Replication of Herpes Simplex Virus DNA: Study of an Origin Binding Protein

The herpes simplex virus type 1 (HSV-1) genome contains two distinct origins of viral DNA replication of related sequence. One (oriL) lies close to the centre of the long unique region whilst two copies of the other (oriS) are present within the inverted repeat regions flanking the short unique region. Previous experiments identified DNA fragments of 100 bp or less which specify a functional oriS and include a 45 bp near perfect palindrome with a central AT-rich region. Moreover, DNase I footprint assays demonstrated the presence of a specific binding site for an HSV-1 encoded polypeptide which overlapped the end of the palindromic sequence. By analogy with other well-characterised origin binding proteins, the HSV-1 encoded origin binding protein was thought likely to play an important role in initiation of HSV DNA synthesis. The work presented in this thesis describes the identification of the HSV-1 gene encoding this origin binding activity and investigates the role of its interaction with oriS. A sensitive gel retardation assay was set up to allow the identification of the virus induced origin binding activity. Incubation of nuclear extract from cells infected with wt HSV-1 with a radio-labelled oriS fragment resulted in the formation of a major specific retarded complex. Experiments with synthetic oligonucleotides demonstrated the presence of two specific binding sites within the origin region, one of which (site I) corresponded to the previously described site whilst the other (site II) was located on the opposite side of the palindromic sequence. While these experiments were in progress, Challberg and colleagues identified a set of seven HSV-1 genes which were necessary and sufficient for HSV DNA synthesis. These genes encoded the viral DNA polymerase (UL30), a single-strand-specific DNA binding protein (UL29), a double-stranded DNA binding protein (UL42) and four less well understood functions (UL5, UL8, UL9 and UL52). It was considered very likely that one of these latter four genes would encode the protein which binds specifically to oriS. Several approaches were used in attempts to identify the gene encoding origin binding activity. These included analysis of origin binding activity induced by ts mutants with defects in DNA synthesis at the non-permissive temperature and transfection of tissue culture cells with fragments encoding individual replication genes. Neither of these approaches however was successful. HSV-1 tsK recombinants which express individual replication genes UL5, UL8, UL9 or UL52 at the non-permissive temperature were available in the laboratory. When these were tested, only the tsK/UL9 recombinant virus expressed origin binding activity at the non-permissive temperature. The protein-DNA complex obtained with extracts from cells infected with the tSK/UL9 recombinant virus exhibited a smeared binding pattern of lower mobility than previously seen with extracts from cells infected with wt HSV-1. Treatment of the tsK/UL9 extract with protease produced a smaller complex of similar mobility to that seen with wt HSV-1 extracts. In addition, antibody reactive with the UL9 protein further retarded the major complex observed with wt HSV-1 extracts, demonstrating that this complex contained a portion of the UL9 polypeptide. Taken together, the above results suggested that a specific domain of the UL9 protein was probably responsible for sequence-specific binding. Fragments of the UL9 gene were therefore expressed as fusion proteins in Escherichia coli and the C-terminal 317 amino acids were found to bind specifically to HSV oriS , indicating that sequence-specific recognition and binding activities resided within the C-terminal 1/3 of the protein. This result was independently confirmed by expressing the C-terminal 1/3 of the UL9 gene in an in vitro transcription and translation system. Of the two UL9 binding sites within oriS , one (site I) contains an 11 bp sequence which is also present in HSV-2 ori S, HSV oriL and varicella-zoster virus oriS, and the other (site II) includes a related sequence element which differs in two positions from the corresponding region of site I. A third 11 bp sequence (motif III) which lies adjacent to site I and differs from the site I element at only a single position was also recognised. Each of the three 11 bp sequences was deleted from within functional copies of HSV-1 oriS and the effect on origin activity and binding of the UL9 protein examined

Military veteran engagement with mental health and well-being services: a qualitative study of the role of the peer support worker

Background: Many UK military veterans experiencing mental health and well-being difficulties do not engage with support services to get the help they need. Some mental health clinics employ Peer Support Workers (PSWs) to help veteran patients engage, however it is not known how the role influences UK veteran engagement. Aims: To gain insight into the role of peer support in UK veteran engagement with mental health and well-being services. Method: A qualitative study based on 18 semi-structured interviews with veterans, PSWs and mental health clinicians at a specialist veteran mental health and well-being clinic in Scotland. Results: Four themes of the PSW role as positive first impression, understanding professional friend, helpful and supportive connector, and an open door were identified across all participants. The PSWs’ military connection, social and well-being support and role in providing veterans with an easily accessible route to dis-engage and re-engage with the service over multiple engagement attempts were particularly crucial. Conclusions: The Peer Support role enhanced veteran engagement in the majority of instances. Study findings mirrored existing peer support literature, provided new evidence in relation to engaging UK veterans, and made recommendations for future veteran research and service provision

Indigenous Australians and Universities: A Study of Postgraduate Students' Experiences in Learning Research

This thesis is concerned with conceptualizing the Social World of Learning of Australian Indigenous postgraduates to determine the quality of research training provided for them. The learning experiences of a group of twenty Indigenous postgraduates in eight Australian universities across ten campuses co-operatively contributed to the information base of this Study. Indigenous entry into the Australian postgraduate sector of education is a recent event, consequently little is known about the area inasmuch as only one Study exists. I used a Democratic Evaluation process to find the answer to the research focus question which guided this Study: How do Australian Indigenous postgraduates experience learning research

Do NGOs Deliver? The Role of NGOs in Responding to the Syrian Refugee Crisis in Jordan

Eleven million Syrians have fled their homes or have been killed as a result of the Syrian Civil War that began in 2011 (Mercy Corps, 2016). The violence, destruction of homes, human rights violations, and lack of access to basic necessities such as food and medication are the motivations for many Syrians to leave their homes and seek refuge in both neighboring countries and Europe. Each year has seen an exponential increase in the number of Syrian refugees; in 2012 there were 100,000 refugees and today there are 4.3 million refugees. This large increase each year contributes to the already significant workload for NGOs responding to the crisis

Straight from the horse's mouth

vi, 137 leaves : ill. ; 29 cm. --The project, Straight from the horse's mouth: Experienced teachers comment on change effectiveness characteristics l. examines the presence or absence of factors which enable or impede the implementation of new primary curriculum. Based on Carney's (1990) research study in Kentucky. this researcher who also teaches at the rural primary school in British Columbia, investigated the presence of Carney's ten change effectiveness characteristics. Research took the form of a case study, basing its results on the interviews conducted with twelve staff members at the school. The staff members spoke of their past experiences of curricular implementation and reflected on the process and professional changes throughout their careers. Hoping to convey a deep understanding of the teachers' dilemma during implementation, the researcher has taken a creative approach and presented information, results and personal reflections in the fonns of free verse, prose, drama, questions and illustrations. This study concludes that some degree of Carney's change effectiveness characteristics were present during the implementation process, that collaboration and teamwork were powerful forces in this study situation and that vision was an important aspect to these participants. Some change effectiveness characteristics impacted on some participants more than others: some participants voiced a need for a positive risk-taking environment whereas communication and information were seldom mentioned. Directions for further investigations are suggested

Dexfenfluramine and the oestrogen-metabolizing enzyme CYP1B1 in the development of pulmonary arterial hypertension

<p>Aims: Pulmonary arterial hypertension (PAH) occurs more frequently in women than men. Oestrogen and the oestrogen-metabolising enzyme cytochrome P450 1B1 (CYP1B1) play a role in the development of PAH. Anorectic drugs such as dexfenfluramine (Dfen) have been associated with the development of PAH. Dfen mediates PAH via a serotonergic mechanism and we have shown serotonin to up-regulate expression of CYP1B1 in human pulmonary artery smooth muscle cells (PASMCs). Thus here we assess the role of CYP1B1 in the development of Dfen-induced PAH.</p> <p>Methods and results: Dfen (5 mg kg−1 day−1 PO for 28 days) increased right ventricular pressure and pulmonary vascular remodelling in female mice only. Mice dosed with Dfen showed increased whole lung expression of CYP1B1 and Dfen-induced PAH was ablated in CYP1B1−/− mice. In line with this, Dfen up-regulated expression of CYP1B1 in PASMCs from PAH patients (PAH-PASMCs) and Dfen-mediated proliferation of PAH-PASMCs was ablated by pharmacological inhibition of CYP1B1. Dfen increased expression of tryptophan hydroxylase 1 (Tph1; the rate-limiting enzyme in the synthesis of serotonin) in PAH-PASMCs and both Dfen-induced proliferation and Dfen-induced up-regulation of CYP1B1 were ablated by inhibition of Tph1. 17β-Oestradiol increased expression of both Tph1 and CYP1B1 in PAH-PASMCs, and Dfen and 17β-oestradiol had synergistic effects on proliferation of PAH-PASMCs. Finally, ovariectomy protected against Dfen-induced PAH in female mice.</p> <p>Conclusion: CYP1B1 is critical in the development of Dfen-induced PAH in mice in vivo and proliferation of PAH-PASMCs in vitro. CYP1B1 may provide a novel therapeutic target for PAH.</p&gt

Gliadin antibody detection in gluten enteropathy

Circulating antigliadin antibody has been described in patients with gluten enteropathy although the prevalence varies in different studies. It has been suggested that the investigation for antigliadin antibody might be useful as a screening test. The object of the present study was to evaluate two different techniques for assaying these antibodies — an indirect immunofluorescent method and an enzyme-linked immunosorbent assay (ELISA). Antibodies were assayed in the sera of 102 patients in whom jejunal biopsies were also obtained. The specificity of both tests was greater than 95%, and the correlation between the presence of antibody and histology was significant (p < 0.005), though the sensitivity of each test was less than 70%

Assessing the feasibility of applying machine learning to diagnosing non-effusive feline infectious peritonitis

Feline infectious peritonitis (FIP) is a severe feline coronavirus-associated syndrome in cats, which is invariably fatal without anti-viral treatment. In the majority of non-effusive FIP cases encountered in practice, confirmatory diagnostic testing is not undertaken and reliance is given to the interpretation of valuable, but essentially non-specific, clinical signs and laboratory markers. We hypothesised that it may be feasible to develop a machine learning (ML) approach which may be applied to the analysis of clinical data to aid in the diagnosis of disease. A dataset encompassing 1939 suspected FIP cases was scored for clinical suspicion of FIP on the basis of history, signalment, clinical signs and laboratory results, using published guidelines, comprising 683 FIP (35.2%), and 1256 non-FIP (64.8%) cases. This dataset was used to train, validate and evaluate two diagnostic machine learning ensemble models. These models, which analysed signalment and laboratory data alone, allowed the accurate discrimination of FIP and non-FIP cases in line with expert opinion. To evaluate whether these models may have value as a diagnostic tool, they were applied to a collection of 80 cases for which the FIP status had been confirmed (FIP: n = 58 (72.5%), non–FIP: n = 22 (27.5%)). Both ensemble models detected FIP with an accuracy of 97.5%, an area under the curve (AUC) of 0.969, sensitivity of 95.45% and specificity of 98.28%. This work demonstrates that, in principle, ML can be usefully applied to the diagnosis of non-effusive FIP. Further work is required before ML may be deployed in the laboratory as a diagnostic tool, such as training models on datasets of confirmed cases and accounting for inter-laboratory variation. Nevertheless, these results illustrate the potential benefit of applying ML to standardising and accelerating the interpretation of clinical pathology data, thereby improving the diagnostic utility of existing laboratory tests