The purpose of this study was to rationally design animal component free, chemically defined serum free media (ACF-CD-SFM) for MRC-5 and Vero cells while adhering to the Quality by Design guidelines. This was achieved by using the Modified Vero Serum Free Medium (MVSFM) as the basal formulation and supplementing it with various combinations of growth factors (LONG® EGF, LONG® R3 IGF-I, rTransferrin, bFGF, TGF-3 and PDGF-AA), lipids (linoleic acid, cholesterol, and dexamethasone), lipid precursors (ethanolamine and phosphoethanolamine) and vitamins (all-trans retinoic acid, -tocopherol and ascorbic acid). Media development was achieved by conducting a series of steps using different experimental methodologies with the end goal of satisfying the requirements of each cell line. MRC-5 and Vero cells were each cultured in specific media containing unique concentrations of supplements that were prepared according to the different statistical design methodologies. The original objective was to create a SFM, however due to the stringent nutritious requirements of anchorage dependent cell lines, only a reduction to 0.5% FBS was achieved. For MRC-5 cells, the one-factor-at-a-time (OFAT) generated the Prototype + 0.5% FBS medium. The Definitive Screening Design (DSD) gave rise to the Delta 1 + 0.5% FBS, which was the optimum medium formulation for MRC-5 cells as it had comparable cell yields to DMEM + 10 % FBS. This result was confirmed by the Genetic Algorithms-Hill Climbing (GA-HC) method. In the case of Vero cells, the OFAT and the DSD confirmed that MVSFM + 0.5 % FBS was the most optimal formulation. The morphology in both media for both cell lines was comparable to that in DMEM-10% FBS. It was concluded that the DSD method successfully achieved a reduction of the serum concentration from 10% to 0.5% FBS. / October 2017

Soyabean meal (SBM), the major protein source in feeds in sub-Saharan Africa, is in short supply. The shortage is a major constraint to intensified animal production to meet increased demand hence the dire need to search for alternatives. Kigelia africana, Mumisops zeyheri, Terminalia sericea and Ximenia caffra are indigenous fruit bearing trees (IFBTs) whose seeds’ potential as alternative protein sources in feeds were evaluated. The evaluation consisted of an initial physico-chemical characterization of the seeds followed by determining in vitro the safety of seed oils on cell lines. Based on the physico-chemical and in vitro evaluation, the most suitable seed was selected, defatted and its meal used as a dietary substitute to SBM in the in vivo trials using adult and weanling male Sprague Dawley rats. The T. sericea seed yield was not viable. Chemically K. africana and X. caffra seed demonstrated potential as protein sources in feeds. M. zeyheri seed demonstrated potential as an energy source. The IFBTs seeds oil yield surpassed that of some traditional oilseed crops. Oleic and linoleic acid were the major fatty acids contained in the oils. In vitro, K. africana, M. zeyheri and X. caffra seed oils suppressed Caco-2 and HEK-293 cell proliferation without causing cell death. X. caffra seed, deemed the most suitable, was defatted and its seed meal used in the in vivo trials. In mature rats, dietary substitution of SBM with the defatted X. caffra seed meal did not affect (P > 0.05) dry matter intake, apparent digestibility of nutrients and nitrogen absorption and retention. In weanling rats, the defatted X. caffra seed meal had no effect on termination (body mass at the end of the feeding trial) and empty carcass mass and linear growth of the rats. Metabolic substrate storage, fasting blood glucose concentration and the general health profile of the growing rats were not altered by dietary X. caffra seed meal. The defatted X. caffra seed meal increased the mass of the stomach and small intestine (P = 0.0071; P = 0.0001) of rats on the test diet where a 100% dietary crude protein (CP) from SBM was substituted by CP from the defatted X. caffra seed meal. Defatted X. caffra seed meal could substitute SBM in rat and possibly monogastrics feeds without compromising digestibility, nitrogen balance, growth and general health.

A Terapia Fotodinâmica (TFP) é uma técnica para tratamento de câncer que usa um fotossensibilizador (FS) na presença de luz e oxigênio gerando espécies altamente reativas de oxigênio que levam as células tumorais à morte. <br />Neste trabalho foi realizado um estudo comparativo com três FSs: Photogem&reg; (PG), um derivado de hematoporfirina que está sendo usado em TFD no Brasil; Photodithazine&reg; (PZ), um derivado hidrossolúvel de mono-L-aspartil clorina, que está na fase clínica para aprovação e Hipericina (HY), um pigmento fotoativo encontrado na planta Hypericum perforatum e usado na medicina popular que está sendo considerado como um promissor agente fotodinâmico para o tratamento de tumores. Este estudo utilizou uma Hipericina sintetizada no Brasil e diversos parâmetros para comparar os três FSs: a concentração inibitória média (IC50) em linhagens celulares; a constante de velocidade de fotoxidação da albumina de soro bovino na presença dos FSs e luz determinada pelo decréscimo na fluorescência da BSA em 340 nm; a fotoxidação do ácido úrico acompanhada pelo decréscimo da banda característica do ácido úrico em 290 nm após irradiação na presença dos FSs como uma estimativa indireta do rendimento quântico de formação de oxigênio singlete (&Delta;&Phi;); o rendimento quântico de fluorescência utilizando rodamina B como padrão; a acumulação dos FSs em células em função do tempo de incubação e a estimativa da quantidade de radicais livres formados após irradiação através da técnica de captura de spins. Todos os resultados obtidos evidenciam uma maior eficiência fotodinâmica da HY seguida pelo PZ e depois por Photogem e, portanto sugerem a Hipericina como o FS de maior potencial para utilização em Terapia Fotodinâmica. / Photodynamic Therapy (PDT) is a technique for the cancer treatment that uses a photosensitizer (FS) in the presence of light and oxygen which combined are able to generate highly reactive oxygen species that lead to tumor cells death. <br />In this investigation, a comparative study with three FSs: Photogem &reg; (PG), a hematoporphyrin derivative being used in PDT in Brazil; Photodithazine &reg; (PZ), a soluble derivative of mono-L-aspartyl chlorin, which is in clinical phase for approval and Hypericin (HY), a photoactive pigment found in the plant Hypericum perforatum and used in popular medicine that is being considered as a promising agent for photodynamic treatment of tumors. The present study used a Hypericin synthesized in Brazil and several parameters to compare these three FSs: the mean inhibitory concentration (IC50) in cell lines; the rate constant for the photooxidation of bovine serum albumin in the presence of light and the FSs determined by the decrease in the fluorescence of BSA at 340 nm; the photooxidation of uric acid assessed by the decrease of the characteristic band of uric acid at 290 nm after irradiation in the presence of the FSs as an indirect estimate of the quantum yield of formation of singlet oxygen (&Delta;&Phi;); the quantum yield of fluorescence using rhodamine B as a standard; the accumulation of FSs in cells as a function of the incubation time, and the estimative of the produced free radicals after irradiation by the technique of spin trapping. All the results show a higher photodynamic efficiency of HY followed by PZ and then by Photogem suggesting Hypericin as the FS with the greatest potential for use in Photodynamic Therapy.

As raízes de Pfaffia paniculata (Ginseng brasileiro) são comercialmente encontradas como cápsulas contendo as raízes pulverizadas, misturadas ou não ao extrato etanólico destas raízes. Estas raízes são popularmente recomendadas para vários propósitos, e têm sido utilizadas na terapia contra o câncer pela medicina popular. Os principais componentes encontrados nestas raízes já isolados incluem o ácido pfáffico e os pfaffosideos A, C, D, E e F; estes componentes inibiram o crescimento de células do melanoma B-16, demonstrado em um outro estudo. O objetivo deste estudo foi investigar os efeitos dos extratos etanólico, butanólico ou aquoso das raízes de Pfaffia paniculata sobre o crescimento das linhagens tumorais de células mamárias humanas MCF-7 e SKBR-3, utilizando método colorimétrico (cristal violeta) e quantificação das células que incorporaram bromodeoxiuridina (BrdU). A coloração por laranja de acridina/brometo de etídeo foi utilizada para avaliar morte celular, e as alterações subcelulares foram avaliadas por microscopia eletrônica. O extrato butanolico apresentou efeitos citotóxicos nas linhagens MCF-7 e SKBR-3. Morte celular foi observada pelo tratamento com o extrato butanólico por 1 h; alterações morfológicas foram observadas com 500&micro;g/mL deste extrato, após 24 h de tratamento. Após o tratamento por 48 h com o extrato butanólico nesta mesma concentração, foi observado degeneração de componentes citoplasmáticos e alterações morfológicas sugestivas de morte celular. O tratamento com 1000&micro;g/mL deste extrato levou a profundas alterações citoplasmáticas e nucleares, incompatíveis com a viabilidade celular. O tratamento com o extrato etanólico não causou efeitos significantes no crescimento das células MCF-7 e SKBR-3; o extrato aquoso, por outro lado, estimulou o crescimento das células MCF-7, após o tratamento por 1 h. Estes resultados indicam efeitos citotóxicos exercidos pelo extrato butanólico das raízes de Pfaffia paniculata em linhagens celulares de tumor de mama humano / The roots of Pfaffia paniculata (Brazilian ginseng) are commercially available as capsules containing the powdered roots, mixed or not with the ethanolic extract of the roots. These roots have been populary recommended for many purposes, and have also been used on cancer therapy by folk medicine. The main components found in these roots that have already been isolated include pfaffic acid and pfaffosides A, C, D, E and F; these components inhibited the growth of melanoma B-16 cells, as shown in another study. The aim of this study was to investigate the effects of ethanolic, butanolic or aqueous extract of the roots of Pfaffia paniculata on the growth of human breast tumor cell lines MCF-7 and SKBR-3, using a colorimetric method (crystal violet) and quantification of bromodeoxiuridine (BrdU) positive cells. Acridine orange/ethidium bromide staining was utilized to evaluate cell death, and subcellular alteration was evaluated by electron microscopy. The butanolic extract presented cytotoxic effects in MCF-7 and SKBR-3 cell lines. Cell death was observed following treatment with the butanolic extract for 1 h; morphologic alterations were observed with 500&micro;g/mL of this extract, after 24 and 48 h of treatment. Following treatment for 48 h with the butanolic extract with this same concentration, degeneration of cytoplasmic components and morphological alterations suggestive of cell death were observed. Treatment with 1000&micro;g/mL of this extract lead to profound cytoplasmic and nuclear alterations, incompatible with cell viability. The treatment with ethanolic extract didn´t lead to significant effects on the growth of MCF-7 and SKBR-3 cells; the aqueous extract, on the other hand, stimulated the growth of MCF-7 cells, following treatment for 1 hour. These results point to cytotoxic effects exerted by the butanolic extract from the roots of Pfaffia paniculata on human breast tumor cell lines

As pesquisas na área da implantologia têm buscado desenvolver superfícies que acelerem o processo de osseointegração adjacente ao implante, permitindo a reabilitação funcional e estética precoce. Modificações da topografia e composição química têm sido propostas com o objetivo de modular as funções celulares na interface do tecido ósseo com o implante e, em última análise, aumentar o contato osso-implante. A deposição iônica por plasma é uma modalidade de tratamento utilizada em implantes ortopédicos. Na odontologia, estudos têm avaliado o efeito das diferentes técnicas de nitretação de superfícies de titânio (Ti) sobre eventos iniciais da osseointegração, como a adesão celular, mas não sobre eventos tardios. O presente estudo avaliou o efeito de superfícies de Ti modificadas por deposição iônica por plasma, pelas técnicas de nitretação por cátodo oco, por 1 e 3 h, e nitretação planar, nas expressões do fenótipo e gênica de osteoblastos derivados de osso alveolar de humanos e de calvária de ratos. Foram realizadas análises de morfologia, adesão e espraiamento celular, síntese e atividade de fosfatase alcalina (ALP), mineralização da matriz extracelular e expressão de genes marcadores do fenótipo osteoblástico. A adesão celular e a atividade de ALP, foram maiores nas superfícies de Ti tratadas comparadas à superfície sem tratamento (controle), enquanto a proliferação celular e a produção de matriz mineralizada não apresentaram diferenças estatisticamente significante entre os grupos. Ainda, as superfícies de Ti tratadas aumentaram a expressão gênica de ALP e diminuíram a de osteocalcina (OC) quando comparadas à superfície controle. Os resultados sugerem que a deposição iônica por plasma pelas técnicas de nitretação avaliadas modifica as características físico-químicas da superfície de Ti e promove atraso e aumento da diferenciação osteoblástica quando comparada à superfície de Ti não tratada. / A current goal in implantology research has been the development of titanium surfaces to enhance the process of osseointegration, allowing functional and aesthetic rehabilitation. Modifications on topography and chemical composition have been proposed in order to regulate cell functions of the osteoblasts in contact with the implant surface and ultimately increase the bone-to-implant contact. Plasma ionic deposition is a treatment usually employed in orthopedic implants and in dentistry some studies have shown that different nitriding techniques of titanium (Ti) surfaces may affect the initial events of osseointegration, such as cell adhesion, without evaluating the late ones. This study evaluated the effect of Ti surfaces modified by plasma nitriding combined with hollow cathode, during 1 and 3 h, and planar nitriding, on the phenotype and gene expression of osteoblasts derived from human alveolar bone and newborn rat calvaria. Cell morphology, adhesion and spreading, alkaline phosphatase (ALP) activity and synthesis, extracellular matrix mineralization and gene expression of bone markers were performed. The cell adhesion and ALP activity were higher on treated Ti surfaces compared with untreated (control) ones, while cell proliferation and extracellular matrix mineralization were not affected by the treatments. Also, compared with control Ti surface, the treated ones increased the gene expression of ALP and reduced osteocalcin (OC). The results suggest that the plasma ionic deposition by nitriding technique modifies the physicochemical features of the Ti surface and promotes a delay and a enhancement of the osteoblastic differentiation compared with untreated Ti surface.

O gênero Ranavirus, especialmente a espécie Frog virus 3 (FV3), é considerado uma ameaça crescente às populações de anfíbios em diversas partes do mundo, desencadeando surtos que frequentemente resultam em mortalidade em massa e perdas econômicas substanciais. Neste sentido, propusemos o isolamento de uma estirpe patogênica de FV3-like, associada a surto com alta mortalidade de anfíbios adultos (Lithobates catesbeianus) em uma ranicultura do Estado de São Paulo, Brasil, bem como a caracterização molecular e fenotípica do vírus isolado. No mais, objetivamos verificar a possível indução de morte celular por apoptose por essa estirpe. O isolamento viral foi realizado a partir de fragmentos de órgãos de animais que vieram à óbito, os quais foram inoculados em células BF-2 (Lepomis macrochirus). A técnica de reação em cadeia da polimerase (PCR) foi conduzida com primers específicos e dirigidos para duas regiões altamente conservadas do genoma dos ranavírus: MCP e 53R. O sequenciamento parcial do gene que codifica a proteína principal do capsídeo (MCP) foi realizado, seguido de alinhamento e análise filogenética com outros ranavírus. Outras técnicas diagnósticas, incluindo microscopia eletrônica de transmissão, imunofluorescência indireta e Western blot foram utilizadas na caracterização e confirmação do isolamento. Dois marcadores apoptóticos foram utilizados para investigar a possível ativação da apoptose em células BF-2 infectadas e amostradas de 4 à 16 horas, incluindo a ativação de caspases efetoras e a fragmentação do DNA celular pelo ensaio de TUNEL. Obtivemos o isolamento de uma estirpe de FV3-like com efeitos citopáticos típicos para ranavírus. A PCR confirmou a presença de DNA viral nas culturas de células BF-2 infectadas, com resultados positivos para os oligonucleotídeos direcionados para MCP e 53R. A análise da sequência nucleotídica obtida para MCP revelou alta homologia (99%) com Frog virus 3, espécie-tipo do gênero Ranavirus (família Iridoviridae) e, na reconstrução filogenética, a cepa isolada mostrou ser intimamente relacionada com outros ranavírus detectados no Brasil. As micrografias eletrônicas mostraram partículas icosaédricas em células BF-2 infectadas, com nucleocapsídeo medindo cerca de 150 &eta;m, semelhante aos ranavírus. No ensaio de imunofluorescência indireta, células BF-2 infectadas com o isolado FV3-like, apresentaram marcação de imunofluorescência positiva para MCP, enquanto que MCP foi demonstrada por um ensaio de Western blot, onde observamos um polipeptídeo com massa molecular estimada em 50 kDa. Por fim, verificamos que o isolado FV3-like é capaz de induzir apoptose em células BF-2, uma vez que foram detectadas caspases efetoras em todos os tempos experimentais, sugerindo ser um mecanismo caspase-dependente. A fragmentação do DNA celular, claramente observada em todos os tempos experimentais, confirmou a indução de apoptose pela estirpe brasileira de FV3-like. Os resultados aqui obtidos tornam-se a base para diversos estudos futuros, podendo ainda contribuir como subsídio para a melhor compreensão dos surtos provocados por estes vírus no país. / The genus Ranavirus, especially the Frog virus 3 (FV3) species, is considered a growing threat to amphibian populations in various parts of the world, triggering outbreaks that often result in mass mortality and substantial economic losses. In this sense, it was proposed the isolation of a pathogenic FV3-like strain associated with an outbreak with high mortality of adult amphibians (Lithobates catesbeianus) in a frog farm in the State of São Paulo, Brazil, as well as the molecular and phenotypic characterization of the isolated virus. In addition, we aimed to verify the possible induction of the mechanism of cell death by apoptosis by this strain. Virus isolation was performed from organ fragments of animals that died, which were inoculated into BF-2 cells (Lepomis macrochirus). The polymerase chain reaction (PCR) technique was conducted with specific primers and directed to two highly conserved genome regions of the ranavirus: MCP and 53R. Partial sequencing of the gene encoding the major capsid protein (MCP) was performed, followed by alignment and phylogenetic analysis with other ranaviruses. Other diagnostic techniques, including transmission electron microscopy, indirect immunofluorescence and Western blot were used in the characterization and confirmation of the isolation. Two apoptotic markers were used to investigate the possible activation of apoptosis in infected BF-2 cells and sampled from 4 to 16 hours, including the activation of effector caspases and the fragmentation of cellular DNA by the TUNEL assay. We obtained the isolation of an FV3-like strain with typical cytopathic effects for ranavirus. PCR confirmed the presence of viral DNA in cultures of infected BF-2 cells, with positive results for MCP and 53R oligonucleotides. Analysis of the nucleotide sequence obtained for MCP revealed high homology (99%) with Frog virus 3, type species member of the genus Ranavirus (family Iridoviridae) and, in phylogenetic reconstruction, the isolated strain showed to be closely related to other ranaviruses detected in Brazil. Electron micrographs showed icosahedral particles in infected BF-2 cells, with a nucleocapsid measuring about 150 &eta;m, similar to ranaviruses. In the indirect immunofluorescence assay, BF-2 cells infected with the FV3-like isolate showed positive immunofluorescence labeling for MCP, whereas MCP was demonstrated by a Western blot assay, where we observed a polypeptide with a molecular mass estimated at 50 kDa. Finally, we verified that the FV3-like isolate is able to induce apoptosis in BF-2 cells, since effector caspases were detected at all experimental times, suggesting to be a caspase-dependent mechanism. The fragmentation of cellular DNA, clearly observed at all experimental times, confirmed the induction of apoptosis by the Brazilian FV3-like strain. The results obtained here become the basis for several future studies, and may contribute as a subsidy to better understand the outbreaks caused by these viruses in the country.

A aquicultura é apontada como um mercado estratégico para o desenvolvimento sustentável, produção de alimentos e ampliação de fronteiras inexploradas no Brasil. No entanto, como outros sistemas de produção animal, este setor enfrenta problemas com doenças resultantes de sua intensificação, como os aspectos sanitários da produção e a falta de estrutura para o diagnóstico das principais enfermidades infecciosas. Durante os últimos 20 anos, os vírus da família Iridoviridae, em especial membros do gênero Ranavirus, têm sido responsáveis por epizootias de grande impacto ecológico e econômico, envolvendo um grande número de espécies de peixes, anfíbios e répteis de importância na aquicultura de várias partes do mundo. No entanto, as informações sobre a ocorrência de infecções de peixes e anfíbios causadas por ranavírus no Brasil são limitadas. O presente projeto compreendeu o isolamento em cultivo celular de estirpe de Frog Virus 3-símile, no Estado de São Paulo, confirmada por sequenciamento nucleotídico do gene MCP e subsequente RFLP, assim como caracterização fenotípica e de cinética de replicação do isolado. Amostras de fígado, baço e rins de rãs-touro gigante provenientes de ranário comercial, positivas ao diagnóstico molecular para Ranavirus, foram utilizadas para isolamento viral em cultivo celular. Efeito citopático foi detectado na segunda passagem em células BF-2 e posterior confirmação do isolamento foi feita por PCR e RFLP. A caracterização fenotípica viral foi feita com microscopia eletrônica de transmissão, a qual permitiu a confirmação de morfologia semelhante às partículas de Ranavirus, além da realização de curva de replicação, indicando maiores títulos virais no quarto dia após inoculação. Também foi feito teste de susceptibilidade a solventes, que confirmou a presença de partículas envelopadas. Os resultados obtidos nesse estudo poderão contribuir para a futura compreensão da biologia dos iridovírus circulantes como agentes etiológicos de ranaviroses em anfíbios no Estado de São Paulo. / Aquaculture is appointed as a strategic market for sustainable development, food production and expansion of unexplored frontiers in Brazil. However, just like other animal production systems, this area faces problems due its intensification, like the production sanitation aspects and the lack of structure for the diagnosis of major infectious diseases. During the last 20 years, viruses from Iridoviridae family, especially Ranavirus genera, have been responsible for major economic and ecological impactant epizootic diseases in a great number of fish, amphibian and reptile species in many parts of the world. However, information on the occurrence of infections in fishes and amphibians caused by Ranavirus in Brazil are limited. In this context, this project aimed to isolate a Frog Virus 3-like strain detected in São Paulo state in cell culture, which was later confirmed by nucleotide sequencing of MCP gene and further RLFP technique and also phenotipical characterization and replication kinetics of the obtained strain. Liver, spleen and kidney samples from american bullfrogs from commercial frog ponds, positive by molecular diagnostic for Ranavirus, were used for viral isolation in cell culture. Citopathic effect was detected during the second passage in cells and later confirmed by PCR and RFLP. The viral characterization was carried out with transmission electronic microscopy, which confirmed similar morphology of viral particles to those of Ranavirus, besides the construction of a growth curve which indicated larger titres on the fourth day post inoculation. A test for solvent sensitivity was also performed and confirmed the presence of enveloped particles. The results obtained in this study will contribute to the understanding of the biology of the circulating iridovirus in São Paulo state as an etiological agent of Ranavirus infection in amphibians.

Diversos estudos têm demonstrado que os implantes de titânio são altamente biocompatíveis e passíveis de osseointegrar, contudo os mecanismos moleculares que atuam por trás da osseointegração permanecem largamente inexplorados. Uma das possibilidades é que o implante exposto ao sangue do paciente durante a cirurgia absorve proteínas relacionadas com a adesão celular, como a fibronectina e vitronectina presentes no plasma. Células como osteoblastos podem então aderir a estas proteínas através dos mecanismos mediadas pelas integrinas. No presente estudo, utilizamos a imunofluorescência para marcação dos anticorpos contra integrinas &#945;2, &#945;3, &#945;5, &#945;6, &#945;v e &#946;1 em células OsA-CL cultivadas sobre lamínulas de vidro e superfície de titânio modificada por radiação à laser no período de 1h à 24 horas. As células aderidas na superfície lisa das lamínulas de vidro tiveram um maior espalhamento durante as primeiras 3 horas, porém nos outros períodos estudados o espalhamento ocorreu de maneira similar em ambas as superfícies. A expressão de integrinas na superfície rugosa dos implantes manteve-se uniforme em todos os períodos estudados, contudo no grupo controle a expressão de integrinas diminuiu na avaliação de 24 horas. Concluiu-se que as características de superfície dos diferentes biomateriais podem afetar o comportamento celular durante a interação inicial das células com a superfície de material utilizado como substrato. / Many studies have been demonstrate that titanium implants are highly biocompatible, however the molecular mechanisms that are behind of the osseointegration process are still largely unexplored. One of the possibilities is that the implant exposed to the patient blood during the surgery, adsorb proteins related to the cellular adhesion. Cells like the osteoblasts can adhere to these proteins trough the mechanisms mediated by integrins. In the present study, we used the immunofluorescence for marking antibodies against &#945;2, &#945;3, &#945;5, &#945;6,, &#945;v and &#946;1 integrins on culture of OsA-CL cells on laminulas of glass and modified titanium surface modified by laser radiation in the period of 1h to 24 hs. Cells adhered in the smooth surface of laminulas of glass had a bigger spreading during the first 3 hours, however in the other studied periods the spreading occurred in a similar way in both surfaces. The expression of integrins in the roughness surface of the implants remained uniform in all the studied periods, but on the control group the integrins expression decreased in the 24-hours evaluation. We concluded that the characteristics of surface of the different biomaterials can affect the cellular behavior during the initial interaction of the cells with the surface of the used material as substratum.

Cell competition leads to elimination of a viable cell population, by fitter cells. Despite over forty years of research, the molecular mechanisms of competition in mammals are poorly understood. During my PhD I have investigated the mechanisms of competition by exploring an established mammalian cell culture system, in which wild-type MDCK cells eliminate scribble-deficient cells, and I have also developed a novel cell culture system to model mammalian competition. My work contributed to the discovery that scribble-deficient cells are eliminated not by biochemical exchange among cells, but by mechanical compaction. We termed this phenomenon mechanical competition. I employed transcriptional profiling to determine the molecular signature of mechanical losers, and identified activation of p53 signalling as their hallmark. My colleagues and I then demonstrated that elevation of p53 is both necessary and sufficient to trigger mechanical competition. In further investigating the mechanisms of mechanical competition, I found that compaction activates ROCK in scribble-deficient cells, and that this is required for their elimination. Inhibition of Src signalling in mechanical losers also protected them form out-competition, and integrin signalling is another pathway likely involved in mechanical competition. While investigating p53 competition, we observed that p53-high and p53-low cells engage in directional migration, with p53-high cells always at the migrating front. As a side-project, I investigated the role of p53 in directional migration, by exploring an established model with a single leader cell and multiple followers. We established a method to generate multinucleated leaders on demand. By creating leaders from p53-deficient cells, I established that p53 signalling is required for some, but not all multinucleated cells to trigger collective migration, thus implicating p53 signalling in a type of migration involved in wound healing. Finally, I successfully modelled p53-driven mechanical competition in a differentiated primary tracheal epithelial cell culture, thereby establishing a novel system to study mammalian competition, and also proving that p53 competition is conserved between different mammalian epithelia. Considering the involvement of p53, mechanical competition may play a major role in cancer.

Biomineralização é o processo em que organismos precipitam materiais sólidos para a formação de estruturas esqueléticas. Nos Echinodermata o esqueleto é composto por CaCO3, com uma organização e morfologia variável entre as classes do filo. Nos Echinoidea, onde o processo de calcificação tem sido mais estudado, os sítios de formação do esqueleto aparentemente são vacúolos sinciciais dos esclerócitos. Pouco tem sido estudado sobre calcificação nas demais classes dos Echinodermata, principalmente em Holothuroidea, onde os elementos esqueléticos têm uma distribuição esparsa, sem a mineralização densa encontrada nas demais classes. Neste grupo os ossículos se encontram em sua maioria agrupados em projeções da superfície corporal, ou papilas, cujas exatas funções na biologia do animal ainda são discutidas. Para um estudo mais detalhado da função destas estruturas e dos mecanismos envolvidos na calcificação, a manutenção das células em ambiente controlado, onde possam ser acompanhadas e manipuladas, seria de grande importância. O conhecimento, no entanto, ainda é bastante restrito, e não existem linhagens celulares estabelecidas de equinodermos. Neste trabalho foram realizados: a descrição morfológica e funcional da estrutura que conecta os ossículos dentro das papilas de Chiridota rotífera; análises da matriz orgânica dos ossículos, envolvidas na calcificação de Holothuria grisea, Synaptula hydriformis e Chiridota rotífera; e desenvolvidos protocolos adequados para manutenção das células de organismos da classe Holothuroidea em cultura. / Biomineralization is a process that organisms precipitate solid materials to the formation of skeletal structures. In Echinodermata the skeleton is composed by CaCO3, with an organization and variable morphology among phylum classes. In Echinoidea, where the calcification process has been hardly studied, apparently the sites of skeleton formation are vacuolar syncytia of sclerocytes. Little is known about calcification process in other Echinodermata classes, mainly in Holothuroidea, where skeletal parts are random distributed, without thick mineralization found in other classes. In this group, ossicles are found in clusters of papillae, that dont have defined functions for biology to animal. To a more specific and detailed study of the structures and mechanisms involved with calcification, it is necessary cell maintenance in a controlled environment, once these points can be accompanied and manipulated. However, the knowledge is extremely restricted and there is no cell lineages established of echinoderms. In this present study was performed the morphological and functional description of the structure that is responsible for the ossicles connection inside papillae of Chiridota rotífera; analyze of ossicles organic matrix involved with the Holothuria grisea, Synaptula hydriformis e Chiridota rotífera calcification, and development of adequate culture protocols to the maintenance of cell from Holothuroidea Class organisms.