It has been repeatedly proven that cell therapies can address many current unmet clinical treatment needs and also improve on current treatment options for various diseases, from neurological disorders to bone repair (Rosset et al. 2014; Corey et al. 2017). Though the potential of cell therapies has been demonstrated at a relatively small scale, the realisation of bringing cell based treatments to a larger market is hindered by the complexity of the product along with safety concerned and high production cost. Safety concerns can be informed with more in-depth analytical analysis of the product, however this in turn increase the costs involved in producing a cell therapy (Davie et al. 2012). Consequently the cost of analytical techniques also needs to be reduced, to address this need the area of microfluidic based bioanalytics holds much promise (Titmarsh et al. 2014). The culturing of human mesenchymal stem cells (hMSC) was used as a proof of concept model to demonstrate where improved bioanalytical and bioassay methods could be utilised in the production of cell therapies. Cells from four donors were cultured under three different oxygen environments and the conditioned medium assessed for pro-angiogenic capabilities using a tube formation bioassay and a proportion of the cytokine secretome profile measured using Luminex technology. Thorough secretome analysis it was shown that predicting cytokine levels based solely on the donor was not possible as the handling of the cells also had an influence on the secretome profile. The donor expression profiles did not behave in the same manner across all oxygen environments, for example in some donors IL-8 levels increased per cell at lower oxygen where as other donors showed a decrease per cell. While the tube formation assay showed some differences between donors in pro-angiogenic capabilities it also highlights the challenges with interpreting large data sets. The feasibility of using a microcapillary film (MCF) based enzyme-linked immunosorbent assay (ELISA) to detected two relevant cytokines, IL-8 and hepatocyte growth factor (HGF) was investigated. Following on from this work the development of a combined MCF ELISA assay with hMSC cell culture to produce a fully closed cell screening system was initiated. It was shown that it was feasible to measure IL-8 and HGF using the MCF ELISA platform but further work would need to be done to make the system more compatible with the manufacturing environment. In order to adapt the MCF to also be an hMSC culture platform the first challenge was to functionalise the Fluorinated Ethylene Propylene (FEP) surface of the MCF. It was concluded that a poly (vinyl- alcohol) (PVA) and gelatin mixture produced a homogenous coating to which a consistent level of hMSC would attach. This work was carried out on a flat surface; therefore steps were taken to adapt this knowledge into the MCF, while there was evidence of hMSCs present inside the MCF more work will need to be done to bring this concept to an established platform.

Orientador: Marco Antonio Alvarenga / Banca: Rogério Martins Amorim / Banca: Cláudia Barbosa Fernandes / Banca: Márcio Teoro do Carmo / Banca: José Antonio Dell'Aqua Junior / Resumo: O trabalho teve por objetivo avaliar a eficiência da terapia celular no tratamento do endométrio de éguas com processo degenerativo crônico caracterizado pela presença de fibrose uterina. Foram utilizadas 10 éguas, de raça Quarto de Milha com idade entre 14 e 23 anos, com massa corpórea entre 400 a 600 Kg, e bom escore corporal com históricos reprodutivos de subfertilidade e grau severo de fibrose uterina detectada previamente pelo exame histológico realizado por meio da biópsia endometrial. O material foi coletada no dia zero (D0) antes do tratamento com células tronco mesenquimais (CTMs) e 15 dias (D15), 30 dias (D30) e 60 dias (D60) após o tratamento. As amostras foram classificadas segundo o modelo proposto por Kenney & Doig (1986) em grau de fibrose: Grau I - normal, Grau IIA - inflamação crônica leve, Grau IIB - inflamação crônica com infiltrado moderado, Grau III - inflamação crônica degenerativa grave. Estas amostras foram submetidas à confecção de lâminas para coloração de Tricrômio de Masson, Coloração de Hematoxilina e Eosina (HE) e Imunohistoquímica. A punção da medula óssea foi realizada em 10 éguas com idade entre 14 e 23 anos e o material coletado foi submetido ao Laboratório. O cultivo celular in vitro foi realizado em meio DMEM alta glicose em estufa com 37,5°C, em atmosfera úmida contendo 95% de ar e 5% de CO2. Após atingir a confluência de 80% na placa de cultivo, estas células foram removidas e injetadas intra-endometrial com uma concentração de 2x107 células\mL em um volume de 0,5 ml em 20 (vinte) diferentes pontos (sitio de administração) espaçados entre um centímetro, com o auxílio de endoscópio flexível, totalizando um volume de 10 mL aplicado. Nas avaliações realizadas, verificou-se diminuição significativa dos ninhos glandulares, mudança siginificativa de células... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The study aimed to evaluate the efficiency of cell therapy in the treatment of endometrial mares with chronic degenerative process characterized by the presence of uterine fibroids. We used 10 mares bred Quarter Horses aged between 14 and 23 years, with body mass between 400 and 600 kg, with good body and reproductive history of subfertility and severe degree of uterine fibrosis detected previously by histological examination performed by endometrial biopsy. The material was collected on day zero (D0) before treatment with mesenchymal stem cells (MSCs) and 15 days (D15), 30 days (D30) and day 60 (D60) post-treatment. The samples were classified according to the model proposed by Kenney & Doig (1986) in fibrosis: Grade I - normal, Grade IIA - mild chronic inflammation, IIB Grade - moderate chronic inflammatory infiltrate, Grade III - severe degenerative chronic inflammation. These samples were submitted for preparation of slides for staining Masson's trichrome, hematoxylin and eosin staining (HE) and immunohistochemistry. A bone marrow puncture was performed in 10 mares aged between 14 and 23 years and the collected material was submitted to the Laboratory. The in vitro cell cultivation was performed in DMEM with high glucose kiln 37.5 ° C in humidified atmosphere containing 95% air and 5% CO2. After reaching 80% confluence on plate culture, these cells were removed and injected intra-endometrial at a concentration of 2x107 cells \ ml in a volume of 0.5 ml for 20 (twenty) different point (site of administration) spaced of an inch, with the aid of flexible endoscope, for a total volume of 10 ml applied. In the assessments, there was a significant reduction in glandular nests, change polymorphonuclear cell siginificativa absent at baseline (D0) to moderate 60 days (D60); amendment of mononuclear cells in going from... (Complete abstract click electronic access below) / Doutor

Embryonic stem (ES) cells have the capacity to form any cell type. However, their propagation and differentiation is limited by current two dimensional (2D) culture techniques which offer little flexibility in terms of surface structure and functionalisation with bioactive molecules. The aim of the current work was to produce a novel scaffold that could manipulate ES cell behaviour using both architectural and biological cues. Electrospinning is a flexible technique that creates nonwoven meshes that mimic the fibrous architecture of the ECM. Initial work focused on investigating the suitability of electrospun poly(lactic-co-glycolic acid) (PLGA) meshes for 2D and three dimensional (3D) culture of mouse ES cells, with the hypothesis that the fibrous architecture would assist in maintaining pluripotency. The study also sought to functionalise the scaffolds with biologically active molecules. Heparan sulphate proteoglycans (HSPGs) reside at the cell surface and within the ECM where they mediate growth factor binding, assist cell attachment and stabilise the ECM. Furthermore, ES cells modulate their own microenvironment by controlling the composition of heparan sulphate (HS), regulating the binding of growth factors such as fibroblast growth factor (FGF) family members. Therefore, we aimed to immobilise HS and heparin (a highly sulphated structural analogue of HS) on the fibre surface in a form that was freely accessible for protein/cell interactions and that retained its biological activity. Electrospinning parameters were optimised to produce microfibre electrospun meshes with an average fibre diameter of 570nm. Cell morphology, proliferation and pluripotency were monitored using an Oct4-GFP reporter cell line and results compared with flat spin coated films. To investigate the potential for 3D culture, spinning parameters were altered to increase fibre diameter to >3micro metre with infiltration assessed using pro-migratory E-cadherin-/- ES cells. Scaffolds were coated with plasma polymerised allylamine (ppAm) to enable non-covalent immobilisation of HS/heparin. Ligand binding assays with the link module of TSG-6 and anti-heparin/HS antibodies were used to probe HS/heparin presentation on the fibre surface. The biological activity of the immobilised HS/heparin was analysed by testing the ability of coated scaffolds to rescue the neural differentiation capacity HS deficient EXT1-/- ES cells. Finally, human ES cells were cultured on the surface of ppAm scaffolds +/- HS in both unconditioned and mouse embryonic fibroblast (MEF) conditioned media for 5 days. Both microfibre meshes and flat spin coated films supported the attachment, growth and pluripotency of mouse ES cells. Cells adopted distinct morphologies, with mouse ES cells aggregating in rounded colonies on microfibre scaffolds and demonstrating increased spreading on spin coated films. Fibres >3micro metre created a thicker mesh with potential for 3D culture supporting the infiltration of E-cadherin-/- ES cells. ppAm enabled non-covalent immobilisation of HS/heparin in a form that was free to participate in protein interactions and which presented essential sulphation motifs within the HS/heparin chains. Bound HS was biologically active and functioned in synchrony with FGF4 to enhance neural differentiation of EXT1-/- ES cells. The constructs also supported the attachment and growth of human ES cells, with HS functionalised scaffolds demonstrating a slight increase in compatibility during culture in unconditioned media. The successful functionalisation of electrospun meshes with HS/heparin creates a highly versatile scaffold for ES cell culture and differentiation. The architecture of the meshes can be manipulated to either serve as a fibrous substrate for maintenance of pluripotency or support the formation of complex cell interactions present in vivo. The immobilisation of HS provides an extra dimension of versatility, as the scaffold can be tailored with specific HS species, potentially enabling the differential regulation of growth factor binding.

The timing of differentiation underlies the development of any organ system. In neural development, the expression of the transcription factor Hes1 has been shown to be oscillatory in neural progenitors, but at a low steady state in differentiated neurons. This change in the dynamics of expression marks the timing of differentiation. We previously constructed a mathematical model to test the experimental hypothesis that the topology of the miR-9/Hes1 network and specifically the accumulation of the micro-RNA, miR-9, could terminate Hes1 oscillations and account for the timing of neuronal differentiation, using deterministic delay differential equations. However, biochemical reactions are the result of random encounters between discrete numbers of molecules, and some of these molecules may be present at low numbers. The finite number of molecules interacting within the system leads to inherent randomness, and this is known as intrinsic stochasticity. The stochastic model predicts that low molecular number causes the time to differentiation to be distributed, which is in agreement with recent experimental evidence and considered important to generate cell type diversity. For the exact same model, fewer reacting molecules causes a decrease in the average time to differentiation, showing that the number of molecules can systematically change the timing of differentiation. Oscillations are important for a wide range of biological processes, but current methods for discovering oscillatory genes have primarily been designed for measurements performed on a population of cells. We introduce a new approach for analysing biological time series data designed for cases where the underlying dynamics of gene expression is inherently noisy at a single cell level. Our analysis method combines mechanistic stochastic modelling with the powerful methods of Bayesian nonparametric regression, and can distinguish oscillatory expression in single cell data from random fluctuations of nonoscillatory gene expression, despite peak-to-peak variability in period and amplitude of single cell oscillations. Models of gene expression commonly involve delayed biological processes, but the combination of stochasticity, delay and nonlinearity lead to emergent dynamics that are not understood at a theoretical level. We develop a theory to explain these effects, and apply it to a simple model of gene regulation. The new theory can account for long time-scale dynamics and nonlinear character of the system that emerge when the number of interacting molecules becomes low. Both the absolute length and the uncertainty in the delay time are shown to be crucial in controlling the magnitude of nonlinear effects.

Periosteum and bone marrow (BM) contain cells that, after isolation and culture-expansion, exhibit properties of mesenchymal stromal/stem cells (MSCs). However, these cells have not been identified and characterised in situ due to the lack of specific markers. This study aimed to identify and phenotypically characterise long-term label-retaining cells (LT-LRCs), thought to include stem cells (SCs), in mouse periosteum and BM. Two mouse models were used: nucleoside-analogue labelling, and doxycycline (Dox)-inducible expression of histone 2B–green fluorescent fusion protein (H2B-GFP). LRCs were identified and phenotypically characterised by immunostaining, and microscopy or by flow cytometry (FCM). LRCs were detected throughout the periosteum with no apparent focal concentration, and subsets of cells displayed a phenotype compatible with MSCs but not pericytes. Osteoblasts were also labelled, but osteocalcin-expressing osteoblasts were distinct from Low-affinity nerve growth factor receptor (LNGFR)/P75-expressing MSCs. Similarly, BM contained LRCs expressing MSC markers that were distinct from pericytes. For FCM analyses, two cell isolation methods were compared, which revealed that crushing and collagenase digestion of long bones yielded a higher percentage of LRCs compared with flushing. BM analysed 40 days after the end of nucleoside administration showed that LRCs both within the CD45- and CD45low population were enriched for cells expressing Platelet-derived growth factor receptor α (PDGFRα) together with Stem cell antigen-1 (Sca-1) as well as cells expressing LNGFR/P75+. Furthermore, the CD45-PDGFRα+Sca-1+ population showed an increase in the percentage of LRCs with an increasing washout period, suggesting PDGFRα together with Sca-1 is most suitable to identify stromal LRCs in mouse BM. Comparison of the nucleoside label-retaining model with the H2B-GFP-label-retaining transgenic model showed a good correlation between nucleoside and H2B-GFP-label retention, suggesting the suitability of the H2B-GFP model for identification of stromal LRCs in BM. Future studies characterising the MSC niche in-vivo could reveal novel therapeutic targets for promoting bone regeneration/repair.

Elongation of the mouse anteroposterior axis depends on stem cell-like axial progenitors including a neuromesodermal (NM) bi-fated population existing in the primitive streak and later in the tail bud. Fate mapping experiments have demonstrated these NM progenitors reside in precise locations of the embryo. At E8.5, these cells are found in the node-streak border (NSB) and anterior epiblast on either side of the primitive streak. At tail bud stages (E10.5-E13.5), these progenitors reside in the chordoneural hinge (CNH). The coexpression of the transcription factors T (brachyury) and Sox2 has been proposed as a good marker to identify NM progenitors in vertebrates. However, this cell signature has never been thoroughly assessed during mouse axis elongation. In this thesis, I performed T and Sox2 double immunofluorescent stainings on different stages of mouse embryos and reconstructed their expression domains in the 3D images to investigate the spatiotemporal dynamics of NM progenitors during axis elongation. The results show the transient existence of T+Sox2+ cells in the posterior progenitor zone, from the headfold stage (E8.0) to the end of axis elongation (E13.5, 65somites). Moreover, the number of T+Sox2+ cells increases between E8.5 and E9.5 but gradually declines afterwards. I then investigated the time points for initiation and loss of NM progenitors by performing a series of heterotopic grafting experiments. It has been previously shown that distal epiblast (Sox2+T- cells) at LS-EB stages (E7.5) are fated to become NSB cells in E8.5 embryos. However, when cells from the distal region of LS-EB stage embryos (E7.5) were grafted to E8.5 NSB, these cells contribute extensity to the notochord but not either neural tissues or paraxial mesoderm. This indicates that NM progenitors may be not yet specified before the onset of T and Sox2 coexpression, while the notochord progenitors are already specified at E7.5. The grafting experiments also show the loss of NM progenitors at E14.5 after the end of axis elongation, which coincides with the disappearance of T+Sox2+ cells in the tail. Collectively, these results indicate that T+Sox2+ cells may represent a distinct cell state that defines NM progenitors. Wnt/β-catenin signalling has been shown to play an important role in maintaining the posterior progenitor zone. However, due to the wide expression of β-catenin and the early lethality of β-catenin null embryos, the exact effect of losing β-catenin in NM progenitors is still unknown. In this study, I took advantage of the Cre-ERT2 system and grafting technique to conditionally delete β-catenin specifically in NM progenitors during ex vivo culture. The results show that Wnt/β-catenin signalling is required cell autonomously for initiating mesoderm fate choice in NM progenitors. In its absence, mesoderm fated NM progenitors convert their fate and differentiate to neural derivatives. Moreover, the interchangeability between neural and mesodermal fate only exists in NM progenitors, as the loss of β-catenin in mesoderm committed progenitors does not affect their fate choice. Using image analysis and quantification software, I also show that Wnt/β-catenin signalling is crucial for the expansion of T+Sox2+ NM progenitors during axis elongation. Due to difficult access and a limited number of NM progenitors in vivo, in vitro generated NM progenitors from pluripotent cells, such as epiblast stem cells (EpiSCs), can offer an insight into the maintenance and differentiation of NM progenitors. Since the in vivo potential of EpiSCs had never been successfully demonstrated before, I first grafted EpiSCs into postimplantation embryos and cultured them ex vivo for 24-48 hours to assess their cell integration. The results show that EpiSCs can integrate successfully in streak stage embryos (E6.5-E7.5), but not at early somite stages (E8.5), when the epiblast has lost its pluripotency. I then further investigated the in vivo potential of EpiSC derivatives. The results show that increasing Wnt signalling in EpiSCs inhibits their ability to generate anterior neural tissues in vivo, which is consistent with the previous in vitro data. Recently, it has been demonstrated that NM progenitors can be derived from EpiSCs. These in vitro derived NM progenitors can incorporate into E8.5 embryos and give rise to both neural and mesodermal derivatives. In this thesis, I show that these in vitro derived NM progenitors do not incorporate successfully in E7.5 embryos. Collectively, by combining grafting experiments with a chimeric embryo formation assay, I can identify the in vivo stage of the in vitro counterparts of the embryonic cell types.

Contrairement aux mammifères adultes, l’amputation d’un membre de vertébrés capables de régénérer, est suivie de la formation d’une structure hautement proliférative et hétérogène : le blastème. Les conditions de formation de ce blastème sont encore mal connues. La sécrétion de facteurs par les cellules dérivées de la crête neurale CCN, seraient à l’origine de la prolifération du blastème. De plus, les macrophages sont recrutés sur le site d’amputation et participeraient à la régénération mais leurs mécanismes d’actions et interactions avec les CCN n’ont jamais été étudiés dans ce contexte. Mon projet de thèse avait pour but d’élucider ces mécanismes en s’appuyant sur deux modèles de régénération : la régénération de la nageoire chez la larve de zebrafish et le membre supérieur de l’embryon de souris au stade E10.5. Ces travaux ont permis :• Chez la larve de zebrafish : d’identifier deux sous-types de macrophages recrutés de manière séquentielle au cours de la régénération, de montrer que l’activation de la voie TNFa/TNFR1 par les macrophages était nécessaire à la prolifération du blastème, d’identifier une population de CCN foxd3+ dans la nageoire dont la présence est indispensable au recrutement et à la polarisation des macrophages ainsi qu’à la prolifération des cellules du blastème.• Chez l’embryon de souris : d’identifier un stade régénératif (E10.5) et non régénératif (E12.5), de montrer l’accumulation de CCN au niveau du site d’amputation au stade E10.5 et de démontrer le rôle de ces cellules dans le processus de régénération. / Unlike in adult mammals, in regenerative species, appendage amputation is followed by the formation of a highly proliferative and heterogeneous structure called the blastema. The required conditions for its formation are still not completely understood. Paracrine factors produced by neural crest derived cells (NCC) have been proposed to be responsible for blastemal cell proliferation. Moreover, macrophages are recruited to the wound site and could participate to the regeneration process. However, their exact functions and interactions with NCC during regeneration have never been investigated. My thesis project consisted in deciphering those mechanisms using two different models: zebrafish larva caudal fin regeneration and forelimb bud regeneration of the E10.5 mouse embryo. This work allowed us:• In zebrafish larva: to identify two subpopulations of macrophages, to highlight their roles during regeneration, to demonstrate the role of the TNFa/TNFR1 axis in the blastemal cell proliferation and to identify a new foxd3+ NCC population in the caudal fin, which is required for macrophage recruitment, polarization and for blastemal cell proliferation. •In mouse embryo: to identify a regenerative (E10.5) and non-regenerative (E12.5) stage of development, to demonstrate the accumulation of NCC at the wound site in E10.5 embryos and demonstrate the crucial role of NCC during epimorphic regeneration in mammals.

Submitted by Luciane Willcox (luwillcox@gmail.com) on 2016-09-01T16:51:25Z No. of bitstreams: 1 TESE AXEL.pdf: 13901759 bytes, checksum: 6f744b4d3c67d9fcc80db57c1c5944a7 (MD5) / Approved for entry into archive by Luciane Willcox (luwillcox@gmail.com) on 2016-09-01T17:02:42Z (GMT) No. of bitstreams: 1 TESE AXEL.pdf: 13901759 bytes, checksum: 6f744b4d3c67d9fcc80db57c1c5944a7 (MD5) / Made available in DSpace on 2016-09-01T17:02:42Z (GMT). No. of bitstreams: 1 TESE AXEL.pdf: 13901759 bytes, checksum: 6f744b4d3c67d9fcc80db57c1c5944a7 (MD5) / Messenger ribonucleoprotein particles (mRNPs) são complexos formados por RNA mensageiro (mRNA) e um pool de diferentes proteínas que se ligam diretamente ou indiretamente ao mRNA. Proteínas diferentes podem formar diferentes mRNPs com diferentes funções. Agregação de mRNPs formam grânulos que são visíveis ao microscópio. Dois desses grânulos comumente encontrados em células eucarióticas são os processing bodies (PB) e os grânulos de estresse (GE) possuem função fundamental na regulação pós-transcricional, mais especificamente, na degradação (PB) e na estocagem (PB e GE) do mRNA. Enquanto os PB são constitutivamente encontrados, GE são vistos somente em condições de estresse. TTP e RCK são componentes presentes em ambos os grânulos enquanto que TIA1 e TIAR são exclusivos de GE e DCP2 exclusivo de PB. Human Adipocyte Derived Stem Cells (ADSCs) são células-tronco adultas multipotentes com capacidade de autorrenovação e diferenciação em células de diversos tecidos. Desde que pouco se sabe sobre a função dos PB e dos GE durante a diferenciação das células-tronco, nosso objetivo é elucidar a dinâmica e função desses grânulos nestas celulas. Foram utilizadas ADSCs derivadas de cirurgia bariátrica e lipoaspiração. Diversos componentes de PB e de GE foram analisados por Imunofluorescência após 1, 4, 7 e 12 dias de diferenciação adipogênica. Oligos de RNA de interferência (siRNA) específicos para RCK, DCP2, TTP, TIAR e TIA1 foram transfectados concomitantemente com a indução à diferenciação adipogênica e as culturas mantidas por 1, 4, 7 e 14 dias de diferenciação. A diferenciação foi mensurada por intensidade de fluorescência após marcação com AdipoRed. GE são ausentes em ADSCs indiferenciadas e durante a diferenciação como determinado por imunomarcação com TIAR que se localiza predominantemente no núcleo. Interessantemente imunomarcação com TIA1/R mostrou que ADSCs possuem grande quantidade de grânulos contendo TIA1/R em células indiferenciadas e não estressadas. Sob estresse oxidativo há um aumento de PB em ADSCs e a formação de GE. Esses grânulos parecem variar de composição, uma vez que eIF4E é ausente e GE imunomarcados com eIF4B são mais perinucleares do que GE imunomarcados com TIAR. PB variam em número e tamanho em células diferenciadas. Notavelmente, em ADSCs possuem poucos PB e até mesmo ausente em algumas células, além disso, TIA1/R e PB significativamente aumentam após 24 horas de diferenciação e após 12 dias o número de PB é similar a células diferenciadas enquanto que os grânulos de TIA1/R praticamente desaparecem. siRNA de componentes essenciais de PB e GE em ADSCs indiferenciadas (tempo 0) mostrou que há um aumento significativo na diferenciação em adipócito após 4, 7 e 12 dias de diferenciação adipogênica. Interessantemente esse aumento se deu pela maturação do adipócito, i.e. tamanho da vesícula de lipídio e não pelo aumento da quantidade de células diferenciadas. Nossos dados mostram que PB estão em baixo número e que existem grânulos de TIA1/R citoplasmárico nunca antes mostrados em ADSCs indiferenciadas. Ainda, esses grânulos aumentam durante a diferenciação e parecem exercer um papel importante na maturação dos adipócitos uma vez que ensaios de siRNA de componentes de PB e GE levam a uma diferenciação mais acelerada. Também, os GE são formados após indução a estresse oxidativo e esses grânulos são praticamente ausentes de eIF4E mostrando que a composição desses grânulos varia em relação a células diferenciadas.

O avanço da ciência regenerativa está se comprometendo com a busca de novas opções terapêuticas no combate as diversas doenças e disfunções orgânicas. Nessas avançadas linhas de pesquisas, as células-tronco são um símbolo dessa evolução. A variedade da aplicação deste novo e experimental método de tratamento está sendo utilizado, pela bioengenharia, para reparar tecidos e órgãos lesados. Defeitos ósseos extensos ocorrem após diversos tipos de injúrias ao esqueleto facial, como traumas faciais, ressecções por lesões agressivas e malformações congênitas. Essas sequelas são tratadas, preferencialmente, através da reconstrução utilizando enxertos ósseos de origem autógena. Entretanto, às desvantagens proporcionadas pela obtenção do tecido ósseo autógeno, lançaram um dos maiores desafios, da bioengenharia que é a busca pelo aprimoramento dos substitutos ósseos. Nesse caminho do aprimoramento dos biomateriais a adição de células-tronco mesenquimais representam a possibilidade da criação de um sinergismo, entre as células e o arcabouço, para otimizar a osteogênese. Nessa linha, esse estudo propõe-se a avaliar o reparo ósseo em estudo experimental, em modelo animal, através do tratamento de defeito ósseos criados em fêmures de ratos. A amostra dessa pesquisa foi composta de 96 ratos albinos da espécie Rattus novergicus albinus, linhagem SHR (Spontaneously Hypertensive Rats), isogênicos. Os animais foram divididos aleatoriamente em quatro grupos de acordo com o tipo de tratamento (Grupo I: α – TCP + ADSCs; Grupo II: α – TCP + ADSCs/ENDO; Grupo III: α – TCP; Grupo IV). A cultura de células tronco tiveram como tecido de origem o tecido adiposo da região abdominal e as células endoteliais formam coletadas da medula óssea. A peças foram avaliadas em 03 períodos de tempo diferentes (07, 14 e 21 dias). A histomorfometria avaliou a área de neoformação óssea dos defeitos bem como a imunohistoquímica com marcação para a proteína VEGF avaliou a eficácia da adição das células endoteliais. Os resultados demonstraram, através dos testes estatísticos que houve uma diferença estatisticamente significante, no reparo ósseo, favorecendo os tratamentos dos defeitos que utilizaram a terapia celular e a vascularização foi também otimizada no grupo que foi tratado com a adição das células endoteliais. Dessa forma conclui-se que nesse modelo de estudo a utilização das ADSCS foram capazes de otimizar o reparo ósseo. / The regenerative medicine has been searching for a new therapeutic options to manage diseases and also organic dysfunctions. The advanced research fields, has enrolled the stem cells to achieve the upgrade in the new treatment objectives. The bioengineering is application of this new and experimental, treatment method to repair damaged tissues and organs using the stem cells, includes the possibility to acelerating and improving the bone repair process. The autogenous bone graft has been considered the gold standart graft material to reconstruction the bone defects, fundamentally because of the osteogenic potential. However, the harvest disadvantages autogenous bone tissue leads to the search for bone substitutes improvement. In this field a promising alternative has been proposed by tissue engineering. The totipotent cells, also called mesenchymal stem cells, which has the cellular plasticity ability, is associated with biomaterials, creating a synergy, between these cells and the scaffold, to optimize the osteogenesis. The tissue engineering application to tissue repair has been extensively researched with the aim of proposing more reliable and more efficient clinical methods. Although the effects of the use of adult stem cells are well known in bone marrow transplants, in some areas, such as bone repair, there is still lack of scientific data. This research was conducted, in animal model, to assessment bone repair in created femural bone defects treated with mesenchymal stem cells. 96 animals (Rattus novergicus albinus - Spontaneously Hypertensive Rats) were randomly divided into four groups (Group I: α - TCP + ADSCs, Group II: α - TCP + ADSCs/Endo, and Group III: α - TCP; (07, 14 and 21 days). The histological sections were stained in H&E and the histomorphometry was used to evaluated the new bone formation area in the defects and also the immunohistochemical expression of VEGF was analysed. Our results suggest that the combination of ADSCs and the scaffold was able to enhance the bone repair in this study model.

Notch signalling is an evolutionary highly conserved signalling pathway. It plays various important roles in the regulation of many fundamental cellular processes such as proliferation, stem cell maintenance and differentiation during embryonic and adult development. Notch signalling has a simple transduction pathway. Upon Notch ligand binding to the receptor, the Notch intracellular domain (NICD) is released into the nucleus. The nuclear NICD interacts with the DNA-binding protein Suppressor of Hairless (Su(H)) to activate the expression of target genes, which are silenced by the Su(H)-corepressor complex in the absence of Notch activity. The functions of Notch are very context-dependent, making it important to identify the Notch regulated genes in different processes. Neural stem cells (NSCs) are cells that can divide and differentiate into all kinds of cells within the brain while they self-renew. Notch signalling is one of the key regulators in maintaining NSCs and performs a similar function in both Drosophila and vertebrate NSCs. Drosophila NSCs serve as an ideal model for studying the relationship between Notch function and stem cell behaviours. Although many target genes, such as the Hes genes, have been identified, they cannot fully account for the diversity of Notch responses. Therefore, further functional study of more potential target genes is needed to gain understanding about Notch-regulated NSC maintenance. In this thesis, a group of potential direct Notch target genes are examined for their responsiveness to Notch regulation and their functions in Drosophila NSCs. Previous genome-wide study in the Bray lab has found a number of potential Notch target genes in the Drosophila larval brain, with the characteristics of Notch transcription factor Su(H) binding and mRNA upregulation by Notch over-activation (Zacharioudaki et al. 2016). I first examined the Notch responsive element (NRE) activity of these potential Notch targets and their regulation by Notch both in vivo and in cell lines. The presented findings validated path, cables and Asph as direct Notch target genes in Drosophila NSCs, while syp, lola and Fer2 do not exhibit characteristics of Notch responsive targets in NSCs. The functional roles of two of the responsive genes, path and cables, were subsequently explored in Drosophila larval brains. Firstly, I found that Path, a potential amino acid transporter, is not only important for protecting NSC proliferation under normal and abnormal conditions through integrating growth pathways, but is also required for protecting brain growth under nutrition deprivation. Secondly, the cables gene was connected to a distal NRE through knocking out the suspected NRE region and the gene itself using the CRISPR/Cas9 technique. Subsequent experiments revealed that cables is also required for NSC proliferation. In summary, a group of direct Notch target genes were validated and as a consequence two genes that are important for protecting NSC proliferation were identified.