A Bayesian system for modeling promoter structure: A case study of histone promoters

Ph.DDOCTOR OF PHILOSOPH

Characterisation of the plasmodium falciparum Hsp40 chaperones and their partnerships with Hsp70

Central to this research, 40 kDa Heat shock proteins (Hsp40s) are known to partner (or cochaperone) 70 kDa Heat shock proteins (Hsp70s), facilitating the selection and transfer of protein substrate to Hsp70 and the stimulation of the protein folding ability of Hsp70. Members of the diverse Hsp70-Hsp40 protein complement of Plasmodium falciparum have been implicated in the cytoprotection of this malaria parasite, and are thought to facilitate the protein folding, assembly and translocation tasks required by the parasite to commandeer the infected human erythrocyte subsequent to invasion. In particular, the parasite has evolved an expanded and specialised 43- member suite of Hsp40 proteins, 19 of which bear an identifiable export motif for secretion into the infected erythrocyte cytoplasm where they potentially interact with human Hsp70. Although type I Hsp40 proteins are representative of typical regulators of Hsp70 activity, only two of these proteins are apparent in the parasite’s Hsp40 complement. These include a characteristic type I Hsp40 termed PfHsp40, and a larger, atypical type I Hsp40 termed Pfj1. Both Hsp40 proteins are predicted to be parasite-resident and are most likely to facilitate the co-chaperone regulation of the highly abundant and stress-inducible Hsp70 homolog, PfHsp70-I. In this work, the co-chaperone functionality of PfHsp40 and Pfj1 was elucidated using in vivo and in vitro assays. Purified recombinant PfHsp40 was shown to stimulate the ATPase activity of PfHsp70-I in in vitro single turnover and steady state ATPase assays, and co-operate with PfHsp70-I in in vitro aggregation suppression assays. In these in vitro assays, heterologous partnerships could be demonstrated between PfHsp70-I and the human Hsp40, Hsj1a, and human Hsp70 and PfHsp40, suggesting a common mode of Hsp70-Hsp40 interaction in the parasite and host organism. The functionality of the signature Hsp40 domain, the Jdomain, of Pfj1 was demonstrated by its ability to replace the equivalent domain of the A. tumefaciens Hsp40, Agt DnaJ, in interactions with the prokaryotic Hsp70, DnaK, in the thermosensitive dnaJ cbpA E. coli OD259 deletion strain. An H33Q mutation introduced into the invariant and crucial HPD tripeptide motif abrogated the functionality of the J-domain in the in vivo complementation system. These findings provide the first evidence for the conservation of the prototypical mode of J-domain based interaction of Hsp40 with Hsp70 in P. falciparum. Immunofluorescence staining revealed the localisation of PfHsp40 to the parasite cytoplasm, and Pfj1 to the parasite cytoplasm and nucleus in cultured intraerythrocytic stage P. falciparum parasites. PfHsp70-I was also shown to localise to the parasite cytoplasm and nucleus in these stages, consistent with the literature. Overall we propose that PfHsp40 and Pfj1 co-localise with and regulate the chaperone activity of PfHsp70-I in P. falciparum. This is the first study to identify and provide evidence for a functional Hsp70-Hsp40 partnership in P. falciparum, and provides a platform for future studies to elucidate the importance of these chaperone partnerships in the establishment and survival of the parasite in the intraerythrocytic-stages of development

Molecular biology and immunology of the 60-kDa stress protein (65-kDa antigen) of Mycobacterium paratuberculosis

M. paratuberculosis infection in sheep causes a chronic granulomatous enteritis, characterised by a massive cellular infiltration of macrophages, epithelioid cells and lymphocytes. Like other mycobacterial infections the predominant immune response is cell-mediated and this response centres on a complex interaction between T cells and macrophages infected with M. paratuberculosis organisms. T cells have been identified as the predominant inducers of the cell mediated immune response to mycobacteria. Stress proteins have been shown to be immunodominant antigens in immune responses to other pathogenic mycobacteria. T cells reactive to stress proteins have been isolated from infected animals. For these reasons, the 60-kDa stress protein of M. paratuberculosis was chosen for the investigation of the immune response to M. paratuberculosis infection in sheep.In this thesis the DNA encoding the 60-kDa stress protein of M. paratuberculosis was amplified by PCR, sequenced and the ORF of this protein expressed as a fusion protein with glutathione S-transferase. Recombinant M. paratuberculosis 60-kDa stress protein was obtained by cleavage of the fusion protein with the proteolytic enzyme thrombin.Recombinant M. paratuberculosis 60-kDa stress protein was used to generate polyclonal T and B cell responses in sheep which were assessed by in vitro proliferation and enzyme-linked immunosorbent assays, respectively. In addition, the recombinant protein was used to generate a monoclonal antibody. The assays and reagents generated in this thesis were subsequently used to investigate the immune response to M. paratuberculosis 60-kDa stress protein in ovine paratuberculosis.This investigation provides preliminary evidence that paratuberculosis-infected animals have T cells that recognise and are capable of responding by proliferation to M. paratuberculosis 60-kDa stress protein in vitro, and that antibodies to this protein could be detected in sera from these animals. Furthermore, M. paratuberculosis 60-kDa stress protein was detected in tissues from infected animals by immunocytochemical analysis. These preliminary findings support a role for M. paratuberculosis 60-kDa stress protein in the ovine immune response to M. paratuberculosis infection

Evaluation of in silico and in vitro screening methods for characterising endocrine disrupting chemical hazards

Anthropogenic activities have drastically altered chemical exposure, with traces of synthetic chemicals detected ubiquitously in the environment. Many of these chemicals are thought to perturb endocrine function, leading to declines in reproductive health and fertility, and increases in the incidence of cancer, metabolic disorders and diabetes. There are over 90 million unique chemicals registered under the Chemical Abstracts Service (CAS), of which only 308,000 were subject to inventory and/or regulation, in September 2013. However, as a specific aim of the EU REACH regulations, the UK is obliged to reduce the chemical safety initiatives reliance on in vivo apical endpoints, promoting the development and validation of alternative mechanistic methods. The human health cost of endocrine disrupting chemical (EDC) exposure in the EU, has been estimated at €31 billion per annum. In light of the EU incentives, this study aims to evaluate current in silico and in vitro tools for EDC screening and hazard characterisation; testing the hypothesis that in silico virtual screening accurately predicts in vitro mechanistic assays. Nuclear receptor binding interactions are the current focus of in silico and in vitro tools to predict EDC mechanisms. To the author’s knowledge, no single study has quantitatively assessed the relationship between in silico nuclear receptor binding and in vitro mechanistic assays, in a comprehensive manner. Tripos ® SYBYL software was used to develop 3D-molecular models of nuclear receptor binding domains. The ligand binding pockets of estrogen (ERα and ERβ), androgen (AR), progesterone (PR) and peroxisome proliferator activated (PPARγ) receptors were successfully modelled from X-ray crystal structures. A database of putative-EDC ligands (n= 378), were computationally ‘docked’ to the pseudo-molecular targets, as a virtual screen for nuclear receptor activity. Relative to in vitro assays, the in silico screen demonstrated a sensitivity of 94.5%. The SYBYL Surflex-Dock method surpassed the OECD Toolbox ER-Profiler, DfW and binary classification models, in correctly identifying endocrine active substances (EAS). Aiming to evaluate the current in vitro tools for endocrine MoA, standardised ERα transactivation (HeLa9903), stably transfected AR transactivation (HeLa4-11) assays in addition to novel transiently transfected reporter gene assays, predicted the mechanism and potency of test substances prioritised from the in silico results (n = 10 potential-EDCs and 10 hormone controls). In conclusion, in silico SYBYL molecular modelling and Surflex-Dock virtual screening sensitively predicted the binding of ERα/β, AR, PR and PPARγ potential EDCs, and was identified as a potentially useful regulatory tool, to support EAS hazard identification

Die Regulation des humanen Lipopolysaccharid bindenden Proteins (hLBP)

Das Lipopolysaccharid Bindende Protein (LBP) ist ein überwiegend in der Leber synthetisiertes Akutphaseprotein. Es bindet den Zellwandbestandteil Lipopolysaccharid (LPS) Gram-negativer Bakterien und transportiert es zu zellulären Rezeptoren, wodurch das angeborene Immunsystem aktiviert wird. In dieser Arbeit wird die Regulation der LBP-Expression in Interleukin (IL)-1, IL-6 und Dexamethason (Dex) stimulierten humanen Hepatomzelllinien HuH-7 und HepG2 untersucht. Der wichtigste Stimulator ist dabei IL-6, dessen Wirkung über die Transkriptionsfaktoren (TF) Stat-3, C/EBP-beta und AP-1 vermittelt wird. Für alle 3 TF konnten aktive Bindungsstellen auf dem LBP-Promotor nachgewiesen werden. Für IL-1-Effekte die u. a. über den TF NF-kappaB vermittelt werden, konnten ebenfalls aktive Bindungsstellen nachgewiesen werden. Die Wirkung von Dex wird über Glucocorticoid Responsive Elements (GREs) vermittelt. Auf dem LBP-Promotor befinden, sich wie gezeigt werden konnte, mehrere aktive GREs, wobei einige verstärkend und einige hemmend wirken. Eine zu beobachtende Synergiewirkung von Dex und IL-6 wird durch die Aufregulation des IL-6-Rezeptors durch Dex verursacht. Die LBP-Expression kann durch TGF (Transforming Growth Factor)-beta gehemmt werden. Der TGF-beta-Signalweg über Smads ist in den Hepatomzellen aktiv, vermittelt aber nicht den TGF-beta-Hemmeffekt, sondern eine geringe stimulierende Wirkung, die bei alleiniger TGF-beta-Inkubation auftritt. Die inhibierende Wirkung von TGF-beta wird durch Gfi-1- und AP-1-Bindungsstellen vermittelt. Die Gfi-1-Bindungsstelle nimmt dabei, wie hier erstmals gezeigt werden konnte, eine herausragende Stellung ein. Die Aufklärung der LBP-Regulation und dabei besonders die Hemmung der LBP-Expression kann mittelfristig dazu beitragen, den klinischen Verlauf von inflammatorischen und infektiösen Erkrankungen zu beeinflussen und bietet daher Potenzial für neue Therapieansätze.Lipopolysaccharide (LPS) binding protein (LBP) is an acute phase protein with the ability to bind and transfer LPS of Gram-negative bacteria. This soluble pattern recognition molecule represents an important defense principle of the host. Regulation of the hepatic acute phase response and its termination are important mechanisms for limiting systemic inflammatory activity of the host. Here were analyze the cooperation of Interleukin (IL)-1, IL-6, and Dexamethasone (Dex) at LBP expression in the hepatoma cell lines HuH-7 and Hep G2. The major inducer of LBP expression is IL-6. Within the LBP promoter numerously highly consensus binding sites such as AP-1, C/EBP-beta? and STAT3 are present, that confer transcriptional activity as shown by truncation and mutation experiments. Additionally, activate NF-kappaB sites activated by IL-1 were detected at the LBP promoter. By mutation experiments of the promoter furthermore were found differentially active glucocorticoid response elements (GREs). The promoter contains GREs enhancing the activity as well as inhibitory ones. The enhancing effect towards LBP expression by Dex was mediated by IL-6. Dex stimulated the expression of the IL-6 receptor and therefore upregulated the IL-6 pathway. Transforming Growth Factor (TGF)-beta is able to inhibit LBP expression in stimulated cells. An AP-1 binding site was identified mediating inhibitory TGF-beta effects towards LBP promoter activity. Furthermore it was shown that a growth factor independence (Gfi)-1 binding site localized near the AP-1 site is essential for mediating the TGF-beta inhibitory effect. The relevancy of the Gfi-1 site fore mediating TGF-beta effects indicates a novel mechanism for understanding inhibitory TGF-beta effects at the transcriptional level. In summary the complex regulation of LBP were elucidate which may help to eventually develop novel intervention strategies for acute phase, sepsis, and septic shock

Endothelial injury in atherosclerosis: identification of mediators and attenuation of inflammation by adenoviral augmentation of elafin and SLPI

Atherosclerosis is a chronic inflammatory process occurring within arterial blood vessels. Clinical sequelae of atherosclerotic plaque build up include angina, myocardial infarction and ischaemic stroke, imposing a massive burden on healthcare provision. The retention and subsequent oxidative modification of lowdensity lipoprotein within the subintimal space provides a persistent stimulus for inflammation throughout atherosclerotic plaque development. Understanding the inflammatory mechanisms of oxidised LDL-induced endothelial cell injury will be necessary before devising therapies that intercept, reverse and prevent atherosclerotic plaque development. The work in this thesis is based on a conviction that strategies aimed at augmenting endogenous anti-inflammatory and repair mediators through local gene delivery have the potential to provide lasting effectiveness with low toxicity.An in vitro model of atherosclerotic endothelial cells was established using cultured human umbilical vein endothelial cells and oxidised LDL. Gene expression profiling identified angiopoietin-2 as a target gene up regulated by oxidised LDL incubation in endothelial cells. High levels of this angiogenic factor were also found in endothelial cell culture supernatants following oxidised LDL incubation and within zones of neointimal angiogenesis in atherosclerotic human coronary arteriesGene expression profiling failed to identify candidate 'endothelial protective' mediators and attention focused on elafin and secretory leucocyte protease inhibitor (SLPI), two low molecular weight elastase inhibitors. Elafin has been demonstrated within human coronary arteries although its function as a locally active antiprotease, antagonising the inflammatory effects of human neutrophil elastase (HNE) and bacterial injury has been characterised best within the lung.Here we have used adenovirus as a vector to deliver elafin and SLPI genes to human endothelial cells and macrophages. We have devised a protocol involving precomplexing of adenovirus with lipofectamine to enhance gene delivery and subsequent gene expression in human endothelial cells and to facilitate gene delivery to human macrophagesElafin and SLPI overexpression were associated with reduced inflammatory cytokine production in endothelial cells and macrophages in response to a range of atherogenic stimuli including oxidised LDL. This anti-inflammatory activity was associated with reduced activation of the transcription factor NF-kB and preservation of its inhibitory sub-unit IkBcl Furthermore, elafin overexpression protected endothelial cells from HNE mediated injury and attenuated FINE mediated impairment of macrophage apoptotic cell recognition.In summary, angiopoietin-2 was identified as a novel mediator produced by endothelial cells in response to oxidised LDL and may contribute to plaque development through facilitation of neointimal angiogenesis. Adenoviral overexpression of elafin and SLPI exhibited therapeutic potential through reducing inflammatory responses and protecting the structure and function of endothelial cells and macrophages in the presence of atherogenic stimuli

Tuberculosis

Data are rapidly accumulating from all over the world regarding the efficacy of standardized treatment regimens for drug-sensitive, drug-resistant TB and latent TB infection. While we are facing the menace of multi drug-resistant TB [MDR-TB], extensively drug-resistant tuberculosis [XDR­ TB] has emerged threatening to undermine global efforts at TB control. Hence we have included chapters to cover all aspects of the diagnosis and management of MDR TB. This book will cover all these developments in great detail. With the widespread availability of internet globally various standard web resources available on TB have also been included so that the readers may get the comprehensive and updated guidelines from these resources. The changing clinical presentation of TB, advances in laboratory, imaging diagnostic modalities, therapeutic measures and emergence of MDR TB all suggest a pressing need to have a updated book on TB. Furthermore, while all physicians encounter the TB disease in their clinical practice, there have been a lot of controversies and misconceptions over various issues for the diagnosis and management of TB. Paucity of a well referenced, updated, standard book of TB has prompted us to undertake this venture sharing the clinical experience of global experts of TB. Our book contains chapters on epidemiology, immune-pathology, diagnosis, treatment and latest advances for TB, highlighting the global perspective of tuberculosis. World-wide resurgence of MDR TB indicates that the battle against this foe of mankind will continue in the coming years. TB still remains to be a research priority of paramount importance from medical, social and financial aspects and we have attempted to highlight all the aspects for the treatment of TB. We believe that this book will serve as a practical guide for the diagnosis and management of TB for practicing physicians (especially pulmonologists and internists) and all those who are involved in the management of TB