The Function of the Halophilic Dodecin

Flavins are physiologically relevant cofactors that catalyze various redox and light-induced reactions. Due to a high intrinsic reactivity, these compounds are found tightly bound to proteins with the chemistry of the flavin either narrowed to a defined reaction channel (flavoenzymes) or reduced to (almost) non-reactivity (flavin binding and carrier proteins). Lumichrome is a product of flavin photodegradation. In spite of the structural similarity to flavins, lumichrome has electronic properties which differ from flavins, preventing this compound from any physiological relevance as a cofactor. The interest in lumichrome is basically focussed on its role as a photosensitizing compound. Lumichrome is excited by the absorption of visible light and relaxes by transferring electrons or electronic energy to surrounding substrates and oxygen, exerting an unspecific toxic effect on the cellular environment. Dodecin is a dodecameric flavin binding protein comprising a novel ligand binding fold. It incorporates dimers of ligands arranged in antiparallel manner within each of the six identical binding pockets. In this thesis, structure and function of dodecin from the archaeal organism Halobacterium salinarum are reported. X-ray structural investigations supplemented with functional data revealed that this protein is an unspecific binder of flavins and binder of the flavin-like compound lumichrome. Dissociation constants were obtained in the nanomolar to micromolar range and found to correlate positively with the ligand size. The preference of dodecin for the small ligands lumichrome and lumiflavin is described as a gated ligand binding mode, based on the low plasticity of the dodecin binding pocket which sterically restricts the bulkier ligands from arranging the flavin aromatic subunit in a high affinity position. Site directed mutagenesis of the halophilic dodecin allowed to spread the idea of dodecin as a small ligand binding particle among homologous proteins. These mutational studies could moreover show that the halophilic type of dodecins is outstanding in additionally exhibiting a high affinity for riboflavin. The stabilization of the ribityl chain by an H-bond network to a single residue was found to suspend restrictions of the gated ligand binding mode and to enable H. salinarum dodecin to exhibit multiple (high) affinity. In Western-Blot and RT-PCR analysis of the dodecin expression level, it could be demonstrated that after a short lag period dodecin is constitutively expressed in light and in dark. In the late stationary phase, a clear influence of dodecin on the riboflavin cellular concentrations could be observed. While high levels of riboflavin were found in H. salinarum wild type cells, in cells of the dodecin deficient strain riboflavin cellular concentrations were depressed. Lumichrome concentrations on the other hand were unaffected from dodecin; however; increased concentrations of lumichrome were found in light, according to a photolytic degradation of riboflavin. In vivo data fully agreed with the deductions from the dodecin structural and functional investigations. Dodecin is a riboflavin binding and carrier protein (RfBP). Its function is to store riboflavin under non-favorable environmental conditions while preventing this flavin from photodegradation. The lumichrome-collecting property represents an extra-feature which allows binding of lumichrome if degradation of riboflavin occurs in order to protect the cellular environment from high amounts of this photo-toxic compound

Topoisomerase-II mediated biochemical mapping of centromeres and nitroreductase mediated drug metabolism in Trypanosomatids

PhDThe protozoan parasites Trypanosoma cruzi and Trypanosoma brucei are the causative agents of Chagas disease and Human African sleeping sickness respectively. Existing therapies are toxic and ineffective against the later stages of the two diseases, consequently safer, improved therapies are urgently required. Here, two areas of trypanosome biology are explored. In the first section, the process of cell division is approached from a fundamental biology perspective. Centromeres are the region of DNA where kinetochore structures form, allowing the attachment of microtubules to facilitate chromosome segregation. In T. brucei we have characterized the nature and location of centromeres by exploiting the localized activity of topoisomerase-II, a cancer chemotherapy target, at the centromere. Etoposide mediated DNA cleavage mapping revealed the presence of signature AT-rich repeat regions coupled with adjacent retrotransposons at the centromere. Further experiments demonstrate that of the two nuclear T. brucei topoisomerase-II isoforms, only topoisomerase-IIα is essential and active at the centromere. The second section centres on pro-drug development against a trypanosome type I nitroreductase. This enzyme has previously been implicated in activation of nifurtimox and benznidazole, the two therapies in clinical use against Chagas disease. Initially we have developed a luciferase based drug assay system in the clinically relevant intracellular T. cruzi stage and rapidly screened a range of nitroaromatic based compounds for trypanocidal activity. A series of derived nitrofuryl compounds previously developed against Chagas disease were also screened against T. brucei, where most demonstrate trypanocidal activities of less than 1 μM. Further we show that these compounds are active substrates of nitroreductase, and act as pro-drugs within the parasite by specific activation of nitroreductase to generate cytotoxic moieties

The genetics of osteoporosis

This thesis presents epidemiological and gene mapping studies of the genetics of osteoporosis and low bone mineral density (BMD). BMD is a highly heritable trait. However the genes that underlie the population variance in BMD remain unknown. Genetic epidemiological studies of families collected for gene mapping investigations demonstrated significantly low BMD in siblings and relatives of probands with osteoporosis. A sibling recurrence risk ratio (Xs) for low BMD was established, with Xs of 6.26 at lumbar spine (LS) and 5.24 at femoral neck (FN), and heritability of BMD estimated at 60% for LS and 48% for FN. There was also evidence of both site- and gender-specific genetic effects. A large candidate gene linkage study demonstrated linkage of BMD with several loci, notably Parathyroid Hormone Receptor Type 1 (PTHRl), type 1 Collagen alpha-1 (COLlAl), type 2 Collagen alpha-1/Vitamin D Receptor, Interleukins 1,4 and 6, Epidermal Growth Factor, RANKL and Estrogen Receptor-alpha. Mutation screening of PTHRl exons and promoter regions revealed both previously described and new polymorphisms. Association of BMD at LS with a polymorphism present in exon M7 of PTHRl was demonstrated in both population-based and within family association studies. A within-family association study of a polymorphism in the first intron of COL1A1 found no evidence of association with BMD. However, when only maternal transmissions were considered, there was evidence of association with BMD at FN, suggesting the possibility of imprinting of this gene. Further genetic studies of PTHR1 and other genes identified as contributing to the population variance of BMD will help clarify their roles in the determination of BMD and the development of osteoporosis

The structural analysis of imidazole-functionalised metallocycles

Thesis (PhD)--Stellenbosch University, 2012.ENGLISH ABSTRACT: The primary objective of this study was to synthesise novel metallocyclic compounds and analyse their crystal structures. To this end seven novel imidazole-functionalised ligands were synthesised and reacted with a variety of transition-metal halide salts. In addition to this study a new, simple, yet robust methodology for the analysis of π···π packing motifs in aromatic molecules is described. Seventeen homeotypic metallocyclic compounds were obtained with a ligand containing a dimethoxyphenyl spacer between imidazole functionalities. These compounds form reliably from a number of solvent systems involving acetonitrile and they all include acetonitrile molecules as part of their host assembly. In each case a second guest molecule is enclosed within the walls of the metallocycles. These compounds are compared by means of thermal analysis, calculated powder X-ray diffraction patterns as well as crystal packing similarity calculations. Metallocycles prepared from a phenanthrene-based ligand form an unprecedented infinite π···π stack, which induces the assembly of an infinite catenane. This catenane forms concomitantly with its topological isomer, which consists of unlinked metallocycles. The intermolecular interactions responsible for the two topologically unique structures were investigated. Finally, a further twelve metallocycles were prepared from four novel imidazolefunctionalised ligands and their structures were analysed for any similarities and/or differences. A few of these crystals showed the release of solvent guest molecules as singlecrystal to single-crystal transformations.AFRIKAANSE OPSOMMING: Die primêre doel van hierdie studie was om nuwe metallosiklieseverbindings te sintetiseer en hul kristalstrukture te ontleed. Vir hierdie doel is sewe nuwe imidasool-gefunksionaliseerde ligande gesintetiseer en gereageer met 'n verskeidenheid van oorgangsmetaalhaliedsoute. In hierdie studie word 'n nuwe, eenvoudige, maar robuuste metode vir die ontleding van π···π verpakkingmotiewe in aromatise molekules beskryf. Sewentien homeotipiese metallosiklieseverbindings is verkry met 'n ligand met 'n dimetoksifeniel spasieerder tussen imidasool funksionaliteite. Hierdie verbindings vorm op betroubare wyse vanuit 'n aantal oplosmiddelsisteme wat asetonitriel behels en almal sluit asetonitriel molekules as deel van hul gasheer samestelling in. In elke geval is 'n tweede gasmolekule ingesluit binne die mure van die metallosiklieseverbindings. Hierdie verbindings is deur middel van termiese analise, berekende poeier X-straaldiffraksie patrone, sowel as kristalverpakking gelykvormigheidsberekeninge vergelyk. Metallosiklieseverbindings wat voorberei is vanaf 'n phenanthrene-gebaseerde ligand, vorm 'n uitsonderlike oneindige π···π verpakking, wat die samestelling van 'n oneindige katenaan tot gevolg het. Hierdie katenaan vorm saam met sy topologiese isomeer, wat bestaan uit ongekoppelde metallosiklieseverbindings. Die intermolekulêre interaksies wat verantwoordelik is vir die twee topologies unieke strukture word ondersoek. Ten slotte, was 'n verdere twaalf metallosiklieseverbindings uit vier voorheen onbekende imidasool-gefunksionaliseerde ligande voorberei en hul strukture is vir enige ooreenkomste en/of verskille ontleed. 'n Paar van hierdie kristalle ondergaan die vrystelling van die oplosmiddelmolekules as 'n enkel-kristal na enkel-kristal omsetting.Doctora

In the pursuit of the fear engram: identification of neuronal circuits underlying the treatment of anxiety disorder

Fear and other anxiety disorders are extraordinarily robust and difficult to treat. Among the most effective treatments for anxiety disorders are exposure-based therapies, during which a patient is repeatedly confronted with the originally fear-eliciting stimulus in a safe environment so that the once fearful stimulus can be newly interpreted as neutral or safe. A fundamental element for successful exposure-based therapies is the reactivation/recall of the traumatic memory, which initiates a time-limited process called memory reconsolidation, during which a memory becomes susceptible to disruption. Presently, the neuronal subpopulations and molecular mechanisms underlying successful fear memory attenuation remain completely unknown, which represents a big gap in memory research. Therefore, the aim of this work is to first identify the neuronal subpopulations that are causally implicated in effective attenuation of remote fear memories. This will help to determine whether the original traumatic memory trace has been permanently modified or a new memory trace of safety has been superimposed over the original one. The second aim is to develop a tool that allows for the isolation of the neuronal subpopulations causally implicated in remote memory attenuation, in order to be able to delineate the epigenetic and transcriptional mechanisms at play within these subpopulations. This will help to identify a molecular signature of effective remote fear memory attenuation. The results of my research suggest for the first time that there is a small population of neurons in the dentate gyrus - that was active during the recall of fear â that needs to be reactivated during extinction to attain successful remote fear attenuation. While the inactivation of such population during extinction impairs fear attenuation, its activation ameliorates behavioral extinction. Furthermore, I have successfully established a method to isolate this neuronal subpopulation from the brain, namely by fluorescence-activated cell sorting. This tool will allow follow up studies to pursue the quest for the molecular signature of successful remote memory attenuation. Overall, these findings could help us to better understand the intricate principles of effective remote fear memory attenuation, and thus to develop new strategies that improve the treatment of anxiety disorder