Stereochemical and steric control of enzymatic glucuronidation : A rational approach for the design of novel Inhibitors for the human UDP-glucuronosyltransferase 2B7

The UDP-glucuronosyltransferases (UGTs) are enzymes of the phase II metabolic system. These enzymes catalyze the transfer of α-D-glucuronic acid from UDP-glucuronic acid to aglycones bearing nucleophilic groups affording exclusively their corresponding β-D-glucuronides to render lipophilic endobiotics and xenobiotics more water soluble. This detoxification pathway aids in the urinary and biliary excretion of lipophilic compounds thus preventing their accumulation to harmful levels. The aim of this study was to investigate the effect of stereochemical and steric features of substrates on the glucuronidation catalyzed by UGTs 2B7 and 2B17. Furthermore, this study relates to the design and synthesis of novel, selective inhibitors that display high affinity for the key enzyme involved in drug glucuronidation, UGT2B7. The starting point for the development of inhibitors was to assess the influence of the stereochemistry of substrates on the UGT-catalyzed glucuronidation reaction. A set of 28 enantiomerically pure alcohols was subjected to glucuronidation assays employing the human UGT isoforms 2B7 and 2B17. Both UGT enzymes displayed high stereoselectivity, favoring the glucuronidation of the (R)-enantiomers over their respective mirror-image compounds. The spatial arrangement of the hydroxy group of the substrate determined the rate of the UGT-catalyzed reaction. However, the affinity of the enantiomeric substrates to the enzymes was not significantly influenced by the spatial orientation of the nucleophilic hydroxy group. Based on these results, a rational approach for the design of inhibitors was developed by addressing the stereochemical features of substrate molecules. Further studies showed that the rate of the enzymatic glucuronidation of substrates was also highly dependent on the steric demand in vicinity of the nucleophilic hydroxy group. These findings provided a rational approach to turn high-affinity substrates into true UGT inhibitors by addressing stereochemical and steric features of substrate molecules. The tricyclic sesquiterpenols longifolol and isolongifolol were identified as high-affinity substrates which displayed high selectivity for the UGT isoform 2B7. These compounds served therefore as lead structures for the design of potent and selective inhibitors for UGT2B7. Selective and potent inhibitors were prepared by synthetically modifying the lead compounds longifolol and isolongifolol taking stereochemical and steric features into account. The best inhibitor of UGT2B7, β-phenyllongifolol, displayed an inhibition constant of 0.91 nM.

Innate immunity and its control in the pathogenesis of inflammatory rheumatic diseases

The pathogenesis of inflammatory rheumatic diseases, including rheumatoid arthritis (RA) and spondyloarthropathies (SpAs) such as reactive arthritis (ReA), is incompletely understood. ReA is a sterile joint inflammation, which may follow a distal infection caused by Gram-negative bacteria that have lipopolysaccharide (LPS) in their outer membrane. The functions of innate immunity that may affect the pathogenesis, prognosis and treatment of these diseases were studied in this thesis. When compared with healthy controls, whole blood monocytes of healthy subjects with previous ReA showed enhanced capacity to produce TNF, an essential proinflammatory cytokine, in response to adherent conditions (mimicking vascular endothelium made adherent by inflammatory signals) and non-specific protein kinase C stimulation. Also, blood neutrophils of these subjects showed high levels of CD11b, an important adhesion molecule, in response to adherence or LPS. Thus, high responsiveness of monocytes and neutrophils when encountering inflammatory stimuli may play a role in the pathogenesis of ReA. The results also suggested that the known risk allele for SpAs, HLA-B27, may be an additive contributor to the observed differences. The promoter polymorphisms TNF 308A and CD14 (gene for an LPS receptor component) 159T were found not to increase the risk of acute arthritis. However, all female patients who developed chronic SpA had 159T and none of them had 308A, possibly reflecting an interplay between hormonal and inflammatory signals in the development of chronic SpA. Among subjects with early RA, those having the polymorphic TLR4 +896G allele (causing the Asp299Gly change in TLR4, another component of LPS receptor) required a combination of disease-modifying antirheumatic drugs to achieve remission. It is known that rapid treatment response is essential in order to maintain the patients work ability. Hence, +896G might be a candidate marker for identifying the patients who need combination treatment. The production of vascular endothelial growth factor (VEGF), which strongly promotes vascular permeability and angiogenesis that takes place e.g. early in rheumatic joints, was induced by LPS and inhibited by interferon (IFN)-alpha in peripheral blood mononuclear cells. These long-living cells might provide a source of VEGF when stimulated by LPS and migrating to inflamed joints, and the effect of IFN-alpha may contribute to the clinical efficacy of this cytokine in inhibiting joint inflammation.

Participation or further exclusion? : Contestations over forest conservation and control in the East Usambara Mountains, Tanzania

This Ph.D. thesis Participation or Further Exclusion? Contestations over Forest Conservation and Control in the East Usambara Mountains, Tanzania describes and analyses the shift in the prevailing discourse of forest and biodiversity conservation policies and strategies towards more participatory approaches in Tanzania, and the changes in the practises of resource control. I explore the scope for and limits to the different actors and groups who are considered to form the community, to participate in resource control, in a specific historical and socio-economic context. I analyse whether, how and to which extent the targets of such participatory conservation interventions have been able to affect the formal rules and practices of resource control, and explore their different responses and discursive and other strategies in relation to conservation efforts. I approach the problematic through exploring certain participatory conservation interventions and related negotiations between the local farmers, government officials and the external actors in the case of two protected forest reserves in the southern part of the East Usambaras, Tanzania. The study area belongs to the Eastern Arc Mountains that are valued globally and nationally for their high level of biodiversity and number of endemic and near endemic species. The theoretical approach draws from theorising on power, participation and conservation in anthropology of development and post-structuralist political ecology. The material was collected in three stages between 2003 and 2008 by using an ethnographic approach. I interviewed and observed the actors and their resource use and control practices at the local level, including the representatives of the villagers living close to the protected forests and the conservation agency, but also followed the selected processes and engaged with the non-local agencies involved in the conservation efforts in the East Usambaras. In addition, the more recent processes of change and the actors strategies in resource control were contextualised against the social and environmental history of the study area and the evolvement of institutions of natural resource control. My findings indicate that the discourse of participation that has emerged in global conservation policy debate within the past three decades, and is being institutionalised in the national policies in many countries, including Tanzania, has shaped the practices of forest conservation in the East Usambaras, although in a fragmented and uneven way. Instrumental interpretation of participation, in which it is to serve the goals of improving the control of the forest and making it more acceptable and efficient, has prevailed among the governmental actors and conservation organisations. Yet, there is variation between the different projects and actors promoting participatory conservation regarding the goals and means of participation, e.g. to which extent the local people are to be involved in decision-making. The actors representing communities also have their diverse agendas, understandings and experiences regarding the rationality, outcomes and benefits of being involved in forest control, making the practices of control fluid. The elements of the exclusive conservation thinking and practices co-exist with the more recent participatory processes, and continue to shape the understandings and strategies of the actors involved in resource control. The ideas and narratives of the different discourses are reproduced and selectively used by the parties involved. The idea of forest conservation is not resisted as such by most of the actors at local level, quite the opposite. However, the strict regulations and rules governing access to resources, such as valuable timber species, continue to be disputed by many. Furthermore, the history of control, such as past injustices related to conservation and unfulfilled promises, undermines the participation of certain social groups in resource control and benefit sharing. This also creates controversies in the practices of conservation, and fuels conflicts regarding the establishment of new protected areas. In spite of this, the fact that the representatives of the communities have been invited to the arenas where information is shared, and principles and conditions of forest control and benefit sharing are discussed and partly decided upon, has created expectations among the participants, and opened up opportunities for some of the local actors to enhance their own, and sometimes wider interests in relation to resource control and the related benefits. The local actors experiences of the previous government and other interventions strongly affect how they position themselves in relation to conservation interventions, and their responses and strategies. However, my findings also suggest, in a similar way to research conducted in some other protected areas, that the benefits of participation in conservation and resource control tend to accrue unevenly between different groups of local people, e.g. due to unequal access to information and differences in their initial resources and social position.