Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-02-21T15:59:01Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_fabio_de_oliveira_gomes.pdf: 605733 bytes, checksum: 828b835237115c312d92d69a1de39520 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-02-22T17:21:29Z (GMT) No. of bitstreams: 2 dissertacao_fabio_de_oliveira_gomes.pdf: 605733 bytes, checksum: 828b835237115c312d92d69a1de39520 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-02-22T17:25:57Z (GMT) No. of bitstreams: 2 dissertacao_fabio_de_oliveira_gomes.pdf: 605733 bytes, checksum: 828b835237115c312d92d69a1de39520 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-02-22T17:38:56Z (GMT). No. of bitstreams: 2 dissertacao_fabio_de_oliveira_gomes.pdf: 605733 bytes, checksum: 828b835237115c312d92d69a1de39520 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-05-20 / Sem bolsa / A Beta amilase é uma enzima do malte que tem a propriedade da hidrolise do amido em açúcares fermentescíveis. Atua hidrolisando ligações alfa-1,4 do amido, mais precisamente nas cadeias lineares da amilose e amilopectina, liberando carboidratos de cadeias menores (maltose, glicose e maltotriose), necessários para processo de fermentação pela levedura cervejeira. Essa quebra ocorre basicamente para disponibilizar açúcares que posteriormente servirão para formação de álcool na cerveja. A atividade enzimática é inicialmente dependente da solubilização das enzimas, como exemplo na mosturação onde os grãos moídos são imersos em água. Existem outros fatores como temperatura, tempo e pH que garantem máxima eficiência enzimática, o que de certa forma é de grande importância para cervejaria na busca de um rendimento otimizado durante a brassagem. Este trabalho propôs um estudo da beta amilase em diferentes períodos de atuação sobre o amido presente no mosto cervejeiro avaliando qual o melhor período de atuação enzimática. Foi utilizada uma única temperatura, diferentes tempos de atuação, que vão de zero até 60 minutos, intercalados em cinco minutos. Todas as amostras foram fermentadas e analisadas individualmente, buscando identificar qual o melhor período de atuação desta enzima e como os mostos produzidos em diferentes períodos de tempo sofreram fermentação, de acordo com a graduação alcoólica, teor alcoólico e extrato real. / Beta amylase is an enzyme of the malt that has the property of breaking down starch into fermentable sugars. Connections acts hydrolyzing alpha -1 ,4 starch, more precisely in the linear chains of amylose and amylopectin, lower releasing carbohydrate chains ( maltose, maltotriose and glucose ) necessary to process the fermentation by the brewing yeast. This breakdown occurs primarily to provide sugars that subsequently serve to formation of alcohol in beer . The enzymatic activity is initially dependent on the solubilization of enzymes , such as the mashing where ground beans are immersed in water . There are other factors such as temperature , time and pH to ensure maximum enzyme efficiency, which is of great importance for brewery in search of a performance optimized during mashing somehow . This paper proposed a study of beta amylase in different periods of activity on the starch present in the beer wort evaluating the best period of enzymatic activity . A single temperature , different operating times , ranging from zero to 60 minutes , interspersed in five minutes was used . All samples were fermented individually analyzed in order to identify the best period of activity of this enzyme and the musts produced in different periods of time have undergone fermentation , according to alcohol content , alcohol content , real extract and apparent extract.

>Magister Scientiae - MSc / The production of reactive oxygen species (ROS) is prominent in all aerobic metabolisms including plants. For this reason, the redox homeostasis of the production and scavenging of these intermediates is imperative for growth, development and survival during unfavourable conditions. In this study, a putative glutathione peroxidase gene (Glyma17g34110) from Glycine max (soybean) was identified and analyzed. The successful characterisation of Glyma17g34110 provided evidence of it being a glutathione peroxidase using glutathione as its preferred electron donor and substrate. Furthermore, it is known that antioxidant enzymes such as GPX exist in various tissues, performing a diverse set of functions. By a bioinformatic analysis of Glyma17g34110 and its promoter region, it was indicated that Glyma17g34110 could be a putative chloroplast protein that could play an important role in photosynthesis.One of the major factors affecting plant growth and development worldwide is abiotic stresses such as salinity. In the presence of salinity the production of harmful ROS is increased, resulting in detrimental reactions with important biological features (DNA, protein and lipid membranes), leading to cell death. The analysis of Glyma17g34110 under salt stress revealed that it is a salt sensitive gene and thus, the down-regulation of Glyma17g34110 could be due to the lack of known defence and response cis-acting elements present in the promoter region. Furthermore, it was proven in previous studies that the application of exogenous nitric oxide (NO) increases the activity of antioxidant enzymes. In this thesis it was observed that the presence of exogenously applied NO increased the expression of Glyma17g34110 tremendously in all soybean tissues (leaves, roots and nodules) investigated.Studies have found numerous cis-acting elements to be NO responsive, however, none of these elements were found in the promoter region upstream of glyma17g34110. This suggests that novel cis-acting elements could be present in the promoter region of Glyma17g34110.Thus, increasing the expression of Glyma17g34110 during salinity in the presence of NO, as well as the identification of these novel cis-acting elements, could lead to the enhancement of the defence mechanisms against ROS, which could lead to increasing plant tolerance to stress.

Melanina é um polímero constituído por uma grande heterogeneidade de monômeros tendo como característica comum a presença de grupos indóis. Por outro lado, a eumelanina produzida pela oxidação enzimática da tirosina é um polímero mais simples constituído principalmente de monômeros 5,6-dihidroxindol (DHI) e de indol-5,6-quinona (IQ). Tirosinase é a enzima chave na produção de melanina, sendo que a sua atividade cinética é medida em função da formação do intermediário dopacroma. Nanopartículas (NPs) de sílica são partículas nanométricas compostas de oxido de silício e são obtidas pelo processo sol-gel desenvolvido por Stöber de hidrólise e condensação de tetraetilortosilicato (TEOS), usando etanol como solvente em meio alcalino. As NPs foram funcionalizadas com 3-Aminopropiltrietoxissilano (ATPES) e depois com glutaraldeído. Este último permitiu a imobilização da tirosinase na superfície da sílica. Caracterizamos as NPs antes e após a reação da enzima, a atividade catalítica da enzima ligada à NP e o mecanismos de formação de melanina na superfície da sílica. As NPs foram caracterizadas por espectrofotometria de absorção e de reflectância, termogravimetria e microscopia eletrônica. A síntese da NP de sílica retornou partículas esféricas com 55nm de diâmetro e a funcionalização da partícula mostrou modificar eficientemente a sua superfície. A imobilização da tirosinase por ligação covalente foi de 99,5% contra 0,5% da adsorção física. A atividade da tirosinase foi caracterizada pela formação de dopacroma. O Km da enzima imobilizada não sofreu alteração em comparação com a tirosinase livre, mas a eficiência catalítica - que considera a eficiência recuperada - foi de apenas 1/3 para a enzima ligada covalentemente, significando que 2/3 das enzimas ligadas não estão ativas. Obtivemos NPs revestidas com melanina a partir de oxidação de tirosina solubilizada em duas preparações: NP com tirosinase ligada covalentemente na superfície e NP funcionalizada com glutaraldeido dispersa em solução de DHI e IQ. O revestimento de melanina foi na forma de um filme fino com espessura ~1,9nm, conferindo perfil de absorção luminosa equivalente ao da própria melanina. Mostramos que o mecanismo de polimerização passa pela oxidação da tirosina pela tirosinase, que gera intermediários oxidados (principalmente DHI e IQ) que vão para solução (mesmo quando a tirosinase está ligada covalentemente na sílica). Estes intermediários ligam-se ao glutaraldeido e a superfície da sílica passa a funcionar como ambiente de polimerização da melanina. / Melanin is a polymer consisting of a large heterogeneity of monomers having as a common feature the presence of indole groups. Contrarily, eumelanin produced by enzymatic oxidation of tyrosine is a simpler polymer consisting mainly of 5,6-dihidroxindol (DHI) and indole-5,6-quinone (IQ) monomers. Tyrosinase is the key enzyme in melanin production, and its kinetic activity is measured by the formation of the intermediate dopacroma. Nanoparticles (NPs) are made of silica nanoparticles of silicon oxide and are obtained by sol-gel method developed by Stöber of hydrolysis and condensation of tetraethylorthosilicate (TEOS), using ethanol as solvent in an alkaline medium. NPs were functionalized with 3-Aminopropyltriethoxysilane (ATPES) and then with glutaraldehyde. The latter allows the immobilization of tyrosinase on the silica surface. We characterized NPs before and after the reaction of the enzyme, the catalytic activity of the enzyme bound to the NP and melanin-forming mechanisms on the silica surface. NPs were characterized by absorption spectrophotometry and reflectance, electron microscopy and thermogravimetric analysis. The synthesis of silica NP returned spherical particles of 55nm diameter and particle functionalization showed efficiently modify its surface. The immobilization of tyrosinase by covalent bond was 99.5% versus 0.5% by physical adsorption. The activity of tyrosinase was characterized by the formation of dopacroma. The Km of the immobilized enzyme did not change compared to the free tyrosinase, but the catalytic efficiency - considering the recovered efficiently - was only 1/3 for the enzyme covalently bound, meaning that 2/3 of the enzymes are not connected active. We obtained melanin coated NPs from tyrosine oxidation in two preparations: NP with covalently bound tyrosinase in the NP surface and NP functionalized with glutaraldehyde dispersed in DHI and IQ solution. The melanin coating was in the form of a thin film with the thickness of ~ 1,9 nm, giving light absorption profile equivalent to that of melanin itself. We showed that the polymerization mechanism involves the oxidation of tyrosine by tyrosinase, which generates oxidized intermediates (especially DHI and lQ) that go into solution (even when tyrosinase is covalently bound to the silica). These intermediates bind the glutaraldehyde and the surface of the silica begins to function as an environment for melanin polymerization.

Tomáš Křížek: Applications of Capillary Electrophoresis in Life Sciences (Dissertation thesis) ABSTRACT This thesis is focused on the applications of capillary electrophoresis in two important areas of life sciences, proteomics and enzyme assays. In the first part, Pluronic F-127 copolymer was studied as a sieving matrix for proteomic applications of capillary gel electrophoresis. The effect of thermoassociation of Pluronic F-127 on the separation selectivity was investigated and no difference in selectivity of the separation below, inside and above the thermoassociation temperature region was observed. The performance of Pluronic F-127 in capillary gel electrophoresis was compared with dextran as a commonly used sieving matrix. The results showed, that Pluronic F-127 offers superior performance for low-molecular-mass proteins because it provides higher separation power than dextran with significantly lower viscosity of the background electrolyte. The lower viscosity makes the polymer easier to replace after each analysis, which leads to remarkably higher repeatability of the experiments. On the other hand, dextran, due to its higher viscosity, was shown to be more convenient for separations of protein digests, where extremely high separation efficiency is required. The second part focuses on...

Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...

Die humanen Noroviren (Familie Caliciviridae, Genus Norovirus) bilden den Haupterreger der nichtbakteriellen Gastroenterotiden. Seit Beginn der 1990er Jahre – besonders seit dem Herbst 2002 – kann weltweit eine sehr starke Zunahme der Norovirus-Infektionen beobachtet werden. Die Mehrheit der Erkrankungen (ca. 80%) ist dabei durch den Genotyp II.4 verursacht, wobei innerhalb dieses Genotyps kontinuierlich „neue“ genetisch veränderte Norovirus-Stämme auftreten, die kontinuierlich in „neue“ Subgenotypen eingeordnet werden. Bis dato wird vermutet, dass die hohe Zunahme der Infektionen durch die subgenotypspezifische Evolution des strukturellen Proteins VP1 bewirkt wurde, da dadurch die Bindung der Viruspartikel an die Rezeptoren der Wirtszelle – die HBGAs – verbessert sein könnte bzw. ein breiteres Spektrum an Rezeptoren erkannt werden könnte. Bisher ist aber weitgehend unbekannt, ob im Bereich der nichtstrukturellen Proteine auch eine subgenotypspezifische Evolution stattgefunden hat und ob diese durch eine verbesserte Enzymaktivität zu der Zunahme der Infektionen beigetragen haben könnte. Im Rahmen dieser Arbeit wurden demnach aus unterschiedlichen Stuhlproben die beiden Enzyme NS6pro und NS7pol von jeweils einem Stamm der epidemiologisch relevanten Subgenotypen II.4-1995, II.4-2002, II.4-2004, II.4-2006a und II.4-2006b rekombinant hergestellt und betreffend ihrer Evolution und Enzymaktivität untersucht. Anhand der durchgeführten Untersuchungen konnte festgestellt werden, dass bei den epidemiologisch relevanten Subgenotypen des Genotyps II.4 im Bereich der NS6pro und der NS7pol keine subgenotypspezifische Evolution stattgefunden hat und daher keine subgenotypspezifische Enzymaktivität vorliegt. Somit hatte die Enzymaktivität der NS6pro und der NS7pol keinen Einfluss auf die Zunahme der Infektionen. Dennoch konnte aber beobachtet werden, dass durch die Mutation einer einzelnen Aminosäure – entsprechend der Position in der räumlichen Struktur der NS6pro bzw. der NS7pol – die Enzymaktivität um bis zu 100 Prozent erhöht bzw. reduziert werden kann. Bei diesen bedeutenden Mutationen ist entweder die Substratbindung oder die Koordination bzw. die Zufuhr der Reaktionskomponenten betroffen. Somit konnte bestätigt werden, dass die Zunahme der Infektionen vermutlich doch durch die Evolution des VP1 bedingt ist. Dies ist auch nachvollziehbar, da das VP1 den Eintritt der Viruspartikel in das Zellplasma erlaubt, in dem danach erst die Virusvermehrung durch die Enzyme stattfindet. Hierbei muss aber erwähnt werden, dass die Evolution des VP1 durch die Mutationsrate der NS7pol bestimmt ist, sodass die NS7pol letztendlich doch für die Zunahme der Infektionen verantwortlich ist. Um aber definitiv abzuklären, ob durch die Evolution des VP1 tatsächlich die Bindung der Viruspartikel an die HBGAs verbessert bzw. ein breiteres Spektrum an Rezeptoren erkannt wird, müssten bspw. Bindungs-Assays zwischen den entsprechenden VLPs (virus-like particles) und bereits typisierten Serum- oder Speichelproben – ähnlich den Untersuchungen von Lindesmith et al. (2008) – durchgeführt werden. Darüber hinaus müsste die Evolution und die Funktionalität der restlichen nichtstrukturellen Proteine untersucht bzw. deren Einfluss auf die Zunahme der Infektionen überprüft werden.

Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...

Mittels immunhistochemischer- und zyto-fotometrischer Verfahren wurden die Änderungen der glykolytischen und der oxidativen Enzymkapazität im Skelettmuskel von Patienten mit T2DM mit der Muskelfasercharakteristik untersucht. Weiterführende Analysen klären den Zusammenhang zwischen der Änderung der Muskelfaserverteilung und der Enzymkapazität. Durch eine perkutane Biopsie des M. vastus lateralis wurden 10 Patienten mit T2DM und 15 Gesunden Probanden Muskelgewebe extrahiert. In der anschließenden Zytophotometrie erfolgte die Bestimmung der glykolytischen und oxidativen Enzymaktivität in Abhängigkeit der Fasercharakteristik nach SO, FOG und FT-Fasern. Die Untersuchung verdeutlichte eine Verminderung der oxidativen Enzymaktivität des M. vastus lateralis im Homogenat bei bestehenden T2DM mit gleichzeitiger Verringerung der SO- Muskelfasern um 16 Prozent und Erhöhung der FT- Muskelfasern um 49 Prozent im Vergleich zur Kontrollpopulation. Bei T2DM ist sowohl die oxidative als auch die glykolytische Enzymaktivität in den FG-Fasern als auch in den FOGMischfasern signifikant erhöht. Zusammenfassend weisen unsere Ergebnisse darauf hin, dass die geringere oxidative Enzymaktivität im Homogenat des Skelettmuskel von Patienten mit T2DM vielmehr durch einen verminderten Anteil von SO-Fasern als durch verminderte oxidative Aktivität in einzelnen Fasern verursacht ist. Die erhöhte glykolytische und oxidative Enzymaktivität in einzelnen Muskelfasern korreliert mit dem Maß langfristiger BZHomöostase und der Insulinempfindlichkeit. Diese Adaptation der Skelettmuskulatur könnte einen kompensatorischen Mechanismus bezüglich des pathologischen Glukosestoffwechsels des T2DM darstellen.:1. Zusammenfassung der Arbeiten 1.1 Hintergrund und Ziel der Arbeit 1.2 Studiendesign und Methoden 1.3 Ergebnisse 1.3.1 Bestimmung der Faserzusammensetzung und der metabolischen Enzymaktivität des M. vastus lateralis bei Typ 2 Diabetes mellitus 1.3.2 Zusammenhang zwischen den Parametern der Hyperglykämie und der Insulinresistenz mit der faserspezifischen Stoffwechselaktivität des M. vastus lateralis bei Typ 2 Diabetes mellitus 1.3.3 Die Stickstoffoxid-Synthase-Aktivität der Skelettmuskelfasern des Typ 2 Diabetes mellitus 1.4 Schlussfolgerungen 1.5 Literaturverzeichnis 2. Publikationen 2.1 Altered Fiber Distribution and Fiber-Specific Glycolytic and Oxidative Enzyme Activity in Skeletal Muscle of Patients With Type 2 Diabetes 2.2 Metabolic profile and nitric oxide synthase expression of skeletal muscle fibers are affected by type 1 and type 2 diabetes 3. Erklärung über die eigenständige Abfassung der Arbeit

Silver nanoparticles (AgNPs) are expected to enter aquatic systems, but there are limited data on how they might affect microbial communities in pathogen impaired streams. We examined microbial community responses to citrate-AgNP (10.9 ± 0.7 nm) and polyvinylpyrrolidone (PVP)-AgNP (11.0 ± 0.7 nm) based on microbial concentration and enzyme activity in sediment from a pathogen impaired stream. Addition of each nanoparticle to sediment caused at least a 69% decrease in microbial concentration (1,264 ± 93.6 to 127 ± 29.5 CFU/g) and a 62% decrease in β-glucosidase activity (11.7 ± 2.1 to 1.3 ± 0.3 μg/g/h). Each AgNP reduced alkaline phosphatase activity but their effects were not statistically significant. Sediment exposed to 0.108 mg Ag/kg of AgNO3 resulted in a 92% decrease in microbial concentration and a reduced enzyme activity which was not statistically significant. Measured total silver in sediments treated with AgNPs which exhibited significant inhibition effects on the microbial community ranged from 0.19 ± 0.02 to 0.39 ± 0.13 mg Ag/kg. These concentrations tested in this study are much lower than the expected concentrations (2-14 mg Ag/kg) in freshwater sediments. The results of this study demonstrate that AgNPs can alter microbial community activity and population size, which may lead to false negative fecal indicator bacteria detection and enumeration using methods that rely on β-glucosidase activity. We conclude that the presence of AgNPs in impaired streams and recreational waters can influence pathogen detection methods, potentially affecting public health risk estimates.

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first protocol tackles this problem by developing a way to obtain active untagged recombinant PRMT proteins. The second protocol describes a fast and efficient method for quantitative measurement of AdoMet-dependent methyltranseferase activity with protein substrates. In addition to being very sensitive, this method decreases the processing time for the analysis of PRMT activity to a few minutes compared to weeks by traditional methods, and generates 3000-fold less radioactive waste. We then used these methods to investigate the effect of truncating the NT of human PRMT1 variant 1 (hPRMT1-V1) on enzyme activity, protein-protein interactions, and substrate specificity. Our studies show that the NT of hPRMT1-V1 influences enzymatic activity and protein-protein interactions. In particular, methylation of a variety of protein substrates was more efficient when the first 10 amino acids of hPRMT1v1 were removed, suggesting an autoinhibitory role for this small section of the N-terminus. Likewise, as portions of the NT were removed, the altered hPRMT1v1 constructs were able to interact with more proteins. Overall, my studies suggest the the sequence and length of the NT of hPRMT1v1 is capable of enforcing specific protein interactions.