The methionine sulfoxide reductase (Msr) enzymes catalyze the reduction of methionine sulfoxide (Met(O)) to methionine. The Msr enzymes protect cells against oxidative stress and may have a role in aging. The MsrA family of enzymes reduces stereospecifically the S epimer of free and protein-bound Met(O) while the MsrB family reduces the R epimer of Met(O) in proteins. It has been generally accepted, primarily from studies on MsrA, that the biological reductant for the Msr enzymes is thioredoxin (Trx), although high levels of dithiothreitol (DTT) can be used as the reductant in vitro. In contrast, certain MsrB enzymes show less than 10% of the activity with Trx as compared to DTT. This raises the possibility that in animal cells Trx may not be the direct hydrogen donor for the MsrB enzymes. Studies with bovine liver extracts have shown that thionein, the apoprotein of metallothionein, can function as a reductant for the Msr proteins. Certain selenium compounds such as selenocystamine and selenocystine can also serve as potent reducing agents for the Msr enzymes. Since an increased activity of Msr enzymes can reduce the level of oxidative damage in tissues, compounds that could activate Msr may have therapeutic potential. A high-throughput screening assay has been developed to screen large chemical libraries to find activators of MsrA, as well as specific inhibitors that could be useful research tools. This study will be done in collaboration with The Scripps Florida Research Institute. Sulindac was originally developed as a non-steroidal anti-inflammatory drug but has also shown efficacy in the treatment of certain cancers. The S epimer of sulindac is known to be reduced by MsrA, but the enzymes responsible for reduction of the R epimer are not known. / An activity has been purified from rat liver which is capable of reducing the R epimers of sulindac, free Met(O) and a dabsylated Met(O) substrate, the latter suggesting that this enzyme may have properties similar t o the MsrB enzymes. The oxidation of the epimers of sulindac to sulindac sulfone has also been characterized, and the members of the cytochrome P450 family involved in the oxidation have been identified. / by David J. Brunell. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

There is no abstract available for this thesis. / Department of Chemistry

This thesis describes the design, synthesis and application of artificial metalloenzymes for transition metal catalysed reactions not performed by natural enzymes. Unique cysteine containing protein templates were covalently modified with transition metal ligand complexes that generate catalytic activity, which allows for the use of virtually any protein template. SCP-2L was selected as template for the linear hydrophobic tunnel that traverses the protein, which has high affinity for linear aliphatic molecules. The use of catalysts based on this protein to induce increased activity in the biphasic hydroformylation of linear α-olefins is investigated in this work. For this purpose, unique cysteine containing mutants of SCP-2L were modified with phosphine ligands by application of a novel bioconjugation procedure. Application of rhodium adducts of the phosphine modified protein constructs led to up to a 100 fold increase of the turn over numbers was measured compared to a Rh/TPPTS model system which is used in industry. Furthermore, good selectivity towards the linear product was observed. If it can be confirmed that the found catalytic results truly are the result of substrate encapsulation by the protein scaffold, this system represents the first rationally designed artificial metalloenzyme which exploits the shape selectivity of the protein scaffold to direct the outcome of a catalytic reaction. In addition, a study was performed for the development of enantioselective artificial metalloenzymes. Nitrogen ligands were covalently introduced in SCP-2L and the obtained conjugates were applied in the copper catalysed Diels-Alder and Michael addition reaction. A promising 25% ee was found for the Diels-Alder reaction between azachalcone and cyclopentadiene using one of the created constructs. Further development of these catalyst systems with the use of both synthetic (e.g. optimisation of ligand structure) and biomolecular tools (e.g. optimisation of protein environment) for optimisation can lead to very efficient and enantioselective conversions in the future.

Drosophila melanogaster encounter periods of increased temperature or decreased oxygen in its native environment. One consequence of these environmental stresses is increased production of reactive oxygen species that damage major molecules within cells. Another consequence is that flies fall into a protective coma where biological functions are minimized to conserve energy expenditures. This biological phenomenon is called spreading depression. The overarching aim of this project is to determine if methionine sulfoxide reductases affect entrance or exit from the protective coma induced by acute thermal stress. The data revealed that complete deficiency of Msr in young flies causes a faster induction of the coma. In both young and old flies, Msr does not affect average recovery time but does affect the pattern of recovery from coma. Entrance into the coma is age dependent with young flies maintaining activity longer than before entering into the coma as compared to old flies. / by Karin Schey. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

by Alexander Kreymerman. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web. / Sulindac is a known NSAID that has also been shown to have anti-cancer activity that is not related to its ability to inhibit COX 1 and 2. During the past 15 years there have been a large number of studies attempting to elucidate its mechanism of action. Our laboratory has shown that sulindac can both protect normal cells and enhance the killing of cancer cells under oxidative stress from H2O2 and TBHP. However, except for mitochondrial dysfunction and ROS production, the mechanism by which sulindac sensitized the cancer cells to oxidative stress remains unknown. Results of this research project suggest that the effect of sulindac and oxidative stress not only involves mitochondrial ROS production, but also aspects of the preconditioning response. In normal cells this leads to survival by a preconditioning pathway, likely involving PKCε. . However, cancer cells react by initiating a pathway leading to apoptosis involving PKCδ.

Oxidative stress is considered a major factor in the etiology of age related diseases and the aging process itself. Organisms have developed mechanisms to protect against oxidative damage resulting from increased production of reactive oxygen species during aging. One of the major antioxidant systems is the methionine sulfoxide reductase (Msr) enzyme family. The two major Msr enzymes, MsrA and MsrB, can stereospecifically reduce the S and R epimers, respectively, of methionine sulfoxide in proteins back to methionine. This study, using Drosophila melanogaster, decribes the first animal system lacking both MsrA and MsrB. The loss of either MsrA or MsrB had no effect on lifespan in Drosophila, but loss of MsrB results in a slight decrease in locomotor activity from middle age onward. Double mutants lacking both forms of Msr have a significantly decreased lifespan and decreased locomotor activity at all ages examined. The double Msr mutants had no detectable increase in protein oxidation or decrease in mitochondrial function and were not more sensitive to oxidative stress. These results suggested that other cellular antioxidant systems were protecting the flies against oxidative damage and the decreased life span observed in the double knockouts was not due to widespread oxidative damage. However, one cannot exclude limited oxidative damage to a specific locus or cell type. In this regard, it was observed that older animals, lacking both MsrA and MsrB, have significantly reduced levels of dopamine, suggesting there might be oxidative damage to the dopaminergic neurons. Preliminary results also suggest that the ratio of F to G actin is skewed towards G actin in all mutants. The present results could have relevance to the loss of dopaminergic neurons in Parkinson’s disease. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Aging is a multifactoral biological process of progressive and deleterious changes partially attributed to a build up of oxidatively damaged biomolecules resulting from attacks by free radicals. Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine (Met) residues found in proteins. Oxidized Met produces two enantiomers, Met-S-(o) and Met-R-(o), reduced by MsrA and MsrB respectively. Unlike other model organisms, our MsrA null fly mutant did not display increased sensitivity to oxidative stress or shortened lifespan, suggesting that in Drosophila, having either a functional copy of either Msr is sufficient. Here, two Msr mutant types were phenotypically assayed against isogenic controls. Results suggest that only the loss of both MsrA and MsrB produces increased sensitivity to oxidative stress and shortened lifespan, while locomotor defects became more severe with the full Msr knockout fly. / by Kelli Robbins. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

The inevitable aging process can be partially attributed to the accumulation of oxidative damage that results from the action of free radicals. Methionine sulfoxide reductases (Msr) are a class of enzymes that repair oxidized methionine residues. The two known forms of Msr are MsrA and MsrB which reduce the R- and S- enantiomers of methionine sulfoxide, respectively. Our lab has created the first genetic animal model that is fully deficient for any Msr activity. Previously our lab showed that these animals exhibit a 20 hour delay in development of the third instar larvae (unpublished data). My studies have further shown that the prolonged third-instar stage is due to a reduced growth rate associated with slower food intake and a markedly slower motility. These Msr-deficient animals also exhibit decreased egg-laying that can be attributed to a lack of female receptivity to mating. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Orientadores: Sergio Marangoni, Luis Alberto Ponce-Soto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T16:19:54Z (GMT). No. of bitstreams: 1 RomeroVargas_FreyFrancisco_M.pdf: 1233125 bytes, checksum: 85502fc7f4b2dd6cdb2a6fc0c220c5b2 (MD5) Previous issue date: 2007 / Resumo: As serpentes do gênero Crotalus incluem várias espécies amplamente distribuídas nocontinente Americano. Os venenos crotálicos das serpentes encontradas no Brasil apresentam características próprias. Entre os componentes bioativos, as fosfolipases A2 (E.C. 3.1.1.4.) destacam-se por ser a principal componente, tais enzimas são dependentes de cálcio e hidrolisam a ligação sn-2 éster dos fosfolipídios e alem disso induz uma variedade de efeitos. Nesta tese, primeiramente nos desenvolvemos um novo protocolo de purificação com uma coluna C18 µ-Bondapack em HPLC de fase reversa destas toxinas, Os perfis obtidos mostraram que o veneno total da espécie Crotalus durissus cumanensis foi fracionado em quatorze picos, onde as frações 9 e 10 apresentam atividade PLA2. Do veneno de Crotalus durissus ruruima foram obtidos dezoito picos, sendo que as frações 12 e 13 apresentaram atividade PLA2 também. Assim, neste trabalho, nos apresentamos o isolamento, purificação determinação de sua estrutura primaria de duas isoformas fosfolipase A2 para cada espécie crotálica: Crotalus durissus cumanensis e Crotalus durissus ruruima denominados como Cdc-9, Cdc-10, Cdr-12 e Cdr-13, respectivamente. Todas elas são formadas por 122 resíduos de aminoácidos. O alinhamento dos aminoácidos, das isoformas Cdc-9, Cdc-10, Cdr-12 e Cdr-13 mostram um alto nível 93.4 % de homologia seqüencial para as duas primeiras e 92.6% para as outras, observaram-se com respeito a distintas PLA2 que diminuíram para cerca de 50 % de homologia. Todas elas foram identificadas como miotoxinas e apresentaram atividade fosfolipásica. Foi possível realizar, a partir das isoformas nativas um processo de modificação química, utilizando-se reagentes seletivos para amino ácidos específicos (His, Tyr e Lys) os quais estão envolvidos na rede catalítica ou na interação com sua interface, logo após, foram analisadas quanto a suas características físico-químicas e biológicas. Nossos estudos de miotoxicidade local e sistêmica de todas as isoformas nativas e modificadas dos venenos crotálicos in vivo, evidenciaram o incremento nos níveis de creatina cinase (CK) séricos e nas isoformas modificadas foram inibidas na sua atividade catalítica ou devido às mudanças estruturais que afetam outras regiões alem do sitio catalítico, revelando a importância de conhecer a relação estrutura-função. Em contraste, o efeito indutor de edema, não pode ser rapidamente correlacionado com suas diferenças estruturais. Os resultados apresentados neste trabalho proporcionam a obtenção de uma ferramenta bioquímica útil para acrescentar nosso conhecimento da relação dos sítios farmacológicos alem da região catalítica nas fosfolipases A2 / Abstract: The serpents of the genus Crotalus include several species widely distributed in the American continent. The poisons crotálicos of the serpents found in Brazil, they present own characteristics. Among the components bioativos, the phospholipases A2 (E.C. 3.1.1.4.) standing out to be the main component, such enzymes are dependents of calcium and hidrolise the bond sn-2 éster of the fosfolipídios and besides that induced a variety of effects. In this thesis, firstly we develop a new purification protocol with a column C18 µ- Bondapack in HPLC of reverse phase of these toxins, The obtained profiles showed that the total poison of the species Crotalus durissus cumanensis was fractioned in fourteen picks, where the fractions 9 and 10 present activity PLA2. Of the poison of Crotalus durissus ruruima they were obtained eighteen picks, and the fractions 12 and 13 also presented activity PLA2. This way, in this work, we present the isolation, purification, determination of his primary structure of two isoforms fosfolipase A2 for each species crotálics: Crotalus durissus cumanensis and Crotalus durissus ruruima denominated like Cdc-9, Cdc-10, Cdr-12 and Cdr-13, respectively. All of them are formed by 122 residues of amino acids. The alignment of amino acids of the isoforms Cdc-9, Cdc-10, Cdr-12 and Cdr-13 it shows a high level of sequential homology 93.4% for the first two and 92.6% for the other ones, they were observed with regard to different PLA2 that decreased for about 50% of homology. All of them were identified as miotoxins and they presented phospholipasic activity. it was possible to carry out starting from the native isoforms a process of chemical modification, being used selective reagents for amino specific acids (His, Tyr and Lys) which are involved in the catalytic net or in the interaction with his interface, soon after, they were analyzed as for their physio-chemical and biological characteristics. Our studies of local and systemic miotoxicity of all the native and modified isoforms in vivo of the poisons crotálicos, evidenced the increment in the levels of creatine kinase (CK) séricos and in the modified isoformas they were inhibited in his activity catalytic or due the structural changes that they affect other regions besides the catalytic place, revealing the importance of knowing the relationship structure-function. In contrast, the effect producing edema, it cannot be correlated quickly with their structural differences. The results presented in this work provide the obtaining of an useful biochemical tool to increase our knowledge of the relationship of the pharmacological sites also of the catalytic region in the phopholipases A2 / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Living cells and their components play a key role within biotechnology industry. Cell cultures and their products of interest are used for the design of vaccines as well as in the agro-alimentary field. In order to ensure optimal working of such bioprocesses, the understanding of the complex mechanisms which rule them is fundamental. Mathematical models may be helpful to grasp the biological phenomena which intervene in a bioprocess. Moreover, they allow prediction of system behaviour and are frequently used within engineering tools to ensure, for instance, product quality and reproducibility.<p> <p>Mathematical models of cell cultures may come in various shapes and be phrased with varying degrees of mathematical formalism. Typically, three main model classes are available to describe the nonlinear dynamic behaviour of such biological systems. They consist of macroscopic models which only describe the main phenomena appearing in a culture. Indeed, a high model complexity may lead to long numerical computation time incompatible with engineering tools like software sensors or controllers. The first model class is composed of the first principles or white box models. They consist of the system of mass balances for the main species (biomass, substrates, and products of interest) involved in a reaction scheme, i.e. a set of irreversible reactions which represent the main biological phenomena occurring in the considered culture. Whereas transport phenomena inside and outside the cell culture are often well known, the reaction scheme and associated kinetics are usually a priori unknown, and require special care for their modelling and identification. The second kind of commonly used models belongs to black box modelling. Black boxes consider the system to be modelled in terms of its input and output characteristics. They consist of mathematical function combinations which do not allow any physical interpretation. They are usually used when no a priori information about the system is available. Finally, hybrid or grey box modelling combines the principles of white and black box models. Typically, a hybrid model uses the available prior knowledge while the reaction scheme and/or the kinetics are replaced by a black box, an Artificial Neural Network for instance.<p><p>Among these numerous models, which one has to be used to obtain the best possible representation of a bioprocess? We attempt to answer this question in the first part of this work. On the basis of two simulated bioprocesses and a real experimental one, two model kinds are analysed. First principles models whose reaction scheme and kinetics can be determined thanks to systematic procedures are compared with hybrid model structures where neural networks are used to describe the kinetics or the whole reaction term (i.e. kinetics and reaction scheme). The most common artificial neural networks, the MultiLayer Perceptron and the Radial Basis Function network, are tested. In this work, pure black box modelling is however not considered. Indeed, numerous papers already compare different neural networks with hybrid models. The results of these previous studies converge to the same conclusion: hybrid models, which combine the available prior knowledge with the neural network nonlinear mapping capabilities, provide better results.<p><p>From this model comparison and the fact that a physical kinetic model structure may be viewed as a combination of basis functions such as a neural network, kinetic model structures allowing biological interpretation should be preferred. This is why the second part of this work is dedicated to the improvement of the general kinetic model structure used in the previous study. Indeed, in spite of its good performance (largely due to the associated systematic identification procedure), this kinetic model which represents activation and/or inhibition effects by every culture component suffers from some limitations: it does not explicitely address saturation by a culture component. The structure models this kind of behaviour by an inhibition which compensates a strong activation. Note that the generalization of this kinetic model is a challenging task as physical interpretation has to be improved while a systematic identification procedure has to be maintained.<p><p>The last part of this work is devoted to another kind of biological systems: proteins. Such macromolecules, which are essential parts of all living organisms and consist of combinations of only 20 different basis molecules called amino acids, are currently used in the industrial world. In order to allow their functioning in non-physiological conditions, industrials are open to modify protein amino acid sequence. However, substitutions of an amino acid by another involve thermodynamic stability changes which may lead to the loss of the biological protein functionality. Among several theoretical methods predicting stability changes caused by mutations, the PoPMuSiC (Prediction Of Proteins Mutations Stability Changes) program has been developed within the Genomic and Structural Bioinformatics Group of the Université Libre de Bruxelles. This software allows to predict, in silico, changes in thermodynamic stability of a given protein under all possible single-site mutations, either in the whole sequence or in a region specified by the user. However, PoPMuSiC suffers from limitations and should be improved thanks to recently developed techniques of protein stability evaluation like the statistical mean force potentials of Dehouck et al. (2006). Our work proposes to enhance the performances of PoPMuSiC by the combination of the new energy functions of Dehouck et al. (2006) and the well known artificial neural networks, MultiLayer Perceptron or Radial Basis Function network. This time, we attempt to obtain models physically interpretable thanks to an appropriate use of the neural networks.<p> / Doctorat en sciences appliquées / info:eu-repo/semantics/nonPublished