer progression. This unique tumor microenvironment contributes to cancer cell dissemination and peritoneal metastasis.The aim of the study was to evaluate the influence of ascites on cancer cell behaviors and to better understand the mechanisms of ovarian cancer cell resistance to the loss of anchorage.We demonstrate that ascites induces a partial epithelial–mesenchymal transition and that the modifications of cell behaviors observed are dependent of alpha-v integrins. A combined purification protocol has been established in order to purify vitronectin and Fibronectin, both ligands of these integrins, from a single pathological sample. These purified ascitic proteins have different molecular features according to the patients and impact the adhesive cell behavior with various degrees.Moreover we showed the importance of alpha-v integrins and MAP Kinases signalling pathways in the anoikis resistance of ovarian cancer spheroids.Our prospects are i) to increase the knowledge of the cellular and molecular processes leading to the intraperitoneal dissemination and to the emergence of chemoresistance and also ii) to determine the potential role of ascitics proteins in these processes. We will expect that these investigations couldlead to the discovery of new therapeutic targets.

O carcinoma adrenocortical (ACC) é um tumor maligno raro. O objetivo foi testar a ação de drogas antitumorais na linhagem H295R, em cultura 2D e 3D, analisadas por MTS, ensaios multiparamétricos, e microscopia confocal. Em monocamada (2D), as drogas tiveram maior efeito quando utilizadas com o mitotano 10 mM, sendo: everolimus 10 mM (50±0,02% p &#8804 0.001), imatinib (10 mM 52±0.01% p &#8804 0.001), sunitinib 5 mM (47±0.02% p &#8804 0.001), e nilotinib 5 mM (59±0,03% p &#8804 0.001), com evidência de apoptose. Nos esferoides (3D) foi necessário mitotano 30 M com everolimus 10 mM para se obter diminuição de 32±0.02% p &#8804 0.001 na viabilidade das células, e com nilotinib 10 mM para redução de 57±0.03% p &#8804 0.001, com evidência de necrose e apoptose. Em resumo, os dados sugerem que os esferoides são mais resistentes aos tratamentos, como ocorre com tumores in vivo, e podem representar uma importante abordagem para estudos de ACC. O nilotinib foi o que induziu as melhores respostas, tanto no modelo em 2D quanto 3D, resultados que se apresentam promissores para o tratamento dos carcinomas adrenocorticais. / Adrenocortical carcinoma (ACC) is a rare malignant tumor. The objective was to test the antitumor drugs action in the H295R cell line, in 2D and 3D culture system, by using MTS, multiparameter assay (High Content Screening) and confocal microscopy. In monolayer, all drugs were more effective in combination with mitotane 10 mM: everolimus 10 mM (50±0.02%), 10 mM imatinib (52±0.01%), 5 mM sunitinib (47±0.02%), and 5 mM nilotinib (59±0.03%). The spheroids required mitotano 30 mM with everolimus 10 mM to decrease cell viability (32±0.02% p &#8804 0.001); and with 10 mM nilotinib to inhibit cell viability in 57±0.03% (p &#8804 0.001), with induction of apoptosis and necrosis. In summary, the spheroids were more resistant to treatment and may represent an important approach for studies of ACC. Nilotinib was the tyrosine kinase inhibitor that, either alone or in combination with mitotane, induced higher cytotoxicity, in both 2D and 3D cell cultures, showing promising results for the treatment of adrenocortical carcinoma, as well as for the studies of its mechanism of action.

Orientador: Willian Fernando Zambuzzi / Resumo: Com o aumento progressivo da expectativa de vida da população, defeitos ósseos se tornou um problema de saúde pública. Por sua vez, o sistema esquelético abriga um conjunto de células que, de maneira hierárquica, sustentam a formação do osso ao longo da vida, sendo sua capacidade regenerativa comprometida durante o processo de envelhecimento. Com o aumento da expectativa de vida, a população idosa vem crescendo nos últimos anos e com ela o aumento eminente de fraturas ósseas. Sabe-se que o desenvolvimento e regeneração ósseos são eventos complexos e controlados por mecanismos parácrinos de sinalização intercelulares, destacando que a osteogênese está acoplada, principalmente, à angiogênese. Embora relatado, este mecanismo de comunicação entre células endoteliais e células osteoprogenitoras não está bem elucidado, sobretudo considerando o repertório de moléculas tróficas envolvidas. A fim de compreender melhor estes mecanismos, o objetivo deste trabalho foi desenvolver metodologias capazes de mimetizar o microambiente endotelial-ósseo, bem como desvendar eventos acoplados entre os diferentes tipos celulares envolvidos no processo, sobretudo gerando tecido endotelial in vitro, equivalente ao original. Para isso, fizemos uso de células humanas primárias, as quais foram submetidas a diferentes protocolos experimentais, onde a mesma densidade de células endoteliais arterial (HCAEC) e venosa (HUVEC) e de musculatura lisa (AoSMC) (cultivo misto) foi plaqueada em tubos cônicos sem tr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: With the progressive increase in the life expectancy of the population, bone defects became a public health problem. In turn, the skeletal system houses a set of cells that, in a hierarchical way, support the formation of the bone throughout life, and its regenerative capacity is compromised during the aging process. With the increase in life expectancy, the elderly population has been increasing in recent years and with it the eminent increase of bone fractures. It is known that bone development and regeneration are complex events and controlled by paracrine mechanisms of intercellular signaling, emphasizing that osteogenesis is mainly coupled with angiogenesis. Although reported, this mechanism of crosstalk between endothelial cells and osteoprogenitor cells is not well elucidated, especially considering the repertoire of involved trophic molecules. In order to better understand these mechanisms, the objective of this work was to develop methodologies capable of mimicking the endothelial-bony microenvironment, as well as unveiling the coupled events between the different cell types involved in the process, especially generating endothelial tissue in vitro, equivalent to the original one. For this, we used primary human cells, which were submitted to different experimental protocols, where the same density of arterial (HCAEC) and venous (HUVEC) endothelial cells and smooth muscle cell (AoSMC) (mixed culture) were plated in tubes (s), and after 72 hours the generated spheroid... (Complete abstract click electronic access below) / Mestre

Within solid tumours, a microenvironment exists that causes resistance to chemotherapy. New drugs that target cells within this microenvironment are required, the first step in this process being the identification of new targets. The aim of this thesis was to characterise changes in the transcriptome and proteome within specific regions of multicell-tumour spheroids (MCTS), an experimental model that mimics many of the features of the tumour microenvironment. HT29 MCTS were separated by sequential trypsinisation into 3 main regions; the outer surface layer (SL) the peri-necroric region (PN) and the necrotic core (NC). Using an iTRAQ quantitative proteomics approach, the proteome of the different MCTS regions was investigated. A 2 dimensional separation approach using Agilent's OffGel system and RP-nano HPLC was incorporated prior to MS analysis. MS analysis was done using both MALDI-TOF-TOF (Bruker Ultraflex II) and ESI-Q-TOF (Agilent 6530 QTOF LC/MS) instruments. Gene expression profiles of the different MCTS were investigated and compared using Agilent's one-color oligonucleotide based microarrays. Transcriptomic and proteomic analysis identified several key differences in the proteins involved in cell metabolism between the SL and PN/NC regions. Similar metabolic changes were also noted between autophagic and normal monolayer cells. Many highlighted proteins represented established cancer associated proteins. Interestingly, a number of proteins were highlighted which have no previous association with cancer and may upon further validation, provide attractive leads for therapeutic intervention.