TRANSCRIPTIONAL ANALYSIS OF HYDROGENASE GENES IN RHODOBACTER SPHAEROIDES O.U.001 In photosynthetic non-sulphur bacteria, hydrogen production is catalyzed by nitrogenases and hydrogenases. Hydrogenases are metalloenzymes that are basically classified into: the Fe hydrogenases, the Ni-Fe hydrogenases and metal-free hydrogenases. Two distinct Ni-Fe hydrogenases are described as uptake hydrogenases and bidirectional hydrogenases. The uptake hydrogenases are membrane bound dimeric enzymes consisting of small (hupS) and large (hupL) subunits, and are involved in uptake and the recycling of hydrogen, providing energy for nitrogen fixation and other metabolic processes. In this study the presence of the uptake hydrogenase genes was shown in Rhodobacter sphaeroides O.U.001 strain for the first time and hupS gene sequence was determined. The sequence shows 93% of homology with the uptake hydrogenase hupS of R.sphaeroides R.V. There was no significant change in growth of the bacteria at different concentrations of metal ions (nickel, molybdenum and iron in growth media). The effect of metal ions on hydrogen production of the organism was also studied. The maximum hydrogen gas production was achieved in 8.4&micro / M of nickel and 0.1 mM of iron containing media. The expression of uptake hydrogenase genes were examined by RT-PCR. Increasing the concentration of Ni++ up to 8.4&micro / M increased the expression of uptake hydrogenase genes (hupS). At varied concentrations of Fe-citrate (0.01 mM-0.1 mM) expression of hupS was not detected until hydrogen production stopped. These results will be significant for the improvement strategies of Rhodobacter sphaeroides O.U.001 to increase hydrogen production efficiency. In order to examine the presence of hupL genes, different primers were designed. However, the products could not be observed by PCR.

Tetrachloroethene (PCE) is a major groundwater contaminant that often persists as dense, nonaqueous phase liquids (DNAPLs) in subsurface environments. Dissolved-phase PCE plumes emanate from DNAPL source zones, which act as continuous sources of contamination for decades. Removal of DNAPL source zones is crucial to achieve lasting remedy of contaminated aquifers. This research explored the contributions of the microbial reductive dechlorination process (i.e., anaerobic bioremediation) to PCE-DNAPL source zone remediation, either in isolation or as a polishing step for the removal of residual DNAPL remaining after application of surfactant enhanced aquifer remediation (SEAR), an emerging physical-chemical source zone treatment. Specific objectives of this research were to: (1) evaluate the ability of microorganisms to dechlorinate in the presence of PCE-DNAPL and at high dissolved-phase PCE concentrations expected near/in DNAPL source zones, (2) assess the distribution and activity of key dechlorinating populations during bioenhanced PCE-DNAPL dissolution in continuous-flow column experiments, (3) determine the influence of Tween 80, a biodegradable surfactant commonly used in SEAR, on the microbial reductive dechlorination process, (4) design and optimize quantitative real-time PCR (qPCR) protocols to detect and enumerate key dechlorinating populations (e.g., Geobacter lovleyi, Sulfurospirillum multivorans), and (5) explore the effects of oxygen on Dehalococcoides viability and biomarker quantification. This research demonstrated that microbial dechlorinating activity within DNAPL source zones promotes bioenhanced dissolution although many dechlorinating isolates cannot tolerate saturated PCE concentrations. Application of newly designed qPCR protocols established a direct link between dissolution enhancement and the distribution of relevant dechlorinating populations in the vicinity of PCE-DNAPL. The limited and reversible impact of Tween 80 on key dechlorinators supported the feasibility of a treatment train approach of SEAR followed by microbial reductive dechlorination to remediate PCE-DNAPL source zones. Finally, experiments with oxygen-exposed, Dehalococcoides-containing cultures suggested limitations of using Dehalococcoides DNA and RNA biomarkers for monitoring bioremediation at field sites. These findings advance the scientific understanding of the microbial reductive dechlorination process and are relevant to environmental remediation practitioners. The advantages and current shortcomings of PCE-DNAPL source zone bioremediation, as well as recommendations for future research, are discussed.

With global freshwater supplies under pressure, viable water reuse methods are being examined to assist in improving water supplies. Municipal effluent is an ideal source for water reclamation as it is consistent in quality and quantity. The health aspects of water reuse have been identified as an issue of concern, in particular the potential presence of enteric viruses. Managed Aquifer Recharge (MAR) is a method that can aid water reclamation by recharging water such as treated effluent into a suitable aquifer. Research into the removal of pathogenic contaminants by natural processes within aquifers, namely the action of autochthonous bacteria, has led to the consideration that MAR could be used to assist in the removal of microbial pathogens. Pathogens have been demonstrated to be removed during residence in groundwater, but the presence of active autochthonous groundwater bacteria are required for significant removal rates to occur. The aim of this research was to investigate the interaction between autochthonous groundwater bacteria (AGB) and the enteroviruses Poliovirus type 1, Coxsackievirus B3 and Adenovirus B41. It was established that these viruses decrease in number in the presence of AGB but the mechanisms causing this decrease are poorly understood. Experiments were designed to examine how the individual AGB caused decay of the viruses. In this study AGB were isolated and tested for their ability in increase the decay of the viruses. It was determined that 27 % (17/63) of the isolated AGB influenced viral decay. The AGB isolates varied in their influence with only 3 out of 17 isolates being able to cause of the decay of both poliovirus and coxsackievirus. Similar variations in decay were observed for adenovirus. Decay times for all three viruses varied amongst the AGB and between the viruses. Experiments were undertaken to characterise the mechanism causing the antiviral activity of four groundwater isolates (1G, 3A, 4B and 9G) under varying conditions and treatments to give insight into the compounds or mechanisms responsible for viral decay. This would indicate whether compounds produced by the AGB responsible for viral decay were closely associated to bacterial cells (perhaps membrane bound), independent of metabolic activity, heat labile or were enzymatic in nature. The influence of enzyme inhibiters and heat treatment indicated that viral degradation is caused by compounds that are enzymatic in nature. As viral numbers were monitored by nucleic acid copy numbers rather than via infectivity assays, the viral protein coats must be the first step in degradation followed by the removal of the viral nucleic acid. This two step process would require both protease and nuclease enzymes to result in loss of viral numbers as measured by RT-PCR/PCR. Further characterisation and identification of these four bacterial isolates was also carried out. Three out of the four isolates were sequenced and analysed using partial 16S rRNA gene sequences to determine their phylogenetic relationships compared to related organisms. Isolate 3A was placed in the order Burkholderiales. Isolate 4B was placed in the family Xanthomonadaceae. Isolate 9G was placed in the family Rhizobiaceae. Isolate 1G was only partially sequenced and preliminary identification placed it in the phylum Bacteriodetes. Understanding of the processes carried out by AGB within an aquifer during MAR using reclaimed waters will aid in increasing the viability of this water reuse process. If important natural processes could be utilised to remediate any potential pathogens, the health concerns with reclaimed waters could be addressed and solved simply through prescribed retention times within the aquifer. Key species of AGB may even be utilised as markers to assess the suitability of an aquifer for MAR.

The effects of caffeine (250 mg) and placebo on healthy controls were studied in a double-blind, cross over study on 24 healthy subjects who performed a working memory n-back task. Reaction time and accuracy levels were tested using the n-back working memory measure in cognitive neuroscience. An experimental study tested on the 1, 2 and 3-back tasks under the placebo/coffee condition. Based on the empirical results obtained in this thesis it can be concluded that changes produced by caffeine ingestion support the hypothesis that caffeine acts as a stimulant. However, it cannot be proven that the stimulant translates into enhanced motor processes with an improvement in performance.

This methodological investigation set out to examine the use of the Internet as a data collection tool: its potential for sampling gay and bisexual men and gathering data on sexual behaviour. The research took place in the context of the Internet and HIV study, an examination of high risk sexual behaviour among gay and bisexual men seeking sex on the Internet. The Internet and HIV study collected quantitative data using selfadministered questionnaires completed online or by pen-and-paper, and qualitative data using one-to-one interviews conducted face-to-face or online. Its design provides an invaluable opportunity for investigating Internet-based research methods. Methodological issues were explored through collection of primary data from respondents who had participated in the Internet and HIV study and secondary analysis of data collected for the study. The first area of exploration was the use of web surveys for sampling gay and bisexual men. The results indicated that participation in the Internet and HIV web survey was driven by an altruistic desire to contribute to research into sexual health promotion and HIV prevention. Drop out was most likely to occur in the early stages of the survey and minor differences in sub-group drop out were identified. Men who participated in the web survey were broadly similar to the MSM drawn from a national probability sample, but were more likely to participate in high risk sexual activity. Differences were more pronounced when the samples were restricted to London men. The second area of exploration was the effect of the Internet mode on data collection. The web survey reduced item nonresponse in comparison to the pen-and-paper survey but did not improve response to sensitive questions over and above this general mode effect. The results indicated that online synchronous interviews may be suited to a more structured format, given the difficulty of expressing complex ideas through typing in real time. It was concluded that the Internet has excellent potential for researching sexual behaviour among gay and bisexual men. It provides effective access to this population and more complete survey data than the traditional alternative. The application of online interviewing requires further exploration.

Submitted by Felipe dos Santos Gomes (felipesantosg@yahoo.com.br) on 2018-06-05T14:18:52Z No. of bitstreams: 1 Dissertação Felipe dos Santos Gomes.pdf: 1189458 bytes, checksum: 7917af6743501bdde9a3c3a0112e23aa (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-06-05T18:53:33Z (GMT) No. of bitstreams: 1 gomes_fs_me_jabo.pdf: 1233173 bytes, checksum: 9619264fd2c88aa95c12e3757010eed9 (MD5) / Made available in DSpace on 2018-06-05T18:53:33Z (GMT). No. of bitstreams: 1 gomes_fs_me_jabo.pdf: 1233173 bytes, checksum: 9619264fd2c88aa95c12e3757010eed9 (MD5) Previous issue date: 2018-05-04 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A infecção persistente ao vírus da diarreia viral bovina (BVDV) pode viabilizar a disseminação do vírus no rebanho, assim como interferir no controle da infecção. Ao mesmo tempo, em suínos, a presença de soropositivos para BVDV pode causar transtornos aos inquéritos sorológicos para a peste suína clássica (PSC). Este trabalho teve como objetivo determinar a condição de persistentemente infectado em leitões nascidos de porcas infectadas experimentalmente pelo vírus da diarreia viral bovina. Foram selecionadas seis porcas prenhes para este estudo que foram divididas em dois grupos, sendo um grupo inoculado com BVDV-2 (G1; n=4) aos 45 dias de gestação, e um grupo controle (G2; n=2). Foram realizadas avaliações clínicas nas porcas diariamente. Os neonatos foram monitorados durante 35 dias, em que foram realizadas avaliações clínicas rotineiras e colheita de suabes nasal dos leitões e de amostras de sangue venoso das porcas e dos leitões para obtenção de sangue total e soro a cada 72 horas. Foram realizados testes de RT-PCR para diagnóstico direto, e virusneutralização para avaliação sorológica. As porcas apresentaram soroconversão entre o 17ºdia pós-infecção (dpi) e o 22ºdpi, mas não foi detectada viremia. Nenhum leitão apresentou títulos de anticorpos ou viremia ao nascimento. Não ocorreu a transmissão transplacentária do vírus, portanto, não foi possível observar animais PI. / The persistently infection to bovine viral diarrhea virus (BVDV) can enable the spread of virus in the herd, as well as interfere in the control of infection. Concurrently, the presence of seropositive pigs may interfere with serological surveys for classical swine fever (CSF). This project aimed to determine the condition of persistently infected in piglets born from gilts infected with bovine viral diarrhea. BVDV-2 was inoculated in four pregnant gilts (G1; n=4), and a placebo was administered in two gilts, which were the control group (G2; n=2). Clinical evaluations were daily performed in the gilts. The newborns were monitored during 35 days, with clinical evaluation and whole blood, serum and nasal swabs sampling every 72 hours. RT-PCR and virus neutralization tests (VN) were performed. The gilts presented seroconversion between 17º dpi and 22ºdpi, but no viremia was detected. No piglets presented antibody titers or viremia at birth. Transplacental transmission of the virus did not occur, therefore, PI animals could not be observed. / FAPESP: 16/214212

Background: There is increasing recognition of the need for service development for patients in the advanced stages of neurological conditions. This study explores the palliative care needs of people with advanced amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease and related disorders, and assesses the impact of a new specialist palliative care service (SPCS) Methods:. A mixed methods approach was adopted: • a qualitative needs assessment using in-depth interviews with patients and their family carers and focus groups of professionals involved in the care of this population. • a quantitative pilot-explorative randomized and controlled trial (RCT) to assess the impact of a new SPCS designed to meet the palliative care needs of the study population – using a waiting list methodology comparing the immediate provision of SPCS with standard care . Results: • Qualitative study: 22 patients, 21 family carers and 11 professionals participated to the needs assessment. The content analysis showed a high prevalence of problems. Professionals confirmed the high burden of problems, were positive about the creation of a new SPCS and their knowledge of specific palliative care topics seemed lacking. • Quantitative study: 50 patients, with 45 carers, participated in the explorative RCT. The comparison between the groups (FT-ST) after 16 weeks revealed significant improvement for the SPCS group for quality of life and in four physical symptoms - pain, breathlessness, sleep disturbance and intestinal symptoms Conclusions: This research confirms the high prevalence of physical symptoms, psychosocial issues and spiritual themes for people severely affected by advanced neurodegenerative disorders and that these can be helped by specialist palliative care. The input of a SPCS caused a significant improvement of the individual quality of life of the patients and improved symptom control for pain, breathlessness, quality of sleep and intestinal symptoms compared to standard best care alone.

In this bachelor thesis, low latency wireless sensor and actuator networks are studied.In particular IEEE 802.15.4e/LLDN and IEEE 802.11g/RT-WiFi protocols. It is in-vestigated if the protocols are suitable for being used in typical industrial automationenvironments with high update frequencies of around 100 Hz. The protocols are exam-ined on a low level to shed lights on the sources of latency and followed by an analysis ofa specific configuration. It is found that LLDN is limited by low transmission rate andRT-WiFi is limited by interference with itself and other appliances. They both work wellfor update frequencies around 100 Hz.

The recent introduction of open formulary non-medical prescribing has created opportunities to radically change health care delivery. This thesis is the first of its kind to document the process of undertaking an examination of NMP in a chemotherapy setting. A purposeful sample of 23 stakeholders connected with the chemotherapy service in a local cancer setting was used including 12 doctors, 5 nurses, 4 pharmacists and 4 managers. A serial data collection technique with individual interviews followed by uni professional focus groups was adopted. Finally a multi professional focus group was held to determine the strategic way forward. A systematic qualitative data management package -NUDIST was used to code and theme data and this data was used as the basis for discussion. This thesis offers an insight into the working of the multidisciplinary team in relation to NMP. It establishes the concerns and issues of the professional stakeholders as well as areas of concurrence. It charts the complexities and difficulties faced when shifting responsibilities of professionals. It has used a novel approach to engage with stakeholders from within the day to day practices of the clinic and developed a framework for monitoring agreed progress. An action oriented approach was inherent to the study and dovetailed to understand the service and concurrently create an opportunity for development. Through its insider perspective it has highlighted the complexes of changing practice and attempted to move forward using a negotiation process. The study illuminated the key features necessary to maximise success of NMP in chemotherapy clinics and captures the importance of good working relationships. Whilst different models of nurse-led practice will emerge, fundamental and core to services is the need for good team working, established and effective communication strategies and perhaps most importantly avoiding isolation in practice. This study additionally reinforced that any evaluation takes place within pre- existing political contexts and in particular medical dominance. Not all medical colleagues agreed with or wanted NMP for their patients highlighting difficulties of developing new models of working within a resisting culture. Medical dominance should be acknowledged and strategies developed to work within this dominance to ensure the unique contribution of nursing is valued and seen. Insights from this study raise the need for further research and exploration of the usefulness and acceptability of NMP in chemotherapy.

When children with cerebral palsy attend a mainstream school, their physical functioning may impact on day-to-day school activities and on their active participation. The Special Educational Needs Code of Practice (DfES 2001b) advocates a multi-agency approach to enable children with SEN to be included within the mainstream school system. Physiotherapists often work with school staff to manage the child’s physical needs within this environment and to deliver therapeutic interventions. Despite numerous government policies endorsing the inclusion of children with SEN within mainstream school, there has been little research into the detail of how this might be achieved for children with physical impairments. This qualitative study explores the physical management of children with cerebral palsy within mainstream school. In the first phase I conducted focus groups and semi-structured interviews exploring the views and experiences of parents of children with cerebral palsy, physiotherapists and school staff regarding the management of the child’s physiotherapy needs. The second phase utilised a case study approach to generate in-depth contextual knowledge of the issues faced when managing the child’s physical needs by exploring individual cases within three mainstream schools using observation, interviews and documents. Thematic analysis was used to analyse these data. Three main themes emerged from the findings of the study: how therapy and education services work together; the delicate balance to achieving participation; and how views of difference impact on the child’s management. I found that the way physical impairments were viewed within the current SEN framework, inhibited a holistic view of the child with physical impairments. It impacted on collaborative practice between agencies affecting how the child’s needs were met. I conclude that a more interactional model of viewing disability is required to ensure that the child’s needs are considered within the context of not only school but the child’s life as a whole.