The interaction behavior of humic acids with uranium(VI) and the influence of humic substances on the migration behavior of uranium was investigated. A main focus of this work was the synthesis of four different humic acid model substances and their characterization and comparison to the natural humic acid from Aldrich. A radiometric method for the determination of humic acid functional groups was applied in addition to conventional methods for the determination of the functionality of humic acids. The humic acid model substances show functional and structural properties comparable to natural humic acids. Modified humic acids with blocked phenolic OH were synthesized to determine the influence of phenolic OH groups on the complexation behavior of humic acids. A synthesis method for 14C-labeled humic acids with high specific activity was developed. The complexation behavior of synthetic and natural humic acids with uranium(VI) was investigated by X-ray absorption spectroscopy, laser-induced fluorescence spectroscopy and FTIR spectroscopy. The synthetic model substances show an interaction behavior with uranium(VI) that is comparable to natural humic acids. This points to the fact that the synthetic humic acids simulate the functionality of their natural analogues very well. For the first time the influence of phenolic OH groups on the complexation behavior of humic acids was investigated by applying a modified humic acid with blocked phenolic OH groups. The formation of a uranyl hydroxy humate complex was identified by laserspectroscopic investigations of the complexation of Aldrich humic acid with uranium(VI)at pH 7. The migration behavior of uranium in a sandy aquifer system rich in humic substances was investigated in column experiments. A part of uranium migrates non-retarded through the sediment, bound to humic colloids. The uranium migration behavior is strongly influenced by the kinetically controlled interaction processes of uranium with the humic colloids. The influence of humic acids on the sorption of uranium(VI) onto phyllite was investigated in batch experiments using two different humic acids. The uranium(VI) sorption onto the rock phyllite is influenced by the pH-dependent sorption behavior of the humic acids.

The interaction behavior of humic acids with uranium(VI) and the influence of humic substances on the migration behavior of uranium was investigated. A main focus of this work was the synthesis of four different humic acid model substances and their characterization and comparison to the natural humic acid from Aldrich. A radiometric method for the determination of humic acid functional groups was applied in addition to conventional methods for the determination of the functionality of humic acids. The humic acid model substances show functional and structural properties comparable to natural humic acids. Modified humic acids with blocked phenolic OH were synthesized to determine the influence of phenolic OH groups on the complexation behavior of humic acids. A synthesis method for 14C-labeled humic acids with high specific activity was developed. The complexation behavior of synthetic and natural humic acids with uranium(VI) was investigated by X-ray absorption spectroscopy, laser-induced fluorescence spectroscopy and FTIR spectroscopy. The synthetic model substances show an interaction behavior with uranium(VI) that is comparable to natural humic acids. This points to the fact that the synthetic humic acids simulate the functionality of their natural analogues very well. For the first time the influence of phenolic OH groups on the complexation behavior of humic acids was investigated by applying a modified humic acid with blocked phenolic OH groups. The formation of a uranyl hydroxy humate complex was identified by laserspectroscopic investigations of the complexation of Aldrich humic acid with uranium(VI)at pH 7. The migration behavior of uranium in a sandy aquifer system rich in humic substances was investigated in column experiments. A part of uranium migrates non-retarded through the sediment, bound to humic colloids. The uranium migration behavior is strongly influenced by the kinetically controlled interaction processes of uranium with the humic colloids. The influence of humic acids on the sorption of uranium(VI) onto phyllite was investigated in batch experiments using two different humic acids. The uranium(VI) sorption onto the rock phyllite is influenced by the pH-dependent sorption behavior of the humic acids.

The gene encoding cholesterol 7α-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt - 149/-118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (-146-TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (-139- AGTTCAaggccGGGTAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)1, binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter- transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site (-175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Supply-demand analysis (SDA) is a tool that allows for the control, regulation and behaviour of metabolic pathways to be understood. In this framework, reactions are grouped into reaction blocks that represent the supply and demand of a metabolic product. The elasticities of these supply and demand blocks can be used to determine the degree of control either block has over the flux in the pathway and the degree of homoeostasis of the metabolic product that links the blocks. Rate characteristic plots, on which the rates of supply and demand blocks are plotted as functions of the concentration of the linking metabolite, represent a powerful visual tool in this framework. Generalised supply-demand analysis (GSDA) allows for the analysis of metabolic models of arbitrary size and complexity without prior knowledge of the regulatory structure of the pathway. This is achieved by performing SDA on each variable metabolite in a pathway instead of choosing a single linking metabolite. GSDA also provides other benefits over SDA as it allows for potential sites of regulation and regulatory metabolites to be identified. Additionally it allows for the identification and quantification of the relative contribution of di erent routes of regulation from an intermediate to a reaction block. Moiety-conserved cycles present a challenge in performing in silico SDA or GSDA, as the total concentration of a moiety must remain constant, thereby limiting the range of possible concentrations of the metabolites between which it cycles. The first goal of this thesis was to develop methods to perform GSDA on two-membered and interlinked moiety-conserved cycles. We showed that by expressing the members of a moiety-conserved cycle as a ratio, rather than individual metabolite concentrations, we can freely vary the ratio without breaking moiety conservation in a GSDA. Furthermore, we showed that by linking the concentrations of the members of two interlinked two-membered moiety-conserved cycles to a “linking metabolite”, we could vary the concentration of this metabolite, within constraints, without breaking moiety conservation. The Python Simulator for Cellular Systems (PySCeS) is a software package developed within our group that provides a variety of tools for the analysis of cellular systems. The RateChar module for PySCeS was previously developed as a tool to perform GSDA on kinetic models of metabolic pathways by automatically generating rate characteristic plots for each variable metabolite in a pathway. The plots generated by RateChar, however, were at times unclear when the models analysed were too complex. Additionally, invalid results where steady-states could not be reached were not filtered out, and therefore appeared together with valid results on the rate characteristic plots generated by RateChar. We therefore set out to improve upon RateChar by building plotting interface that produces clear and error-free rate characteristics. The resulting RCFigure class allows users to interactively change the composition of a rate characteristic plot and it includes automatic error checking. It also provides clearer rate characteristics with e ective use of colour. Using these tools two case studies were undertaken. In the first, GSDA was used to investigate the regulation of aspartate-derived amino acid synthesis in Arabidopsis thaliana. A central result was that the direct interaction of aspartate-semialdehyde (ASA), a metabolite at a branch point in the pathway, with the enzyme that produces it only accounts for 7% of the total response in the flux of supply. Instead, 89% of the observed flux response was due to ASA interacting with of the downstream enzymes for which it is a substrate. This result was unexpected as the ASA producing enzyme had a high elasticity towards ASA. In a second case study moiety-conserved cycles in a model of the pyruvate branches in lactic acid bacteria were linearised using the above mentioned method. This served to illustrate how multiple reaction blocks are connected by these conserved moieties. By performing GSDA on this model, we demonstrated that the interactions of these conserved moieties with the various reaction blocks in the pathway, led to non-monotonic behaviour of the rate characteristics of the supply and demand for the moiety ratios. An example of this is that flux would increase in response to an increase in product for certain ranges. This thesis illustrates the power of GSDA as an entry point in studying metabolic pathways, as it can potentially reveal properties of the regulation and behaviour of metabolic pathways that were not previously known, even if these pathways were subjected to previous analysis and a kinetic model is available. In general it also demonstrates how e ective analysis tools and metabolic models are vital for the study of metabolism. / AFRIKAANSE OPSOMMING: Vraag-en-aanbod analise (VAA) is ’n analisemetode wat mens in staat stel om die beheer, regulering en gedrag van metaboliese paaie beter te verstaan. In hierdie raamwerk word reaksies gegroepeer as reaksieblokke wat die aanbod (produksiestappe) en die aanvraag (verbruik-stappe) van ’n metaboliese produk verteenwoordig. Vanaf die elastisiteite van hierdie aanbod- en aanvraag-blokke kan die graad van beheer van elkeen van die blokke oor die fluksie, asook die graad van homeostase van die metaboliese koppelingsintermediaat, bereken word. Snelheidskenmerk-grafieke, waarop die snelhede van die vraag- en aanbod-blokke as funksies van die konsentrasie van die koppelingsmetaboliet uiteengesit word, verteenwoordig ’n kragtige visuele hulpmiddel in hierdie raamwerk. Veralgemeende vraag-aanbod analise (VVAA), die veralgemeende vorm van VAA, maak dit moontlikommetaboliese modelle van arbitrêre grootte en kompleksiteit te analiseer sonder enige vooraf-kennis van die regulatoriese struktuur van die paaie. Die prosedure is om VAA op elk van die veranderlike metaboliete in die pad uit te voer, eerder as om ’n enkele koppelingsmetaboliet te kies. VVAA het ook ander voordele bo VAA aangesien dit potensiële setels van regulering en regulatoriese metaboliete kan identifiseer. Daarbenewens kan dit die relatiewe bydrae van verskillende regulerings-roetes van vanaf ’n intermediaat na ’n reaksieblok identifiseer en hulle kwantifiseer. Groep-gekonserveerde siklusse bied ’n uitdaging vir in silico VAA of VVAA, aangesien die totale konsentrasie van die gekonserveerde groep konstant moet bly. Dit beperk die waardes van moontlike konsentrasies van die metaboliete wat die siklus uitmaak. Die eerste doelstelling van hierdie tesis was dus om metodes te ontwikkel waarmee VVAA op tweeledige en saamgebonde groep-gekonserveerde siklusse uitgevoer kan word. Deur die lede van groep-gekonserveerde siklusse eerder as verhoudings uit te druk in plaas van as individuele metabolietkonsentrasies, het ons gewys dat ons hierdie verhouding vrylik kan varieer sonder om die groep-konservering te breek in ’n VVAA. Ons het ook gewys dat die konsentrasies van die lede van ’n saamgebonde groep-gekonserveerde siklus gekoppel kan word aan ’n “koppelingsmetaboliet”, waarvan die konsentrasie dan binne perke gevarieer kan word sonder om die groep-konservering te breek. Die “Python Simulator for Cellular Systems” (PySCeS) is ’n programmatuur-pakket wat binne ons navorsingsgroep ontwikkel is met die doel om sellulêre sisteme numeries te analiseer. Die RateChar module vir PySCeS was reeds voor die aanvang van hierdie projek ontwikkel om VVAAop kinetiese modelle van metaboliese paaie uit te voer deur outomaties snelheidskenmerke vir elke veranderlikke metaboliet te genereer. Die grafieke wat deur RateChar gegenereer is, was egter soms onduidelik wanneer die modelle te groot of kompleks geraak het. Daarbenewens is ongeldige resultate, waar ’n bestendige toestand nie bereik kon word nie, nie uitgefiltreer nie, en het dus saam met geldige resultate op die snelheidskenmerke verskyn. Een van die doelstellings was dus om RateChar te verbeter deur ’n koppelvlak vir grafieke te ontwikkel wat duidelike en foutlose snelheidskenmerke kon produseer. Dit het gelei tot die RCFigure klas wat outomatiese foutopsporing uitvoer en gebruikers in staat stel om op ’n interaktiewe wyse die samestelling van ’n snelheidskenmerkgrafiek te verander. Dit bied ook duideliker snelheidskenmerke deur e ektief van kleur gebruik te maak. Met hierdie ontwikkelde gereedskap is twee gevallestudies onderneem. In die eerste is VVAA gebruik om die regulering van aspartaat-afgeleide aminosuursintese in Arabidopsis thaliana te bestudeer. Die belangrikste resultaat was dat die direkte interaksie van aspartaat-semialdehied (ASA), ’n metaboliet by ’n vertakkingspunt in die pad, met die ensiem wat dit produseer, slegs vir 7% van die totale respons in die aanbod-fluksie verantwoordelik was. Daarteen was 89% van die waargenome fluksierespons die gevolg van die interaksie van ASA met drie van die stroomafensieme, waarvoor dit ’n substraat is. Hierdie resultaat was onverwag aangesien die ensiem wat ASA produseer ’n hoë elastisiteit teenoor ASA toon. In ’n tweede gevallestudie is die groep-gekonserveerde siklusse in ’n model van die pirovaat-takke in melksuurbakterie-metabolisme gelineariseer deur gebruik te maak van die bo beskrewe metode. Dit illustreer hoe verskeie reaksieblokke verbind word deur hierdie gekonserveerde groepe. M.b.v. ’n VVAA van hierdie model het ons gedemonstreer dat die interaksies van die gekonserveerde groepe met die verskeie reaksieblokke in die pad kan lei tot nie-monotoniese gedrag van die snelheidskenmerke van die vraag- en aanbod-reaksies vir die verhouding van die gekonserveerde groep-komponente. ’n Voorbeeld hiervan is die onverwagte waarneming dat die fluksie toeneem met toenemende produk-konsentrasie oor sekere gebiede. Hierdie tesis illustreer die krag van VVAA as ’n beginpunt vir die studie van metaboliese paaie, aangesien dit onbekende regulatoriese eienskappe en gedragspatrone kan ontbloot, selfs al is die paaie vantevore m.b.v. kinetiese modelle geanaliseer. Oor die algemeen demonstreer dit die noodsaaklikheid van e ektiewe analisegereedskap en metaboliese modelle vir die bestudering van metabolisme. / National Research Foundation

The carbonylation of methanol to acetic acid is one of the most important applications in homogeneous catalysis. The first chapter comprises a review on the mechanistic studies into the catalytic cycle of the ‘Monsanto process’ and includes some of the most prominent studies into the use of phosphines in the rhodium-catalysed carbonylation of methanol. The second chapter of this thesis reports on an investigation into the application of rhodium complexes containing several C4 bridged diphosphines, namely BINAP, dppb, dppx and dcpb as catalysts for hydrogen tolerant methanol carbonylation. An investigation into the structure, reactivity and stability of pre-catalysts and catalyst resting states of these complexes has also been carried out. The origin of this hydrogen tolerance is explained based on the differing reactivities of the Rh acetyls with hydrogen gas, and by considering the structure of the complexes. In the third chapter I report on an investigation into how electronic properties and coordination mode affect the elimination of phosphonium salts from rhodium complexes. The stability of a range of monodentate, bidentate and tridentate rhodium-phosphine complexes was tested. I also report on the formation of a novel bidentate complex containing a partially quaternised TRIPHOS ligand and investigate the mechanism of formation using 13CH3I. Strong evidence is also presented supporting a dissociative mechanism as the means of phosphine loss from the rhodium centre. In the final chapters I report an investigation into the stability of rhodium-aminophosphine ligand complexes and into increasing the solubility of potential rhodium pre-catalysts through the use of amine-containing phosphine ligands.

<p>This thesis describes stereoselective syntheses of four amino acids, three of which are C-glycosidic analogues of glycosylated amino acids. The overall goal of the project was to probe the interactions between MHC molecules, glycopeptide antigens and T cell receptors, that are essential for development of collagen induced arthritis. Collagen induced arthritis is a frequently used mouse model for rheumatoid arthritis, an autoimmune disease that attacks joint cartilage and leads to a painful and eventually crippling condition.</p><p>The thesis is based on four studies. The first study describes the synthesis of hydroxylysine, an amino acid that is found in collagen and is an important constituent of the glycopeptide proposed as an antigen in collagen induced arthritis. During the synthesis of hydroxylysine some new insight into the mechanism of the reductive opening of <i>p</i>-methoxybenzylidene acetals was obtained.</p><p>The remaining three studies deals with the synthesis of C-glycosidic analogues of glycosylated amino acids, hydroxy norvaline, threonine and hydroxylysine.The synthesis of each amino acid required control of several stereogenic centra and utilizes a variety of approaches such as use of stereoselective reactions, chiral auxilaries, chiral templates and asymmetric catalysis.</p><p>The C-glycosidic analogues of galactosylated hydroxynorvaline and hydroxylysine were incorporated in glycopeptides from type II collagen and evaluated in T cell response assays. It was found that the T cells were stimulated by the C-glycopeptides, but that higher concentrations were required than for the native O-glycopeptide</p>

This thesis describes stereoselective syntheses of four amino acids, three of which are C-glycosidic analogues of glycosylated amino acids. The overall goal of the project was to probe the interactions between MHC molecules, glycopeptide antigens and T cell receptors, that are essential for development of collagen induced arthritis. Collagen induced arthritis is a frequently used mouse model for rheumatoid arthritis, an autoimmune disease that attacks joint cartilage and leads to a painful and eventually crippling condition. The thesis is based on four studies. The first study describes the synthesis of hydroxylysine, an amino acid that is found in collagen and is an important constituent of the glycopeptide proposed as an antigen in collagen induced arthritis. During the synthesis of hydroxylysine some new insight into the mechanism of the reductive opening of p-methoxybenzylidene acetals was obtained. The remaining three studies deals with the synthesis of C-glycosidic analogues of glycosylated amino acids, hydroxy norvaline, threonine and hydroxylysine.The synthesis of each amino acid required control of several stereogenic centra and utilizes a variety of approaches such as use of stereoselective reactions, chiral auxilaries, chiral templates and asymmetric catalysis. The C-glycosidic analogues of galactosylated hydroxynorvaline and hydroxylysine were incorporated in glycopeptides from type II collagen and evaluated in T cell response assays. It was found that the T cells were stimulated by the C-glycopeptides, but that higher concentrations were required than for the native O-glycopeptide

The disease malaria afflicts more than a billion people and kills almost one to three million of them every year. Of the four species of Plasmodium affecting man viz., P. falciparum, P. vivax, P. ovale and P. malariae, Plasmodium falciparum is the deadliest as it causes cerebral malaria. The situation has become worse with the continuous emergence of drug resistance in the parasite. Therefore, improving existing drugs and deciphering new pathways for drug development are the need of the hour. The discovery of the type II fatty acid biosynthesis pathway in Plasmodium falciparum (Surolia and Surolia, 2001) has opened up new avenues for the development of new antimalarials as this pathway is entirely different from the human host in which type I pathway exists. Although many biochemical pathways such as the purine, pyrimidine and carbohydrate metabolic pathways, and the phospholipid, folate and heme biosynthetic pathways operate in the malaria parasite and are being investigated for their amenability as antimalarial therapeutic targets, no antimalarial of commercial use based on the direct use of these biochemical pathways as targets has emerged so far. This is due to the fact that the structure and function of the targets of these drugs overlaps with that of the human host. A description of such pathways forms the Chapter 1 of the thesis. This is followed by a description of the discovery and the importance of fatty acid biosynthesis pathway and the available literature on the various enzymes that are targets of potential antimalarials. Three isoforms are known for condensing enzymes - FabH which functions in initiation, and FabB and FabF which function in elongation. These isoforms differ in their biochemical properties and have unique roles to play in deciding the membrane composition of any organism. This aspect is also discussed in this chapter. Cloning and expression of -ketoacyl-ACP synthase, FabB/F from Plasmodium falciparum is described in Chapter 2. PfFabB/F is coded by the nuclear genome and is targeted to the apicoplast. The gene is coded by the locus MAL6P1.165 and the putative amino acid sequence of the protein exists in PlasmoDB. All apicoplast targeted proteins have a characteristic bipartite leader sequence consisting of a signal and a transit peptide sequence (Waller et al., 1998). Since the mature protein start site was not known and none of the software packages could predict the site, I aligned the PfFabB/F sequence with the sequences of other -ketoacyl-ACP synthases. On the basis of similarity with E. coli synthases and the mature protein start site of plant synthases, I cloned the first construct of PfFabB/F. The sequence was amplified by PCR and ligated in pET as well as pGEX vector. Expression in various hosts under different temperature and induction conditions could not solubilize the protein in significant quantities and most of the protein was found in inclusion bodies. Next I expressed the sequence with five more amino acids towards the N-terminal and expressed it as an N- terminal NusA fusion. The protein was purified by single step Ni-NTA affinity chromatography. Along with the full length protein (108 kDa), a truncated version of the protein was also obtained. The identity of the protein was confirmed by western blotting using anti-His antibody and anti-FabB/F antibody. In Chapter 3, the substrate specificity of PfFabB/F has been elucidated. PfFabB/F condenses malonyl-ACP with a range of acyl-ACPs. In vivo, acyl carrier protein (ACP) shuttles the acyl substrates between various enzymes of the fatty acid biosynthesis pathway. Enzymes of the pathway other than synthases can accept substrate analogs like acyl-CoA and acyl-NAC’s also in vitro. Acyl-ACPs are not very stable species and thus are not commercially available. Therefore, they have to be synthesized. Since malonyl-ACP could not be synthesized by chemical means, enzymatic synthesis of acyl-ACPs was done. Acyl-ACP synthetase (Aas) or holo-ACP synthase (ACPS) can be used for enzymatic synthesis. Aas is specific only for longer chain substrates; therefore, I decided to use holo-ACP synthase, an enzyme responsible for converting apo-ACP to holo-ACP in the presence of CoA in vivo (Lambalot and Walsch, 1995). When acyl-CoAs are supplied in place of CoA, acyl-ACP is produced. Malonyl-ACP and acyl-ACPs (C4-C16:1) were thus synthesized using holo-ACP synthase from E. coli. The reaction went to almost 95% completion, indicating broad substrate specificity of this enzyme. Bacterial or plant acyl-ACPs of different chain lengths can be resolved by Conformation Sensitive PAGE (Heath and Rock, 1995, Post- Beittenmiller et al., 1991). However, Pfacyl-ACPs synthesized using ACPS did not show any significant shift on CS-PAGE. Therefore I resorted to MALDI-TOF (Matrix Assisted Laser Desorption and Ionization- Time Of Flight) for monitoring the PfFabB/F condensation reactions. PfFabB/F condensed C4-C12-ACPs with malonyl-ACP to their corresponding -ketoacyl-ACP products, with C6, C8 and C10-ACPs being most readily elongated. C14-ACP was very sluggishly elongated, and C16 and C16:1-ACPs were not elongated at all. The condensation reaction was also followed by autoradiography using14C labeled malonyl-ACP, exploiting the clear mobility shift between malonyl-ACP and the other acyl-ACPs. The inhibitory effect of cerulenin, a known inhibitor of condensing enzymes was also checked. PfFabB/F also exhibited malonyl decarboxylase activity resulting in the production of acetyl-ACP in the absence of any significant condensation activity. All the enzymes of fatty acid synthesis pathway required to complete a cycle were assembled together for the in vitro reconstitution of Plasmodium fatty acid synthesis cycle which is described in Chapter 4. Earlier studies of Surolia & Surolia have shown that C12 and C14 fatty acids are the major constituents of Plasmodium lipids. One of my objectives was to determine the maximum length of the acyl ACP product that is synthesized when all the functionally active enzymes of fatty acid synthesis are put together (Kapoor et. al, 2001, Sharma et al., 2003, Karmodiya and Surolia, 2006). Condensing enzymes have a deterministic role in the fatty acid composition as they catalyze the only irreversible step in fatty acid biosynthesis. By analyzing products of the elongation cycle by mass spectrometry it was apparent that C14-ACP is the longest species formed. As already mentioned, PfFabB/F readily elongates C12-ACP but C14-ACP is weakly elongated. Thus the end product of the Plasmodium FAB pathway is influenced by the substrate specificity of PfFabB/F. This confirms the role of PfFabB/F as a decisive enzyme in determining the length of fatty acids synthesized. The inhibition of the cycle by cerulenin and triclosan is also described in this chapter. Chapter 5 describes the ability of the PffabB/F gene to complement for the mutation of condensing enzymes in CY244 cells (fabBtsfabF-, Yasuno et al., 2004). CY244 cells were transformed with pBAD alone or PfFabB/F cloned in pBAD vector (pBADPffabB/F) and the growth was monitored at non-permissive temperature. The product of PfFabB/F could rescue the growth of mutant cells at high temperature but only in the presence of oleic acid. FabB and FabF are the isoforms of condensing enzymes involved in elongation of the fatty acid synthesis cycle but they have a unique role to play (Garwin et al., 1980). FabB is responsible for unsaturated fatty acid synthesis, and fabB-mutants require oleic acid supplementation for growth. FabF is utilized in temperature regulation of membrane fluidity and E. coli FabF elevates the level of C18:1 or cis-vaccenic acid at lower growth temperature but FabF-mutants have no growth phenotype (Ulrich et al., 1983). Rescue of CY244 cells in the presence of oleic acid supplementation indicated that the PffabB/F gene behaves like FabF and not FabB. Analysis of the fatty acid composition of membrane lipids of CY244 cells transformed with pBAD vector or pBADPffabB/F by GC-MS demonstrated no elevated levels of cis-vaccenic acid in transformed cells. This observation is in agreement with the in vitro determined substrate specificity data which shows that PfFabB/F does not elongate C16:1ACP. The thesis ends with a summary of the findings in Chapter 6 in the context of FabB and FabF enzymes known from other sources. 2, 4, 4’-Trichloro-2’hydroxydiphenylether, commonly known as triclosan, has been used as a topical antibacterial agent for decades. I determined its efficacy in treating acute systemic bacterial infection in mouse model. Triclosan, as compared to other well known antibiotics, could extend the survival time of mice by 48 hours. This work is described in Appendix I. (Sharma et al., 2003)

The thesis deals with X-ray crystallographic analysis of two enzymes involved in the fatty acid biosynthesis pathway, known as Fatty Acid Synthase or FAS, of the malarial parasite, Plasmodium falciparum, in order to understand their functions at the atomic level and to provide structural basis for the rational design of antimalarial compounds. Targeting highly specific and well-characterized biochemical pathways to develop effective therapeutic agents has the advantage of designing new drugs or modifying the existing ones based on the details of the known features of the processes. Knowledge of the three-dimensional structures of the molecules involved in the reactions will enhance the capabilities of this procedure. The recently identified fatty acid biosynthesis pathway in Plasmodium falciparum is undoubtedly an attractive target for drug development as it differs from that in humans. In the malarial parasite, each reaction of the pathway is catalyzed by a specific enzyme whereas in humans, the synthesis is carried out by a single multidomain enzyme. Essentially each step in the FAS of P. falciparum can be a potential target to prevent the growth of the parasite as the fatty acids are essential for the formation of the cell membrane which is vital for its survival. All the reactions of this pathway have been well-characterized. Nevertheless, there is a dearth of structural information of the proteins involved in performing various functions in this pathway. When this work was initiated, crystal structures of none of these proteins were reported. The current work on the plasmodial FAS proteins has been undertaken with a view to obtain precise structural details of their reaction and inhibition mechanisms. The introductory chapter of the thesis includes a discussion on malaria, the fatty acid biosynthesis in various organisms and an overview of the structural features of the enzymes involved in the pathway that have been characterized from other organisms.The second chapter includes the tools of X-ray crystallography that were used for structural studies of the present work. It also discusses the other computational and biophysical methods used to further characterize the enzymes under study. FabI, the enoyl acyl carrier protein reductase, that regulates the third step in FAS has been crystallized as a binary complex with its cofactor NADH and as a ternary complex with NAD+and triclosan. The crystal structures of the binary and the ternary complexes have been determined at 2.5 and 2.2 ˚A, respectively. The mode of binding of the cofactor and the inhibitor triclosan to the enzyme with details of the interactions between them could be clearly obtained from these structures. Each subunit of the tetrameric FabI has the classical Rossmann fold. We carried out a thorough analysis of this structure and compared it with the FabI structures from various sources, four microbial (Escherichia coli, Mycobacterium tuberculosis and Helicobacter pylori) and one plant (Brassica napus), and arrived at a number of significant conclusions: Though the tertiary and the quaternary structures of the enzymes from different sources are similar, the substrate binding loop shows significant changes. The position and nature of the loop are strongly correlated to the affinity of triclosan to the enzyme. Small to major changes in the structure of the enzyme occur to enhance ligand binding. Water molecules play an important role in enzyme-ligand interactions. The crystal structure has also confirmed our previous prediction based on modeling studies of the enzyme that the introduction of bulkier groups at carbon 4’ of triclosan is likely to improve its efficacy as an inhibitor of FabI of P. falciparum. It has also provided the structural basis for its preference to bind to the coenzyme NADH but not to NADPH which was also predicted by our modeling studies. Chapters 3 and 4 discuss the structure solution and a comparative analysis of the crystal structures of FabIs from various sources. The crystal structure of FabZ, the β-hydroxyacyl acyl carrier protein dehydratase of P. falciparum, has been determined at a resolution of 2.4 ˚A. Each subunit of FabZ has a hotdog fold with one long central α-helix surrounded by a six-stranded antiparallel β-sheet. FabZ has been found to exist as a homodimer in the crystals of the present study in contrast to the hexameric form which is a trimer of dimers crystallized in a different condition, reported while we completed the structure of the dimeric form. In the dimeric form, the architecture of the catalytic site has been drastically altered with two catalytic histidine residues moving away from the catalytic site caused by two cis to trans peptide flips compared to the hexameric form. These alterations not only prevent the formation of a hexamer but also distort the active site geometry resulting in a dimeric form of FabZ that is incapable of substrate-binding. The dimeric state and an altered catalytic site architecture make the dimeric FabZ presented in the thesis distinctly different from the FabZ structures described so far. This is the first observation and report of the existence of an inactive form of the enzyme and its unique structural features. Further analysis using dynamic light scattering and size exclusion chromatographic studies have shown that a pH-related conformational switching occurs between the inactive dimers and active hexamers of FabZ in P. falciparum. These findings open alternate and entirely new strategies to design inhibitors to make FabZ inactive. FabZ crystals show polymorphism with varying length of its longest cell axis. We could collect X-ray diffraction data for three of these forms. An analysis of these forms revealed that three flexible loops of the structure including those containing the peptide flips compete for the space between two symmetry-related molecules. In the form with the longest cell axis, the loops have the highest stability resulting in a better diffraction from the crystal. We also performed docking studies with two previously characterized inhibitors of FabZ. The docking showed that the inhibitors bind strongly at the active site each one making a number of different interactions with the catalytic residues. Observations from our docking studies are in excellent agreement with and strongly supported by chemical modification and fluorimetric analysis of the wild type enzyme and its mutants. Chapters 5 and 6 explain in detail about the structure solution of dimeric form of PfFabZ, the pH induced conformational flipping of two cis-trans peptide flips that lead to different oligomeric states, and the structural basis for these oligomeric shifts. The mechanism of action of PfFabZ inhibitors NAS-21 and NAS-91 are also discussed in detail. Intrigued by the hot dog fold of the Fab enzyme, we have analyzed and compared proteins having this fold in their structures. It has been observed that the fold is often associated with fatty acids. However, the sequences, the quaternary structures, substrate specificities and the reactions that the proteins catalyze are quite diverse. The consensus sequence motifs lining the interface of quaternary association and at active site clearly indicated that the information for different modes of quaternary associations is embedded in the sequences itself. The diversity in function and quaternary association of hot dog fold proteins and their structure-function relationships are discussed in chapter 7. Malaria affects hundreds of millions of people worldwide causing about two million deaths every year. In spite of the commendable success of the available antimalarials, it has not been possible to contain the disease completely as the protozoan has become resistant to a majority of frontline drugs. The structural studies presented here should enhance the current biochemical knowledge to develop selective and more potent inhibitors of the pathway and contribute to the ongoing efforts to fight the disease.

Made available in DSpace on 2014-12-17T14:07:18Z (GMT). No. of bitstreams: 1 AnaCMC_DISSERT.pdf: 3568281 bytes, checksum: 78b0d72ff4bc1b9774bb0c806de32258 (MD5) Previous issue date: 2013-11-11 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / With the advances in medicine, life expectancy of the world population has grown considerably in recent decades. Studies have been performed in order to maintain the quality of life through the development of new drugs and new surgical procedures. Biomaterials is an example of the researches to improve quality of life, and its use goes from the reconstruction of tissues and organs affected by diseases or other types of failure, to use in drug delivery system able to prolong the drug in the body and increase its bioavailability. Biopolymers are a class of biomaterials widely targeted by researchers since they have ideal properties for biomedical applications, such as high biocompatibility and biodegradability. Poly (lactic acid) (PLA) is a biopolymer used as a biomaterial and its monomer, lactic acid, is eliminated by the Krebs Cycle (citric acid cycle). It is possible to synthesize PLA through various synthesis routes, however, the direct polycondensation is cheaper due the use of few steps of polymerization. In this work we used experimental design (DOE) to produce PLAs with different molecular weight from the direct polycondensation of lactic acid, with characteristics suitable for use in drug delivery system (DDS). Through the experimental design it was noted that the time of esterification, in the direct polycondensation, is the most important stage to obtain a higher molecular weight. The Fourier Transform Infrared (FTIR) spectrograms obtained were equivalent to the PLAs available in the literature. Results of Differential Scanning Calorimetry (DSC) showed that all PLAs produced are semicrystalline with glass transition temperatures (Tgs) ranging between 36 - 48 ?C, and melting temperatures (Tm) ranging from 117 to 130 ?C. The PLAs molecular weight characterized from Size Exclusion Chromatography (SEC), varied from 1000 to 11,000 g/mol. PLAs obtained showed a fibrous morphology characterized by Scanning Electron Microscopy (SEM) / Com os avan?os na medicina, a expectativa de vida da popula??o mundial vem crescendo consideravelmente nas ?ltimas d?cadas. Pesquisas v?m sendo realizadas visando manter a qualidade de vida da popula??o atrav?s do desenvolvimento de novas drogas e novos procedimentos cir?rgicos. Os biomateriais s?o estudados atualmente, e sua utiliza??o vai desde a reconstru??o de tecidos e ?rg?os afetados por patologias ou outros tipos de falha, ? utiliza??o em sistema de libera??o de f?rmacos capazes de prolongar o tempo do medicamento no organismo e aumentar a sua biodisponibilidade. Os biopol?meros consistem em uma classe de biomateriais muito visada pelos pesquisadores, uma vez que apresentam propriedades ideais para tal aplica??o, como alta biocompatibilidade e biodegradabilidade. O Poli(?cido l?ctico) (PLA) ? um biopol?mero usado como biomaterial, e seu mon?mero, o ?cido l?ctico, ? eliminado pelo ciclo do ?cido c?trico (ciclo de Krebs). ? poss?vel sintetizar o PLA atrav?s de v?rias rotas de s?ntese, no entanto, a policondensa??o direta ? a rota mais barata devido ao uso de poucas etapas de polimeriza??o. Neste trabalho foi utilizada a ferramenta de planejamento fatorial para produzir PLAs com diferentes massas molares, a partir da policondensa??o direta do ?cido l?ctico, com caracter?sticas adequadas para a utiliza??o em sistema de libera??o de f?rmacos (SLFs). Por meio do planejamento observou-se que o tempo de esterifica??o ? o est?gio mais importante na obten??o de uma maior massa molar na rota de policondensa??o. Os Infravermelhos por Transformada de Fourier (FTIR) obtidos apresentaram espectrogramas equivalentes ao de PLAs dispon?veis na literatura. Resultados de Calorimetria Explorat?ria Diferencial (DSC) mostraram que todos os PLAs produzidos s?o semicristalinos, com temperaturas de transi??o v?treas (Tgs) variando entre 36 48?C e temperaturas de fus?o (Tm) variando entre 117 130?C. As massas molares, caracterizadas a partir de Cromatografia por Exclus?o de Tamanho (SEC), variaram entre 1000 11000 g/mol. Os PLAs obtidos apresentaram uma morfologia fibrosa caracterizada por meio de Microscopia Eletr?nica de Varredura (MEV)