The intracellular proteolysis of ingested meal plays an essential role in tick development. The thesis focuses on the factors influencing the expressions and activities of digestive enzymes in Ixodes ricinus females during the feeding and post-feeding period. We have revealed the effect of fertilization on blood feeding and digestion. The females cannot reach the rapid engorgement phase without being fertilized. The rate of mated females in the nature proved the presumption that mating can occur even off the host. Implementation of in vitro feeding technique further extended our current knowledge about tick digestive apparatus. Adult females were fed on hemoglobin-rich and hemoglobin-poor diet and the mRNA expression levels of digestive proteases were determined. In line with obtained data, we assumed that albuminolysis is conducted by the same or similar pathway as hemoglobinolysis. The gene silencing method and protein immuno-detection were used to unequivocally identify the isoforms of 'early expressed' IrCL1 and 'late expressed' IrCL3 isoform of cathepsin L.

Submitted by RONIVALDO RODRIGUES DA SILVA (rds.roni@yahoo.com.br) on 2016-03-01T13:46:53Z No. of bitstreams: 1 Tese Doutorado RONIVALDO R. SILVA.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-01T18:27:48Z (GMT) No. of bitstreams: 1 silva_rr_dr_sjrp.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) / Made available in DSpace on 2016-03-01T18:27:48Z (GMT). No. of bitstreams: 1 silva_rr_dr_sjrp.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) Previous issue date: 2016-02-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Aspártico (E.C. 3.4.23), cisteíno (E.C. 3.4.22) e serino peptidases (E.C. 3.4.21) são endopeptidases, cujos modos de ação são dependentes de resíduos de ácido aspártico, cisteína e serina presentes no sítio catalítico, respectivamente. Atualmente, vários estudos são realizados na busca por novas enzimas com relevantes propriedades bioquímicas para aplicação industrial. Neste contexto, nós propomos a produção de enzimas em bioprocesso submerso, purificação, estudo das propriedades bioquímicas e determinação da especificidade catalítica das peptidases secretadas pelos fungos filamentosos Rhizomucor miehei, Phanerochaete chrysosporium e Leptosphaeria sp. Inicialmente, após produção por bioprocesso submerso, estas enzimas foram purificadas utilizando cromatografias de exclusão molecular e troca iônica. Em ensaios de inibidores na atividade enzimática, notamos inibição das peptidases por pepstatina A (R. miehei), ácido iodoacético/N-Etilmaleimida (P. chrysosporium) e fluoreto de fenil metil sulfonila (Leptosphaeria sp), sendo então definidas como aspártico, cisteíno e serino peptidases, respectivamente. Por SDS-PAGE (12%), as massas moleculares foram estimadas em 37 kDa (aspártico), 23 kDa (cisteíno) e 35 kDa (serino). O máximo de atividade proteolítica foi alcançado em pH 5,5 e 55 ºC para peptidase aspártica secretada por R. miehei; pH 7 e faixa de temperatura 45-55 ºC para cisteíno peptidase secretada por P. chrysosporium, e pH 7 e 45 ºC para serino peptidase secretada por Leptosphaeria sp. Sob efeito de incubação a diferentes pH, a peptidase aspártica mostrou-se estável em condições ácidas (pH 3-5); cisteíno peptidase foi estável em ampla faixa de pH (pH 4-9), e serino peptidase mostrou-se mais estável em condições com tendências alcalinas e pH ligeiramente ácido (pH 5-9). Em todas estas faixas de pH citadas, as peptidases apresentaram atividade proteolítica acima de 80% por 1 hora de incubação. Quanto à estabilidade térmica, a cisteíno peptidase mostrou-se mais termoestável dentre as três enzimas e serino peptidase descreveu a menor tolerância à temperatura. Em incubação com agentes desnaturantes, observamos redução na atividade proteolítica sob efeito de surfactantes iônicos (0,02-1%): dodecil sulfato de sódio (SDS) e brometo de cetil-trimetil amônio (CTAB); íon cobre II (5 mM); Ditiotreitol (DTT) e guanidina (ambos na faixa de 10-200 mM) para todas as peptidases. Por último, em estudo de especificidade catalítica destas enzimas, observamos a preferência por aminoácidos aromáticos (F e W), básicos (K e R) e apolares (em particular, resíduo de metionina) para peptidase aspártica. Alta especificidade descrita por cisteíno peptidase, cuja preferência catalítica é notória por aminoácidos básicos (K, H e R), especialmente na posição P3 e lisina-dependência para catálise na posição P'3. Em serino peptidase, notamos maior aceitação por aminoácidos apolares (G, I, L, M e V), básicos (H e R) e polares neutros (N e Q) para as diferentes posições avaliadas no substrato. / Aspartic (EC 3.4.23), cysteine (EC 3.4.22) and serine peptidases (EC 3.4.21) are endopeptidases whose modes of action are dependent on aspartic acid, cysteine and serine residues present in the catalytic site, respectively. Currently, several studies are conducted in the search for new enzymes with relevant biochemical properties for industrial application. In this context, we propose the production of enzymes in submerged bioprocess, purification, the study of biochemical properties and determining the catalytic specificity peptidases secreted by the filamentous fungus Rhizomucor miehei, Phanerochaete chrysosporium and Leptosphaeria sp. Initially, after production submerged bioprocess, these enzymes have been purified using size-exclusion and ion exchange chromatographies. In the effect of inhibitors on enzyme activity, we note peptidase inhibition by pepstatin A (R. miehei), iodoacetic acid/ N-Ethylmaleimide (P. chrysosporium) and phenyl methyl sulfonyl fluoride (Leptosphaeria sp), suggesting that these enzymes are aspartic, cysteine and serine peptidases, respectively. For SDS-PAGE (12%), molecular weights were estimated at 37 kDa (aspartic), 23 kDa (cysteine) and 35 kDa (serine). Maximum proteolytic activity was achieved at pH 5.5 and 55 °C for aspartic peptidase secreted by R. miehei; pH 7 and temperature range 45-55 °C for cysteine peptidase secreted by P. chrysosporium and pH 7 and 45 °C for serine peptidase secreted by Leptosphaeria sp. Under incubation at different pH effect, aspartic peptidase was stable under acidic conditions (pH 3-5); cysteine peptidase was stable in wide pH range (pH 4-9), and serine peptidase was more stable under alkaline conditions and pH slightly acidic (pH 5-9). In all these pH ranges mentioned, peptidases showed proteolytic activity above 80% by 1 hour incubation. As regards the thermal stability, cysteine peptidase was more thermostable enzyme and serine peptidase described the lowest temperature tolerance. In incubation with denaturing agents, we observed a decrease in proteolytic activity under the effect of ionic surfactant (0.02-1%) sodium dodecyl sulfate (SDS) bromide and cetyl-trimethyl ammonium bromide (CTAB); copper (II) ion (5 mM); Dithiothreitol (DTT) and guanidine (both in the range of 10-200 mM) for all peptidases. Finally, the study of catalytic specificity of these enzymes, we found a preference for aromatic amino acids (F and W), basic (K and R) and nonpolar (in particular, methionine residue) to aspartic peptidase. High specificity described by cysteine peptidase, which a catalytic preference is notorious for basic amino acids (K, R and H), especially in position P3 and lysine-dependence for catalysis at position P'3. In serine peptidase, for different evaluated positions, we noticed greater acceptance by nonpolar amino acids (G, I, L, M and V), basic (M and R) and neutral polar (N and Q).

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Computer simulations within the Density Functional Theory (DFT) formalism were accomplished to find the structural, electronic and vibrational properties of aspartic acid (Asp) crystals in the L-anhydrous, L-monohydrated, and DL-anhydrous phases. Aspartic acid is a non-essential amino acid with a role in the fadigue resistance mechanism. It also works as an excitatory neurotransmitter in the brain, contributes to eliminate any excess of toxins from the cells and is capable to affect RNA synthesis. The computer codes CASTEP (for crystals) and GAUSSIAN (for molecules) were employed in the present study. For the aspartic acid crystals, LDA and GGA-PBE exchange-correlation functional were used in the simulations, the last one taking into account empirical corrections for dispersive forces (PBE+TS) following the scheme proposed by Tkatchenko and Scheffler. Molecular calculations were carried out using the Gaussian09 program with the hybrid exchange-correlation functional B3LYP and the 6-311+G(d,p) basis set. The molecules were simulated in the gaseous phase and solvated in water within the Polarizable Continuum Model (PCM). Crystalline (optimized unit cells) and molecular (smallest energy conformations) structures obtained from the calculations were compared with experimental results and other theoretical computations. For the L-Asp anhydrous crystal, the optical and vibrational infrared (IR) and Raman spectra were contrasted with experimental measurements, and its band structure suggests it is a semiconductor. For the monohydrated L-Asp crystal, an indirect gap 0,1 eV larger than the gap of the anhydrous crystal is caused due to the role of water in its electronic structure. The DL-Asp anhydrous crystal, on the other hand, exhibits a wide direct band gap, which suggests possible optoelectronic uses. Effective masses obtained for all crystals exhibit an anisotropy which must affect their electronic transport properties, with electric conduction more likely along a direction parallel to the molecular planes for the L-Asp anhydrous system. The analysis of the density of electronic states revealed the contributions per atom and per functional group to the valence and conduction band states. A nice agreement was found between the theoretical IR and Raman spectra and the experimental data for the L-Asp anhydrous crystal, allowing for an adequate interpretation of the normal modes involved in each spectral feature. / SimulaÃÃes computacionais no formalismo DFT (Density Functional Theory) foram realizadas para a determinaÃÃo das propriedades estruturais, eletrÃnicas e vibracionais de cristais de Ãcido aspÃrtico (Asp) nas formas cristalinas L-Asp anidro, L-Asp monohidratado e DL-Asp anidro. O Ãcido aspÃrtico à um aminoÃcido nÃo essencial com papel na fisiologia da resistÃncia fÃsica, atuando como neurotransmissor excitatÃrio no cÃrebro, contribuindo para a eliminaÃÃo do excesso de toxinas nas cÃlulas, alÃm de ser capaz de afetar a sÃntese de RNA. Os cÃdigos CASTEP (para cristais) e GAUSSIAN (para molÃculas) foram utilizados no presente estudo. Para os cristais de Ãcido aspÃrtico, os funcionais de troca e correlaÃÃo LDA e GGA-PBE foram empregados nas simulaÃÃes, este Ãltimo incluindo correÃÃes empÃricas para interaÃÃes dispersivas (PBE+TS) de acordo com o esquema de Tkatchenko e Scheffler. Os cÃlculos moleculares foram realizados utilizando o programa Gaussian09 com o funcional hibrido de troca e correlaÃÃo B3LYP e o conjunto de base 6-311+G(d,p). As molÃculas foram simuladas em fase gasosa e em meio aquoso (modelo de solvataÃÃo contÃnuo PCM). As estruturas cristalinas (cÃlulas unitÃrias otimizadas) e moleculares (conformaÃÃes de menor energia) obtidas nos cÃlculos foram comparadas com resultados experimentais e outros cÃlculos teÃricos. No caso do cristal L-Asp anidro, a absorÃÃo Ãptica e os espectros vibracionais IR e Raman tambÃm foram confrontados com medidas experimentais, e sua estrutura de bandas sugere um possÃvel carÃter semicondutor. No caso do cristal L-Asp monohidratado, um gap indireto 0,1 eV maior do que o do cristal anidro reflete o impacto da Ãgua sobre a estrutura eletrÃnica desse cristal. O cristal DL-Asp anidro, por outro lado, exibe um gap direto largo, o que sugere possÃveis usos em optoeletrÃnica. As massas efetivas obtidas para todos os cristais revelam uma anisotropia das propriedades de transporte eletrÃnico, sendo a conduÃÃo elÃtrica mais favorÃvel na direÃÃo paralela aos planos moleculares para o caso L-Asp anidro. A anÃlise da densidade de estados eletrÃnicos permitiu estabelecer a contribuiÃÃo por Ãtomo e por grupo funcional para os estados das bandas de valÃncia e conduÃÃo e um Ãtimo acordo foi obtido entre os espectros vibracionais IR e Raman teÃricos e experimentais do cristal L-Asp anidro, permitindo uma adequada interpretaÃÃo dos modos normais envolvidos em cada pico espectral.

Orientador: Sonia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-06T20:08:56Z (GMT). No. of bitstreams: 1 Bresolin_IgorTadeuLazzarotto_M.pdf: 4686617 bytes, checksum: ed7a688421b5d854024279523878c12a (MD5) Previous issue date: 2006 / Resumo: Anticorpos monoclonais são imunoglobulinas secretadas por clones de linfócitos B, normais, tumorais ou obtidos pela tecnologia de hibridomas. Os anticorpos monoclonais têm sido utilizados nas áreas analítica e terapêutica, o que implica na necessidade de sua obtenção com pureza superior a 95%. Muitos estudos têm sido realizados visando à purificação de anticorpos monoclonais, destacando-se as técnicas de adsorção seletiva, como as cromatografias de troca iônica, hidrofóbica e de afinidade. Neste trabalho aplicou-se a cromatografia de afinidade em membranas de álcool polietileno-vinílico (PEVA), comparando o desempenho dos agentes quelantes (AQ) ácido iminodiacético (IDA), ácido aspártico carboximetilado (CM-Asp) e tris-2(aminoetil)amina (TREN) com íons metálicos imobilizados na purificação de anticorpos monoclonais anti-TNP, isotipo IgG1 a partir de sobrenadante de cultura celular precipitado e dialisado. Para determinar as melhores condições de adsorção e eluição na presença de diferentes sistemas tamponantes, foram testados os quelatos AQ-Ni2+, AQ-Zn2+ e AQ-Co2+, bem como os agentes quelantes sem metal imobilizado como grupos ionogênicos. De acordo com eletroforeses SDS-PAGE e análises ELlSA das frações dos picos de proteína obtidos, as melhores condições utilizadas para a purificação foi o uso de membranas PEVA-IDA-sem metal e PEVA-CM-Asp-Zn2+, em presença de tampão Tris-HCI 50 mM a pH 7,0 e eluição por aumento de concentração de Tris, atingindo fatores de purificação de 138,9 e 103,8 e pureza de 92,3% e 68,1%, respectivamente. A partir das isotermas de adsorção, determinou-se a capacidade máxima de adsorção e a constante de dissociação dos complexos IDA-lgG1 e CM-Asp-Zn2+ -lgG1 que, de acordo com o ajuste dos parâmetros pelo modelo de Langmuir, mostraram alta capacidade de adsorção e constantes de dissociação características de sistemas de afinidade média. Com o módulo de fibras ocas, construído em nosso laboratório, determinaram-se as curvas de ruptura por meio de experimentos de filtração para os processos propostos, e os resultados obtidos demonstraram que os sistemas de fibras ocas PEVA-IDA e PEVA-GM-Asp-Zn2+ são factíveis para a purificação de anticorpos monoclonais do isotipo IgG1 / Abstract: Monoclonal antibodies are immunoglobulins produced by normal, tumoral or hybrids Iymphocytes 8 obtained by hibridoma technology. Hybridomas are resulted from the fusion of Iymphocytes B with malignant myeloma cells which express both the Iymphocyte's property of specific-antibody production and the immortal characteristic of the myeloma cells. Monoclonal antibodies have been used in analytic and therapeutic areas. This application needs highly pure antibodies. Many techniques have been studied focusing monoclonal antibodies purification. These techniques include ion exchange, hydrophobic and affinity chromatography. In this study, we applied polyethylenevinyl alcohol (PEVA) membranes in the purification of monoclonal antibody from cel! culture supematant comparing the chelating agents Iminodiacetic Acid (IDA), Carboxy-methyl Aspartic Acid (CM-Asp) and Tris-2(aminoethyl)amine) (TREN) with different immobilized metal ios, Ni2+, Zn2+ and C02+, and with different buffers. We also evaluated the adsorption and purification of monoclonal antibodies using the chelating agents as ionogenic groups. According to SDS-PAGE electrophoresis and ELlSA analysis, the higher selectivity was obtained in the presence of 50 mM Tris-HCI buffer, pH 7,0 and with elution by increasing Tris concentration, with CM-Asp-Zn2+ and IDA as ionogenic group, which provided the purification of IgG with traces of albumin, reaching purification factors of 138.9 and 103.8 and purities of 92.3% and 68.1, respectively. The adsorbent capacity and the dissociation constant of the complexes IDA-lgG1 e CM-Asp-Zn2+ -lgG1 were determinate from adsorption isotherms. According to Langmuir model, the results indicated that the matrix presents high adsorption capacity and a dissociation constant characteristic for intermediate affinity systems. We also evaluated the breaktrough curves for the proposed processes. These breaktrough curves are important to scale up procedure / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Nesta dissertaÃÃo sÃo apresentados resultados de espalhamento Raman em cristais de Ãcido DL-aspÃrtico sob diversas condiÃÃes de temperatura. O Ãcido DL-aspÃrtico (C4H7NO4) cristaliza-se no grupo espacial C2h6 com oito molÃculas por cÃlula unitÃria, existindo portanto 128 Ãtomos na cÃlula unitÃria que darÃo origem a 384 modos normais de vibraÃÃo. Destes um total de 192 modos sÃo Raman ativos, que poderiam ser observados nos espectros nÃo polarizados, mas que por diversos fatores apenas parte desses modos à observada. Fez-se a identificaÃÃo tentativa de todos os modos normais de vibraÃÃo que aparecem no intervalo espectral entre 50 e 3200cm-1 e um estudo com variaÃÃo de temperatura entre 10 e 433K. O intervalo compreendido entre 0 e 150 cm-1 à de extrema importÃncia para detecÃÃes de transiÃÃes de fase estrutural pois contÃm os modos de vibraÃÃo da rede. No caso do Ãcido DL-aspÃrtico ocorreu uma inversÃo de intensidade para os modos em 82 e 87 cm-1, considerando os extremos do intervalo de temperatura medido. Tal inversÃo foi interpretada como uma pequena mudanÃa conformacional, nada associado a transiÃÃo. Com exceÃÃo desse fato nÃo ocorreram anomalias, nem aparecimento ou surgimento de modos nessa regiÃo, o que apontou para a estabilidade do material. Outro evento ocorreu nessa regiÃo: as bandas em 116 e 132 cm-1, bastante distintas a baixÃssimas temperaturas (< 150 K) tornam-se indistinguÃveis a 200 K. Tal fato nÃo pode ser associado a uma transiÃÃo de fase pois o prÃprio alargamento das linhas, consequÃncia do aumento da temperatura, implica a superposiÃÃo dos modos. Some-se a isso o fato de que nas vibraÃÃes de torÃÃo do NH3 e rocking do CO2â (modos associados Ãs ligaÃÃes de hidrogÃnio) ter-se observado linearidade nas curvas frequÃncia-temperatura. Por fim realizou-se um estudo de calorimetria diferencial de varredura, confirmando-se o que havia sido observado pela espectroscopia Raman â a estabilidade da estrutura em todo o intervalo de temperatura investigado. / This dissertation presents the results of Raman scattering in crystals of DL-aspartic acid under various temperature conditions. The DL-aspartic acid (C4H7NO4) crystallizes in space group C2h6 with eight molecules per unit cell, so there are 128 atoms in the unit cell that give rise to 384 normal modes of vibration. Of these modes a total of 192 modes are Raman active, which could be observed in not polarized spectra, but by various factors only some of these modes are observed. We did an attempt identification of all normal modes of vibration that appears in the spectral range between 50 and 3200cm-1 and a study with variation in temperature between 10 and 433 K. The interval between 0 and 150 cm-1 is extremely important for detection of phase transitions because it contains the structural modes of vibration of the lattice. In the case of DL-aspartic acid there was a reversal of intensity for the modes at 82 and 87 cm-1, in considering the extremes of temperature interval measured. This reversal was interpreted as a small conformational change, not associated with a phase transition. With exception of this reversal there were not anomalies, not appearance or disappearance of modes in this region, which pointed to the stability of the material. Another event occurred in this region: the bands at 116 and 132 cm-1, very different at very low temperatures (< 150 K) become indistinguishable at 200 K. This fact can not be associated with a phase transition because the broadening of bands, arising from increasing temperatures, implies the superposition of modes. Added to this there is the fact that the torsional vibrations of the NH3+ and rocking of CO2- (modes associated with hydrogen bonds) behaved linearly in frequency-temperature curves. Finally we did a study of differential scanning calorimetry, which confirmed what had been observed by Raman spectroscopy - the stability of the structure throughout the temperature range investigated.

Esta pesquisa bibliográfica teve o propósito de elucidar a via do acido aspártico, apontando como fonte deste estudo os cereais. O objetivo principal desta pesquisa consistiu em estudar a via do acido aspártico, visando descrever uma variedade de técnicas de engenharia genética e bioquímicas que podem ser empregadas para aumentar a qualidade nutricional de cereais, podendo, assim, compreender o que acarreta o aumento do acumulo de lisina, metionina e treonina nos grãos para suprir essa necessidade na formação de uma proteína balanceada nutricionalmente. Foi realizada uma busca exaustiva em bases de dados Google Scholar, Portal Capes, ISI web of Science, no período de publicação de 1970 a 2012. Foram adotados textos de referencia internacional e nacional. Esta pesquisa foi dividida em três etapas: via do acido aspártico e seus aminoácidos derivados em plantas superiores de 1970 a 1997, via metabólica do acido aspártico no período de 1997 a 2006 e estratégias interessantes para aumentar o nível dos aminoácidos essenciais da via do acido aspártico em plantas no período de 2006 ate o momento. A primeira etapa foi desenvolvida relatando o acido aspártico como precursor dos aminoácidos essenciais: metionina, lisina, treonina e isoleucina. Entre os essenciais, a lisina e um dos mais estudados devido a escassez em muitos cereais, o que contribuiu para o estudo extensivo da via do acido aspártico, revelando, assim, a importância da aspartato quinase (AK), homoserina desidrogenase (HSDH) e dihidrodipicolinato sintase (DHDPS) como enzimas chaves para a regulação da síntese de lisina. A aspartato quinase (AK) exerce um controle sobre via do acido aspártico. A enzima dihidrodipicolinato sintase (DHDPS) regula a síntese de lisina. Na segunda etapa foi apresentada a importância dos aminoácidos sintetizados nas plantas através de complexas vias metabólicas que são controladas por enzimas, intermediários, substratos e aminoácidos. Este estudo também relata os aspectos importantes para uma melhor compreensão da síntese e o acumulo de aminoácidos solúveis e incorporados em proteínas. A terceira etapa foi apresentar estratégias interessantes para utilização em estudos, visando aumentar o nível de aminoácidos essenciais através da manipulação de genes já existentes, como também a introdução de genes estranhos nas plantas. Devido a importância nutricional, essa via tem sido extensivamente estudada, utilizando técnicas de engenharia genética e bioquímica. Pesquisadores tem apresentado esforços considerados no estudo desta via a fim de contribuir para futuras manipulações genéticas, cujo objetivo e produzir plantas com alto conteúdo de lisina, metionina e treonina. / The aim of this research was to elucidate the aspartate metabolic pathway using grains of cereal as a source of study. Therefore, it was necessary to understand the aspartate metabolic pathway in order to depict various biochemical and genetic techniques which can be used to enhance the nutritional value in cereals. After studying theses issues, it was possible to understand the results of having cereals with a high lysine, methionine, and threonine content, so that grains can have balanced protein content. For that reason, an exhaustive research was done by using international and national scientific data published in 1970 to 2012. These data were found in Google Scholar, Portal Capes, and ISI Web of Science. This research was divided in three parts: studies of aspartate metabolic pathway and their essential amino acids derived from plants published in 1970 to 1997, studies of aspartate metabolic pathway published in the period of 1997 to 2006, and interesting strategies to enhance the level of essential amino acids of the aspartate metabolic pathway in plants from 2006 to this moment. Firstly, this investigation reported about the aspartic acid as a precursor of essential amino acids such as methionine, lysine, threonine, and isoleucyne. Among the essential amino acids, lysine has been the most researched due to its lack in many kinds of grains. Needless to say, it has contributed to the intensive study showing the relevancy of aspartate kinase (AK), homoserine dehydrogenase (HSDH), dihydrodipicolinate synthase (DHDPS), as key enzymes for lysine regulation. The aspartate kinase (AK) has an important role on the aspartate metabolic pathway, meanwhile the dihydrodipicolinate synthase (DHDPS) is intrinsically involved on lysine synthesis regulation. Secondly, this investigation presented the importance of the amino acids which are synthesized by plants through metabolic pathways that are controlled by enzymes, intermediates, substrates, and amino acids. In addition, this research reported relevant aspects whereby scientists can improve their understanding about the synthesis and accumulation of soluble amino acids which are incorporated in proteins. Finally, the third part showed interesting strategies which can be used in future researches in order to increase not only the level of essential amino acids by manipulating genes, but also the introduction of odd genes in plants. Given the nutritional relevancy, this pathway has been extensively investigated by using techniques used by biochemical and genetic engineering. Hence, researchers have demonstrated a considerable effort on this matter contributing for future genetic manipulations, so that plants with high lysine, methionine, and threonine content can be produced.

L'élimination des protéines est une étape clé lors de la production du vin blanc afin d'éviter l'apparition éventuelle d'un voile inoffensif mais inesthétique. Des solutions de rechange à l'utilisation de la bentonite sont activement recherchées en raison des problèmes technologiques, organoleptiques et de durabilité associés à son utilisation. Dans cette étude, MpAPr1, une protéase aspartique extracellulaire préalablement isolée et partiellement caractérisée à partir de la levure Metschnikowia pulcherrima, a été clonée et exprimée de manière hétérologue dans la levure Komagataella pastoris. Les propriétés enzymatiques de MpAPr1 ont été initialement caractérisées dans un extrait brut. Après plusieurs essais faisant appel à différentes techniques, MpAPr1 a été purifié avec succès par chromatographie échangeuse de cations. Son activité contre les protéines de raisin a été initialement testée dans une solution modèle dans des conditions environnementales optimales pour l'activité de MpAPr1 puis dans celles qui règnent lors de la vinification. Ensuite, l'activité de MpAPr1 a été évaluée dans du moût de raisin et au cours de la fermentation alcoolique. La présence de MpAPr1, supplémenté au moût de raisin, a entraîné une dégradation partielle des protéines de raisin tout au long de la fermentation et une légère différence dans la composition en composés volatils du vin. L'étude a confirmé que les protéases aspartiques pourraient représenter une alternative à la bentonite pour l'industrie du vin et que les levures non-Saccharomyces telles que M. pulcherrima pourraient avoir un impact bénéfique sur les propriétés du vin. / Protein removal is a key step during the production of white wine in order to avoid the possible appearance of a harmless but unsightly haze. Alternatives to the use of bentonite are actively sought because of technological, organoleptic and sustainability issues associated with its use. In this study, MpAPr1, an extracellular aspartic protease previously isolated and partially characterised from the yeast Metschnikowia pulcherrima, was cloned and expressed heterologously in Komagataella pastoris. Enzymatic properties of MpAPr1 were initially characterised in a crude extract. After several attempts using different techniques, MpAPr1 was successfully purified via cation exchange chromatography. Its activity against haze-forming grape proteins was initially tested in a model solution under optimal environmental conditions for MpAPr1 activity and under those occurring during winemaking. Thereafter, MpAPr1 activity was evaluated in grape must and throughout alcoholic fermentation. The presence of MpAPr1, supplemented to grape must, resulted in the partial degradation of grape proteins throughout fermentation and ultimately in a slight difference in the wine’s composition in volatile compounds. The study provides further evidence that aspartic proteases could represent a potential alternative to bentonite for the wine industry and that non-Saccharomyces yeasts such as M. pulcherrima could have a beneficial impact on wine properties.

Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...

The goal of cancer chemotherapy is to develop effective, safe, and well-tolerated medications. The over-expression of certain receptors on cancer cell membrane provides a basis for active targeting by not only specific interaction between drug delivery system and cells, but also facilitated cellular uptake via receptor-mediated endocytosis. In this study, LDV oligomers up to six LDV repeating units were synthesized via solid phase peptide synthesis method, and evaluated as drug carrier as well as targeting moiety to deliver doxorubicin (Dox) to human malignant melanoma cells (A375), which over-express integrin α 4 β 1 . Cells expressing different levels of integrin α 4 β 1 or modulated using integrin α 4 -specific siRNA knock-down technique were verified by western blot and PCR. Magnetic beads with tripeptides LDV, VDL, or LNV on the surface were used in the binding specificity studies. Results verified that LDV was the minimally required ligand sequence for the specific binding to integrin α 4 β 1 , of which the interaction depends on the amount of integrin and can be utilized for the design of targeted drug delivery. The studies on A375 cells uptake of FITC-labeled LDV oligomers examined the effects of EDTA, temperature, endocytosis inhibitor, and competitive ligand. Cellular uptake mechanism was revealed to be temperature-dependent, receptor-mediated endocytosis, involving the specific interaction between LDV and integrin α 4 β 1 . The internalization extent of LDV monomer was the highest and was also inhibited to the most by the addition of free LDV when compared to other LDV oligomers. Cytotoxicity profiles of Dox-conjugated LDV oligomers were obtained on wild-type A375, integrin α4 knock-down A375, and normal human epithelial keratinocytes (NHEK) using SRB assay. A significant decrease (3∼6 folds) in the cytotoxicity of oligo(LDV)-Dox on A375 cells were observed when the integrin α4 expression was knocked down by ∼50%. Cytotoxicity further decreased on NHEK, which has the lowest integrin α4 expression among three cell lines. In contrast to oligo(LDV)-Dox, free Dox was not able to differentiate between cancerous and normal cells. This result demonstrated the potential of oligo(LDV) as targeting ligand. However, increase of repeating LDV unit did not lead to any apparent trend in cytotoxicity capacity. To facilitate the intracellular Dox release, hydrazone bond (HYD) was introduced between LDV and Dox. In vitro Dox release profiles in pH 6.0, 7.4, and rat plasma proved the pH-sensitivity of LDV-HYD-Dox. Cytotoxicity studies showed an increased cytotoxic effect of LDV-HYD-Dox when compared with LDV-Dox on wild-type A375 (2.5 times), knock-down A375 (1.5 times); while no significant difference in cytotoxicity on NHEK was observed. In vivo animal study supported the in vitro findings on LDV-HYD-Dox, which showed a significant inhibition of tumor growth and longest mice life span when compared to free Dox, poly(L,D,V)-Dox, and LDV-Dox, with averagely only ¼ of the tumor size and almost twice the life span of that from the free Dox group. In conclusion, based on the concept of specific interaction between LDV and integrin α 4 β 1 , oligo(LDV)-Dox targeted drug delivery system was developed and proved to be effective in the delivery of Dox to melanoma cells.

This diploma thesis deals with the efficiency and factors affecting the adsorption of AOM (Algal Organic Matter) amino acids (AAs) arginine (Arg), phenylalanine (Phe) and aspartic acid (Asp) onto granular activated carbon (GAC) Picabiol 12x40 (PIC). The efficiency of AOM AAs removal was studied in laboratory equilibrium and kinetic experiments and it was shown that the adsorption efficiency of the selected AAs is dependent on the structure of the molecule of AAs and the nature of the functional groups of their side chain, and more particularly to solution pH, which determines the nature and size and surface charge of AAs and GAC. In contrast to this, the ionic strength (IS) of solution had relatively low effect on the AAs adsorption. Arg adsorption efficiency increased with increasing pH and reached a maximum at pH 9, where AAs and GAC were oppositely charged, and this leads to attractive electrostatic interactions. In the case of Asp adsorption on PIC practically did not work. The reason is that under all experimental conditions Asp molecules and the surface of the PIC carried identical negative charge. This led to the strong electrostatic repulsion between Asp and PIC which prevented effective adsorption. In the case of Phe the adsorption decreases with increasing pH. Maximum adsorption...