A mutagênese química utilizando o agente mutagênico N-ethyl-N-nitrosourea (ENU) seguida da observação do fenótipo deu origem a um mutante Anêmico. O tipo de herança é autossômica dominante, com morte intra útero dos mutantes homozigotos. O mapeamento genético foi feito utilizando-se marcadores microssatélites, sendo selecionados marcadores polimórficos entre as linhagens BALB/c e C57BL/6 envolvidas no mapeamento. Estabeleceu-se um painel de microssatélites distribuídos por todo o genoma do camundongo, que permitisse a localização do cromossomo portador da mutação. O gene mutante foi localizado no cromossomo 7 entre os marcadores D7Mit301 e D7Mit131 delimitando um intervalo entre 46,5cM e 51cM de 4,5cM. Através das análises fenotípicas do mutante Anêmico e estudo dos genes candidatos neste intervalo, foi selecionado o gene Hbb responsável pela síntese das globinas b-major e b-minor , sendo o gene que mais se identifica com as características do mutante, localizado a 50cM. A deficiência deste gene leva a uma das mais severas anemias humana, a b-Talassemia major. / Chemical mutagenesis, using the mutagenic agent N-ethyl-N-nitrosourea (ENU), and followed by observation of the phenotype, originated in an Anaemic mutant. The inheritance-type is dominant auto-somic, with intra-uterus death of the homozygotic mutants. Genetic mapping was undertaken by means of micro-satellite markers, polymorphic markers being selected from among the BALB/c and C57BL/6 lineages involved in the mapping itself. A panel was established of the micro-satellites distributed throughout the whole mouse genome, thereby permitting localization of the mutation bearing chromosome. The mutant gene was located in chromosome 7 between markers D7Mit301 and D7Mit131, these delimiting an interval between 46,5cM and 51cM of 4,5cM. Selection of the Hbb gene responsible for synthesis of the b-major and b-minor globins came about through phenotypic analysis of the Anaemic mutant and a study of candidate genes within this interval, the selected gene being that which was most identified with the mutants characteristics and located at 50cM. A deficiency in this gene leads to one of the most severe forms of human anaemia, b-Talhassemia major.

Made available in DSpace on 2016-05-02T13:54:46Z (GMT). No. of bitstreams: 1 AdrianaPoncianoFernandes-dissertacao-completa-PDF.pdf: 757559 bytes, checksum: e6a59737e6f245fe2c768705d6b780ac (MD5) Previous issue date: 2008-10-13 / This study analyzed the effects of the hydroalcoholic extract of Pyrostegia venusta, popularly known as cipó-de-são-joão , on various types of Gram negative and Gram positive bacteria, and on yeasts. It also evaluated the effects of the extract on the growth and cell differentiation in Herpetomonas samuelpessoai in vitro , and the in vivo mutagenic effect by the micronucleus test. The antimicrobial activity of the extract was evaluated by two methods: agar diffusion test, and tube dilution test. The growth and cell differentiation of H. samuelpessoai occurred in chemically defined medium after incubation at 28°C, for 48 hours. Growth was calculated by cell count in a Neubauer chamber, and differentiation was measured by observing cells stained by the panoptic method to calculate the percentages of the pro-, para- and opistomastigote forms. To determine the LD 50, groups of female albino Swiss mice received a single oral dose of different extract concentrations (300 mg/kg and 2000 mg/kg). For mutagenic evaluation, Swiss albino mice, aged approximately12 weeks, were used. Each trial was carried out in five groups of animals, each group consisting of 3 males and 3 females: negative control (0.9% NaCl); positive control (50 mg/kg of ENU) treatments 1, 2 and 3 (1000, 1500 and 2000 mg/kg of extract, respectively). The micronucleus test in mouse bone marrow erythrocytes was done 24 and 28 hours after treatment. The polychromatic erythrocytes (PCEs) were observed through an optical microscope and counted with the help of a digital cell counter. The results showed that the hydroalcoholic extract of Pyrostegia venusta leaves at the concentrations of 72.6 mg/mL and 145.2 mg/mL had no antimicrobial activity on the 19 strains of bacteria and yeasts tested. With regard to LD50, the extract did not show median lethal dose at the concentrations of 300 mg/kg and 2000 mg/kg. The micronucleus test showed statistically significant differences in the number/percentage index of micronucleated PCEs between the positive control group (ENU 50mg/kg) and negative control (0.9% NaCl), and positive control and extract treatments. But these differences were not observed either between the negative controls and the extract-treated group, or between the sexes and times of treatment (24 hr-48hr), thus suggesting that the hydroalcoholic extract of P. venusta leaves does not exhibit either clastogenic or aneugenic potentials. / Este estudo teve por objetivos analisar os efeitos do extrato hidroalcoólico de Pyrostegia venusta, conhecida popularmente como cipó-de-são-joão, sobre diversos tipos de bactérias Gram-negativas e Gram-positivas e também sobre leveduras, além de analisar seu efeito no crescimento e diferenciação celular em Herpetomonas samuelpessoai in vitro e mutagênico in vivo , através do Teste do Micronúcleo. A atividade antimicrobiana do extrato foi verificada por dois métodos: teste de difusão em ágar e teste de diluição em tubo. Os experimentos de crescimento e diferenciação celular de H. samuelpessoai foram realizados em meio quimicamente definido, após incubação a 28 °C, por 48 horas, sendo o crescimento estimado pela contagem das células em câmara de Neubauer e a diferenciação pela observação das células coradas pelo método Panótico em microscopia óptica, objetivando estimar os percentuais de formas pró, para e opistomastigota. Para a determinação da DL 50 foram utilizados grupos de camundongos Swiss albinos fêmeas que receberam, por via oral, dose única de diferentes concentrações do extrato (300 e 2000 mg/Kg). Para a avaliação mutagênica foram utilizados camundongos Swiss albinos, com idade aproximada de 12 semanas. Cada ensaio foi realizado empregando-se cinco grupos de animais, cada grupo constituído por 3 machos e 3 fêmeas, sendo assim tratados: controle negativo (NaCl 0,9%); controle positivo (50 mg/kg ENU); tratamento 1, 2 e 3 (1000, 1500 e 2000mg/Kg do extrato, respectivamente). O teste do micronúcleo em eritrócitos da medula óssea de camundongos foi realizado 24 e 48 horas após o tratamento, e os eritrócitos policromáticos (PCEs) foram observados em microscopia óptica e contados com o auxílio de um contador de células digital. Os resultados evidenciaram que o extrato hidroalcoólico da folha de Pyrostegia venusta, nas concentrações testadas (72,6 mg/mL e 145,2 mg/mL), não possui atividade antimicrobiana para as dezenove cepas testadas de bactérias e fungos. No que se refere à DL50, o extrato não apresentou dose letal média nas concentrações testadas de 300mg/kg e 2000mg/kg. Ao teste de micronúcleo, os resultados revelaram diferenças estatísticas significativas do número/índice percentual de PCEs micronucleados entre o grupo de animais do controle positivo (ENU 50mg/Kg) e controle negativo (NaCl 0,9%), bem como controle positivo e tratamentos com o extrato. Entretanto, essas diferenças não foram observadas entre o grupo de animais do controle negativo e o grupo de animais tratados com o extrato e, ainda, entre os sexos e os tempos de tratamento (24-48h), sugerindo que o extrato hidroalcoólico de folhas de P. venusta não apresenta potencial clastogênico e/ou aneugênico.

Made available in DSpace on 2016-05-02T13:54:48Z (GMT). No. of bitstreams: 1 AdrianaPoncianoFernandes-dissertacao.pdf: 757676 bytes, checksum: 9e7b2d453d791c3e8b207f675e650f36 (MD5) Previous issue date: 2008-10-13 / This study analyzed the effects of the hydroalcoholic extract of Pyrostegia venusta, popularly known as cipó-de-são-joão , on various types of Gram negative and Gram positive bacteria, and on yeasts. It also evaluated the effects of the extract on the growth and cell differentiation in Herpetomonas samuelpessoai in vitro , and the in vivo mutagenic effect by the micronucleus test. The antimicrobial activity of the extract was evaluated by two methods: agar diffusion test, and tube dilution test. The growth and cell differentiation of H. samuelpessoai occurred in chemically defined medium after incubation at 28°C, for 48 hours. Growth was calculated by cell count in a Neubauer chamber, and differentiation was measured by observing cells stained by the panoptic method to calculate the percentages of the pro-, para- and opistomastigote forms. To determine the LD 50, groups of female albino Swiss mice received a single oral dose of different extract concentrations (300 mg/kg and 2000 mg/kg). For mutagenic evaluation, Swiss albino mice, aged approximately12 weeks, were used. Each trial was carried out in five groups of animals, each group consisting of 3 males and 3 females: negative control (0.9% NaCl); positive control (50 mg/kg of ENU) treatments 1, 2 and 3 (1000, 1500 and 2000 mg/kg of extract, respectively). The micronucleus test in mouse bone marrow erythrocytes was done 24 and 28 hours after treatment. The polychromatic erythrocytes (PCEs) were observed through an optical microscope and counted with the help of a digital cell counter. The results showed that the hydroalcoholic extract of Pyrostegia venusta leaves at the concentrations of 72.6 mg/mL and 145.2 mg/mL had no antimicrobial activity on the 19 strains of bacteria and yeasts tested. With regard to LD50, the extract did not show median lethal dose at the concentrations of 300 mg/kg and 2000 mg/kg. The micronucleus test showed statistically significant differences in the number/percentage index of micronucleated PCEs between the positive control group (ENU 50mg/kg) and negative control (0.9% NaCl), and positive control and extract treatments. But these differences were not observed either between the negative controls and the extract-treated group, or between the sexes and times of treatment (24 hr-48hr), thus suggesting that the hydroalcoholic extract of P. venusta leaves does not exhibit either clastogenic or aneugenic potentials. / Este estudo teve por objetivos analisar os efeitos do extrato hidroalcoólico de Pyrostegia venusta, conhecida popularmente como cipó-de-são-joão, sobre diversos tipos de bactérias Gram-negativas e Gram-positivas e também sobre leveduras, além de analisar seu efeito no crescimento e diferenciação celular em Herpetomonas samuelpessoai in vitro e mutagênico in vivo , através do Teste do Micronúcleo. A atividade antimicrobiana do extrato foi verificada por dois métodos: teste de difusão em ágar e teste de diluição em tubo. Os experimentos de crescimento e diferenciação celular de H. samuelpessoai foram realizados em meio quimicamente definido, após incubação a 28 °C, por 48 horas, sendo o crescimento estimado pela contagem das células em câmara de Neubauer e a diferenciação pela observação das células coradas pelo método Panótico em microscopia óptica, objetivando estimar os percentuais de formas pró, para e opistomastigota. Para a determinação da DL 50 foram utilizados grupos de camundongos Swiss albinos fêmeas que receberam, por via oral, dose única de diferentes concentrações do extrato (300 e 2000 mg/Kg). Para a avaliação mutagênica foram utilizados camundongos Swiss albinos, com idade aproximada de 12 semanas. Cada ensaio foi realizado empregando-se cinco grupos de animais, cada grupo constituído por 3 machos e 3 fêmeas, sendo assim tratados: controle negativo (NaCl 0,9%); controle positivo (50 mg/kg ENU); tratamento 1, 2 e 3 (1000, 1500 e 2000mg/Kg do extrato, respectivamente). O teste do micronúcleo em eritrócitos da medula óssea de camundongos foi realizado 24 e 48 horas após o tratamento, e os eritrócitos policromáticos (PCEs) foram observados em microscopia óptica e contados com o auxílio de um contador de células digital. Os resultados evidenciaram que o extrato hidroalcoólico da folha de Pyrostegia venusta, nas concentrações testadas (72,6 mg/mL e 145,2 mg/mL), não possui atividade antimicrobiana para as dezenove cepas testadas de bactérias e fungos. No que se refere à DL50, o extrato não apresentou dose letal média nas concentrações testadas de 300mg/kg e 2000mg/kg. Ao teste de micronúcleo, os resultados revelaram diferenças estatísticas significativas do número/índice percentual de PCEs micronucleados entre o grupo de animais do controle positivo (ENU 50mg/Kg) e controle negativo (NaCl 0,9%), bem como controle positivo e tratamentos com o extrato. Entretanto, essas diferenças não foram observadas entre o grupo de animais do controle negativo e o grupo de animais tratados com o extrato e, ainda, entre os sexos e os tempos de tratamento (24-48h), sugerindo que o extrato hidroalcoólico de folhas de P. venusta não apresenta potencial clastogênico e/ou aneugênico

A l’arrivée d’un potentiel d’action au niveau d’une synapse neuronale, des ions calcium (Ca2+) pénètrent dans le neurone, permettant aux protéines SNAREs (N-ethylmaleimide-sensitive factor activating protein receptor) de s’assembler entièrement, engendrant la fusion des vésicules synaptiques contenant les neurotransmetteurs avec la membrane plasmique du neurone. Des protéines régulatrices telles que la Complexine et la Synaptotagmine sont étroitement couplées aux SNAREs et permettent une fusion rapide et synchrone. La Synaptotagmin-1 (Syt1), une protéine transmembranaire localisée sur les vésicules synaptiques, est le senseur calcique de la neurotransmission. Syt1 possède deux domaines de liaison au Ca2+, C2A et C2B, un domaine flexible reliant la région membranaire au C2A, ainsi qu’un court lien entre C2A et C2B. Il a été montré qu’une région polybasique dans le C2B se liait aux lipides anioniques tels que phosphatidylserine (PS) et phosphatidylinositol-4,5-bisphosphate (PIP2) en l’absence de Ca2+. A l’entrée du Ca2+, les ions Ca2+ se lient au C2A et au C2B. La liaison de Syt1 aux ions Ca2+ permet aux résidus non polaires à proximité des sites de liaison au Ca2+ de s’insérer dans la membrane. Si ces mécanismes sont relativement bien acceptés, les mécanismes biochimiques et biophysiques précis du déclenchement de la fusion induit par la liaison de Syt1 au Ca2+ restent flous. Dans ce travail, nous mesurons directement les interactions de Syt1 liée à une membrane avec des membranes anioniques comprenant des lipides PS et PIP2 par un appareil à force de surface (SFA), afin d’imiter la membrane d’une vésicule synaptique contenant Syt1 interagissant avec la membrane plasmique anionique. Nous réalisons une mutagénèse dirigée sur les sites de liaison au Ca2+ de C2A et C2B, ainsi que sur le site polybasique de C2B, pour entièrement cartographier les énergies de liaison à la membrane relatives à ces sites, à la fois en présence et en l’absence d’ions divalents. Nous trouvons que Syt1 se lie avec une énergie de ~6 kBT dans l’EGTA, ~10 kBT dans le Mg2+, et ~18 kBT dans le Ca2+. Des réarrangements moléculaires mesurés pendant le confinement de Syt1 entre les membranes prévalent dans le Ca2+ et dans le Mg2+, et suggèrent que Syt1 se lie initialement via le C2B puis réoriente ses domaines C2 dans la conformation de liaison privilégiée. La neutralisation des sites de liaison au Ca2+ de C2B engendre une réduction radicale de l’énergie de liaison de Syt1 dans le Ca2+, alors que la même mutation dans le C2A a un effet plus nuancé. Ces résultats éclairent sur la coopérativité de C2A et C2B dans leur liaison à la membrane, et montrent un rôle apparent prédominant de C2B. / Upon arrival of an action potential at the neuronal synapse, calcium ions (Ca2+) enter the neuron, allowing soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins to fully zipper, leading to the fusion of pre-docked synaptic vesicles containing neurotransmitters with the plasma membrane of the neurone. Regulatory proteins such as Complexin and Synaptotagmin are closely coupled to SNAREs during synaptic vesicle fusion and lead to synchronous, fast fusion. Synaptotagmin-1 (Syt1) is a transmembrane protein found in synaptic vesicles and is the Ca2+ sensor for synaptic transmission. Syt1 has two Ca2+ binding domains, C2A and C2B, with a flexible linker domain from the membrane region to C2A, and a short linker between C2A and C2B. A polybasic patch in C2B has been shown to bind to anionic lipids such as phophidylserine (PS) and phosphisotinol (PIP2) in the absence of Ca2+. Upon Ca2+ influx, Ca2+ ions bind in C2A and C2B. Ca2+ binding to Syt1 allows non-polar residues nearby the Ca2+ binding sites to insert into the membrane. While these mechanisms are relatively well-accepted, the precise biochemical and biophysical mechanisms for the Syt1 Ca2+ trigger remain unclear. In this work, we directly measure the interactions of Syt1-coated membranes with anionic membranes including PS and PIP2 lipids by the surface forces apparatus (SFA) technique, in order to mimic a Syt1-coated synaptic vesicle membrane interacting with the anionic plasma membrane. We perform site directed mutagenesis of the Ca2+ binding sites of C2A and C2B, along with the polybasic patch in C2B, to fully map the site-binding energetics of Syt1 with membranes, both in the absence and presence of divalent ions. We find that Syt1 binds with ~6 kBT in EGTA, ~10 kBT in Mg2+, and ~18 kBT in Ca2+. Molecular rearrangements measured during confinement of Syt1 between membranes are more prevalent in Ca2+ and Mg2+ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. Neutralization of C2B Ca2+ binding site leads to a drastic decrease of Syt1 binding energy in Ca2+, while the same mutation in C2A has a milder effect. These results illuminate that C2A and C2B cooperate in membrane binding, with an apparent predominant role of C2B.

No description available.

This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.

MSCAGR (Plant Production) / Department of Plant Production / Tepary bean (Phaseolus acutifolius) is an important food legume originating from South America and the South-western parts of the United States. The crop is produced in many countries worldwide including South Africa. It is highly tolerant to drought and the seed contains a wide range of vitamins, minerals and protein of high nutritional quality. The genetic base of tepary bean is narrow but can be widened by chemical mutagenesis. However, there are no reports on the impact of chemical mutagenesis on the root nodulation and seed storage proteins in tepary bean. Therefore, this study was designed to examine root nodulation attributes and seed storage proteins of three tepary bean genotypes in the early mutagenic generations (M2 to M4) derived through treatment with varying doses (0.0, 0.5, 1.0, 1.5 and 2.0 v/v) of ethyl methanesulfonate (EMS). The experiment on root nodulation attributes was laid out as a 3 x 5 x 3 (genotypes x EMS doses x mutant generations) factorial design replicated three times. At harvest, shoot height (SHT), primary root length (PRL), dry weights (shoot, root and nodule), number of nodules per plant (NNP) and grain yield components such as the number of pods per plant (NPP) and number of seeds per pod (NSP) were measured. Highly significant (P≤0.01) dose effects were observed for SHT, PRL, shoot dry weight (SDW) and root dry weight (RDW). Highly significant (P≤0.01) interaction effects of mutant generation x genotype x dose were observed for NSP. A highly significant (P≤0.01) positive linear relationship was observed between the NNP and nodule dry weight (NDW). Increase in the PRL suggested that tepary bean mutants could be important in drought tolerance. EMS treatment led to an enhanced partitioning of dry matter (assimilates) to the shoots and roots. There was a three fold increase in most of the root nodulation traits at the 0.5% EMS dose.The Kjeldahl method was used for crude protein determination whereas the sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS PAGE) was utilized in determining the protein banding patterns of the bean. There were highly significant (P≤0.01) differences among the genotypes in crude protein accumulation. Highly significant (P≤0.01) mutant generation x genotype x dose were observed for seed protein accumulation. ‘Genotype 3’ attained the highest protein content (24.23%) at 1.5% EMS dose in the M4 generation. EMS doses ≥0.5% positively stimulated protein accumulation in all genotypes but high EMS doses (2.0%) depressed protein content. There were significant variations in seed storage protein profiles among the genotypes and mutant generations. ‘Genotype 6’ showed a distinct 15.0kDa protein fragment which was absent in the majority of the remaining genotypes. The presence of distinct protein subunits in the three genotypes could be used in varietal / NRF

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to c-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0–10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.

Kallikrein-related peptidases KLK5, KLK7 and KLK14 are important proteases in skin desquamation and aberrant KLK activity is associated with inflammatory skin diseases such as Netherton syndrome but also with various serious forms of cancer. Previously, we have identified KLK7 as the first protease target of vaspin (Serpin A12). Here, we report KLK14 as a second KLK protease to be inhibited by vaspin. In conclusion, vaspin represents a multispecific serpin targeting the kallikrein proteases KLK7 and KLK14, with distinct exosites regulating recognition of these target proteases and opposing effects of heparin binding on the inhibition reaction.

Glycosidases (EC 3.2.1.) alias glycoside hydrolases are enzymes that catalyze the cleavage of a glycosidic bond between two carbohydrates or between a carbohydrate and an aglycone. Under suitable conditions (especially reduction of water activity in the reaction mixture), these enzymes are also able to synthesize a glycosidic bond. By targeted mutagenesis of the catalytic centre of the enzymes, it is possible to suppress or completely abolish their hydrolytic activity. Enzyme synthesis using glycosidases makes it possible to prepare bioactive galactosides, for example galectin ligands. The present work deals mainly with β-galactosidase from Bacillus circulans, its recombinant expression and mutagenesis. In the first part of the work, the commercially prepared plasmid of -galactosidase from B. circulans isoform A that I designed was used for recombinant expression in E. coli. It was necessary to optimize the conditions of the enzyme production. As it is a large protein (189 kDa), the expression vector pCOLD II and cold production at 15 ř C were used. The enzyme is specific for the formation of the β-1,4 glycosidic bond and has been used to synthesize complex tri- and tetrasaccharide ligands that cannot be prepared with a crude commercial preparation containing undesirable enzyme activities....