This thesis deals mainly with two oxidation reactions: water oxidation and aerobic oxidation, both of which have been applied in a biomimetic fashion. In the former reaction molecular oxygen is generated whereas in the latter it was used as terminal oxidant in oxidation reactions. The first part of this thesis describes the synthesis of different ruthenium and manganese complexes that could potentially act as catalysts for water oxidation. This part includes a discussion of the stability and reactivity of a new manganese(III) amide-type complex, that has been used as a catalyst for both epoxidation of stilbene and alcohol oxidation. The second part of this thesis discusses the synthesis of two new hybrid catalysts consisting of hydroquinone linked cobalt(II) salophen and cobalt(II) salmdpt, which have been used as oxygen-activating catalysts in aerobic oxidation reactions. The former catalyst was applied to the Pd-catalyzed reactions such as 1,4-diacetoxylation of cyclohexadiene whereas the latter was applied to the Ru-catalyzed oxidation of secondary alcohols to ketones. Moreover, these two hybrid catalysts could be used in the Pd-catalyzed carbocyclization of enallenes. In all cases molecular oxygen was used as the stoichiometric oxidant.

Platelets are small anucleate cell fragments found in blood whose physiological role is important in maintaining haemostasis. In vivo, platelet surface glycoproteins mediate the mechanistic roles of platelets, and polymorphic changes to these glycoproteins have been observed to have significant effects on the platelet cellular function and such changes may include over-expression, under-expression and antigenicity of the protein. Human platelet antigens (HPA) are a result of polymorphic differences in platelet surface glycoproteins which have been found to be variably expressed in the population. Foetal maternal alloimmune thrombocytopaenia (FMAIT) is a condition that is observed in the unborn foetus and neonates due to HPA incompatibility between the mother and the foetus. HPA incompatibility accounts for a majority of severe thrombocytopaenic cases in neonates, and delayed diagnosis and treatment of such a condition often lead to intracranial haemorrhage. The risk in neonates diagnosed with FMAIT becomes increasingly significant in cases where intra-uterine (during pregnancy) platelet transfusion is the only effective therapeutic option. There are currently no antenatal screening programs for this condition, and laboratory diagnosis of FMAIT relies on the detection of maternal alloantibodies and parental HPA typing. For these reasons a significant amount of research is currently being invested into the isolation of recombinant antibodies with specific reactivity against FMAIT-related platelet antigens. Stable and specific recombinant platelet antibodies have great potential as a diagnostic agent in antenatal screening and broad-scale HPA typing of blood donors for platelet transfusion. Further characterisation of the isolated antibody may lead to a possible therapeutic agent. Studies by previous researchers have shown that the traditional methods (ie. Mouse monoclonal and EBV transformation) of obtaining monoclonal antibodies against FMAIT-related antigens have proven unsuccessful. The continuing progress in the discipline of phage display has produced several novel antibodies against self and non-self antigens. A further advantage in the application of phage display technology for the isolation of novel antibodies is the easy transition from bacterial to mammalian expression for the characterisation of glycosylated antibodies. The main focus of this project was to create and isolate a recombinant human anti-HPA-3a antibody using phage display for its possible application as a therapeutic or diagnostic agent.

Platelet-derived microparticles, released as a result of platelet activation, promote coagulation through the surface exposure of phosphatidylserine, acting as the catalytic site for the conversion of prothrombin to thrombin by the activated coagulation factors X and V. Although elevated numbers of circulating platelet-derived microparticles can be detected in a number of clinical disorders, the methods for the detection of these microparticles are far from standardised. In addition, recent reports have also speculated that not all microparticles may expose phosphatidylserine, demonstrating that the binding of Annexin V, a phosphatidylserine-specific binding protein, is not detectable on a population of microparticles. The initial stage of this thesis was to establish a flow cytometric method for the detection and enumeration of microparticles based on their capacity to bind Annexin V and to utilise this assay to investigate a number of the issues that have limited assay standardisation. The assay could be performed on either stimulated or unstimulated plasma or whole blood samples. Interestingly, plasma microparticle counts were significantly higher than whole blood microparticle counts. The effects of centrifugation alone could not be attributed as the sole source of this discrepancy. The antigenic characteristics of platelet-derived microparticles were also investigated, with platelet-derived microparticles demonstrated to express the platelet glycoproteins CD31, CD41a, CD42a and CD61. Platelet-derived microparticles also expressed CD42b, and this expression was significantly decreased when compared to their progenitor platelets. The expression of the platelet activation markers CD62p, CD63, CD40L and PAC-1 was dependent upon the sample milieu, suggesting that the centrifugation conditions required to generate platelet-poor plasma may lead to artefactual increases in the expression of platelet activation markers. An investigation of the role of the GpIIb/IIIa complex on the formation of platelet-derived microparticles was also performed. A monoclonal antibody to the GpIIb/IIIa complex (Abciximab) significantly inhibited in vitro collagen-stimulated platelet-derived microparticle formation. Interestingly, platelets obtained from two subjects with impaired GpIIb/IIIa activation, demonstrated normal microparticle formation following collagen stimulation, suggesting that the presence of GpIIb/IIIa complex, but not its activation, is required for collagen-induced microparticle formation. A novel mechanism for microparticle formation was also investigated, with platelet-derived microparticles demonstrated to form in response to the sclerosing agents sodium-tetradecyl sulphate and polidocanol. Interestingly, the removal of plasma proteins by the washing of platelets left platelets more susceptible to sclerosant-induced microparticle formation, suggesting that plasma proteins may protect platelets from microparticle formation. The procoagulant activity of platelet-derived microparticles was also investigated using a novel coagulation assay (XACT) specific for the procoagulant phospholipid. An evaluation of this assay demonstrated a significant correlation between Annexin V binding microparticle counts and procoagulant activity in both whole blood and plasma samples. There was more procoagulant activity in whole blood samples than in plasma samples, suggesting that the procoagulant phospholipid activity was also associated with erythrocytes or leukocytes. To further investigate this phenomenon, a whole blood flow cytometric assay was developed to assess Annexin V binding to erythrocytes, leukocytes, platelets and microparticles. This assay demonstrated that a large proportion of Annexin V binding (51.0%) was associated with erythrocytes. Interestingly, a proportion of the Annexin V binding erythrocytes (24.5%) and leukocytes (78.8%) were also associated with platelet CD61 antigen, suggesting that they also bound a platelet or platelet-derived microparticle. The effect of sample anticoagulant on microparticle procoagulant activity was investigated. Microparticle counts were most stable in EDTA anticoagulated samples, but were stable in sodium citrate for up to 15 minutes following sample collection. The procoagulant activity of microparticles was significantly inhibited by EDTA in collagen-stimulated platelet-rich plasma samples, when compared to sodium citrate anticoagulated samples. Although the initial method used to investigate microparticles was based upon their ability to bind Annexin V, it was consistently observed that a large proportion of events in the size region of a microparticle were Annexin V negative. An investigation was therefore commenced into the procoagulant activity of microparticles based on their capacity to bind Annexin V. The presence of Annexin V negative microparticles was confirmed by flow cytometry and the proportion of microparticles that bound Annexin V was dependent upon type of agonist used to stimulate microparticle formation. Varying the assay constituents (calcium concentration / Annexin V concentration / buffer type) did not alter the proportion of Annexin V binding microparticles. When compared to Annexin V positive microparticles, Annexin V negative microparticles expressed significantly higher levels of CD42b on their surface, but possessed significantly decreased expressions of CD62p, and CD63. A significant correlation between the percentage of Annexin V binding and XACT procoagulant activity was found (p=0.03). Furthermore, Annexin V binding inhibited greater than 98% of procoagulant phospholipid activity, suggesting that Annexin V binding was a true reflection of procoagulant activity. Microparticles could be sorted using either a flow cytometric or magnetic sorting strategy. By electron microscopy, Annexin V negative events isolated following magnetic sorting were vesicular structures and not small platelets or the remnants of activated platelets. In summary, this thesis has demonstrated the ability of the flow cytometer and XACT assays to detect microparticles and their procoagulant activity. It has also shown that the use of Annexin V to detect microparticles may warrant further investigation.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph.D. in Cell Biology, Stem Cells, and Development) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 105-116).

Thesis (Ph.D.)--McMaster University, 2003. / Advisor: David W. Andrews. Includes bibliographical references (leaves 122-141) Also available via World Wide Web.

RAGs (Recombination Activating Genes) are responsible for generation of antigen receptor diversity in case of B-cells and T-cells, through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. Each of these segments are flanked by recombination signal sequences (RSS), which consist of a conserved heptamer and nonamer separated by a less conserved spacer of 12 or 23 bp. RAGs recognize and cleave at the 5’ end of heptamer, leading to the formation of hairpin coding ends and blunt signal ends. The coding ends are joined through the process of no homologous DNA end joining (NHEJ), leading to the rearrangement of variable region of antigen receptors. Apart from its physiological property, RAGs can also act as a structure-specific nuclease. Previously, it has been shown that inadvertent action of RAGs on cryptic RSS and non B-DNA structures can lead to the generation of genomic instability and cancer. A very coordinated expression of RAGs has been observed in pro- and pre-B cells of the lymphoid system, which overlaps with the window of productive rearrangement during V(D)J recombination. Besides, studies by us and others have shown that RAG cleavage at altered DNA structures and cryptic RSS leads to chromosomal translocations resulting into cancer. However, several questions related to regulation of RAG expression and its activity in lymphoid cells remains to be answered. Previous studies have suggested regulation of RAG expression at different levels, such as methylation, ubiquitination, phosphorylation and by coordinate action of various transcription factors. In the present study, we evaluate the potential role of miRNAs in the regulation of RAG expression and its function in lymphoid cells. miRNAs are small, single-stranded non-coding RNAs, which play an important role in the regulation of gene expression. They play a critical role in the regulation of different cellular functions. Although there are miRNAs identified to play critical role during development of immune system, several key questions such as its role in the regulation of RAGs is yet to be addressed. In the current study, we have used bioinformatics approach to extract potential miRNAs that bind to 3’UTR of RAG1 and RAG2. miRNA expression datasets were downloaded from NCBI SRA database and extensive evaluation was done using various bioinformatics tools such as Bowtie, Sam tools, Bam tools, Bed tools and R package. We screened the miRNA expression profile across different stages of B-cell development (pro, pre, immature and mature B-cells), which overlap with the narrow window of RAG expression. The shortlisted miRNAs were further analyzed using miRNA databases such as miRBase, Targetscan and EMBL. Results showed that 33 miRNAs were specific to RAG1, among that one (miRNA1) followed RAG expression profile in B-cells. Besides miRNA2, which is a novel miRNA, was selected only on the basis of RAGs expression profile in a stage specific manner and the complementarity of the seed sequence of miRNA2 to the 3’UTR of RAG1 was checked manually. Interestingly, we observed that RAG1 expression was significantly down regulated in the presence of these miRNAs. However, there was no significant difference in the levels of other genes analysed. Further, semi-quantitative RT-PCR analysis confirmed the endogenous processing of pre-miRNA into mature miRNA using the cellular machinery. Besides, enrichment of 3’UTR of seed region of these miRNAs, enhanced the expression level of RAG1. Importantly, the enhancement in RAG1 expression level was limited in case of mature B-cells, where RAG expression is normally not observed. Further, transfection of lymphoid cells with miRNA inhibitors, specific to the miRNAs under study, showed the enhancement in RAG1 expression in lymphoid cells. In addition to this, specificity of selected miRNAs was confirmed by performing 3’UTR reporter assays, where enhanced luciferase expression was observed in case of mutant 3’UTR, while it was minimal in case of wild type constructs. Endogenous expression levels of selected miRNAs were evaluated in both lymphoid and nonlymphoid normal tissues and cancer cells using RT-PCR. Interestingly, we observed inverse correlation of expression levels of miRNA and RAG expression in all the cells tested. Besides, miRNA expression levels were less in pre-B cells and T-cells, owing to the increased expression of RAGs. Apart from this, recombinogenic potential of candidate miRNAs was assessed using episomal based V(D)J recombination assays. Interestingly we observed significant decrease (2-4 fold) in the V(D)J recombination efficiency when miRNA1 or 2 constructs were transfected in Nalm6 cells, as compared to that of controls, where no miRNAs were used. However, in case of Reh cells upon transfection with miRNA1construct, the decrease in recombination potential was upto 9 fold. Hence, we identify two miRNAs that can play an important role in the regulation of RAG1 expression and its physiological activity. Further, studies are being carried out to confirm their role in the regulation of RAG1 during different developmental stages of lymphoid cells in mice. As stated above, in addition to the sequence-specific activity, RAG possesses structure-specific nuclease activity as well. It has been shown that RAGs can cleave different types of altered DNA structures. Studies from our laboratory showed that even when RAGs act as a structure-specific nuclease there is a sequence bias. Presence of cytosine and thymine at the single-stranded region of heteroduplex DNA is important for RAG nicking and double-strand break (DSB) formation. In addition, proximity of a nonamer to bubble structures can enhance RAG cleavage. However, the role of immediate flanking sequences in the RAG mediated cleavage at heteroduplex regions is not understood. We investigated the role of flanking double-stranded DNA sequences in the regulation of RAG cleavage on non-B DNA structures. We found that RAG binding and cleavage on heteroduplex DNA is dependent on the length of double-stranded flanking region. Besides, immediate flanking regions of the heteroduplex DNA affected the RAG binding and cleavage in a sequence dependent manner. Interestingly, we also observed that the cleavage efficiency of RAGs at heteroduplex region was influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights into regulation of the pathological action of RAGs. Previous studies have shown that in addition to formation of coding and signal joints during V(D)J recombination, nonstandard V(D)J recombination products known as hybrid joints and open-shut joints may be formed, particularly in certain aberrant conditions such as defective NHEJ machinery. Interestingly, the hybrid and open-shut joints closely resemble the transposition mechanisms associated with transposons oretroviruses. Studies have also shown that RAGs possess structural similarity with integrases in domain organization. Both the proteins have Zinc Finger Binding domain (ZFB) which helps in multimerization of the protein, a central catalytic core domain comprising three acidic amino acids D, D and E essential for enzymatic activity and C-terminal domain (CTD) responsible for nonspecific binding to the DNA. Previous studies from our laboratory showed that, Elvitegravir, an inhibitor of integrase could interfere with the biochemical functions of the RAGs in vitro. Specifically, it inhibited the RAG binding and cleavage at RSS, hairpin formation, post-cleavage complex formation involving 12RSS and 23RSS. Using the episomal assay system that mimics signal joints (pGG49) and coding joints (pGG51), we show that Elvitegravir can inhibit V(D)J recombination inside cells. Interestingly we observed 3-6 fold decrease in the recombination frequency in signal ends joining, when treated with increasing concentrations (100, 500 and 1000 nM) of Elvitegravir. A 5-8 fold decrease in coding joints formation was also observed upon treatment with the inhibitor. The presence of recombination was confirmed by restriction digestion followed by sequencing analysis. Further analysis of recombination junctions revealed extensive deletion before joining in the case of Elvitegravir treated samples. Insertions or substitutions near to the recombination junctions were also prominent in treated samples. In depth analysis of sequenced junctions showed the presence of sequence having the features to form hairpins both upstream and downstream to the RSS sequences and was the site of cleavage in cases were higher deletion was observed. The analyzed recombinants did not show any signal joints or coding joints formation in treated samples. This suggests that Elvitegravir affects the physiological function, the V(D)J recombination of RAGs inside the cells. Thus, in the present study, we show that RAGs can be regulated by specific miRNAs. We have identified two potential miRNAs, which can regulate the RAG expression as well as its function in different stages of B- and T-cell development. Further, we also identify a novel regulatory mechanism for the structure-specific activity of the RAG complex. In addition to this, we find that integrase inhibitor, Elvitegravir, affects V(D)J recombination within B-cells, indicating its potential deleterious impact in HIV patients, which needs to be further evaluated.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-02-10T16:31:37Z No. of bitstreams: 1 Vinicius Costa Souza Ferreira, Papel do fator... 2014.pdf: 10087481 bytes, checksum: 8c34a10bbc4c46304eaf9166412e6f0f (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-02-10T16:31:51Z (GMT) No. of bitstreams: 1 Vinicius Costa Souza Ferreira, Papel do fator... 2014.pdf: 10087481 bytes, checksum: 8c34a10bbc4c46304eaf9166412e6f0f (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-02-10T16:32:18Z (GMT) No. of bitstreams: 1 Vinicius Costa Souza Ferreira, Papel do fator... 2014.pdf: 10087481 bytes, checksum: 8c34a10bbc4c46304eaf9166412e6f0f (MD5) / Made available in DSpace on 2015-02-10T16:32:18Z (GMT). No. of bitstreams: 1 Vinicius Costa Souza Ferreira, Papel do fator... 2014.pdf: 10087481 bytes, checksum: 8c34a10bbc4c46304eaf9166412e6f0f (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A leishmaniose visceral é causada pelo parasita Leishmania infantum. A infecção ocorre quando flebótomos infectados se alimentam na derme do hospedeiro vertebrado, inoculando o parasita. A infecção produz uma resposta com diversas moléculas inflamatórias, como os mediadores lipídicos. O fator de ativação de plaquetas (PAF) é um potente mediador lipídico derivado de um lisofosfolipídio. PAF participa da fisiologia normal da célula e possui um perfil pró-inflamatório. A participação de mediadores lipídicos, como eicosanóides e PAF, já foi identificada na imunopatogênese das leishmanioses. PAF gerado pelo hospedeiro tem efeito leishmanicida e de controle da infecção por L. amazonensis. PAF-acetilhidrolases (PAF-AH) são fosfolipases A2 que hidrolisam PAF e foi demonstrado que PAF-AH podem ser um fator de virulência devido a essa habilidade. O objetivo desse estudo foi avaliar o papel do PAF e de uma PAF-AH na infecção de macrófagos por L. infantum. Foi observado que PAF 1μM, quando adicionado durante e após a infecção, foi capaz de diminuir 50% da infecção após 72 horas, bem como a viabilidade dos parasitas dentro dos macrófagos num mecanismos independente do seu receptor PAFR e da produção de óxido nítrico. PAF 10μM interrompeu o crescimento de promastigotas de L. infantum em cultura axênica. Uma PAFAH, com elevada identidade e semelhança com PLA2/PAF-AH de outros tripanossomatídeos, foi identificada no genoma de L. infantum. A clonagem e expressão recombinante produziu uma proteína de cerca de 69kDa, com atividade PAF-AH. Frações celulares do parasita, enriquecidas com estruturas de membrana também apresentaram atividade PAF-AH. Os resultados indicam que PAF é capaz de diminuir a infecção de macrófagos por L. infantum e que o parasita possui uma PAF-AH funcional possivelmente envolvida com sua virulência. / Visceral leishmaniasis is caused by Leishmania infantum parasites. Infection occurs when infected sandflies feed on vertebrate host skin delivering the parasite which survive, multiply and spread on the parasitophorous vacuoles of macrophages. The inflammatory response during the infection leads to the production of diverse bioactive molecules, as lipid mediators. The platelet activating factor (PAF) is a lipid mediator derived from a lysophospholipid. PAF has a role in normal cellular physiology, acting as proinflamatory molecule. The participation of some lipid mediators, as eicosanoids and PAF has been identified in leishmaniasis. PAF produced by the host is able to kill the parasite and control the infection by L. amazonensis. PAF-acetylhydrolases (PAF-AH) are phospholipases A2 (PLA2) that hydrolyse PAF, and possibly involved in pathogen virulence. The aim of this study was to evaluate the role of PAF on macrophages infection by L. infantum and identify a PAF-AH expressed by the parasite. PAF 1μM, added during and after the infection, was able to reduce approximately 50% of infection, as well as, the viability of parasites inside macrophages. Apparently this reduction occurs by an classical PAF receptor and nitric oxide production independent mechanism. PAF 10μM inhibited L. infantum promastigotes growing in axenic culture. A PAF-AH with high identity to PLA2/PAF-AH of others trypanosomatids was identified in L. infantum genome. The cloning and recombinant expression produced a 69kDa protein with PAF-AH activity. Cellular fractions from parasites, with membrane structures also presented PAF-AH activity. The results suggest that PAF is able to decrease machophage infection by L. infantum witch has a functional PAFAH possibly related to its virulence

Although much progress has been made in understanding the events that lead to successful cell division, many details of this process remain a mystery. This dissertation presents findings which help to explain events that occur in the latest stages of cytokinesis, with an emphasis on the role of centrosome proteins. The first chapter introduces the novel centrosome protein centriolin. We show that this protein is localized specifically to the subdistal appendages of the maternal centriole in interphase, and it localizes to the midbody during cytokinesis. Disruption of this protein results in a unique cytokinesis defect in which cleavage furrow formation and ingression appear normal, but the cells remain connected by a thin intracellular bridge for extended periods of time. These results lead us to the conclusion that centriolin has an important function in cytokinesis. The second chapter describes our attempt to identify centriolin interacting partners. A yeast two hybrid screen was performed, and the results of this screen revealed an interaction between centriolin and proteins involved in vesicle target specificity and fusion. Further studies of these proteins revealed a novel localization to the midbody in cycling cells and a novel function in the final stages of cytokinesis, similar to centriolin. The third chapter discusses my attempts to clone and characterize a novel GTPase Activating Protein (GAP), which was also discovered in the screen for centriolin interacting proteins.

Dans le cadre du conditionnement des déchets nucléaires, les géopolymères représentent une alternative potentielle aux liants hydrauliques classiques. A ce jour, il est admis que la formation des géopolymères procède selon un mécanisme de dissolution - restructuration - polymérisation conduisant à un matériau solide. Cependant, les liens précis entre la composition de la solution d’activation, le déroulement des réactions de géopolymérisation et la microstructure finale doivent être déterminés du point de vue de la texturation et de l’évolution structurale du matériau. Ainsi plusieurs paramètres de formulation ont été étudiés : la nature de l’activateur alcalin, la concentration en silice et l’ajout d’une source d’aluminium. Il apparait que l’environnement chimique des précurseurs silicatés ou alumino-silicatés est contrôlé par les paramètres de formulation constitutifs de la solution d’activation. Lors de la formation du réseau géopolymérique, ces précurseurs jouent un rôle primordial sur la cinétique de dissolution du métakolin, la taille, l’arrangement et l’agrégation des oligomères alumino-silicatés participant à la formation du réseau poreux. A plus long terme, certaines caractéristiques du réseau poreux qui sont-elles même contrôlées par la taille et l’agencement des oligomères, varient légèrement au cours du temps. Le réarrangement de la structure poreuse associé d’une part à la densification du réseau solide, et d'autre part à la fermeture partielle de la porosité conduit à une légère augmentation de la taille des pores et à une diminution du volume poreux accessible. / In the context of conditioning of nuclear waste, geopolymers represent a potential alternative to conventional hydraulic binders. Currently, it is recognized that the formation of geopolymer proceeds according to a mechanism of dissolution - restructuring - polymerization resulting in a solid material. Despite the availability of many studies to understand these mechanisms, the precise relationship between the chemical nature of the activating solution, the geopolymerization process and the final microstructure should be determined from the point of view of the texturing and the structural evolution of the material. In this work, several parameters were studied: the nature of the alkali activator, the silica concentration and the addition of an aluminum source. It appears that the chemical environment of the silicate or aluminosilicate precursors is controlled by the activating solution composition. During the consolidation of the geopolymer network, these precursors play an important role on the dissolution rate of metakaolin, the size, the rearrange¬ment and the aggregation of aluminosilicate oligomers involved in the porous solid network formation. Subsequently, some characteristics of the porous network which are controlled by the size and the rearrangement of oligomers vary slightly over time. The refinement of the porosity associated to first, a densification of the solid network, and secondly, a partial closure of the porosity at the nanometer scale leads to an increase in the pore size and a decrease of the pore volume.