The North American elk or wapiti is an indigenous species to Canada. Understanding of the reproductive physiology of elk is limited, as little research has been conducted in this field as compared to domestic farmed species. In order to make available the tools to study reproductive physiology of the elk this thesis describes the cloning and expression of elk pituitary glycoprotein hormone cDNAs. The common gonadotropin a-subunit, and FSH, LH and TSH b-subunit elk cDNAs were amplified by reverse transcription and polymerase chain reaction (RT-PCR). There was a high degree of nucleotide similarity between the elk a and b subunits when compared with reported sequences from other species. The cDNAs for the pituitary glycoprotein hormone genes were used as probes to investigate seasonal expression in the female elk pituitary gland. Steady state levels of the common a-subunit mRNA was observed regardless of the reproductive season, but a significant increase in expression occurred during the breeding season. The FSH and LH b-subunit genes were expressed at low levels in pituitary glands of animals during presumed anestrous and pregnancy, but levels considerably increased during estrus. In contrast, levels of TSH b-subunit mRNA were similar regardless of the reproductive status. The FSH cDNAs were also transfected into a Chinese hamster ovary (CHO) mammalian expression system, aimed at the production of recombinant elk FSH. Transfected CHO cell lines were screened for expression of a- and FSH b-subunit mRNA by Northern blot. Activity of FSH was equivalent to ~100 mIU/ml of recombinant human FSH (Gonal-FTM), identified by FSH receptor signaling in an in vitro cell based assay. In conclusion, this work represents an advance towards understanding the molecular basis of seasonal reproduction in elk. This information and the availability of elk recombinant FSH will be useful for the application of advanced reproductive technologies required for the rapid expansion of healthy, disease resistant, and genetically superior animals, which are important for domestic production and wildlife management.

This research investigated the existence of <10 £gm nitrogen-fixing unicellular cyanobacteria in the South China Sea. The surveys covered the period from February 2004 to January 2005 and a total of seven cruises. The unicellular cyanobacteria that express orange-yellow from cellular phycoerythrin were observed under a fluorescence microscope. Their expressions in nitrogen-fixation were confirmed by the results from reverse transcription polymerase chain reaction (RT-PCR) and whole cell fluorescence immunolocalization of nitrogenase. The nifH gene sequences of the unicellular cyanobacteria collected from the South China Sea was with >90% identities of their nucleotides similar to the nifH gene sequences of unicellular diazotrophs from ALOHA (Hawaii) as well as Synechocystis sp. WH 8501, Cyanothece sp. ATCC 51142, Cyanothece sp. WH 8902, Cyanothece sp. WH 8904 and Synechococcus sp. RF-1. Positive reactions of fluorescence immunolocalization of nitrogenase were only observed in some, not all, of <10 £gm unicellular cyanobacteria, suggesting that cell counting alone can not be used to estimate nitrogen fixation rate. There was great seasonal and spatial variation in the unicellular cyanobacteria cell density. There was, however, no significant relationship between cell density and the investigated environmental factors. Cell density was high when temperature was high or where stratification index of water column was high, such as in summer or in basin in contrast to other seasons or the shelf-slope regions.

The effect of heat shock was observed on the expression of hilA in Salmonella Typhimurium by creating a fluorescence-based reporter strain of Salmonella and by realtime reverse transcriptase polymerase chain reaction (RT-PCR). The hilA gene in Salmonella is known to play an important role in its pathogenesis. hilA is known to be activated when the bacteria encounter stress-inducing conditions. A number of factors have been identified that affect hilA expression, such as, pH, osmolarity, oxygen tension. When Salmonella enter their warm-blooded hosts, they encounter an increase in temperature. Therefore, heat is another stressor that is encountered by Salmonella during infection of their hosts. A fluorescence-based strain of Salmonella was created to study the effect of heat shock. The gene for green fluorescent protein (gfp) was placed under the control of the promoter of hilA on a plasmid. This plasmid was used to transform Salmonella cells to create a fluorescent strain. In this strain, when the hilA promoter is activated, gfp is transcribed, which encodes the green fluorescent protein. This protein can be measured by a fluorescence assay. The results of this study indicated that at 45ºC, hilA is activated. RT-PCR was used to look at hilA expression at different temperature. The results of this study indicated that, compared to 37ºC, higher temperatures like 45ºC and 55ºC significantly activate hilA.

MicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan® Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.

In 1975, the medical staff at Glasgow's Western Infirmary suggested a study of patients making frequent and inappropriate use of the hospital IS acute receiving area. The staff claimed that these patients seldom had acute medical problems; they wasted the doctors I time; and consumed a substantial amount of hospital resources. The staff wondered what prompted the behaviour of these patients and whether they could be better managed in the future. In order to identify the frequent and inappropriate users of the acute receiving area, we reviewed the past acute attendances of the 3,284 patients in our patient sample. As we had no objective measures of inappropriate patient behaviour, we used the frequency of presentation as our sole selection criterion. knowinq that any frequent and inappropriate users would thereby be included. After samplinq the patient records. we defined frequent attendance as six or more acute attendances between 1st January 1970 - 31st July 1975, a 5 year 7 month study period. We found 150 (5%) of the 3,284 patients studied had been frequent attenders. We looked for ways to identify the frequent attenders at presentation but found no significant difference in age. sex. or presenting complaint between the frequent attenders and the 3.284 patients sampled. We then used these characteristics (age. sex, and presenting complaint) to select matched controls from the patient sample for each of the 120 frequent attenders who proved available for interview. We found inappropriate use of the acute receiving area mentioned in all but two of the 120 frequent attender records and 68% of the frequent attenders' acute presentations were attributed to inappropriate patient behaviour. While few (12%) frequent attenders were diagnosed as having greater medical problems than their matched controls, psychological problems were diagnosed in 77% of the frequent attenders in contrast to only 22% of the controls. The patients· answers at interview showed frequent attendance strongly associated with a number of background variables, with no single variable proving pathognomonic of frequent attenders. We found that, on average, the frequent attenders had greater health, housing, and employment problems, greater difficulties in relating to others, and were more accident-prone than their matched controls. We found most of the acute receiving area costs to be fixed costs and thought little would have been saved had the frequent attenders not presented. We thought the amount of hospital resources consumed by the frequent attenders was too small to. adversely affect other patients. Our study showed that a small number of frequent and inappropriate users of the acute receiving area indeed existed at the Western Infirmary. However, we thought that little could be done either to alter the social and psychological factors we found associated with frequent attendance or to prevent future acute attendances by these patients. At a hospital level, we thought the costs and risks involved in excluding the frequent attenders were outweighed by the benefits of simply treating these patients. At a community level, we thought that seeing the frequent attenders on demand in the acute receiving area was an efficient and relatively inexpensive way of supporting and maintaining these patients in the community.

No description available.

Neotyphodium endophytes (Claviceptaceae) are asexual filamentous fungi found living between the cells of many cool season forage grasses including tall fescue, meadow fescue and perennial ryegrass. They produce a range of alkaloids, including ergovaline and lolitrem B, which have been shown to be directly associated with the livestock disorders fescue toxicosis and ryegrass staggers syndrome, while others, including peramine and the lolines, have been linked to increased insect and drought resistance of the grass host. In the past decade, the Neotyphodium strains AR1, MaxQ and MaxP were selected because they did not produce the alkaloids associated with livestock disorders. Subsequently, artificial associations were established between them and commercial forage grass cultivars. The slow growth rate of Neotyphodium endophytes in vitro and the low success rate of the present methods for establishing artificial associations between endophytes and grass hosts are limiting the rate at which new novel endophytes can be incorporated into plant breeding programs and eventually commercialised. In this thesis, the type and concentration of the growth medium was shown to affect radial growth rate, colony appearance and mycelial morphology of three strains of Neotyphodium endophytes. The floret inoculation of meadow fescue with the U2 strain of N. uncinatum using several techniques involving liquid culture was attempted but was unsuccessful in creating any artificial associations. Neotyphodium endophytes are unstable in stored seed. In New Zealand, it is critical that pastures are infected with protective Neotyphodium endophytes to ensure that they will not be destroyed by exotic pests. The present methods for determining the percentage of viable endophyte infection of a seed lot are too slow for efficient use in the commercial seed industry. In this thesis, primers specific to the â-tubulin gene of N. coenophialum, N. lolii and N. uncinatum were designed and successfully used to detect these species in planta. However, using these primers to develop a method to accurately determine the viable endophyte infection rate of a seed lot using RT-qPCR was unsuccessful.

This study examined the ability of reaction time (RT) and Event-Related Potentials (ERP) to detect malingered neurocognitive deficit (MNCD)in two new tasks compared to the TOMM (N = 47). Honest (HON), malingering (MAL), and traumatic brain injury (TBI) groups were compared on accuracy, RT and ERP measures. Overall, the Test of Memory Malingering (TOMM) accuracy was the most effective at classifying groups (hit rate = 100%). Several non-TOMM accuracy variables and RT variables reached hit rates in the range of 71%-88%. The TOMM RT variable had an unlimited time for participants to respond and was the most successful RT variable compared to the Old/New and Repetition Priming tasks that had a short time limit for participants to respond (approximately 1.5 seconds). The classic old/new effect RT pattern was evident for both the HON and TBI groups with significantly faster RTs for old items compared to new items. A logistic regression was employed to see if a RT and/or ERP variable added any unique prediction power in detecting malingering. The frontal-posterior ERP difference score had unique prediction power to detect malingering when classifying MAL vs. TBI (hit rate = 86%). In the Old/New task, ERP responses of HON produced greater activity in the frontal region compared to the posterior region. The opposite trend was found in TBI (posterior activity andgt; frontal) and MAL showed no significant difference.

This thesis describes an action research study that took place in the context of increasing intervention by UK central government in the shaping and delivery of health services, and broadening expectations about who could deliver services. The study was aimed at exploring the issues arising from the development of the interprofessional care co-ordinator (IPCC) role in an acute in-patient setting. The role was new, introduced with an inherent flexibility that enabled IPCCs to speed patients through their in-patient stays as fast as clinically possible. None of the four IPCCs appointed held a registrable qualification in health or social care. A review of the literature identified that very little is known about care co-ordinator roles in practice, particularly those held by non-registered workers. The study reported in this thesis began two years after the IPCCs took up post. The study’s objectives were to describe the characteristics, impact, issues and influences on the role. A wide range of qualitative and quantitative data were gathered and analysed between October 1998 and July 2000 within the framework of an action research approach. The findings identified that the IPCC role had informally shifted over time to take up the complex discharge planning work previously carried out by nurses. This shift was not reflected in Trust policy and had not been accompanied by a review of training, regulation or supervision. This had led to situations of risk for some patients. The findings threw light on contextual factors that enabled the role shift and disrupted the reflective leadership and long-term overview needed to monitor and respond to the shift. These factors included nursing staff shortages and a turbulent environment for managers characterised by multiple pressures, top-down targets particularly for acute efficiency, and high managerial turnover. In addition, nurses did not perceive that they had an influence on the ongoing development of the IPCC role. The findings support Abbott’s (1988) theory that an occupational group can take up the discarded work of a higher status occupational group, but challenge the theory that the work discarded is always more routine than the work retained. They support theories of a growing challenge to the primacy of professional knowledge and the existence of an organisational culture in the NHS in which there are broader expectations of who can deliver which health services. They also indicate that role substitution can lead to the routinisation and marginalisation of aspects of patient care. The findings also illustrate how an innovation can continue to be re-invented following its establishment into routine practice, and how the journey of an innovation can be influenced by its context. The findings throw light on a role in practice that is a cameo of current policy on new roles and have a number of implications for practice, policy, education and research.

Im Graduiertenkolleg „Schlachttierbelastung und Produktsicherheit“ der Veterinärmedizinischen Fakultät der Universität Leipzig sollten in interdisziplinärer Zusammenarbeit Erkenntnisse zum Verhalten transportbelasteter Schlachtschweine in Bezug auf bakterielle Translokationsprozesse erarbeitet werden. Es wurden Mastschweine aus herkömmlichen Mastbetrieben definierten Belastungssituationen ausgesetzt , geschlachtet und untersucht. Dabei sollten physiologische, pathologisch-anatomische, immunologische, lebensmittel- und fleischhygienische, bakteriologische, virologische sowie ethologische Fragestellungen bearbeitet werden. Ziel war es festzustellen, ob eine Belastung der Tiere (u.a. durch den Transport) Auswirkungen auf die Produktqualität hat und ob durch eine Translokation pathogener Erreger ein Risiko für die Gesundheit des Verbrauchers besteht. Im Teilprojekt des Institutes für Virologie wurde untersucht, ob Rotavirus-Infektionen von Schlachtschweinen unter der Problematik der Belastung ein mögliches Infektionsrisiko für den Menschen darstellen. Um die zu erwartende niedrige Konzentration von Rotaviren in Organen von Schlachtschweinen nachweisen zu können, wurde eine kompetitive RT-PCR zum Nachweis von Rotaviren der Gruppe A verschiedener Spezies entwickelt. Dazu wurde ein sogenannter Kompetitor synthetisch hergestellt, welcher als interne oder externe Reaktionskontrolle eingesetzt wurde. Zum einen diente er der Überprüfung des ordnungsgemäßen Verlaufes einer RT-PCR, zum anderen wurde er zur Herstellung von Standards verwendet. Die RT-PCR wurde anschließend in eine „Real time“ RT-PCR umgewandelt. Sowohl mit der herkömmlichen als auch mit der „Real-time“ RT-PCR konnten 10 spezifische RNA-Moleküle in einer Probe nachgewiesen werden. In einer SPF-Schweineherde, welche einer Belastung infolge eines Tiertransports ausgesetzt war, konnten mit Hilfe der RT-PCR klinisch gesunde intermittierende Rotavirus-Ausscheider entdeckt werden. Bei einigen dieser Tiere gelang der Nachweis der Virusausscheidung über einen Zeitraum von drei Monaten. Nach der Schlachtung wurden in Organen des lymphatischen Systems bei zwei Schweinen sehr geringe Konzentrationen an rotavirus-spezifischer RNA detektiert. Infektiöses Virus konnte daraus allerdings nicht isoliert werden. Auch in einer Mastschweineherde konnte bei einigen Tieren Rotavirus-spezifische RNA im Kot nachgewiesen werden. Ein Infektionsversuch dieser Tiere mit Salmonella typhimurium konnte keine Reaktivierung der Rotavirus-Infektion auslösen. Aufgrund des Zoonose-Potentials von Rotaviren kann nach den Untersuchungen ein Infektionsrisiko für den Verbraucher durch eine endogene Kontamination von Schlachttieren mit Rotaviren nicht sicher ausgeschlossen werden. Die Untersuchungen zeigten auch, dass intermittierende Rotavirus-Ausscheider ein Infektionsrisiko für den Verbraucher darstellen, wenn z.B. bei der Schlachtung der Tiere oder bei der Verarbeitung des Fleisches dieser Tiere hygienische Grundregeln verletzt werden. Besonders gefährdet wären hierbei Neugeborene, Kinder, Senioren und immunsupprimierte Personen. Ein Ort einer Viruspersistenz in Organen konnte auch nach diesen Untersuchungen nicht gefunden werden. Dennoch scheint es, dass Rotaviren in der Natur oder in einer Population von Menschen oder Tieren persistieren. Durch ständige Neuinfektionen bzw. Reinfektionen empfänglicher Organismen haben Rotaviren so ihre Erhaltung gesichert. / Within the graduate programme “Schlachttierbelastung und Produktsicherheit” of the Veterinary Faculty of the University of Leipzig, the behaviour of slaughter swine exposed to the stress of transport was observed in an interdisciplinary collaboration concerning the translocation processes of bacteria. Fattened pigs from conventional pig fattening units were exposed to particular stress situations, and then slaughtered and examined. The following aspects of this process were investigated: physiology, pathological-anatomy, immunology, food and meat hygiene, bacteriology, virology and ethology. The aim of this study was to verify whether exposing the animals to stress situations (such as transport) influences the quality of the product and whether the translocation of pathogens represents a risk for consumer health. Within the sub-project of the Institute for Virology, analyses were made to verify whether rotavirus infections of slaughter swine exposed to stress situations represents a potential contamination risk for humans. In order to detect the expected low concentration of rotaviruses in the organs of slaughtered pigs, a competitive RT-PCR was developed as a test of rotaviruses for various group A species. To do this, a so-called competitor was synthetically created, which was used as an internal and external reaction control. On one hand, it was used to verify the regular course of an RT-PCR reaction, and on the other hand, it was implemented to develop standards. RT-PCR was then modified by means of a real time RT-PCR. Both with the conventional and with the real time RT-PCR, it was possible to detect 10 specific RNA molecules/ sample.With this new very sensitive and specific amplification process, it was possible to detect rotavirus-specific RNA in the excrement of people and of pigs, cows, horses, rabbits and monkeys. the evidence of the virus excretion was produced over a time period of three months. After slaughtering, low amounts of rotavirus specific RNA were found in the organs of the lymphatic system. There were no indications that any of these organs were infectious. Rotavirus specific RNA was also found in the excrement of some fattened pigs. An attempt to infect these animals with Salmonella typhimurium was unable to cause any reactivation of the rotavirus infection. An infection risk for the consumer through an endogenous rotavirus contamination of fattened pigs cannot be excluded with any degree of certainty on the basis of these analyses due to the zoonotic potential of rotaviruses. The analyses also showed that intermittent rotavirus excretors represent an infection risk for the consumer, if for example basic hygiene rules are broken during the slaughter or meat processing of these animals. At special risk may be new-borns, children, youth, the elderly and people suffering from immunodeficiency. These examinations could not find a specific place in the organs where the virus persists. Nevertheless, it seems that rotaviruses persist in the environment or in a population of people or animals. With constant new infections or re-infections of receptive organisms, rotaviruses seem to have assured their survival.