The cell wall is essential for fungal survival in natural environments. Galactofuranose (Galf) decorates certain carbohydrates and lipids of Aspergillus cell wall, is absent in humans and appears to play a role in fungal cell wall maturation. Previous studies in our lab showed that deletion of any of three sequential-acting genes (ugeA, ugmA, and ugtA) of Galf pathway caused substantially reduced growth and spore production. Two genes upstream of the Galf pathway, galD and galE are essential for galactose metabolism in many systems including the budding yeast, Saccharomyces cerevisiae. Interestingly, characterization of galD and galE in A. nidulans using cell and molecular techniques showed that unlike yeast, neither of these genes was essential for growth at physiological pH 7.5. Nevertheless for each case, their expressions were up-regulated by growth on galactose, revealing the relative complexity of galactose metabolism in A. nidulans. Our study also showed that repression of the three sequentially acting Galf pathway genes by conditional promoters phenocopied previously characterized deletion morphology. Using anti-Galf (L10) we also showed that deletion and repression of these genes caused no Galf in the hyphal wall. Gene deletion or repression also increased sensitivity to the wall-targeting drug, caspofungin. Related results from qPCR showed that deletion or repression of ugmA increased gene expression of α-glucan synthase agsB and decreased that of β-glucan synthase fksA. Therefore, Galf is non-essential but important for many aspects of Aspergillus growth, sporulation, and wall maturation. Aspergillosis, the most common airborne systemic fungal disease, is typically caused by Aspergillus fumigatus. Several A. fumigatus UgmA (AfUgmA) mutants with altered enzyme activity due to single amino acid changes were used to assess their effect on growth and wall composition in A. nidulans. Wild type AfugmA complemented the phenotypic defects in an A. nidulans ugmAΔ strain, consistent with these two genes being homologous. The AfUgmA crystal structure has been solved, and the in vitro enzymatic effects of specific mutations in the enzyme active site have been published. AfUgmA mutated strains with reduced activity in vitro impaired A. nidulans growth in a manner substantially similar to gene deletion and gene down-regulation. Site directed mutagenesis showed that AfUgmA residues R182 and R327 were critical for Galf generation both in vivo and in vitro. This supports previous results showing that UgmA is essential for Galf biosynthesis. Using fluorescent latex beads, we showed that reduction of wall Galf increased hyphal surface adhesion. Consistent with qPCR studies, immunofluorescence and ELISA results showed that loss or absence of Galf increased wall α-glucan but reduced wall β -glucan. Galf is important for wall surface integrity and for maintaining dynamic co-ordination with other pathways. To begin to assess this dynamic co-ordination, Tandem Affinity Purification (TAP) tagging combined with LC-MS/MS was used to identify the interacting partners of UgmA. Our results showed that UgmA interacted with proteins that are involved in cytoskeleton generation, osmotic adaptation, and cell signalling pathway. Further study will help us to understand the dynamic coordination of Galf biosynthesis pathway with other wall carbohydrate polymers for Aspergillus wall formation. In summary, my thesis results have clearly shown that Galf plays important roles in Aspergillus growth, and wall surface integrity. We also showed that Galf deficient strains are hypersensitive to wall-targeting drugs, indicating that Galf biosynthesis pathway could be potential target for combination therapy. The Galf pathway also maintained a dynamic co-ordination with alpha-glucan and beta-glucan carbohydrate pathways. Future study may include developing an inhibitor against UgmA and exploring the relationship of Galf pathway with alpha-glucan and beta-glucan carbohydrate pathways.

No description available.

The capacity of pea root plastids for galactolipid synthesis was investigated utilizing radiolabelled acetate and UDP-galactose. Galactolipid biosynthesis was completely dependent on an exogenous supply of UDP-galactose. UDP-galactose stimulated both total lipid biosynthesis from acetate and the proportion of radioactivity accumulated in monogalactosyldiacylglycerol (MGDG). The proportion of MGDG synthesized was saturated at 30$ mu$M UDP-galactose and represented approximately 30% of the total lipid radioactivity after a one hour incubation. However, total lipid biosynthesis continued to increase with concentrations of UDP-galactose up to 75$ mu$M while the proportion of radioactivity in MGDG remained at 30%. MGDG biosynthesis was always accompanied by a corresponding decrease in the amount of diacylglycerol (DAG) accumulated. Digalactosyldiacylglycerol (DGDG) synthesis was not routinely observed in these experiments. These results suggest that the in vitro pathway for MGDG synthesis in the root plastids of pea (an 18:3 plant) is similar to 16:3 plants (FFA's$ to$PA$ to$DAG$ to$MGDG). The endogenous lipids, consistent with the thought of pea as an 18:3 plant, contained 80% C$ sb{18}$ in the fatty acids of MGDG, DGDG, TG and PC. However, in labelled acetate experiments palmitate was the predominately labelled fatty acid in all lipids except PC (where 80% was 18:1). The precursors PA and DAG had ratios of 16:0, 18:0, and 18:1 similar to that of MGDG. 70-80% of the label was associated with the sn-2 position of glycerolipids. The cofactors required for fatty acid synthesis were generally not as required for galactolipid synthesis. The results suggest that galactolipid synthesis relies primarily on endogenous DAG and only partly involves de novo fatty acid synthesis. (Abstract shortened by UMI.)

No description available.

Xenotransplantation is the final frontier of using vascularised organs or cellular grafts to treat end-organ disease and offers a potential solution to the worldwide shortage of human tissue available for transplantation. The main immunological barrier to xenografting from pig-to-primate is the antigen, Galactose-α1,3-Galactose (Gal) which is found in all species except humans and other higher primates. Even with the major advancement of deleting Gal from the potential pig donor species with the aid of cloning technology, complete elimination may be elusive as alternative genes yet to be fully characterised, may still produce Gal at low levels. Thus, the human immune response against Gal may continue to be a barrier to successful xenotransplantation. The aim of this project was to develop small animal models of the important components of xenograft rejection that largely relate to the anti-Gal immune response. These include models of hyperacute, acute vascular and chronic xenograft-like rejection that in turn, provide new insights in the immune mechanisms of the rejection processes. The role of antibody and both innate and cognate cellular immunity are explored. Both vascularised heart grafts and non-vascularised skin graft models are examined as rejection of solid organs may differ from cellular transplantation. The project also provides a platform for future studies in testing genetic and pharmacotherapeutic strategies to overcome the rejection processes uncovered.

A targeted, stimuli-responsive, polymeric drug delivery vehicle has been developed to help alleviate the severe side-effects caused by narrow therapeutic window drugs. Doxorubicin, a commonly used chemotherapeutic agent has been conjugated to dextran by two different techniques. In the first method, doxorubicin and hepatocyte-targeting galactosamine were attached to dextran through amine bonds. Conjugation efficiency based on the amount loaded of each reactant varied from 1% to 50% for doxorubicin and from 2% to 20% for galactosamine, depending on various synthesis parameters. For the second conjugate, doxorubicin was attached to dextran through an acid-labile hydrazide bond. Fluorescence quenching indicated that all our conjugates can bind to DNA. The degree of binding was improved with increasing polymer molecular weight and substitution of doxorubicin, and also with hydrazide-bonded conjugate. In cell culture experiments, we have found that the uptake of conjugates was much lower than that of free doxorubicin. Lower uptake of conjugates decreased the toxicity of doxorubicin. Also, the uptake of non-galactosylated conjugate was lower than that of the galactosylated conjugate. Microscopy studies indicated that doxorubicin was localized almost exclusively at the nucleus, whereas the amine-bonded conjugates were present throughout the cell. Targeted amine-linked conjugates and hydrazide-bonded conjugates achieved greatly improved cytotoxicity. Following uptake, the doxorubicin was dissociated from the hydrazide conjugate in an endosomal compartment and diffused to the nucleus. The LC₅₀ values of non-targeted amine-linked, targeted amine-linked, and hydrazide-linked doxorubicin were 19.81 μg/mL, 7.33 μg/mL and 4.39 μg/mL, respectively. The amine-linked conjugates were also tested on a multidrug-resistant cell line; the LC₅₀ values of doxorubicin and the non-targeted amine-linked conjugate were 8.60 μg/mL and 36.02 μg/mL, respectively. / Singapore-MIT Alliance (SMA)

Dissertação apresentado ao Programa de Pós-graduação em Ciências da Saúde, da Universidade do Extremo Sul Catarinense, UNESC, para obtenção do título de Mestre em Ciências da Saúde. / O envelhecimento é uma etapa do desenvolvimento humano em que ocorre um processo de transformação do organismo, caracterizada por alterações físicas, psicológicas e sociais do indivíduo. Essas alterações podem desencadear neurodegeneração, como na doença de Alzheimer (DA), caracterizada por perda progressiva de memória e diminuição de várias funções cognitivas, sendo um processo associado à perda de neurônios e perda sináptica, com consequentes respostas inflamatórias, anormalidades mitocondriais e plasticidade neuronal marcadamente reduzida, que levam a prejuízos na memória. Perda da plasticidade neuronal e danos de memória são características comuns no envelhecimento e na DA, e podem estar associados à redução nos níveis de neutrofinas. Contudo, pouco se sabe o papel das neurotrofinas no processo de envelhecimento e DA. Dessa forma, o objetivo do presente estudo foi avaliar os níveis das neurotrofinas fator neurotrófico derivado do cérebro (BDNF), fator neurotrófico derivado de células gliais (GDNF) e fator de crescimento do nervo (NFG), além da memória de ratos submetidos ao modelo animal de DA pela administração crônica oral, diariamente uma vez ao dia de D-galactose (D-gal) (100 mg/kg) durante 1, 2, 4, 6 e 8 semanas. Para tal, foram utilizados ratos Wistar machos adultos. Para avaliação da memória, os animais foram submetidos ao teste de esquiva inibitória de múltiplos treinos ao final de 1, 2, 4, 6 e 8 semanas de tratamento com D-gal e 24hs após a última administração de D-gal, os animais foram submetidos à eutanásia por decapitação com guilhotina e foram dissecadas as estruturas cerebrais hipocampo e córtex pré-frontal, para avaliação dos níveis de neurotrofinas. Os resultados obtidos demonstraram que os animais tiveram redução de memória apenas na curva de 4 semanas de tratamento com D-gal, quando comparado ao seu grupo controle. Os níveis de BDNF aumentaram em hipocampo e córtex pré-frontal na 1 e 2 semanas de tratamento com D-gal. Porém, houve uma diminuição dos níveis desta neurotrofina em córtex pré-frontal em 4, 6 e 8 semanas de tratamento quando comparado aos seus respectivos controles. Os níveis de NGF encontraram-se aumentados após 1 e 2 semanas de tratamento com D-gal nas duas estruturas e diminuíram apenas em córtex préfrontal após 6 e 8 semanas de tratamento. Os níveis de GDNF em hipocampo encontraram-se aumentados em 1 semana e diminuídos após 8 semanas de tratamento com este monossacarídeo. Em córtex préfrontal, houve diminuição deste fator neurotrófico apenas após 4, 6 e 8 semanas de tratamento com D-gal, quando comparado aos controles. Com isso, pode-se sugerir que a administração oral de D-gal pode causar comprometimento cognitivo e redução dos níveis de fatores neurotróficos em animais, podendo mimetizar alterações semelhantes ao envelhecimento e DA.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-Graduação em Química / Made available in DSpace on 2013-07-16T02:17:49Z (GMT). No. of bitstreams: 0 / No presente trabalho, foram sintetizados e caracterizados novos complexos mononucleares de cobre(II), zinco(II), manganês(III), gálio(III), índio(III) e ferro(III). As estruturas cristalinas dos complexos 1, 2, 3, 5, 7, 8 e 12 foram obtidas através da análise de monocristais dos respectivos complexos pelo método de difração de raios X. Praticamente todos os complexos apresentam um ambiente de coordenação tetragonalmente distorcido para os íons metálicos, sendo a geometrias observadas nos complexos de cobre muito similares as do sítio ativo da enzima GAO. Através da oxidação dos complexos 1 a 10, via processos químicos e/ou eletroquímicos foram obtidos complexos radicalares (metalo-fenoxil) do tipo [MII(HL)]2+", [MII(HL)]3+"", [MII(L)]+", [MII(L)]2+"", [MIII(L)]2+", [MIII(L)]3+"" e [MnIV(L)]4+"", os quais foram devidamente caracterizados pelas espectroscopias de UV-VIS, RPE, assim como técnicas espectroeletroquímicas e coulométricas. As propriedades eletroquímicas e eletrônicas dos complexos metalo-fenoxil apresentam bastante semelhança com às encontradas na GAO e em outros complexos modelo metalo-fenoxil, o que coloca estes complexos como bons modelos funcionais para a enzima GAO e para espécies metalo-radicalares. Alguns dos complexos metalo-fenoxil obtidos são moderadamente estáveis à temperatura ambiente e mostraram-se efetivos na oxidação do substrato 2,4,6-tri-terc-butilfenol.

Tese (Doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. / Made available in DSpace on 2012-10-18T20:30:05Z (GMT). No. of bitstreams: 0Bitstream added on 2016-01-09T02:52:57Z : No. of bitstreams: 1 141953.pdf: 25714431 bytes, checksum: 7760a9ba9c4a5fbf040ce062f39c0ebb (MD5)

A β-galactosidase (E.C 3.2.1.23) é uma das enzimas mais empregadas na indústria de alimentos sendo utilizada na hidrólise da lactose. Neste trabalho foram utilizadas duas metodologias para imobilização desta enzima. Na primeira delas foi empregado como suporte um material híbrido à base de sílica que possui um grupo orgânico catiônico covalentemente ligado. A adsorção da enzima a este material apresentou eficiência que variou de 74 a 53% com o aumento da quantidade de enzima aplicada ao suporte. A baixa estabilidade térmica da enzima imobilizada obtida e as prováveis fracas interações envolvidas na sua adsorção a este suporte podem explicar o decréscimo de atividade observada durante as sucessivas bateladas de hidrólise da lactose. Na primeira batelada o grau de hidrólise foi de 90,9% e no final da última batelada (4ª), a enzima foi capaz de converter apenas 13% do substrato. A segunda metodologia utilizada foi imobilização covalente da enzima em um filme de celulose/líquido iônico modificado com uma poliamina e ativado com glutaraldeído. A presença da poliamina foi confirmada por análises de infravermelho. Após a imobilização, a enzima reteve 60% de sua atividade inicial. Bons resultados de hidrólise da lactose em batelada foram obtidos tanto a 7ºC como a 35ºC e foi possível reutilizar a enzima imobilizada por 16 ciclos consecutivos, a 7ºC, sem mudanças significativas na atividade enzimática. O valor de Km para a enzima imobilizada no material híbrido à base de sílica foi de 9,17 mM e para a enzima imobilizada nos filmes de celulose foi de 11,22 mM, ambos apresentaram um acréscimo quando comparados ao Km enzima livre (1,25 mM), devido à dificuldade de acesso do substrato ao sítio ativo da enzima. Não houve mudança no pH e temperatura ótimos da enzima imobilizada em relação à enzima livre em nenhum dos métodos testados. / β-galactosidase (E.C 3.2.1.23) is the most widely used enzymes in the food industry and its employed in the lactose hydrolysis process. In this study, two methodologies were used to test their immobilization. In the first, the enzyme was immobilized by adsorption in one silica based hybrid material that contains a cationic organic group covalently linked. The efficiency of immobilization showed a decrease of 74 to 53% by increasing the protein load applied to the support. The low thermo stability of the immobilized enzyme and the probable weak interactions involved in their adsorption, could explain the decrease in enzyme activity observed in the successive batch hydrolysis of lactose. In the first run, the degree of lactose hydrolysis was 90.9% and, at the end of the last run (4th), the enzyme was able to convert only 13% of the substrate. The second methodology used was the covalent immobilization of the enzyme on a cellulose/ionic liquid film, modified with a polyamine and activated using glutaraldehyde. The presence of a polyamine was confirmed by infrared analysis. After immobilization, the enzyme retained 60% of its initial activity. Highly efficient lactose conversion was achieved in a batch process at 7ºC and 35ºC and was possible to reuse the immobilized enzyme in 16 repeated cycles, at 7ºC, without any drastic decrease in enzyme activity. Km value for the immobilized enzyme in silica based hybrid material was 9.17 mM and for the enzyme immobilized in the film of cellulose/ionic liquid was 11.22 mM, both showing an increase compared with the Km value for free enzyme (1.25 mM), due to the difficulty of access of the substrate to the active sites of the enzyme. The immobilized enzyme did not show any changes in the optimal pH and temperature when compared to the free enzyme in both methods tested.