Made available in DSpace on 2014-06-11T19:23:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-27Bitstream added on 2014-06-13T19:30:09Z : No. of bitstreams: 1 pires_ea_me_jabo.pdf: 426419 bytes, checksum: b57d15a1404fbc5b5fdc2c88bcefb224 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo desta pesquisa foi avaliar os efeitos da suplementação de cisteína e cisteamina no desenvolvimento meiótico de oócitos caninos durante o processo de maturação ín vítro. Os oócitos foram coletados de sete cadelas hígidas em fase pré-ovulatória imediata, submetidas à ovario-histerectomia. Os COC's selecionados foram cultivados por um período de 72 horas em quatro meios diferentes: A (controle) - TCM199 suplementado com BSA (3 mg/mL) + FSH (5 J.Lg/mL) + LH (10 f.Lg/mL) + progesterona (2 f.Lg/mL) + estradiol (2 f.Lglml); 8 - controle + 0,1mM de cisteína; C - controle + 100J.1M de cisteamina; D - controle + 0,1 mM de cisteína + 100J.1M de cisteamina. Os resultados demonstraram que não houve diferença significativa entre os tratamentos (p<0,05), ou seja, a suplementação de compostos antioxidantes no meio de maturação não favoreceu a competência meiótica. Além disso, neste estudo pode-se inferir que para cada fase do ciclo estral, talvez seja necessário um período de maturação diferenciado. / The aim of this research was to evaluate the effects of the cysteine and cysteamine supplementation on meiotic deveropment of canine oocytes dunng the process of in vitro maturation. The oocytes were collected atter ovanohysterectomy from seven healthy bitches in immediate preovulatory stage. The selected COC's were cultured by a period of 72 hours in four different media: A (control) - TCM199 supplemented with BSA (3 mg/mL) + FSH (5 J-Lg/mL) + LH (10 J-Lg/mL) + progesterone (2 f.Lg/mL) + estradiol (2 f.LglmL); 8 - control + 0,1mM of cysteine; C - control + 100JlM of cysteamine; O - control + 0,1 mM of cysteine + 100J,lM of cysteamine. The present study demonstrated that there was not significant difference among the treatments (p<0,05), in other words, the supplementation of antioxidant in the medium of maturation didn't favor the meiotic competence. Besides, in this study it can be inferred that for each stage of the oestrus cycle, perhaps it is necessary a different maturation penod.

>Magister Scientiae - MSc / This study investigated the effects that drought stress imposed on the growth and development of soybean plants. Soybeans were initially observed at the whole-plant level in order to identify the physical changes that had taken place in response to drought. Further investigation of the effects of drought stress on Soybean plants were quantified at the molecular level. Physical changes of soybeans in response to drought stress were typified by the change in leaf morphology and pigmentation. At the molecular level, it was observed that drought stress resulted in the accumulation of hydrogen peroxide in soybean leaves, which was met by elevated levels of lipid peroxidation. The effects of drought on the modulation of (and interplay between cystatins) cysteine protease (caspase-like) activity and programmed cell death (PCD) were also investigated. Total caspase-like activity and cell death were enhanced in response to water deficit despite the up-regulation in gene expression of the cystatin Glyma14g04250. The cystatin Glyma18g12240 was not expressed in soybean leaves, whilst the gene expression of the cystatin Glyma20g08800 remained unchanged in response to drought. This study was aimed at the characterization of two single domain soybean cystatins, namely, Glyma14g04250 and Glyma20g08800 which could potentially be overexpressed in transgenic soybean plants in an attempt to alleviate the effects of drought stress. / National Research Foundation (NRF)

Plasmodium is a genus of parasites causing malaria, a virulent protozoan infection in humans resulting in over a million deaths annually. Treatment of malaria is increasingly limited by parasite resistance to available drugs. Hence, there is a need to identify new drug targets and authenticate antimalarial compounds that act on these targets. A relatively new therapeutic approach targets proteolytic enzymes responsible for parasite‟s invasion, rupture and hemoglobin degradation at the erythrocytic stage of infection. Cysteine proteases (CPs) are essential for these crucial roles in the intraerythrocytic parasite. CPs are a diverse group of enzymes subdivided into clans and further subdivided into families. Our interest is in Clan CA, papain family C1 proteases, whose members play numerous roles in human and parasitic metabolism. These proteases are produced as zymogens having an N-terminal extension known as the prodomain which regulates the protease activity by selectively inhibiting its active site, preventing substrate access. A Clan CA protease Falcipain-2 (FP-2) of Plasmodium falciparum is a validated drug target but little is known of its orthologs in other malarial Plasmodium species. This study uses various structural bioinformatics approaches to characterise the prodomain‟s regulatory effect in FP-2 and its orthologs in Plasmodium species (P. vivax, P. berghei, P. knowlesi, P. ovale, P. chabaudi and P. yoelii). This was in an effort to discover short peptides with essential residues to mimic the prodomain‟s inhibition of these proteases, as potential peptidomimetic therapeutic agents. Residues in the prodomain region that spans over the active site are most likely to interact with the subsite residues inhibiting the protease. Sequence analysis revealed conservation of residues in this region of Plasmodium proteases that differed significantly in human proteases. Further prediction of the 3D structure of these proteases by homology modelling allowed visualisation of these interactions revealing differences between parasite and human proteases which will lead to significant contribution in structure based malarial inhibitor design.

Thesis advisor: Eranthie Weerapana / Disulfide-bonding cysteine residues perform critical roles in the structural stabilization and redox regulation of protein function. Secreted proteins are often enriched for structural disulfide bonds conferring conformational stability in the oxidizing extracellular environment. The controlled formation of disulfide bonds in secreted proteins is regulated in the endoplasmic reticulum (ER) by the protein disulfide isomerase (PDI) family. To investigate disulfide-bond formation in the ER, quantitative chemical-proteomic methods were coupled to subcellular-fractionation-based ER enrichment. Cysteine reactivity studies identified highly reactive post-translationally modified cysteine residues including disulfide-bonding cysteines. Upon discovering a highly reactive population of traditionally oxidized cysteines, the percentage of oxidation for cysteines localizing to the ER was determined. Next, ER function was chemically perturbed to evaluate changes to cysteine oxidation following upregulation of the unfolded protein response (UPR). Disulfide bond formation was specifically disrupted in the ER by CRISPR-Cas9-mediated PDIA1 and PDIA4 knockout. The effects of PDI knockout on cancer cell phenotype and changes to cysteine oxidation states were evaluated. Finally, in vitro studies were performed to evaluate PDIA4 oxidase activity and identify potential PDIA4-selective inhibitors. In the future, the platforms developed within may be applied to profiling changes to cysteine oxidation in other biological systems such as other organelles and disease states. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The reduction of ferricytochrome c ( cyt c^III), as well as some iron ligand complexes and chemically modified derivatives of cyt c^III, by two molybdenum(V)-cysteine complexes has been investigated as a model for electron transfer in molybdenum enzymes. The reduction of cyt c^III by di-μ-oxo-bis[oxo(L-cysteinato)molybdate(V)] (I) is first order in cyt c^III and zero order in I over a wide range in concentration of I at neutral pH. The reduction of cyt c^III by μ-oxo-bisfoxodihydroxo(L-cysteinato) molybdate(V)] (II) is first order in both reactants and has a rate several orders of magnitude greater than the rate of reduction by I. Activation parameters have been determined for both reactions. The reduction of two pyridoxal phosphate derivatives of cyt c^III by I proceeds at the same rate as the reduction of native cyt c^III, while the mononitromonotyrosyl derivative is reduced somewhat faster. The reduction of the N-formyltryptophyl derivative of cyt c^III by I is biphasic, with the fast phase proceeding with a rate similar to the native cyt c^III and the slow phase being somewhat slower. The reduction by I of all derivatives studied is first order in cyt c^III and zero order in I, suggesting the mechanism does not change with these chemically modified forms of cyt c^III. The pyridoxal phosphate and mononitromonotyrosyl derivatives of cyt c^III are reduced by II with rates similar to the native cyt c^III and by the same mechanism. The reduction of the N-formyltryptophyl derivative by II has complicated kinetics which suggest the presence of at least two formyl-cyt c^III species. The reduction of the cyanide, azide and imidazole complexes of cyt c^III by II occurs at a greatly reduced rate and by a different mechanism than the reduction of native cyt c^III The rate of reduction of the cyanide and azide complexes is dependent on the rate of dissociation of the ligand cyt c^III complex and independent of the concentration of II. The reduction of the imidazole complex by II is much more complicated, with the rate independent of II at very low concentrations but shows a dependence on the concentration of II at higher concentrations. Rate constants have been determined for the reactions and possible mechanisms have been developed. The results have been discussed with regard to electron transfer in molybdenum enzymes and electron transfer to cyt c^III This study is especially valuable as a model for the hepatic sulfite oxidase enzyme.

Le cuivre est un micronutriment essentiel qui participe à de nombreux processus biologiques. Cependant, le cuivre libre est toxique pour l’organisme parce qu’il catalyse une réaction de type Fenton formant des espèces réactives de l’oxygène. Par conséquent la concentration en cuivre est finement régulée dans tous les organismes vivants. Les maladies de Menkes et de Wilson sont dues à des dérèglements de l’homéostasie du cuivre qui se manifestent respectivement par une déficience ou une accumulation de cuivre dans l’organisme. La maladie de Wilson est traitée avec des chélateurs du cuivre, qui provoquent des effets secondaires importants chez certains patients.Mon projet de doctorat consiste en l’élaboration de trois familles de peptides qui contiennent des acides aminés cystéines et en l’étude de leurs complexes de Cu(I) pour déterminer s’ils sont des candidats adaptés pour le traitement de la maladie de Wilson. L'interaction de certains peptides avec les ions Hg(II) ou Zn(II) a également été étudiée. En effet, le Hg(II) est un cation métallique possédant des propriétés similaires au Cu(I) et donc souvent utilisé pour modéliser le Cu(I) qui est sensible à l'oxygène et se dismute dans l’eau. Le Zn(II) est quant à lui omniprésent dans les cellules et un compétiteur intracellulaire potentiel du Cu().Les séquences des peptides ont été choisies selon trois stratégies différentes. Dans la première, des séquences inspirées de la boucle de liaison du cuivre de la protéine bactérienne CueR (copper efflux regulator), contenant deux cystéines, ont été étudiées afin de bénéficier de la sélectivité et de la sensibilité de ce régulateur. Dans une deuxième approche, des peptides contenant trois cystéines dans les motifs CxCxxC et CxCxC ont été étudiés pour combiner les avantage des peptides (bonne internalisation dans les cellules hépatiques quand ils sont judicieusement fonctionnalisés) et des tripodes (très forte affinité pour le Cu(I)) de l’équipe CIBEST. Finalement, la pré-organisation a été exploitée dans un tétrapeptide rigide où les deux cysteines sont liées dans un coude β préformé.Les trois peptides modèles du régulateur CueR miment la capacité de la protéine à accueillir exclusivement un ion Cu(I) dans des conditions d'excès de ligand et une forte affinité et sélectivité par rapport au Zn(II). Ces caractéristiques sont avantageuses dans la perspective du développement de nouveaux chélateurs du Cu(I).Les peptides contenant trois cystéines s’avèrent trop flexibles pour contrôler la spéciation des complexes du Cu(I). Par ailleur, ces peptides sont bien adaptés pour une coordination efficace du Hg(II) par trois groupes thiolates. Les différences structurales n’ont qu’une influence modeste sur les stabilités des complexes. La différence dans la coordination des peptides vis-à-vis des deux ions mous Hg(II) et Cu(I) démontre que l'utilisation du Hg(II) comme ion modèle pour la coordination du Cu(I) avec des peptides ou des protéines riches en soufre dans des conditions physiologiques n’est pas toujours appropriée.La pré-organisation de la structure peptidique est un élément clé du contrôle de la spéciation du complexe Cu(I) et de l’affinité des ligands pour le Cu(I). Le peptide CDPPC forme uniquement le cluster Cu4P3 avec une grande stabilité et une bonne sélectivité Cu(I)/Zn(II). Au contraire, les données expérimentales avec le tétrapeptide plus flexible CPGC montrent la formation d’un mélange de complexes polymétalliques de Cu(I). Il est intéressant de noter que le peptide simple CDPPC est capable d’imiter la formation des clusters Cu(I)-thiolates identifiés dans de nombreuses protéines impliquées dans l’homéostasie du cuivre, comme Cox17 ou Ctr1. CDPPC est intéressant pour mettre au point un chélateur intracellulaire du Cu(I), et sa fonctionnalisation afin de pouvoir cibler les cellules hépatiques pour le traitement de maladie Wilson sera donc pertinente dans le futur. / The essential micronutrient copper participates in several biological processes, like respiration, iron homeostasis, antioxidant defense or pigment formation. However, excess of copper can promote ROS formation and thus induce oxidative damages. Therefore, intracellular copper concentration is under strict control. Menkes and Wilson’s diseases are genetic disorders causing impairment in copper homeostasis leading to copper deficiency or overload, respectively. Wilson’s disease is treated by chelation therapy, but the presently used drugs have several adverse side effects.The aim of my Ph.D. work consisted of the design of three groups of cysteine containing peptides and the characterization of their Cu(I) complexes to determine whether they are appropriate candidates for the treatment of Wilson’s disease.The peptides were designed following three different approaches. In a first strategy, we attempted to take advantage of the outstanding selectivity and sensitivity of the bacterial copper efflux regulator protein CueR by studying oligopeptides based on the metal binding motif of CueR involving two cysteine residues. Second, three-cysteine containing linear and cyclic peptides were designed with the aim of merging the better internalization of peptides by hepatocytes and the high Cu(I) affinity of tripods previously studied in the Delangle’s lab. Finally, the advantages of a highly preorganized peptide structure were exploited in a short, rigid tetrapeptide where two cysteines were linked by a turn motif (CDPPC). For comparative purposes studies were also performed with another, less rigid tetrapeptide ligand containing the PG unit as a turn inducing motif.The three CueR model peptides resemble the ability of the protein to exclusively accommodate one metal ion under ligand excess conditions. This, combined with the large affinity and high selectivity vs. Zn(II), are the features that are advantageous in the view of the development of new Cu(I) chelators.The three-cysteine-containing peptides proved to be too flexible to control the speciation and hereby leading to the formation of several species. On the other hand, they are well adapted for an efficient trithiolate coordination of the thiophilic cation Hg(II). Structural differences in the three-cysteine containing peptides have minor effect on the affinity of the ligands towards Cu(I) and Hg(II) ions. The striking difference in the behavior of the peptides towards the two soft metal ions demonstrate that the use of Hg(II) as a probe for Cu(I) coordination with sulfur-rich peptides or proteins in physiological conditions may not always be fully appropriate.Preorganization of the peptide structure is a key element in the control of Cu(I) complex speciation and in the affinity of the ligands for Cu(I).CdPPC forms a single Cu4P3 cluster with high stability and displays large selectivity for Cu(I) with respect to the ubiquitous Zn(II). In contrast, The CPGC-Cu(I) system is characterized by a more complicated complex formation. It is worth to note, that the simple CdPPC peptide is able to mimic the Cu(I)-thiolate cluster formation that are typical in proteins like Ctr1 or Cox17. CDPPC is an interesting simple peptide candidate to be targeted to the liver cells for the localized treatment of Cu overload in Wilson’s disease.

Trichomonas vaginalis and Tritrichomonas foetus are the etiologic agents of human and bovine trichomoniasis, respectively. As microaerophilic protozoans; both share a wide array of clinical manifestations ranging from vaginitis to abnormal pregnancies. Human trichomoniasis receives minimal public health attention despite of its worldwide high prevalence rate. Emerging evidence of metronidazole-resistant T vaginalis strains facilitates a concern to understand this protozoan. Cysteine proteases have been implicated as important virulence factors produced by T vagina/is. This study explores the expression of one particular legumain-like cysteine protease known as Tv AE 1. Furthermore, it highlights the relationship between inhibitory effects of trichomonal cells caused by sanguinarine and chelerythrine. A system for obtaining legumains by expressing it in methylotrophic yeast, Pichia pastor is, has been described. The recombinant legumains were produced and processed by the yeast to their inactive and mature forms. Secondly, T foetus cells were transfected with TvAEl construct. Localization and enzymatic studies on legumains will provide evidence into the pathogenicity ofT vagina/is. This study revealed the vesicularization of recombinantly unprocessed TvAEl proteins. Thirdly, plant derived compounds, sanguinarine (SA) and chelerythrine (CHE) were assessed in vitro for their inhibitory effects against T vagina/is and T. foetus. Treatment of SA and CHE for 24 h led to a significant inhibitory growth of in vitro cultures for all three trichomonal strains, G3, Tl and Dl, compared to untreated cells. For these bovine and human trichomonal strains, SA was slightly more effective inhibitor than CHE. With IC5o values between 3 - 8 micromolar for the alkaloids, CHE had less inhibitory effect compared to SA. These findings are significant considering the association between cysteine pro teases and trichomoniasis. Further elucidation of the exact anti protozoal mechanism of both compounds toward legumains may lead to the development of these potent agents against trichomonads.

Trichomoniasis is a common STD caused by the parasitic protozoan Trichomonas vaginalis. The parasite is estimated to have infected roughly 3.7 million Americans. Complications from trichomoniasis can lead to cervical cancer in women and prostate cancer in men. One of the mechanisms of the parasite employs is using cysteine proteases to break down the cellular matrix of its host. However, three endogenous phytocystatin-like protease inhibitors have been found within the parasite’s genome. By recombinantly expressing these cystatins we have been able to test their ability to inhibit cysteine proteases such as papain and those found in T. vaginalis to find their effectiveness. By characterizing these inhibitors, it appears that they are effective at reducing the ability of T. vaginalis cysteine proteases and thus could be useful against the pathogenicity of the parasite.

<p> Bcl-2 proteins play a critical role in the regulation of apoptosis, a form of programmed cell death. Apoptosis is important during development to facilitate the elimination of supernumerary, damaged or harmful cells in multicellular organisms. Altered regulation of apoptosis is associated with many diseases such as several forms of cancer as well as autoimmune and degenerative disorders. The way in which Bcl-2 proteins regulate apoptosis is unknown and much research is focused on elucidating the molecular mechanism of their function. Bcl-2, an anti-apoptotic member of this family, is localized to the mitochondria, endoplasmic reticulum and nuclear envelope. In healthy cells, Bcl-2 adopts a typical tail-anchored topology in which the carboxyl-terminal helix (a9) is inserted into the membrane, anchoring the protein, leaving the majority of the protein in the cytosol. Previous results from our lab have shown that after the induction of apoptosis, Bcl-2 undergoes a conformational change in which the endogenous cysteine residue, C158, in the a5 helix becomes protected from a membrane impermeant cysteine specific labelling reagent, IASD (4-acetamido-4' ((iodoacetyl)amino)-stilbene-2,2'disulfonate). Modification of cysteine residues results in a change in migration ofBcl-2 in an isoelectric focusing, IEF, gel system. To investigate the nature of this conformational change, cysteine scanning mutagenesis was used to determine the topology of Bcl-2 in the late stages of apoptosis. The results from the current study showed that in rat 1 myc ERTM fibroblasts, a discontinuous sequence of residues in the a5 and a6 helices of Bcl-2 become protected from IASD labelling after the induction of apoptosis by etoposide or serum starvation. The data support a model topology in which, during apoptosis, Bcl-2 undergoes a functionally significant conformational change, going from a single spanning transmembrane protein to a polytopic membrane protein in which three helices span the membrane, a5, a6 and a9. </p> / Thesis / Master of Science (MSc)

The process of xylem tracheary element differentiation involves the coordination of vascular cambium activity, cell fate determination, cell expansion/elongation, secondary wall synthesis, programmed cell death, and cellular autolysis. The end result of tracheary element differentiation is a cellular corpse lacking a protoplast and consisting of a thickened cell wall composed mostly of lignin and cellulose. Little is known about the genetic mechanisms regulating the process of tracheary element differentiation. XCP2 expression localizes to tracheary elements according to two independent methods of analysis: promoter reporter experiments and immunogold localization by electron microscopy. XCP2 may be involved in catalyzing the degeneration of the protoplast during the final autolytic stages of tracheary element differentiation. To this date XCP2 function has not been directly demonstrated. In principle, any tracheary element-specific markers can be linked to upstream regulatory genes with roles in tracheary element differentiation. To develop the XCP2 promoter as a tool for identification of transacting factors, a promoter deletion analysis was carried out. Utilizing information from 5â and 3â deletion constructs, a 70-bp region upstream of the XCP2 translational start site is both necessary and sufficient for TE-specific expression of the UidA reporter gene. Mutational analysis of the ACTTTA element at position -113-bp strongly suggests it is a cis element required for XCP2 expression. In silico analysis of an 18-bp promoter region located within 200-bp of the translation start site and including the ACTTTA element revealed high indentity shared between xylem-specific XCP2 homologs from Zinnia elegans, Populus trichocarpa, and XCP1 from Arabidopsis thaliana. / Master of Science