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Sulfur-containing compounds such as thiamin, biotin, molybdopterin, lipoic acid, and [Fe-S] clusters are essential for life. Sulfurtransferases are present in eukaryotes, eubacteria, and archaea and are believed to play important roles in mobilizing sulfur necessary for biosynthesis of these compounds and for normal cellular functions. The rhodanese homology domain is a ubiquitous structural module containing a characteristic active site cysteine residue. Some proteins containing a rhodanese domain display thiosulfate:cyanide sulfurtransferase activity in vitro. However, the physiological functions of rhodaneses remain largely unknown. YrkF, the first rhodanese to be characterized from Bacillus subtilis, is a unique protein containing two domains, an N-terminal Ccd1 domain and a C-terminal rhodanese domain. Ccd1 (conserved cysteine domain 1) is a ubiquitous structural module characterized by a Cys-Pro-X-Pro sequence motif. Thus, YrkF contains two cysteine residues (Cys¹⁵ and Cys¹⁴⁹), one in each domain. Biochemical, genetic, and bioinformatic approaches were used in order to characterize YrkF. First, YrkF was overexpressed and assayed for rhodanese activity to show that the protein is a functional rhodanese. A variant protein, YrkF^C15A, containing a cysteine to alanine substitution in the Ccd1 domain was created to determine if the Ccd1 cysteine is essential for rhodanese activity. The variant protein was overexpressed and rhodanese assays showed that YrkF^C15A is also a functional rhodanese. Inherent structural and catalytic differences were observed when comparing YrkF and YrkF^C15A, which may reflect the importance of the Ccd1 cysteine residue to normal enzymatic function and structural stability. Initial kinetic studies identified differences in activity between YrkF and YrkF^C15A. Cross-linking experiments showed a propensity for the formation of inter- and intramolecular disulfide bonds between the two cysteine residues and indicated that Cys¹⁵ and Cys¹⁴⁹ are located near one another in the 3-dimensional structure of the protein. Analysis of the proteins by mass spectrometry suggested YrkF contains a stable persulfide sulfur, whereas YrkF^C15A showed no evidence of a stable persulfide sulfur and was prone to oxidation and other active site modifications. A homology model of YrkF was created using structures of a rhodanese homolog and a Ccd1 homolog as templates. The model was used to predict the structure of YrkF based on the results of the cross-linking experiments. A strain containing a yrkF chromosomal deletion could be constructed, indicating YrkF is not essential for survival. Phenotypic analysis of the yrkF mutant revealed that YrkF is not needed for biosynthesis of sulfur-containing cofactors (thiamin, biotin, molybdopterin, or lipoic acid) or amino acids. The characterization of YrkF could lead to the discovery of novel physiological roles for rhodaneses and may give insight into possible roles for the Ccd1 module. / Master of Science

Made available in DSpace on 2015-04-17T14:49:20Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1613275 bytes, checksum: 00b0097dfdb9c99181e93e899c5d60df (MD5) Previous issue date: 2011-11-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Recent years have seen a significant increase in demand for starches with specific properties, leading to the modification of these properties in order to get ideal for your application. The objective of this study was to obtain information about the properties of starch from avocado seed cv. "Butter" and submit it to the modification by acetylation and cross-linking, to provide rheological characteristics applicable in obtaining food. The native starch showed starch content of 74.47 % in the physical-chemical analysis and performance of the extraction process used in 11.36%. The degree of substitution (GS) of native starch by acetylation was 0.007 and 0.006 for cross-linking. The native starch granules and had modified oval and circular, their dimensions ranging from 26 to 26.34 μm for the smaller diameter and 35.75 to 36.85 μm for the larger diameter. The pattern of crystallinity shown by X-ray diffraction was type C. The swelling power and solubility at temperatures 75, 85 and 95 °C was maximum for the native starch and modified by acetylation in relation to cross-linking. The changes reduced the ability of native starch hydrophilic and hydrophobic increased their capacity. The native starch gel showed an opaque folder, and with a tendency to retrogradation, modified with acetic anhydride showed no difference compared to the native whereas modification by cross-linking improved the clarity of the folder and decreased the tendency to retrogradation. Stability to freezing and thawing all samples released water in the first cycle, but with each passing day there was a decrease in syneresis. The acetylated starch did not differ from native in the analysis of viscosity has low viscosity and a greater tendency to retrogradation. The starch is modified by cross-linking showed efficacy by introducing cross-links in native starch granule and its peak viscosity was well below the value obtained in native, just had a lower tendency to retrogradation. In Differential Scanning Calorimetry (DSC) was an initial decrease in temperature for starch modified by cross-linking, and all samples did not differ significantly in peak temperature. / Nos últimos anos houve um aumento expressivo na procura por amidos com propriedades específicas, levando à modificação dos mesmos a fim de obter propriedades ideais para sua aplicação. O objetivo deste trabalho foi obter informações a respeito das propriedades do amido da semente de abacate cv. Manteiga e submetê-lo à modificação por acetilação e cross-linking, a fim de oferecer características reológicas aplicáveis na obtenção de produtos alimentares. O amido nativo apresentou teor de amido de 74,47 % na análise físico-química e rendimento para o processo de extração utilizado de 11,36 %. O grau de substituição (GS) do amido nativo por acetilação foi de 0,007 e por cross-linking de 0,006. Os grânulos de amido nativo e modificado apresentaram formato oval e circular, suas dimensões variam de 26 a 26,34 μm para o diâmetro menor e de 35,75 a 36,85 μm para o diâmetro maior. O padrão de cristalinidade mostrado por difratometria de raios-X foi do tipo C. O poder de intumescimento e a solubilidade nas temperaturas 75, 85 e 95 ºC foi máxima para o amido nativo e modificado por acetilação em relação ao cross-linking. As modificações reduziram a capacidade hidrofílica do amido nativo e aumentaram a sua capacidade hidrofóbica. O gel do amido nativo apresentou uma pasta opaca e com tendência a retrogradação, a modificação com anidrido acético não apresentou diferença em relação ao nativo enquanto que, a modificação por cross-linking melhorou a claridade da pasta e diminuiu a tendência a retrogradação. Na estabilidade ao congelamento e descongelamento todas as amostras liberaram água no primeiro ciclo, mas com o passar dos dias houve uma diminuição da sinérese. O amido acetilado não divergiu do nativo na análise de viscosidade apresentando baixa viscosidade e maior tendência a retrogradação. O amido modificado por cross-linking apresentou eficácia com a introdução de ligações cruzadas no grânulo de amido nativo e seu pico de viscosidade foi bem abaixo do valor obtido no nativo, logo teve menor tendência a retrogradação. Na Calorimetria Exploratória Diferencial (DSC) houve uma diminuição da temperatura inicial para o amido modificado por cross-linking, sendo que todas as amostras analisadas não diferiram significativamente na temperatura de pico.

Selective gas-phase dissociation strategies have been developed for the characterization of cross-linked peptides and proteins in quadrupole ion trap mass spectrometers. An infrared chromogenic cross-linker (IRCX) containing a phosphotriester afforded rapid differentiation of cross-linked peptides from unmodified ones in proteolytic digests of cross-linked proteins by selective infrared multiphoton dissociation (IRMPD). Only the cross-linked peptides containing the chromogenic phosphate underwent IRMPD and unmodified peptides were not affected by IR irradiation. IRMPD of IRCX-cross-linked peptides yielded uncross-linked y-ion sequence tags of the constituent peptides due to secondary dissociation of all primary product ions which contained the chromophore, thus allowing successful de novo sequencing of the cross-linked peptides. Peptides cross-linked via a two-step conjugation strategy through the formation of a bis-arylhydrazone (BAH) bond were selectively dissociated by ultraviolet radiation at 355 nm. The BAH-cross-linked peptides could be distinguished from not only unmodified peptides but also dead-end modified peptides based on the selectivity of ultraviolet photodissociation. In a complementary approach, electron transfer dissociation of BAH-cross-linked peptides resulted in preferential cleavage of the hydrazone bond which produced two modified peptides. These modified peptides were subsequently interrogated by CID which allowed for the original site of cross-linking to be pinpointed. IRMPD was implemented in a dual pressure linear ion trap to demonstrate successful photodissociation of peptides having modest absorptivities. Peptides were observed to efficiently dissociation by IR irradiation exclusively in the low pressure cell whereas no dissociation was observed in the high pressure cell due to extensive collisional cooling. IRMPD provided greater sequence coverage of the peptides than CID and yielded product ion mass spectra which were predominantly composed of singly charged product ions which simplified spectral interpretation. IRMPD was further applied for the sequencing of small-interfering RNA. Complete sequence coverage was obtained and the results were compared to CID. / text

O desenvolvimento de nanocarreadores a base de albumina são considerados biocompatíveis e biodegradáveis, e tem sido extensivamente estudada com objetivo de novas alternativas de tratamento para inúmeras doenças. A característica mais relevante reside no fato de que estes sistemas proteicos serem capazes de atravessar a barreira Hematoencefálica (BHE) e atingir as células-alvo, a partir de sinalizações por canais específicos na barreira cerebral. Por serem proteínas com ligações covalentes, pode-se afirmar que o processo de clivagem proteolítica tende a ser realizado pelas enzimas pertencente à família das proteases. Dada à importância desses sistemas de entrega de fármacos (DDS) e sua eficaz permeação através da BHE, propôs-se um desenvolvimento científico multidisciplinar combinando-se protocolos, técnicas e ensaios experimentais das áreas de tecnologia farmacêutica, nanotecnologia e química para realização da incorporação do fotoativo ou outros compostos, como a ftalocianina de cloro alumínio (AlClPc ou Pc) livre e/ou pré encapsuladas e as nanopartículas magnéticas, nestes sistemas de DDS conhecidos como nanopartícula de albumina (NpA). Dentre as técnicas usadas nestes estudos destaca-se tanto o método de cross-linking térmico (via Térmica) quanto o químico (via Química), sendo que no segundo, foram utilizados 2 reagentes distintos, o glutaraldeído e o gliceraldeído, os quais foram analisados e comparados neste projeto de pesquisa. Análises de Microscopia Eletrônica de Varredura (MEV), Microscopia de Força Atômica (MFA), medidas de estabilidade por ZetaSizer demonstraram claramente que as nanopartículas preparadas pelos diferentes reagentes possuíam formato esféricos, diâmetro médio em torno de 200 nm e eram homogêneas, entretanto, apenas os nanocarreadores preparados com GU apresentaram elevada repulsão eletrostática (prevenindo a agregação das mesmas). Após caracterização, os estudos foram baseados na utilização da AlClPc como fotoativo aplicado a terapia fotodinâmica (TFD) para o tratamento in vitro das doenças que acometem o sistema nervoso central (SNC). / The development of albumin-based nanocarriers, which are nontoxic and biocompatible and biodegradable, have been extensively studied for seeking new alternatives of the treatment for numerous diseases. The most relevant characteristic is that these protein-based systems could across the blood-brain barrier (BBB) and selectively achieve the target cells within of the brain. These nanocarriers are proteins-based and have covalent bonds, and consequently it can be digested by a class of enzymes belonging to the protease family, which rapidly degrade the protein-based nanoparticles through of the proteolytic cleavage process. Given the importance of these drug delivery systems (DDS) and their effective permeation through BBB, it was proposed a multidisciplinary scientific development combining protocols, techniques and experimental tests of the areas of pharmaceutical technology, nanotechnology and chemistry to carry out the incorporation of the photoactive or another compound, as aluminum chlorine phthalocyanine (AlClPc or Pc), free and/or pre-encapsulated or magnetic nanoparticles in these albumin-based DDS systems known as albumin nanoparticles (NpA). Among the techniques used in these studies we highlight by thermal cross-linking method (via Thermal) and chemistry (via Chemistry), in this second, it was used 2 reagents, glutaraldehyde and glyceraldehyde, that were analyzed and compared in this research project. From Scanning Electron Microscopy, Atomic Force Microcopy, Zeta potential measurements, we have clearly shown that the elaborated nanoparticles (NPs) have a smaller size with a spherical shape and are more homogeneous, however only the nanoparticles prepared with glutaraldehyde showed greater electronic repulsion (preventing their aggregation). After the characterization, the studies were based on the use of AlClPc as a photoactive applied in the photodynamic therapy (PDT) for the treatment of central nervous system (CNS) diseases.

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Die Arbeit umreißt das Krankheitsbild der Myopie (Kurzsichtigkeit) und deren unterschiedliche Ausprägungen, im Speziellen der progressiven und pathologischen Myopie. Hierbei wird ein Einblick in die Symptomatik, die anatomischen Ursachen und die heutigen medizinischen Interventionen gegeben. Hierdurch wird die Problematik einer zu „weichen“ Sklera (Lederhaut des Auges) und des damit einhergehenden fortschreitenden Augenwachstums deutlich. Im Zentrum der Arbeit steht ein neuer therapeutischer Ansatz zur vorbeugenden Behandlung der pathologischen Myopie; das Riboflavin/Blaulicht Cross-Linking der Sklera des Kaninchenauges. Dessen Wirkungsweise ermöglicht die biomechanische Versteifung von kollagenem Gewebe. Aus diesem Sachverhalt ergibt sich die Fragestellung der Arbeit: Ist das sklerale Riboflavin/Blaulicht Cross Linking geeignet das Augenwachstum im Tiermodell (junge Kaninchen) verträglich zu hemmen? Operationsbeeinflussende Parameter wie die Riboflavin-Durchdringungsdauer der Sklera und die sklerale Lichtdurchlässigkeit werden untersucht und für die Optimierung der Operationsmethode herangezogen und diskutiert. Zur Einschätzung des Versuchsansatzes werden die im Methodikteil dargelegten Anwendungen an adulten und jungen Kaninchen/Kaninchenaugen durchgeführt. In Tierversuchen wird die Schadensschwelle in Abhängigkeit der Blaulichtintensität, des Alters und der Pigmentierung untersucht, wobei histologische, immunhistochemische und elektronenmikroskopische Verfahren angewendet werden. Der inhibitorische Einfluss des Riboflavin/Blaulicht Cross-Linkings auf das Augenwachstum kann im Jungtiermodell durch verschiedene metrische Verfahren und MRT-Untersuchungen belegt werden.