FREITAS, M. F. M. Produção de lipases por leveduras isoladas do bagaço de caju utilizando fontes alternativas de Carbono e Nitrogênio. 2017. 103 f. Tese (Doutorado em Engenharia Química)-Centro de Tecnologia, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by pgeq ufc (pgeq@ufc.br) on 2017-11-21T19:19:47Z No. of bitstreams: 1 2017_tese_mfmfsales.pdf: 1907549 bytes, checksum: 22fee806e690e6f63b1598b0a1542349 (MD5) / Rejected by Marlene Sousa (mmarlene@ufc.br), reason: Prezada Fátima: Vc realizou muitas alterações sugeridas, mas faltam ainda algumas para vc corrigir. Por esse motivo, sugerimos consultar o modelo de template, para ajudá-la nesta tarefa, disponível em: http://www.biblioteca.ufc.br/educacao-de-usuarios/templates/ Vamos agora as correções sempre de acordo com o template: 1. Na capa, na parte inferior da folha coloque a informação da cidade e o ano em negrito. 3. Na ficha Catalográfica, o seu nome deve estar de acordo com a folha de rosto e capa. Voce suprimiu o ultimo nome. Sendo assim a entrada: Sales, Maria de Fátima Matos de Freitas (Não de Freitas) No corpo da ficha coloque seu nome completo por extenso. 4. Retire os hifens de separação na LISTA DE ABREVIATURAS E SIGLAS. 7. No sumário na seção terciária (3 dígitos) a partir do 4.2.1 os numerais não estão em itálico. Favor colocar em toda a lista. A palavra CONCLUSÃO Por ser seção primária deve ser toda maiúscula. Retire o numeral 7 da palavra REFERÊNCIAS 8. Na lista de referencias, A palavra que deve ser usada é REFERÊNCIAS (sem o numeral 7), em negrito e centralizada na folha. 9. Na lista de REFERENCIAS, dê uma revisada em toda a lista para colocar o espaço depois do ponto de abreviação (v. n. p. ) nos artigos de revistas. Ex; v.2, n. 3, 2012. Coloque espaço após v. 2, n. 3, 2012 Reenvie novo arquivo para a secretaria com as correções que enviaremos o nada consta por e-mail. Att. Marlene mmarlene@ufc.br 3366-9620 on 2017-11-22T11:23:36Z (GMT) / Submitted by pgeq ufc (pgeq@ufc.br) on 2017-11-24T19:13:19Z No. of bitstreams: 1 2017_tese_mfmfsales.pdf: 1891030 bytes, checksum: b360634c75b70dda334f156745c6be57 (MD5) / Rejected by Marlene Sousa (mmarlene@ufc.br), reason: Alteração de Maria de Fátima Matos de Freitas Sales Prezada Fátima: Vc realizou muitas alterações sugeridas, mas faltam ainda algumas para vc corrigir. Por esse motivo, sugerimos consultar o modelo de template, para ajudá-la nesta tarefa, disponível em: http://www.biblioteca.ufc.br/educacao-de-usuarios/templates/ Vamos agora as correções sempre de acordo com o template: 3. Verifiquei que houve alteração no seu nome em relação ao arquivo passado, portanto vc tem que alterar em todas as folhas em que é citada. Faltou alterar na folha de aprovação, pois seu nome ainda permanece com SALES. Na ficha então deve ficar: Freitas, Maria de Fátima Matos de (Não de Freitas). No corpo da ficha coloque seu nome completo por extenso. 7. No sumário na seção terciária (3 dígitos) a partir do 4.2.1 os numerais não estão todos em itálico. Favor colocar em toda a lista. Faltou vc colocar em itálico: 5.9.1 Efeito da temperatura e pH na atividade da enzima produzida. 5.9.2 Eletroforese. 5.9.3 Processo de purificação 9. Na lista de REFERENCIAS, dê uma revisada em toda a lista para colocar o espaço depois do ponto de abreviação (v. n. p. ) nos artigos de revistas. Ex; v.2, n. 3, 2012. Coloque espaço após v. 2, n. 3, p. 513-523, 2012 . Vc ainda não corrigiu o espaçamento solicitado em toda a lista. Reenvie novo arquivo para a secretaria com as correções que enviaremos o nada consta por e-mail. Att. 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No. of bitstreams: 1 2017_tese_mfmfreitas.pdf: 1894579 bytes, checksum: 225db412b0fb510a23717698c92cd485 (MD5) Previous issue date: 2017-06-13 / Lipolytic enzymes can catalyse a wide variety of reactions and are highly specific. For biotechnology, the need arises for the search for new microrganisms for this production. The objective of this work was to evaluate lipase production by two yeasts isolated from cashew bagasse, Candida tropicalis URM 7057 and Meyerozyma caribbica LABIOTEC 4 and to compare it with C. rugosa yeast NRRL Y-95. Synthetic culture media were initially tested for production of the enzyme. Synthetic medium C (containing glucose, KH2PO4, yeast extract, MgSO4 .7H2O, peptone and oleic acid) showed the best intracellular activity results (282 ± 14 in 48 h of cultive). Then, alternative medium culture was formulated with residues, in addition to ammonium sulfate and peptone, and evaluated the production of the enzymes of interest. Medium culture 01 contained molasses, medium culture 02, corn steep liquor (CSL), medium culture 03, olive mill wastewater (OMW) and medium culture 04, all of them. Medium 04 was better because it allowed the production of larger amounts of lipase, probably because it had a higher amount of nutrients for yeast. An experimental design was conducted to verify the influence of the concentrations of residues on lipase production. Corn steep liquor (CSL) had a positive effect on this production, providing extracellular lipase activity of 587 ± 5 U/L, in 24 h of cultive. The use of stirred tank bioreactors favored the production of lipases, since the activities of the lipase produced by C. rugosa in bioreactor were higher than those produced in flasks (600 U/L in bioreactor and 400 U/L in flasks, after 48 h of cultive) and for yeast C. tropicalis the enzymatic activity also presented in a larger quantity (close to 500 U/L, compared to 400 U/L in flasks). Biomass growth of yeast biomass C. tropicalis, in flasks and in bioreactor, was similar up to 48 h. The enzyme produced by C. rugosa has a molar mass close to 60 KDa, with an optimum pH of 7.0 and an optimum temperature of 40 °C. The partial biochemical characterization of the lipase produced by C. tropicalis showed that it presents an optimum temperature of 60 °C for the intracellular extract and 70 °C for the extracellular extract, optimum pH equal to 7.0. After adsorption of the enzyme in octyl-agarose, it was possible to identify, by zymogram analysis and by electrophoresis, bands close to those obtained for C. rugosa commercial lipase (60 KDa, 45 KDa and 22 KDa). / As enzimas lipolíticas são capazes de catalisar uma grande variedade de reações e são altamente específicas. Para a biotecnologia surge a necessidade da busca de novos micro-organismos para essa produção. O objetivo desse trabalho foi avaliar a produção de lipases por duas leveduras isoladas do bagaço de caju, Candida tropicalis URM 7057 e Meyerozyma caribbica LABIOTEC 4 e comparar pela levedura C. rugosa NRRL Y-95. Inicialmente foram testados meios de cultura sintéticos para a produção da enzima. O meio sintético C (contendo glicose, KH2PO4, extrato de levedura, MgSO4.7H2O, peptona e ácido oleico) foi o que apresentou melhor resultados de atividade intracelular (282 ± 14 U/L em 48 horas de cultivo). Em seguida, 4 meios de cultura alternativos foram formulados com resíduos, além de sulfato de amônia e peptona, e avaliados na produção da enzima de interesse. O meio 01 continha melaço, o meio 02, milhocina (CSL - corn steep liquor), o meio 03, águas residuais da produção do azeite (olive mill wastewater - OMW) e o meio 04, todos eles. O meio 04 se mostrou melhor por permitir a produção de maiores quantidades da enzima lipolítica, provavelmente por apresentar uma quantidade maior de nutrientes para levedura. Um planejamento fatorial fracionado foi conduzido para se verificar a influência das concentrações dos resíduos na produção da lipase. A milhocina apresentou efeito positivo nessa produção, propiciando atividade extracelular de lipase de 587 ± 5 U/L, em 24 horas de cultivo. O uso de biorreator tipo tanque agitado favoreceu a produção de lipases, visto que as atividades da lipase produzida por C. rugosa em biorreator foram maiores que as produzidas em frascos (600 U/L em biorreator e 400 U/L em frascos, após 48 horas de cultivo), e para levedura C. tropicalis a atividade enzimática também se apresentou em quantidade maior (próximo a 500 U/L, comparado a 400 U/L em frascos). O crescimento da biomassa da levedura C. tropicalis em frascos e em biorreator foi similar até 48 horas. A enzima produzida por C. rugosa tem massa molar próxima a 60 KDa, com pH ótimo em torno de 7,0 e temperatura ótima de 40 °C. A caracterização bioquímica parcial da lipase produzida por C. tropicalis mostrou que ela apresenta temperatura ótima de 60 ºC para o extrato intracelular e 70 °C para o extracelular, pH ótimo igual a 7.0. Após adsorção da enzima em octil-agarose, foi possível identificar, por análise de zimograma e por eletroforese, bandas próximas às obtidas para a lipase de C. rugosa comercial (60 KDa, 45 KDa e 22 KDa).

Made available in DSpace on 2014-06-11T19:35:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-28Bitstream added on 2014-06-13T19:44:49Z : No. of bitstreams: 1 dovigo_ln_dr_arafo.pdf: 3524091 bytes, checksum: bd9c194e1468a342205c6be45f7e5d62 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Esta investigação teve como objetivo avaliar: 1. a viabilidade de utilização da curcumina como agente fotossensibilizador (PS) em Terapia Fotodinâmica (PDT) para inativação de uma cepa padrão de Candida albicans e seu efeito tóxico sobre culturas celulares de macrófagos; 2. a efetividade da PDT mediada pela curcumina para inativação de suspensões planctônicas e biofilmes formados por diferentes isolados clínicos de C. albicans, Candida tropicalis e Candida glabrata; 3. a fotoinativação de C. albicans, presente em candidose induzida em camundongos, por meio da utilização de diferentes concentrações de curcumina. No primeiro estudo, suspensões planctônicas de C. albicans (ATCC 90028) foram expostas a nove concentrações de curcumina (0,005; 0,01; 0,05; 0,1; 0,5; 1, 5, 10 e 20μM) e oito doses de luz (1,32; 2,64; 3,96; 5,28; 6,60; 13,20 e 26,4J/cm2). O efeito de diferentes tempos de pré-irradiação (PIT), a possível captação de curcumina pelas células fúngicas e participação do oxigênio singlete na fotoinativação também foram avaliados. Adicionalmente, a PDT mediada pela curcumina foi avaliada em biofilmes formados pela cepa de referência de C. albicans e sobre culturas celulares de macrófagos. Além disso, as características ópticas da solução de curcumina foram investigadas em função da dose de luz utilizada. Os resultados foram submetidos à análise descritiva, Análise de Variância (ANOVA) e Kruskal-Wallis (α= 5%). As suspensões planctônicas de C. albicans foram completamente inativadas com a utilização de 20μM de curcumina e 5,28J/cm2. Além disso, foi observada redução significativa na viabilidade celular dos biofilmes de C. albicans. O efeito fotodinâmico foi acentuado pela presença da curcumina durante a iluminação... / The aim of this investigation was to evaluate: 1. the Photodynamic Therapy (PDT) mediated by curcumin for in vitro inactivation of a reference strain of Candida albicans and its citotoxic effects against a macrofage cell line; 2. the effectiveness of PDT mediated by curcumin to inactivate clinical isolates of C. albicans, Candida tropicalis and Candida glabrata, both in planktonic and biofilm phases; 3. the photoinactivation of C. albicans in a murine model of oral candidiasis using different curcumin concentrations. In the first study, suspensions of C. albicans were treated with nine curcumin concentrations (0.005; 0.01; 0.05; 0.1; 0.5; 1, 5, 10 and 20μM) and exposed to LED light at different fluences (1.32; 2.64; 3.96; 5.28; 6.60; 13.20 and 26.4J/cm2). The effect of the pre-irradiation time (PIT) on PDT effectiveness, the uptake of curcumin by C. albicans cells and the possible involvement of singlet oxygen in the photodynamic action was also evaluated. In addition, curcumin-mediated PDT was assessed against biofilms. Similar protocols were tested on a macrophage cell line and the effect was evaluated by MTT and SEM analysis. The optical properties of curcumin were investigated as a function of illumination fluence. Data were analyzed with Analysis of Variance (ANOVA), and Kruskal-Wallis test (α= 5%). The results showed a statistically significant reduction in C. albicans viability after PDT (p<0.05), for both planktonic and biofilm cultures. Photodynamic effect was greatly increased with the presence of curcumin in the surrounding media and the PIT of 20 minutes improved PDT effectiveness. Although PDT was phototoxic to macrophages, the therapy was more effective in inactivating the yeast cell than the defense cell. The spectral changes showed a high photobleaching rate of curcumin. In the second study, planktonic... (Complete abstract click electronic access below)

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-04-25T22:57:38Z No. of bitstreams: 1 DianaLuziaZuzaAlves_DISSERT.pdf: 2035213 bytes, checksum: 1604df1e90593ddd103ab0202e591e9e (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-27T22:44:59Z (GMT) No. of bitstreams: 1 DianaLuziaZuzaAlves_DISSERT.pdf: 2035213 bytes, checksum: 1604df1e90593ddd103ab0202e591e9e (MD5) / Made available in DSpace on 2016-04-27T22:44:59Z (GMT). No. of bitstreams: 1 DianaLuziaZuzaAlves_DISSERT.pdf: 2035213 bytes, checksum: 1604df1e90593ddd103ab0202e591e9e (MD5) Previous issue date: 2015-03-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / V?rios estudos t?m sido desenvolvidos com rela??o aos riscos ? sa?de humana associados ao uso recreativo de praias contaminadas com esgotos dom?sticos. Esses res?duos cont?m v?rios micro-organismos, incluindo Candida tropicalis, agente etiol?gico tanto de infec??es superficiais quanto sist?micas, al?m de indicador de contamina??o fecal do meio ambiente. Nesse contexto, o presente trabalho teve como objetivo caracterizar isolados de C. tropicalis oriundos da areia da Praia de Ponta Negra, Natal, Rio Grande do Norte, Brasil, quanto a express?o de fatores de virul?ncia in vitro, a adapta??o ao estresse osm?tico e a susceptibilidade ?s f?rmacos antif?ngicos. Foram analisados 62 isolados ambientais de C. tropicalis, observando-se grande varia??o entre os mesmos para os diversos fatores de virul?ncia avaliados. Em geral, os isolados ambientais foram mais aderentes a c?lulas epiteliais bucais humanas (CEBH) do que a cepa de refer?ncia de C. tropicalis ATCC13803, al?m de serem altamente produtoras de biofilme. Em rela??o ? morfog?nese, a maioria dos isolados exibiu fen?tipo rugoso em meio Spider (34 isolados, 54,8%). Na avalia??o da atividade enzim?tica, a maioria dos isolados teve maior produ??o de proteinase do que a cepa de refer?ncia de C. tropicalis ATCC13803. Adicionalmente, 35 isolados (56,4%) tiveram alta atividade hem?litica (?ndice de hem?lise > 55). Com rela??o ? resist?ncia de C. tropicalis ao estresse osm?tico, 85,4% dos isolados foram resistentes em meio contendo 15% de cloreto de s?dio, o que corrobora com a alta capacidade de sobreviv?ncia descrita para essa levedura no ambiente mar?timo. Finalmente, no que diz respeito ? sensibilidade aos antif?ngicos foi observada elevada resist?ncia aos az?licos testados (fluconazol, voriconazol e itraconazol), com ocorr?ncia do fen?meno ?Low-high? e de efeito semelhante ao crescimento paradoxal que ocorre para as equinocandinas. As cepas resistentes aos tr?s az?licos testados foram 15 (24,2%). Para a anfotericina B tamb?m ocorreu resist?ncia (14 isolados, 22,6%), ao passo que para as equinocandinas todas as cepas foram sens?veis. Portanto, nossos resultados demonstram que isolados de C. tropicalis oriundos da areia de praia do nordeste brasileiro podem expressar plenamente atributos de virul?ncia e apresentam alta capacidade de persist?ncia no ambiente costeiro, al?m de serem significativamente resistentes aos antif?ngicos mais empregados na pr?tica cl?nica atual. Isso constitui potencial risco ? sa?de dos frequentadores desse ambiente, especialmente indiv?duos imunocomprometidos e em extremos et?rios. / Several studies have been developed regarding health risks associated with the recreational use of beaches contaminated with domestic sewage. These wastes contain various microorganisms, including Candida tropicalis, etiologic agent of both superficial infections such as systemic, as well as indicator of fecal contamination for the environment. In this context, the objective of this study was to characterize C. tropicalis isolates from the sandy beach of Ponta Negra, Natal, Rio Grande do Norte, Brazil, regarding the expression of in vitro virulence factors, adaptation to osmotic stress and susceptibility to antifungal drugs. We analyzed 62 environmental isolates of C. tropicalis and observed a great variation between them for the various virulence factors evaluated. In general, environmental isolates were more adherent to CEBH than C. tropicalis ATCC13803 reference strain, besides the fact they were also highly biofilm producers. In relation to morphogenesis, most isolates presented wrinkled phenotype in Spider medium (34 isolates, 54.8 %). When assessing enzyme activity, most isolates had higher proteinase production than C. tropicalis ATCC13803 reference strain. In addition, 35 isolates (56.4 %) had high hemolytic activity (hemolysis index > 55). With regard to C. tropicalis resistance to osmotic stress, 85.4% of the isolates were able to grow in a liquid medium containing 15% sodium chloride, corroborating to high survival capacity described for this yeast at marine environment. Finally, with regard to sensitivity to antifungal drugs, it was observed high resistance to the azoles tested, with the occurrence of the "Low-high" phenomenon and similar effect to the paradoxical growth which occurs to the echinocandins. For the three azoles tested we verified that 15 strains were resistant (24.2 %). Some strains were also resistant to amphotericin B (14 isolates, 22.6 %), while all of them were sensitive for the echinocandins tested. Therefore, our results demonstrate that C. tropicalis isolated from the sand of northeast of Brazil can fully express virulence attributes and showed a high persistence capacity on the coastal environment, in addition of being significantly resistant to most applied antifungals in current clinical practice. This constitutes a potential health risk to visitors of this environment, especially immunocompromised individuals and those with extreme age range.

Orientador: Altair Antoninha Del Bel Cury, Renata Cunha Matheus Rodrigues Garcia / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-05T13:21:12Z (GMT). No. of bitstreams: 1 Moura_JulianaSilva_D.pdf: 1782557 bytes, checksum: 062461fb23404728637a2298815f99c6 (MD5) Previous issue date: 2005 / Resumo: O objetivo deste trabalho foi avaliar a influência do método de polimerização e a presença da saliva humana na aderência de Candida spp. às superfícies de resinas acrílicas. Duzentas e cinqüenta e seis amostras retangulares (2,5 x 1,2 x 0,2 cm), confeccionadas com resinas polimerizadas por banho de água ou microondas foram avaliadas para rugosidade e energia livre de superfície e em seguida utilizadas para o ensaio de aderência de Candida spp. Para este propósito, as amostras foram aleatoriamente divididas em 8 grupos por resina, sendo quatro expostos durante 30 min a saliva humana. Em seguida, as amostras foram posicionadas verticalmente em tubos de plásticos estéreis contendo meio de cultura Sabouraud e uma entre quatro suspensões de Candida: Candida albicans, Candida tropicalis, Candida dubliniensis e Cândida glabrata (1 a 5 x 106 células / mL). A contagem das células aderidas foi realizada em microscópio óptico com 400 x de aumento. Os dados de rugosidade superficial e energia de superfície foram submetidos a ANOVA um fator e teste t. Não houve diferença estatística significante para rugosidade (p > 0,05), enquanto maiores valores de energia livre de superfície foram encontrados para a resina polimerizada por banho de água (p < 0,05). Para aderência de Cândida spp., os testes Wilcoxon-Mann-Whitney, Kruskal Wallis e Qui-quadrado demonstraram não haver influência dos valores de rugosidade e energia livre de superfície na aderência de Candida, enquanto houve uma diminuição geral na contagem de microrganismos em grupos expostos à saliva (p < 0,05). Concluiu-se que a saliva foi capaz de reduzir a aderência total de Candida, enquanto não houve correlação destes valores com rugosidade e energia de superfície / Abstract: The aim of this work was to evaluate the influence of polymerization methods and presence of human clarified saliva on Candida spp. adherence to acrylic resins surfaces. Two hundred and fifty six rectangular acrylic resins samples (2.5 x 1.2 x 0.2 cm) polymerized by water bath or microwave were evaluated for surface roughness and surface free energy and used in an adherence assay for Candida spp. For this purpose, acrylic samples were randomly divided into 8 groups for each resin, where 4 were exposed to saliva. For the adherence assay, the samples were placed vertically on test tubes containing yeast Sabouraud broth medium and one of the four Candida suspensions: Candida albicans, Candida tropicalis, Candida dubliniensis and Candida glabrata (1 to 5 x 106 cells / mL). Adhered yeasts were counted using an optic microscope at 400 x. Data from surface roughness and surface free energy were submitted to one-way ANOVA ant t test and Candida spp. adherence values were evaluated by the Wilcoxon-Mann-Whitney, Kruskal Wallis and Chi-square tests. No statistical significance was found for roughness (p > 0.05), while higher surface free energy values were found for water bath resin (p < 0.05). Roughness and surface free energy did not influence Candida adherence, while saliva generally decreased yeast counts (p < 0.05). It was concluded that polymerization methods did not interfere with adherence assay while saliva was capable of reducing Candida spp. adherence, while roughness and free energy did not influence the adherence rates / Doutorado / Protese Dental / Doutor em Clínica Odontológica

Martínez Macías, F. (2015). Nuevos genes reguladores de la tolerancia a estrés abiótico en Arabidopsis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48560 / TESIS

African clawed frogs (Xenopus) represent an ideal model organism for study of regeneration mechanisms. In frogs, complete regeneration occurs in the tadpole stage. In later stages the regeneration capacity is lost. The Laboratory of Developmental biology was successful in establishment of cell culture called Xenopus tropicalis immature Sertoli cells (XtiSCs) derived from X. tropicalis testes. These cells are common progenitors of Sertoli cells and peritubular myoid cells. XtiSCs show similar characteristics as mesenchymal stem cells. MSCs hold interest of scientists for their immunomodulatory properties and multipotent differential and regeneration potential. In this thesis, we studied regeneration and migration potential of XtiSCs after X. tropicalis tadpole's tail amputation in developmental stage 47 - 50. Transgenic XtiSCs culture expressing RFP was prepared to facilitate transplantation experiments. Transplantation experiments showed preferential migration of XtiSCs into the site of injury. XtiSCs transplantations in X. laevis tadpoles with downregulated NO synthases eNOS and nNOS revealed their migratory dependence on nitric oxide signalization. Imunocytochemical staining of XtiSCs in vitro showed positive iNOS, nNOS and Pax7 expression. Imunohistochemical staining of tadpole's tail vibratome...

Naphthenic acids (NAs) are oil-derived mixtures of carboxylic acids and are aquatic contaminants of emerging concern. The objective of the research presented in this thesis was to investigate the toxicity of NAs in tadpoles of the frog Silurana (Xenopus) tropicalis. Using electrospray ionization high-resolution mass spectrometry (ESI-HRMS), I determined that the proportions of O2 (presumably carboxylic acid moiety) species were 98.8, 98.9 and 58.6% respectively, for two commercial extracts (S1 and S2), and acid extractable organics (AEOs) from oil sands process-affected water (OSPW). The rank order potency based on the lethal concentration fifty (LC50) and effect concentration fifty (EC50) with and without normalization for the quantity of O2 species was S1 > S2 > AEO. The main effects observed were reduced body size, edema, and cranial, cardiac, gut and ocular abnormalities. Oligonucleotide microarray technology was used to determine the transcriptomic responses in developing S. tropicalis embryos following exposure to S1 and S2 at a sub-lethal concentration of 2 mg/L. Some of the significantly enriched pathways (p < 0.05) included metabolism and cell membrane depolarization, and some were related to observed abnormalities including edema, gastrointestinal system, and cartilage differentiation. I established and validated a derivatization method for NAs using pentafluorobenzyl bromide (PFBBr) prior to gas chromatography-electron impact mass spectrometry (GC-EIMS) to increase chromatographic resolution, and sensitivity, compared to boron trifluoride-methanol (BF3/MeOH) and N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). Solid-phase microextraction of volatiles originating from S1, S2, Merichem NAs and an AEO mixture led to the identification of 54, 56, 40 and 4 compounds, respectively. The compounds identified in the mixtures included aliphatic and cyclic hydrocarbons, carboxylic acids, alkyl-benzenes, phenols, naphthalene and alkyl-naphthalene, and decalin compounds. To determine the chemical nature of the toxic compounds in NA mixtures, the S2 and AEOs preparations were fractionated using open column chromatography. A non-polar and a polar fraction were obtained from S2. Overall, the toxicity of the polar fraction was not significantly different from whole S2 (p > 0.05). Six fractions of AEOs were obtained, however because of limited material, only the toxicities of F3 and F4 were assessed. The toxicity of F3 was significantly lower than AEOs (p < 0.05) and F4 was not toxic for S. tropicalis (p > 0.05). These results suggest that during fractionation, toxic compounds were lost or that the toxicity of AEOs results from the combined effects of the compounds present in the whole extract. The toxicological dose descriptors, morphometric, transcriptomic and chemical analysis herein presented may contribute to the development of environmental guidelines for NAs and AEOs.

Candida tropicalis yeast is a microorganism that possesses high tolerance for phenol and strong phenol degrading activity. This yeast is capable of utilizing phenol as the sole source of carbon and energy without formation of any secondary waste product. Catechol-1,2- dioxygenase was isolated from cytosolic fraction of this yeast by the procedure consisting of chromatography on DEAE-Sepharose and gel permeation chromatography on Sephadex G- 100. The catechol-1,2-dioxygenase was purified to homogeneity. The enzyme activity was followed by HPLC (catechol consumption and/or cis,cis-muconic acid formation). The activity profiles at different temperatures showed temperature optimum of 30řC. Kinetic characterizations were studying in different values of pH. The values of Km and Vmax of 0,52 mM and 17,2 nM/min for consumption of catechol, respectively, and 0,34 mM and 12,6 nM/min for formation of cis,cis-muconic acid, respectively, were found at optimum pH of the reaction, pH 7,6.

In our Laboratory of Developmental Biology there was established a long term culture derived from Xenopus tropicalis testes. It contains pre-Sertoli cells mostly. They compose a feeder layer allowing cultivation of stem cells, revealing the morphology of spermatogonial stem cells. This diploma thesis was focused on a preparation of two growth factors, FGF2 (fibroblast growth factor 2) and GDNF (glial cell line-derived neurotrophic factor), with the subsequent characterization of their influence at cell culture mentioned above. Factors were selected on the basis of the microenvironmental niche theory, according which FGF2 and GDNF are the most important factors for spermatogonial stem cells proliferation and self-renewal. FGF2 recombinant factor was gained using the expression plasmid pET-15b. Its characterization in the testicular culture brought surprising result. Even a low concentration of FGF2 factor (2.5ng/ml) caused cell detaching and dying. Similar result was previously shown in differentiating osteoblast culture only. More experiments need to be done to prove if apoptose take place and why do testicular cells act this way. Key words: Xenopus tropicalis, FGF2, GDNF, SSC, pre-Seroli cells

The study of Xenopus tropicalis testis-derived stem cells Nguyen Thi Minh Xuan Abstract The substances secreted by Sertoli cells (SCs) are crucial to determine male sex characteristics in embryos and regulate spermatogenesis in adulthood. The failure in SC maturation can cause sterility in men. Before puberty, SCs keep the ability to proliferate and have been considered as immature cells. They differ remarkably from mature cells in connection with their morphology and biochemical activity and thus they probably play a part in maintaining spermatogonia stem cells in an undifferentiated stage. The transient presence of cytokeratin in immature SCs has been reported in many species, but not in Xenopus yet. We investigated which molecules are expressing only in immature Sertoli cells of X. tropicalis testes. The regulation of cytokeratin and β-catenin was revealed by fluorescent immunostaining. Cytokeratin and membrane β-catenin co- expressed in X.tropicalis juvenile testes and in cultured SC progenitors, called XtiSCs, but they were absent in adulthood. There was no signal of cytokeratin in migrating SCs (pre-SCs) located outside the seminiferous tubules. The suppression of cytokeratin along with the breakdown of β-catenin-based cell contacts have been observed in XtiSCs after the treatment with a small...