Factors and mechanisms of archaeal transcription termination and DNA repair

2022 Spring.Includes bibliographical references.RNA synthesis by RNA polymerase (RNAP) is an essential process and must be properly regulated both temporally and spatially to ensure cellular health in dynamic environments. Regulation of RNA synthesis in response to internal and environmental stimuli is typically achieved through interactions with RNAP at all stages of the transcription cycle- initiation, elongation, and termination. While studies of transcription initiation and elongation have identified multiple regulatory transcription factors and defined mechanisms, only a handful of protein factors able to terminate transcription have yet been described, and the general mechanism of transcription termination is still highly debated. We previously identified the first two factors capable of terminating transcription elongation complexes (TECs) in Archaea from the genetically tractable Thermococcus kodakarensis, and use both factors as models to explore the molecular mechanisms involved in collapse of the TEC. The Factor that terminates transcription in Archaea (FttA), a close homolog of the human CPSF subunit CPSF73, is completely conserved throughout Archaea, and appears to act analogously to the bacterial termination factor Rho, terminating transcription after the uncoupling of transcription and translation at the end of protein coding genes. We employed a novel genetic screen to verify the role of FttA in the polar repression of transcription, a phenomenon specific to regulation of genes contained within operons in prokaryotes. Eta, a euryarchaeal-specific superfamily 2 (SF2) helicase, appears to terminate transcription in a more specialized context, potentially terminating transcription of TECs arrested at sites of DNA damage while concurrently recruiting appropriate DNA repair enzymes, akin to the bacterial termination factor Mfd. A structure-function study of Eta employing select mutations derived from a crystallographic structure was conducted to elucidate the Eta-TEC contacts and various activities of Eta required for Eta-mediated termination. Further, many efforts were directed at establishing a role of Eta as an archaeal transcription-repair coupling factor (TRCF), and while this was not achieved, a state-of-the-art next-generation sequencing based approach to monitor nucleotide excision repair (NER) and the sub pathway transcription-coupled repair (TCR) genome-wide was developed and verified in E.coli. The work in this dissertation adds valuable insight to multiple fields of research. First, exploration into the mechanism of Eta-mediated transcription termination reveals a potential shared susceptibility of core RNAP subunits to transcription termination while elucidating activities of SF2 helicases- enzymes which are ubiquitously distributed in multiple essential cellular pathways. Second, our genetic screen identifies FttA as the archaeal polarity factor, shedding light on functions of an ancestral factor indispensable in mammalian transcription termination pathways. Establishment of the novel RADAR-seq/RNA-seq measurement of NER genome-wide will likely prove instrumental in future studies of archaeal DNA repair, and potentially presents a new paradigm in research of eukaryotic-like NER by use of Archaea as a advantageous model organism

Effects of uric acid on endothelial function and dysfunction

The association between elevated uric acid (UA) concentrations and cardiovascular disease is well established in epidemiology, but the possibility that UA plays a specific role in the pathophysiology of cardiovascular disease remains a matter of debate. Although there are putative mechanisms by which UA could injure the cardiovascular system, it also has a number of properties that might be considered as protective. Most notably, its role as a radical-scavenging antioxidant might be expected to mitigate the effects of increased oxidative stress, which is characteristic of most risk factors for cardiovascular disease and is an important precipitant of endothelial dysfunction. The aim of the studies described in this thesis was to examine the effects of UA on endothelial function and investigate whether UA has the potential to reverse endothelial dysfunction induced by low-density lipoprotein (LDL).Mesenteric arteries were isolated from Wistar-Kyoto rats and the responses to a vasoconstrictor (PE, phenylephrine) and endothelium-dependent (ACh, acetylcholine) and -independent (SNP, sodium nitroprusside) vasodilators examined using perfusion myography. This model was considered advantageous because it enabled the measurement of pharmacological responses in the presence of different luminal solutions in an experimental environment that most closely mimics the conditions found in vivo. Luminal perfusion with L-NAME and/or indomethacin demonstrated that nitric oxide synthase (NOS) -derived nitric oxide (NO) was the major vasodilator released by the endothelium in response to ACh in this experimental model.Exposure of the vascular lumen to increasing concentrations of UA (200, 400, 600pM) or vehicle solution had no effect upon the responses to PE, ACh or SNP. This implied that, in this model, acute exposure to elevated UA does not impair endothelial function. In contrast, when the lumen was perfused with increasing concentrations of LDL (250, 500 and lOOOμg/ml), maximal vasodilatation towards resting diameter in response to ACh was reduced to 42.6, 33.6 and 21.7% respectively. The failure of the NOS inhibitor LNAME to further impair vasodilatation implied that major effect of LDL was to abolish endothelium-dependent NO-mediated vasodilatation. Supplementing the perfusing LDL solution with ImM L-arginine restored endothelium-dependent responses, implying that the LDL-induced endothelial dysfunction was in part explained by a disruption of Larginine metabolism. Supplementation with the extracellular O₂ scavenger, superoxide dismutase (SOD), did not prevent the deleterious action of LDL.Supplementation of 250μg/ml LDL with increasing concentrations of UA (200, 400, 600μM) partially reversed the inhibition of maximal vasodilatation towards resting diameter in response to ACh to 62.2, 69.5 and 74.4% respectively. No such improvement could be achieved in the presence of L-NAME. The beneficial effect of 400μM UA upon LDL-induced endothelial dysfunction contrasted with the lack of effect of two other water-soluble antioxidants, ascorbic acid (AA) and glutathione (GSH), at the same concentration.The experiments then focused on investigating the potential mechanism by which UA prevented LDL-induced endothelial dysfunction. Isolated rings of thoracic aorta from Wistar-Kyoto rats were mounted in a wire myograph and exposed to ACh in the presence of varying concentrations of UA and 250pg/ml LDL. The superfusate was then transferred to endothelium-denuded rings and caused significant vasodilation in previously unresponsive ring segments. The extent of the vasodilatation in response to the transferred solution was dependent on the concentration of UA and ox-Hb sensitive. The decay in vasodilator response if the exposure of the denuded ring was delayed had a half-life of 29 minutes. These results implied that the stimulation of an endotheliumintact vessel by ACh in the presence of both UA and LDL results in the formation of an endothelium-independent vasodilator that releases NO and may be a derivative of UA.In summary, the results of these experiments suggest that acute exposure to UA in physiological concentrations does not impair either endothelium-dependent or - independent vascular responses. Conversely, UA reverses the impairment of AChinduced vasodilatation caused by LDL and does so more effectively than other high concentration hydrophilic antioxidants. Furthermore, UA appears to enable the formation of an NO-releasing compound when it is present with LDL and endothelial cells VI stimulated by ACh. The presence and nature of a possible NO-donor compound formed in these circumstances requires further investigation. Taken together, this work implies that UA exposure is not directly injurious to vascular function and may protect against the effects of LDL on the vascular endothelium. This might offer a physiological role for a compound which is found in much higher concentration in the extracellular fluids of humans than almost any other species

Levels of industrial militancy and the political radicalization of the Durham miners 1885-1914

To date there have been several works written on the Durham Miners' Association, the most notable of which have been G Metcalfe's 'A History of the Durham Miners’ Association' and E Melbourne's 'The Miners' Unions of Northumberland and Durham'. These works have tended to concentrate on the growth of the D.M.A. itself, rather than studying any of the pressure groups which were active within the coalfield. This work is split into two sections. The first alms to discover the levels of industrial militancy and the degree of political radicalisation in the coalfield between 1885 and 1914. In order to do this a careful study has to be made of the official and unofficial disputes in the period, and of the policies put forward by different lodges in the D.M.A. council. As yet there has been little work on the growth of the Labour Party in the North East, an omission which this work starts to remedy by a study of the political fortunes of the miners in certain selected parts of the coalfield. The second section of the work moves away from the ascertaining of the levels of industrial militancy and political radicalisation towards an analysis of factors which influenced their respective levels. In this section four separate influences are studied in some detail, the economic condition of the coalfield, the position of the Methodists regarding both militancy and radicalism, the level of migration and its influence and the relationship which existed between the union leaders and the rank and file. The findings of these studies go some way towards an explanation of the actions of the miners

Structural and biochemical studies on three Aspergillus fumigatus proteins that present as targets for novel antifungal drugs

The increasing use of aggressive immunosuppressive regimes over the last half-century has been accompanied by an increased incidence of opportunistic invasive fungal infections, with Aspergillus fumigatus emerging as one of the main etiologic agents. A. fumigatus is now the major airborne fungal pathogen and is a pathogen to be feared; it is difficult to diagnose early and accurately, a detailed understanding of the factors regulating its growth and pathogenesis is lacking, and invasive infections are associated with high mortality rates, longer hospital stays and high treatment costs. In addition, the number of antifungals effective against it is limited and resistance is increasing, highlighting the need for the characterisation of novel targets for new antifungals. A detailed knowledge of the structure of a protein is essential for understanding its underlying molecular mechanism and enabling structure-based drug design. This dissertation reports the application a structural genomics-style approach to a pilot set of proteins that are involved in a range of processes essential for the growth, cellular homeostasis and survival of A. fumigatus in the human host. Following on from preliminary experiments to identify proteins amenable to study by X-ray crystallography, three A. fumigatus proteins were selected for comprehensive structural and biochemical characterisation: (1) Crystal structures of cytosolic thiolase (afCT), which catalyses the first step in isoprenoid synthesis, were solved to resolutions between 1.7 and 2.25 Å. One of these crystals trapped two previously unobserved tetrahedral reaction intermediates, providing the first direct structural observation of the full thiolase reaction cycle. Unexpectedly, afCT is more similar structurally and biochemically to human mitochondrial thiolase II (hT2) than it is to human cytosolic thiolase (hCT). Here it is shown that, like hT2, afCT is strongly activated by monovalent cations, with a preference for K⁺ ions. Additionally, structural comparisons and in silico docking experiments suggest that afCT has a substrate specificity more similar to hT2 than hCT. (2) The structure of proliferating cell nuclear antigen (AfumPCNA), which is essential for DNA replication and repair, was solved to 2.6 Å. Strong structural similarities between AfumPCNA and human PCNA at their main protein-protein interaction site suggest that the structural basis by which PCNA functions is conserved. Consistent with this, it is shown herein that the PCNA-interacting peptide of the human tumour-suppressor protein p21 binds AfumPCNA with only ten-fold lower affinity than for hPCNA, presenting AfumPCNA as a target for potential peptide-based antifungals. (3) Thioredoxin reductase (AfTrxR) catalyses the transfer of reducing equivalents from NADPH to a number of important biosynthetic and redox-active enzymes. The structure of AfTrxR was solved to 3.2 Å, and shows high structural similarity to bacterial, plant and yeast TrxRs. It is also shown herein that ebselen (EbSe), a small drug-like molecule, is a nanomolar inhibitor of AfTrxR in vitro and a potent inhibitor of A. fumigatus growth. Further investigation by mass spectrometry revealed that EbSe interacts covalently with a redox-active cysteine at the AfTrxR catalytic site. In silico docking experiments were used to define key enzyme-inhibitor interactions that will be important to consider when designing potent and specific antifungal drugs targeting TrxR in the future.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Biological Sciences, 201

The provision and justification of a volunteer-based religious education programme

This study considers voluntary religious education for adolescents, provided by Christian Churches in England. It provides justification for such programmes through the comprehensive demonstration of an urgent need; it argues that existing provision in the churches is generally poor; finally, it shows that a solution to the challenge is possible, with a detailed analysis of one particular programme. Since the emergence of schools as a public, rather than a church responsibility, there has been tension and controversy over religious education. Various historical developments have been important in shaping the present unsatisfactory situation. The nature of religion and education ensures that state schools will never be able to provide a complete religious education, satisfactory to everyone; however in the past they have provided a comprehensive -basic grounding in Protestant Christian theology. As a result of changing educational philosophy and practice, they no longer do so. Thus the churches have inherited a large gap in the religious education of their youth, which has only recently been fully recognised. The response to this by the churches is generally inadequate, and their focus tends to be with infants and children. Some advances have been seen in curriculum and teacher training courses, but generally the provision for teenagers is poor. The Seminary programme of the LDS Church has operated in this country since 1968, and continues to expand. It serves the fourteen to seventeen age group, and was originally devised to replace school-based religious education. Success is partly because of the commitment of the Church, for historical and doctrinal reasons, to religious education, though many other strengths and weaknesses were identified, the most encouraging of which is the high level of personal motivation shown by the teenagers involved

VALIDITY AND RELIABILITY OF FINAL SAY DECISION MEASURES OF MARITAL POWER

The final say decision measure of marital power has fared poorly in several studies which have examined its validity. This research was designed to explore two possible explanations for these findings: (1) inadequate or inappropriate procedures in previous validity studies, and (2) weaknesses of the final say measure itself, including insensitivity to item saliency and the discrepancies in responses between family members. Several US samples and one from India provided the data for this study. These samples had been gathered in other studies in which husbands\u27, wives\u27 and/or children had responded to the final say decision index. Up to three versions of the final say index were computed for each respondent. In the first version, decision items were unweighted (FSD index). In the other two versions, items were weighted by their relative importance (WFSD index) or by the amount of conflict associated with the particular decision area (CFSD index). The reliability and validity of these measures and of measures based on the responses of husbands, wives and children was assessed through analytical procedures such as (1) analysis of item-removed alpha coefficients and (2) external criterion correlation analysis. The major findings of the study are: (1) The final say decision measure has cross-cultural validity, evidenced by consistent patterns among the validity coefficients across samples (though the coefficients were generally low). (2) Weighting the final say decision measure by importance or conflict does not improve validity and reliability. (3) The reliability of power measures based on wives\u27 reports is higher than measures using husbands\u27 reports. (4) The validity of measures derived from husbands\u27 and wives\u27 reports is basically equivalent. (5) Although there is some indication that power measures based on children\u27s reports have the highest reliability and validity, the small sample size and other problems make such comparisons tenuous. The results indicate that the simple unweighted final say measure is a more valid and reliable instrument than previous research has suggested

KINETIC MONTE CARLO SIMULATION OF BINARY ALLOYS

There are many tools to simulate physical phenomena. Generally, the simulation technique is defined by the size of the simulation area. Two well know techniques for simulating atom dynamics are kinetic Monte Carlo (kMC) and molecular dynamics (MD). In this work we simulate physical vapor deposition of binary metallic systems using the kMC technique. A sufficient quantity of atoms are deposited so that morphological features can be observed. Where kMC has fallen short we have used MD to supplement our results

Moral Status and the Oversight of Research Involving Chimeric Animals

The use of nonhuman animals in research has long been a source of bioethical and scientific debate. We consider the oversight and use of nonhuman animals in chimeric research. We conducted interviews with twelve members of embryonic stem cell research oversight committees, nine members of institutional animal care and use committees, and fourteen scientists involved in human–nonhuman-animal chimeric research in different areas of the United States. Interviews addressed animal welfare and conceptual issues associated with moral status and humanization of nonhuman animals that contain human cells. Our findings suggest that concepts of enhanced moral status and consciousness are not very useful in human–nonhuman-animal chimeric research in part because their meanings are not easily defined, which presents challenges to applying the concepts in research. Instead, scientists and oversight committee members we interviewed seemed to rely on standard assessments of changes in animal welfare when focusing on the ethics of human-animal chimeric research