No description available.

Drosophila nonclaret disjunctional (Ncd) is a kinesin-like C-terminal motor protein that is involved in spindle assembly in oocytes during meiosis and in spindle maintenance in early embryos during mitosis. Ncd interacts with both "highway" and "cargo" microtubules (MTs) in meiotic and mitotic spindles through the action of ATP-dependent and ATP-independent MT binding sites in the head and tail domains, respectively. Through the action of these binding sites, Ncd bundles and, perhaps, slides MTs relative to each other. These functions are important for the in vivo role of Ncd in the formation of the bipolar spindle and maintenance of the spindle assembly. Despite the high homology of the Ncd head domain to the kinesin head domain, the Ncd tail domain is unique among kinesin-like motor proteins. Characterization of ATP-independent interactions of Ncd with cargo MTs and identification of MT binding sites (located in amino acid residues 83-100 and 115-187) in the tail region by MT co-sedimentation assays revealed that the Ncd tail has functional similarities to microtubule-associated proteins, especially to tau and MAP2, that regulate MT assembly. Like tau MT binding motifs, MT binding sites of the tail domain are rich in basic amino acids that are flanked by proline residues. Cross-linking and MT co-sedimentation assays with subtilisin-digested MTs demonstrated that Ncd tail binding sites (located at the extreme C-terminus and in the H11-H12 loop / H12 helix of each tubulin monomer) on tubulin correspond to tau binding sites. Further, the Ncd tail domain, like tau, can promote and stabilize MT assembly under conditions that induce MT disassembly. Taken together, these results suggest that the Ncd tail functions both in the transport of cargo MTs to spindle poles for the formation of the spindle assembly during meiosis, and in maintenance of spindle assembly during mitosis. How these different functions of Ncd are regulated still remains unknown, however further understanding of the regulation of Ncd function should contribute to our knowledge of cell cycle regulation in both meiotic and mitotic cells. / Ph. D.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23433号 / 農博第2464号 / 新制||農||1086(附属図書館) / 学位論文||R3||N5348(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 谷 史人, 教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Protein engineering has been used to alter the stability of proteins for several decades withmuch success, one approach being to introduce two cysteine residues that together form adisulfide bridge. The disulfide bridge can increase the Gibbs free energy of the transitionstate, thus increasing energy difference between the folded state and the unfolding transitionstate, leading to increased kinetic stability of the protein. Subtilisin Carlsberg is a serineprotease that has widespread applications within the industry but has also been tried in biogasprocesses to increase the biomethane yield from proteinaceous substrates. Subtilisin’s activitylifetime was found to be short in the biogas process, which prompted the need to increase theenzyme’s kinetic stability, meaning that the introduction of a disulfide bridge could be asolution. The aim of this project was to increase the kinetic stability of Subtilisin Carlsbergwith the use of introduced disulfide bridges.The production of Subtilisin Carlsberg has traditionally been done using the source organismBacillus Licheniformis, but here a successful method for expressing Subtilisin, and fourdisulfide variants of it, as an inclusion-body protein is presented. Also, a method forpurifying and refolding the protein under denaturing conditions is presented with a significantprotein yield.Thermal stability analysis of the WT enzyme and its four variants (A24C/S86C,N122C/A227C, K12C/E270C, V26C/A231C) was performed using NanoDSF, and showedthat the thermal stability was practically unchanged for A24C/S86C at 67.9 ℃, decreased by5.6 ℃ for N122C/A227C, increased by 8.2 ℃ for K12C/E270C, and increased by 11.5 ℃ forV26C/A231C.The kinetic stability of Subtilisin and its variants was analysed using stopped-flowmeasurements of the proteins’ denaturation rate at various GuHCl concentrations. The resultsshowed that N122C/A227C and V26C/A231C were more kinetically stable than the WTenzyme, while A24C/S86C and K12C/E270C were less stable. N122C/A227C had anactivation energy for unfolding of 5.217 kJ/mol higher than WT Subtilisin. V26C/A231C hadan activation energy for unfolding of 1.220 kJ/mol higher than WT Subtilisin. The resultsthereby show that two disulfides bond mutations achieved the desired outcome of increasedkinetic stability. Thereby, the aim of the project was fulfilled.

The colonization of immersed surfaces by a myriad of marine organisms is a complex, multi-stage, species-specific process giving rise to economic and environmental costs. This unwanted accumulation of organisms in the marine environment, called biofouling, has been attacked from different fronts, going from the ‘problem-elimination-as-problem-solving’ strategy (essentially through the use of biocides) to more elaborated and environmentally-friendly options based on the principle of ‘non-stick’ or ‘easy foul-release’ surfaces, which do not jeopardize marine life viability. Several marine organisms rely on proteinaceous adhesives to secure a holdfast to surfaces. Proteolytic enzymes have been demonstrated to be effective agents against settlement and settlement consolidation onto surfaces of marine bacteria, algae, and invertebrates, their proposed mode-of-action being the enzymatic degradation of the proteinaceous components of the adhesives. So far, however, the evidence remains inconclusive since most of the published investigations refer to commercial preparations where the enzyme is mixed with other components, like additives, which obviously act as additional experimental variables. This work aims at providing clear, conclusive evidence about the potential of serine proteases to target the adhesives produced by a group of model marine biofoulers. The strategy towards the goal consisted in the preparation and characterization of maleic anhydride copolymer nanocoatings modified by a surface-bound enzyme, Subtilisin A, the active constituent of the commercial preparations reported as effective against biofouling. The enzyme-containing maleic anhydride copolymer films were characterized (enzyme surface concentration, activity, stability, roughness and wettability) and thereafter tested in biological assays with three major biofoulers: spores of the green alga Ulva linza, cells of the pennate diatom Navicula perminuta, and cyprid larvae of the barnacle Balanus amphitrite. The purpose of the biological assays was to elucidate the efficacy of the immobilized catalyst to discourage settlement and/or to facilitate removal of these organisms from the bioactive layers. Results confirmed the initial hypotheses related to the enzymatic degradation of the biological adhesives: the immobilized protease was effective at reducing the adhesion strength of Ulva spores and Navicula diatoms in a manner that correlated with the enzyme activity and surface concentration, and deterred settlement of Balanus amphitrite barnacle cyprids even at the lowest surface activity tested. By facilitating the removal of biofilm-forming diatoms and of spores of the troublesome alga Ulva linza, as well as by interfering with the consolidation of adhesion of the calcareous Balanus amphitrite macrofouler, the enzyme-containing coatings here disclosed are considered to constitute an appealing and promising alternative to control marine biofouling without jeopardizing marine life.

The colonization of immersed surfaces by a myriad of marine organisms is a complex, multi-stage, species-specific process giving rise to economic and environmental costs. This unwanted accumulation of organisms in the marine environment, called biofouling, has been attacked from different fronts, going from the ‘problem-elimination-as-problem-solving’ strategy (essentially through the use of biocides) to more elaborated and environmentally-friendly options based on the principle of ‘non-stick’ or ‘easy foul-release’ surfaces, which do not jeopardize marine life viability. Several marine organisms rely on proteinaceous adhesives to secure a holdfast to surfaces. Proteolytic enzymes have been demonstrated to be effective agents against settlement and settlement consolidation onto surfaces of marine bacteria, algae, and invertebrates, their proposed mode-of-action being the enzymatic degradation of the proteinaceous components of the adhesives. So far, however, the evidence remains inconclusive since most of the published investigations refer to commercial preparations where the enzyme is mixed with other components, like additives, which obviously act as additional experimental variables. This work aims at providing clear, conclusive evidence about the potential of serine proteases to target the adhesives produced by a group of model marine biofoulers. The strategy towards the goal consisted in the preparation and characterization of maleic anhydride copolymer nanocoatings modified by a surface-bound enzyme, Subtilisin A, the active constituent of the commercial preparations reported as effective against biofouling. The enzyme-containing maleic anhydride copolymer films were characterized (enzyme surface concentration, activity, stability, roughness and wettability) and thereafter tested in biological assays with three major biofoulers: spores of the green alga Ulva linza, cells of the pennate diatom Navicula perminuta, and cyprid larvae of the barnacle Balanus amphitrite. The purpose of the biological assays was to elucidate the efficacy of the immobilized catalyst to discourage settlement and/or to facilitate removal of these organisms from the bioactive layers. Results confirmed the initial hypotheses related to the enzymatic degradation of the biological adhesives: the immobilized protease was effective at reducing the adhesion strength of Ulva spores and Navicula diatoms in a manner that correlated with the enzyme activity and surface concentration, and deterred settlement of Balanus amphitrite barnacle cyprids even at the lowest surface activity tested. By facilitating the removal of biofilm-forming diatoms and of spores of the troublesome alga Ulva linza, as well as by interfering with the consolidation of adhesion of the calcareous Balanus amphitrite macrofouler, the enzyme-containing coatings here disclosed are considered to constitute an appealing and promising alternative to control marine biofouling without jeopardizing marine life.

No description available.

Orientador: Prof. Dr. Luciano Puzer / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / Myxobacterias sao bacterias Gram-negativas com uma peculiaridade no ciclo de vida em comparacao a outros procariotos. Em resposta a periodos de baixa disponibilidade de nutrientes, inicia-se um processo de morfogenese cooperativa que culmina na formacao de corpos de frutificacao, um agregado de celulas que contem estruturas de resistencia caracteristicas. Subtilisinas sao serino proteases da familia S8A, inicialmente encontrada no Bacillus subtilis e atualmente identificada em uma ampla variedade de organismos procarioticos e eucarioticos. Essas enzimas apresentam um grande mercado de aplicacoes biotecnologicas e industriais. O objetivo deste trabalho foi clonar aqualisina, uma subtilisina extracelular da Myxococcus xanthus, uma especie de myxobacterias que preda outros microrganismos, e submete-la a um sistema de expressao heterologa em celulas E. coli BL2 (DE3). A proteina permaneceu na fase insoluvel apos as lises; portanto, foi necessario otimizar protocolos de solubilizacao e purificacao da aqualisina dos corpos de inclusao da celula, para que fosse possivel a caracterizacao cinetica. A solubilizacao utilizando tampao 0,1M de tris e pH 12 mostrou-se a mais eficiente em solubilizar os corpos de inclusao, o que permitiu obter os valores cineticos Km de 0,6 e 1,3 ¿ÊM, para aqualisina purificada (AQN) e para nao retida pela coluna (FT), respectivamente, que demonstram uma alta afinidade pelo substrato utilizado. No entanto, valores obtidos de kcat, e kcat/Km apontam a presenca de duas populacoes de proteinas, nas quais a populacao inativa esta presente em maior quantidade. Embora o sistema de expressao da aqualisina tenha ocorrido com sucesso, a solubilizacao dos corpos de inclusao sem utilizacao de agentes desnaturantes, e posterior purificacao ainda necessitam ser otimizadas, a fim de aumentar a recuperacao de enzima ativa, possibilitando que sua caracterizacao cinetica seja realizada sem a influencia desta populacao de enzima inativa. / Despite its status as one of the oldest registered diseases, rabies remains a public health challenge. This zoonosis affects the central nervous system (CNS) of all mammals and is usually considered fatal. However, some cases in which the disease was reportedly cured have been registered worldwide, leading to renewed interest in researching potential antiviral mechanisms against the rabies virus (RABV). The aim of this work was to evaluate the antiviral activity of the hydroethanolic plant extract Dalbergia variabilis against different genetic lineages of RABV. In the first step, the maximum tolerated concentration (MTC) of plant extract in murine neuroblastoma cells (N2a) was determined to be 1.56 mg/ml. The antiviral activity of plant extract Dalbergia variabilis was evaluated to determine the difference presence and absence of the plant extract in four samples of RABV (IP4005- sample of RABV with the genetic lineage characteristic of haematophagous bat, Desmodus rotundus; IP964- sample of RABV with the genetic lineage characteristic of insectivorous bat, Eptesicus furinalis; IP4871- sample of RABV with the genetic lineage characteristic of wild dog, Cerdocyon thous; and a standard sample of RABV, "Pasteur vírus" - PV). Since there was a difference in the infectious viral titer between the RABV samples analysed in the presence of the plant extract, subsequent experiments were carried out to better understand the performance of this plant extract against RABV. The antiviral activity of plant extract Dalbergia variabilis against samples of RABV was analysed, and the interference in viral replication was more significant against samples of RABV with genetic lineage characteristic of bats.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-02-05T23:00:04Z No. of bitstreams: 1 IsabelAndradeLopesDeSousa_DISSERT.pdf: 1629932 bytes, checksum: 69b762646403d829620b6a8a168e9988 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-02-16T21:49:50Z (GMT) No. of bitstreams: 1 IsabelAndradeLopesDeSousa_DISSERT.pdf: 1629932 bytes, checksum: 69b762646403d829620b6a8a168e9988 (MD5) / Made available in DSpace on 2016-02-16T21:49:50Z (GMT). No. of bitstreams: 1 IsabelAndradeLopesDeSousa_DISSERT.pdf: 1629932 bytes, checksum: 69b762646403d829620b6a8a168e9988 (MD5) Previous issue date: 2015-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / A cana-de-a??car ? uma planta monocotiled?nea cultivada em regi?es tropicais e subtropicais, sendo o Brasil o maior produtor mundial. Apesar de sua import?ncia econ?mica, muito pouco ? conhecido molecularmente sobre o processo de flora??o em cana-de-a??car. Este processo fisiol?gico pode promover uma perda de at? 60% na produtividade de a??car ou bioetanol. Desta forma, este trabalho teve como objetivo caracterizar um gene hom?logo ao gene que codifica a prote?na HINT1, identificado anteriormente em bibliotecas subtrativas de flora??o. An?lises gen?micas da estrutura g?nica e regi?o promotora permitiram observar que existem pelo menos dois genes distintos hom?logos ? HINT em cana-de-a??car. An?lises de bioinform?tica mostraram a conserva??o do dom?nio proteico caracter?stico da superfam?lia HIT e indicam uma rela??o filogen?tica associada ? localiza??o celular. Al?m disso, foi observada uma poss?vel rela??o com as prote?nas da fam?lia SBTILISIN-like por meio das informa??es dispon?veis em interatomas. Isto sugere que o gene HINT de cana-de-a??car pode estar relacionado ao desenvolvimento vegetal, havendo v?rias possibilidades de intera??es para a regula??o do processo de indu??o floral, pois as sequ?ncias presentes nas regi?es regulat?rias indicam que a express?o diferencial de HINT seria relacionada tanto com fatores clim?ticos presentes na regi?o Nordeste do Brasil quanto ? estresse bi?tico e fito-horm?nios. Al?m disso, os fen?tipos relacionados ao florescimento tardio indicam que a influ?ncia de HINT pode ocorrer devido ao ac?mulo de produtos na sua atua??o enzim?tica. Por estas caracter?sticas este gene pode ser utilizado como um marcador na sele??o de novos cultivares. / The sugarcane is a monocot plant grown in tropical and subtropical regions, with Brazil being the largest producer. Despite its economic importance, little is known about the molecular flowering process in sugarcane. This physiological process can promote a loss up to 60% in sugar or bioethanol. Thus, this work had as objective characterize a HINT1 homologous gene previously identified in subtractive libraries of flowering. Genomic analysis of gene and promoter region structure allowed the observation that there are at least two distinct genes homologous to HINT on sugarcane. Bioinformatics analyses showed the conservation of the characteristic protein domain of HIT superfamily and indicate a phylogenetic relationship associated to cell location. Moreover, a possible relation with the SBTILISIN-like protein family through the information available in interatomas was observed. This suggests that the HINT gene of sugarcane can be related to plant development, there are several possibilities of interactions in the regulation of floral induction process, because the sequences present in regulatory regions indicate that differential expression of HINT was related to with climatic factors in the Northeast region of Brazil as well as to biotic stress and phytohormones. Furthermore, the sugarcane phenotypes indicate that the influence of HINT may happen due to product accumulation of its enzymatic activity. For these characteristics this gene can be used as a marker in the selection of new varieties.

No description available.