O conhecimento da dinâmica do potássio no sistema solo-planta é importante para entender a disponibilidade desse nutriente as culturas. O uso de isótopos possibilita especificar a quantidade do nutriente recuperada pela planta proveniente do fertilizante. O 42K, isótopo radioativo do potássio, apresenta meia-vida de 12,4 horas o que impossibilita o seu uso em pesquisas relacionadas à eficiência dos fertilizantes. Como alternativa há o Rb que é análogo ao K, tem propriedades químicas semelhantes, e realiza algumas funções do K nas plantas. Entretanto, por não se tratar do mesmo elemento, persiste a dúvida em relação à quantidade de Rb que não prejudique o desenvolvimento das culturas a fim de usá-lo como marcador. Essa pesquisa tem como objetivo identificar as relações de massa entre o K e o Rb em estudos de aproveitamento do nutriente. O experimento foi desenvolvido em casa de vegetação com delineamento experimental em blocos ao acaso e quatro repetições. Para verificação do objetivo estudou-se dois métodos, da substituição de K por Rb e da adição de Rb. No método da substituição foram utilizados os seguintes tratamentos: sem Rb, 10%, 20%, 40%, 50%, 70%, 85% e 100% de Rb; no método de adição utilizou os seguintes tratamentos: sem Rb, 10%, 20%, 40%, 50%, 70%, 85% e 100% de Rb adicionado ao total aplicado de K (150 mg dm-3). Para avaliação dos tratamentos o milho foi conduzido até o estádio V8, ao final do qual determinaram-se atributos relacionados ao desenvolvimento e a fotossíntese, os teores de Rb, de macronutrientes (K, N, P, S, Ca e Mg) e micronutrientes (Mn, Fe, Zn e Cu) na parte aérea e nas raízes da planta. A substituição do K pelo Rb prejudicou o desenvolvimento do milho, sem haver dano à planta na adição. Os teores de nutrientes na parte aérea e nas raízes variaram diferenciadamente em cada método. A eficiência de recuperação do K foi superior à do Rb. O desenvolvimento da planta foi limitado pela substituição de 10% do K por Rb. A adição máxima aceitável de Rb foi 30%, estabelecida pelo decréscimo nos teores de nutrientes. / The knowledge of the potassium dynamics in the soil-plant system is important to understand the nutrient availability in cultures. The use of isotopes allows to specify the amount of nutrient recovered by the plant from the fertilizer. The 42K, radioactive isotope of potassium has a half-life of 12.4 hours, which precludes its use in research related to the efficiency of fertilizers. Alternatively, Rb is analogous to K and has similar chemical properties, thus, it can perform certain K functions in plants. However, because it is not the same element, questions arise regarding the amount of Rb that does not harm the plant development when used for K labeling. This research aims to identify the relationship between the mass of K and Rb in nutrient utilization studies. The experiment was conducted in a greenhouse in a randomized block design with four replications. Two methods, replacing K by Rb and adding Rb, were used. In the replacement method, the following treatments were used: without Rb, 10%, 20%, 40%, 50%, 70%, 85% and 100% of Rb, and in the addition method: without Rb, 10%, 20%, 40%, 50%, 70%, 85% and 100% of the Rb added to total applied K (150 mg dm-3). For the evaluation of treatments, maize plants were grown until the V8 stage, after which the attributes related to development and photosynthesis, contents of Rb, macronutrients (K, N, P, S, Ca and Mg) and some micronutrients (Mn, Fe, Cu and Zn) were determined in the shoots and roots of the plant. The replacement of K by Rb hindered the corn development, and no damage was observed to the plant with the addition of Rb. Nutrient concentration in the shoots and roots varied differently in each method. The efficiency of recovery was higher for K. The plant growth was limited by the replacement of 10% of K by Rb. The maximum acceptable addition of Rb was 30% established by the decrease in nutrient content.

Strontium-82 (t1/2 = 25.5 d) is one of the medical isotopes produced on a large scale at the Isotope Production Facility (IPF) of the Los Alamos National Laboratory (LANL), employing a high intensity 100 MeV proton beam and RbCl targets. A constant increase in the 82Sr demand over the last decade combined with an established thermal limit of molten RbCl salt targets [1,2] has challenged the IPF’s world leading production capacity in recent years and necessitated the consideration of low-melting point (39.3 °C) Rb metal targets. Metal targets are used at other facilities [3–5] and offer obvious production rate advantages due to a higher relative density of Rb target atoms and a higher expected thermal performance of molten metal. One major disadvantage is the known violent reaction of molten Rb with cooling water and the potential for facility damage following a catastrophic target failure. This represents a significant risk, given the high beam intensities used routinely at IPF. In order to assess this risk, a target failure experiment was conducted at the LANL firing site using a mockup target station. Subsequent fabrication, irradiation and processing of two prototype targets showed a target thermal performance consistent with thermal modeling predictions and yields in agreement with predictions based on IAEA recommended cross sections [6]. Target failure test: The target failure test bed (FIG. 1) was constructed to represent a near replica of the IPF target station, incorporating its most important features. One of the most vulnerable components in the assembly is the Inconel beam window (FIG. 2) which forms the only barrier between the target cooling water and the beam line vacuum. The test bed also mimicked relevant IPF operational parameters seeking to simulate the target environment during irradiation, such as typical cooling water flow velocities around the target surfaces. While the aggressive thermal effects of the beam heating could not be simulated directly, heated cooling water (45 °C) ensured that the rubidium target material remained molten during the failure test. A worst case catastrophic target failure event was initiated by uncovering an oversized predrilled pinhole (1 mm Φ) to abruptly expose the molten target material to fast flowing cooling water. Prototype target irradiations: Two prototype Rb metal target containers were fabricated by machining Inconel 625 parts and by EB welding. The target containers were filled with molten Rb metal under an inert argon atmosphere. Follow-ing appropriate QA inspections, the prototype targets were irradiated in the medium energy slot of a standard IPF target stack using beam currents up to 230 µA. After irradiation the targets were transported to the LANL hot cell facili-ty for processing and for 82Sr yield verification. During the target failure test, cooling water conductivity and pressure excursions in the target chamber were continuously monitored and recorded at a rate of 1 kHz. Video footage taken of the beam window and the pinhole area combined with the recorded data indicated an aggressive reaction between the Rb metal and the cooling water, but did not reveal a violent explosion that could seriously damage the beam window. These observations, together with thermal model predictions, provided the necessary confidence to fabricate and fill prototype targets for irradiation at production-scale beam currents. X-ray imaging of filled targets (FIG. 3) shows a need for tighter control over the target fill level. One prototype target was first subjected to lower intensity (< 150 µA) beams before the second was irradiated at production level (230 µA) beams. During irradiation, monitoring of cooling water conductivity indicated no container breach or leak and, as anticipated given the model predictions, the post irradiation target inspection showed no sign of imminent thermal failure (see FIG. 4). Subsequent chemical processing of the targets followed an established procedure that was slightly modified to accommodate the larger target mass. TABLE 1 shows that post chemistry 82Sr yields agree to within 2 % of the in-target production rates expected on the basis of IAEA recommended cross sections. The table also compares 82Sr yields from the Rb metal targets against yields routinely obtained from RbCl targets, showing an increase in yield of almost 50 %.

O conhecimento da dinâmica do potássio no sistema solo-planta é importante para entender a disponibilidade desse nutriente as culturas. O uso de isótopos possibilita especificar a quantidade do nutriente recuperada pela planta proveniente do fertilizante. O 42K, isótopo radioativo do potássio, apresenta meia-vida de 12,4 horas o que impossibilita o seu uso em pesquisas relacionadas à eficiência dos fertilizantes. Como alternativa há o Rb que é análogo ao K, tem propriedades químicas semelhantes, e realiza algumas funções do K nas plantas. Entretanto, por não se tratar do mesmo elemento, persiste a dúvida em relação à quantidade de Rb que não prejudique o desenvolvimento das culturas a fim de usá-lo como marcador. Essa pesquisa tem como objetivo identificar as relações de massa entre o K e o Rb em estudos de aproveitamento do nutriente. O experimento foi desenvolvido em casa de vegetação com delineamento experimental em blocos ao acaso e quatro repetições. Para verificação do objetivo estudou-se dois métodos, da substituição de K por Rb e da adição de Rb. No método da substituição foram utilizados os seguintes tratamentos: sem Rb, 10%, 20%, 40%, 50%, 70%, 85% e 100% de Rb; no método de adição utilizou os seguintes tratamentos: sem Rb, 10%, 20%, 40%, 50%, 70%, 85% e 100% de Rb adicionado ao total aplicado de K (150 mg dm-3). Para avaliação dos tratamentos o milho foi conduzido até o estádio V8, ao final do qual determinaram-se atributos relacionados ao desenvolvimento e a fotossíntese, os teores de Rb, de macronutrientes (K, N, P, S, Ca e Mg) e micronutrientes (Mn, Fe, Zn e Cu) na parte aérea e nas raízes da planta. A substituição do K pelo Rb prejudicou o desenvolvimento do milho, sem haver dano à planta na adição. Os teores de nutrientes na parte aérea e nas raízes variaram diferenciadamente em cada método. A eficiência de recuperação do K foi superior à do Rb. O desenvolvimento da planta foi limitado pela substituição de 10% do K por Rb. A adição máxima aceitável de Rb foi 30%, estabelecida pelo decréscimo nos teores de nutrientes. / The knowledge of the potassium dynamics in the soil-plant system is important to understand the nutrient availability in cultures. The use of isotopes allows to specify the amount of nutrient recovered by the plant from the fertilizer. The 42K, radioactive isotope of potassium has a half-life of 12.4 hours, which precludes its use in research related to the efficiency of fertilizers. Alternatively, Rb is analogous to K and has similar chemical properties, thus, it can perform certain K functions in plants. However, because it is not the same element, questions arise regarding the amount of Rb that does not harm the plant development when used for K labeling. This research aims to identify the relationship between the mass of K and Rb in nutrient utilization studies. The experiment was conducted in a greenhouse in a randomized block design with four replications. Two methods, replacing K by Rb and adding Rb, were used. In the replacement method, the following treatments were used: without Rb, 10%, 20%, 40%, 50%, 70%, 85% and 100% of Rb, and in the addition method: without Rb, 10%, 20%, 40%, 50%, 70%, 85% and 100% of the Rb added to total applied K (150 mg dm-3). For the evaluation of treatments, maize plants were grown until the V8 stage, after which the attributes related to development and photosynthesis, contents of Rb, macronutrients (K, N, P, S, Ca and Mg) and some micronutrients (Mn, Fe, Cu and Zn) were determined in the shoots and roots of the plant. The replacement of K by Rb hindered the corn development, and no damage was observed to the plant with the addition of Rb. Nutrient concentration in the shoots and roots varied differently in each method. The efficiency of recovery was higher for K. The plant growth was limited by the replacement of 10% of K by Rb. The maximum acceptable addition of Rb was 30% established by the decrease in nutrient content.

Strontium-82 (t1/2 = 25.5 d) is one of the medical isotopes produced on a large scale at the Isotope Production Facility (IPF) of the Los Alamos National Laboratory (LANL), employing a high intensity 100 MeV proton beam and RbCl targets. A constant increase in the 82Sr demand over the last decade combined with an established thermal limit of molten RbCl salt targets [1,2] has challenged the IPF’s world leading production capacity in recent years and necessitated the consideration of low-melting point (39.3 °C) Rb metal targets. Metal targets are used at other facilities [3–5] and offer obvious production rate advantages due to a higher relative density of Rb target atoms and a higher expected thermal performance of molten metal. One major disadvantage is the known violent reaction of molten Rb with cooling water and the potential for facility damage following a catastrophic target failure. This represents a significant risk, given the high beam intensities used routinely at IPF. In order to assess this risk, a target failure experiment was conducted at the LANL firing site using a mockup target station. Subsequent fabrication, irradiation and processing of two prototype targets showed a target thermal performance consistent with thermal modeling predictions and yields in agreement with predictions based on IAEA recommended cross sections [6]. Target failure test: The target failure test bed (FIG. 1) was constructed to represent a near replica of the IPF target station, incorporating its most important features. One of the most vulnerable components in the assembly is the Inconel beam window (FIG. 2) which forms the only barrier between the target cooling water and the beam line vacuum. The test bed also mimicked relevant IPF operational parameters seeking to simulate the target environment during irradiation, such as typical cooling water flow velocities around the target surfaces. While the aggressive thermal effects of the beam heating could not be simulated directly, heated cooling water (45 °C) ensured that the rubidium target material remained molten during the failure test. A worst case catastrophic target failure event was initiated by uncovering an oversized predrilled pinhole (1 mm Φ) to abruptly expose the molten target material to fast flowing cooling water. Prototype target irradiations: Two prototype Rb metal target containers were fabricated by machining Inconel 625 parts and by EB welding. The target containers were filled with molten Rb metal under an inert argon atmosphere. Follow-ing appropriate QA inspections, the prototype targets were irradiated in the medium energy slot of a standard IPF target stack using beam currents up to 230 µA. After irradiation the targets were transported to the LANL hot cell facili-ty for processing and for 82Sr yield verification. During the target failure test, cooling water conductivity and pressure excursions in the target chamber were continuously monitored and recorded at a rate of 1 kHz. Video footage taken of the beam window and the pinhole area combined with the recorded data indicated an aggressive reaction between the Rb metal and the cooling water, but did not reveal a violent explosion that could seriously damage the beam window. These observations, together with thermal model predictions, provided the necessary confidence to fabricate and fill prototype targets for irradiation at production-scale beam currents. X-ray imaging of filled targets (FIG. 3) shows a need for tighter control over the target fill level. One prototype target was first subjected to lower intensity (< 150 µA) beams before the second was irradiated at production level (230 µA) beams. During irradiation, monitoring of cooling water conductivity indicated no container breach or leak and, as anticipated given the model predictions, the post irradiation target inspection showed no sign of imminent thermal failure (see FIG. 4). Subsequent chemical processing of the targets followed an established procedure that was slightly modified to accommodate the larger target mass. TABLE 1 shows that post chemistry 82Sr yields agree to within 2 % of the in-target production rates expected on the basis of IAEA recommended cross sections. The table also compares 82Sr yields from the Rb metal targets against yields routinely obtained from RbCl targets, showing an increase in yield of almost 50 %.

Obwohl der Bedeutung von hyporheischen Zonen als Übergangsbereiche zwischen Flüssen und angrenzenden alluvialen Aquiferen eine wachsende Anerkennung zuteilwird, sind dynamische hyporheische Reaktionen auf instationäre hydrologische Bedingungen weiterhin signifikant untererforscht. Um diese Lücke zu schließen, liegt der Fokus dieser Doktorarbeit insbesondere auf den Effekten transienter Abflussverhalten und Temperaturschwankungen in Flüssen auf die raumzeitliche Variabilität von hyporheischen Austauschprozessen. Unter Beachtung dieser Ziele wird ein neues physikalisch basiertes numerisches Modell vorgeschlagen und schließlich angewandt, um systematisch die hyporheischen, durch Sedimentoberflächenstrukturen ausgelösten Reaktionen auf eine Reihe von künstlichen und natürlichen Abflussregimen abzuschätzen. Parameter wie das räumliche Ausmaß der hyporheischen Zone, hyporheische Austauschrate, mittlere Aufenthaltszeit, Temperatur des hyporheischen Flusses sowie das Denitrifikationspotenzial werden definiert, um den Einfluss der Antriebskräfte und Regulatoren auf dynamische hyporheische Reaktionen zu quantifizieren. Die Ergebnisse zeigen, dass mit zunehmendem Abfluss generell das räumliche Ausmaß der hyporheischen Zone vergrößert wird; jedoch bestimmen geomorphologische Bedingungen und Grundwasserflüsse erheblich das Ausdehnen und Zusammenziehen hyporheischer Zonen zusammen mit Strömungen, Wärme- und Stoffaustausch zwischen Fluss und Grundwasser. Temperaturvariabilität, ein wichtiger Faktor, welcher oft in hydrodynamischen Studien vernachlässigt wird, zeigt direkte kontrollierende Effekte beim Bestimmen hyporheischer Austauschraten und mittlerer Aufenthaltszeiten. Weiterhin spielt die Dynamik von Grundwasserständen eine entscheidende Rolle bei hyporheischen Austauschprozessen. Das Optimieren der Terminierung von Grundwasserförderung ist ausschlaggebend für die Regulierung von Wasserqualität, Nährstoffkreisläufen und der Entstehung thermischer hyporheischer Refugien. / Although there is a growing recognition of the importance of hyporheic zones as transitional areas connecting rivers and adjacent alluvial aquifers, the dynamic hyporheic responses to unsteady hydrological conditions are still significantly understudied. To bridge this gap, the present PhD thesis primarily focuses on the effects of transient river discharge and temperature fluctuations on the spatiotemporal variability of hyporheic exchange processes. With these objectives in mind, a novel physically based numerical model is proposed and then applied to systematically evaluate bedform-induced hyporheic responses to a series of synthetic and natural hydrological regimes. Metrics including spatial hyporheic extent, hyporheic exchange rate, mean residence time, temperature of hyporheic flux, and denitrification potential are defined to quantify the impact of drivers and modulators of dynamic hyporheic responses. Results indicate that increasing river discharge generally enlarges the spatial hyporheic extent; however, geomorphological settings and groundwater fluxes substantially modulate the expansion and contraction of hyporheic zones along with flow, heat and solute exchange between river and groundwater. Temperature variability, an important factor which is often neglected in hydrodynamic studies, displays direct controlling effects in determining hyporheic exchange rates and mean residence times. Groundwater table dynamics also play a critical role in hyporheic exchange processes. Optimizing the timing of aquifer pumping is crucial for regulation of water quality, nutrient cycling, and the formation of thermal hyporheic refugia. The findings largely advanced our mechanistic understandings of dynamic hyporheic responses to varying transient flow and temperature conditions, and therefore shed lights on improving river management and restoration strategies.

Clostridium difficile, a Gram positive, anaerobic, spore-forming bacterium is the commonest cause of hospital acquired infection in the UK. The organism initiates infection through spore formation and attachment, germination in the gut and then the production of two potent cytotoxins; toxins A and B. While the contribution of toxins A and B to infection is beyond dispute the relative importance of each toxin is a subject of debate. Thus diagnostic assays capable of rapidly detecting the presence of both toxins are needed. To develop such an assay we first characterised the structure of C. difficile spores to better understand their role in pathogenicity and adherence to organic and inorganic surfaces. Following attachment the spore germinates and the resulting vegetative bacteria express toxins. To facilitate the development of an assay capable of detecting both toxins, we employed a bioinformatics based approach which identified highly conserved nucleotide sequences within regions of each toxin which we hypothesised were under strict selective pressure. The specificity of the probes identified was confirmed using a panel of 58 clinical C. difficile isolates, related Clostridium isolates, non-related species and human gut metagenomic DNA samples. Selected probes were incorporated into a metal enhanced fluorescent assay platform and their ability to detect the organism in various organic backgrounds was determined. We were able to detect as few as 10 bacteria in 500 μl of human faecal material within 40 seconds, suggesting that this approach has the potential to be developed into a commercial assay. To support the development of this assay we sought to develop an insect infection model using the worm Manduca sexta. Our inability to initiate infection, inspite of the fact that bioinformatic analysis revealed the presence of genes with homology to known insect virulence factors, suggests that C. difficile may have potential evolutionary association to invertebrates.

Osteosarcoma (OS) is the most common bone cancer in dogs. It is biologically aggressive and <20% survive >2 years with standard therapy. Hence, new approaches must be considered. TP53 is altered in ~50% of human and canine cancers, including OS, making it a candidate for targeted suicide gene therapy strategies. Canine OS is considered to be a good model for human OS. The aims of this study were to:  examine the site incidence of canine OS retrieved from Glasgow University Veterinary School (GUVS) histology database;  perform TP53 mutational analysis in canine OS cases diagnosed at GUVS;  investigate delivery of exogenous wild-type canine TP53 into D17, CMT3, CMT7 and CMT8 canine OS cell lines;  design and construct vectors for a TP53-targeted suicide gene strategy, which can selectively target canine OS cells containing accumulated TP53, and initially analyse using Dual-Luciferase® reporter assays (DLR);  perform suicide gene/prodrug assays using nitroreductase (NTR) in combination with CB1954 or nitrofurazone (NFZ) in several canine cell lines;  replace luciferase with NTR in vectors for TP53-targeted suicide gene strategy, and with CB1954, determine if survival of CMT7 cells possessing accumulated TP53 are reduced, in comparison to D17 cells, containing wild-type TP53. OS were most commonly found in appendicular areas, followed by axial and extraskeletal sites; this agrees with published findings. No TP53 mutations were found in 7 biopsies removed from 4 dogs, 5 were OS, due to analysis of a small sample number, but still fits within published data. TP53 expression did not have a significant negative effect on canine OS cell growth. Contrasting results have been shown in canine and human OS cells. Luciferase expression levels following transfection with designed constructs were higher in CMT7 cells, than in D17 cells. Similar results were shown in NTR/CB1954 assays as reductions in cell survival only occurred in CMT7 cells but not in D17 cells. NFZ was not suitable as a prodrug for NTR in canine cells as there were no differences in cell survival with cells not expressing NTR. Hence, the TP53-targeted suicide gene therapy strategy appears to selectively reduce survival of canine OS cells possessing accumulated TP53, warranting further investigation as a treatment modality for OS, in both dogs and humans.

Breast cancer is the most common female cancer in Scotland, in common with many Western countries. This thesis aimed to analyse changes in the incidence and molecular epidemiology of breast cancer in Scotland. Part 1 concentrated on epidemiological research, with data derived from various agencies, and Part 2 on a laboratory project aimed at looking at changes in the molecular profile of breast cancers in two cohorts of patients in Glasgow. The period between 1987 and 1994 in which coverage of the country by the breast screening programme gradually increased was expected to raise incidence rates as seen in studies in Scandinavia and elsewhere; after 1994, incidence rates should have returned to normal in women aged 55-64, with incidence in women aged 50-54 remaining slightly above pre-existing rates. An observed/expected analysis of breast cancer incidence rates after 1994 was performed; this showed a 58% increase in rates in women aged 50-54 above that which would have been expected had the trends continued as expected in the absence of screening. In 55-59 year olds and 60-64 year olds there were 42% and 40% increases, respectively, above expected rates. Reproductive risk factors such as low parity and late age at first pregnancy are important risk factors in breast cancer. Reproductive risk factors are likely to affect the ‘birth-cohort’ incidence of breast cancer but the temporal effects of breast screening make this difficult to interpret. Breast cancer incidence in Scotland by year of birth was examined using a Lexis diagram. In women aged 50-54, 55-59 and 60-64, breast cancer incidence rates increased by birth cohort during the presence of the prevalent round of screening, a finding which is likely to have been due to detection of large numbers of asymptomatic tumours. However, in women who were offered screening after the prevalent round, incidence continued to rise with successive birth year, suggesting a contribution from risk factors. This is the first study of birth cohort incidence of breast cancer and its relation to screening (published in Breast Cancer Research and Treatment). The contribution of screening and risk factors to breast cancer incidence in Scotland was also assessed. A small rise in screening uptake between 1990 and 2001 and an increase in standardised detection ratio may indicate that screening improvements could be contributing to increasing incidence. The number of first pregnancies to women in Scotland aged 35-59 has risen from several hundred in 1976 to 2000 in 2001. A plot of completed family size in Scotland against maternal birth year shows that a steadily declining trend has been developing since the 1935 birth cohort. Based on data from the Scottish Health Surveys, the percentage of women with a BMI of over 25 has increased from 47.2% to 57.3% between 1995 and 2003. Mean BMI in women has increased from 25.7 to 26.9 over the same period. It is likely that the observed changes have contributed to changes in breast cancer incidence in Scotland. Using prescription and population data, the prevalence of HRT use in women aged 40-64 in Scotland was estimated; this estimated prevalence has increased from 13.8% in 1993 to 17% in 2001. It is difficult to know if this small increase in prevalence of HRT could have influenced breast cancer epidemiology. A study of breast cancer incidence by deprivation quintile showed that breast cancer incidence between 1991 and 2000 rose in all quintiles. Interaction analysis suggested that breast cancer incidence is rising to the same extent in deprived and affluent women. The risk factor analyses above were also applied to women of different socioeconomic standing (the results were published in Breast Cancer Research and Treatment). A laboratory project was carried out to assess whether increasing survival from breast cancer could be a result of changing molecular epidemiology. This project was an comparison of the prevalence of breast cancers which were ER, PR and Her2 positive and of different grades in two cohorts of Glasgow patients, from 1984-86 and 1996-1997. The application of current molecular techniques to stored tissue aimed to improve the quality of data compared to previous studies based on clinical databases using heterogeneous techniques. There were significant differences in grade distribution of tumours in the two cohorts (p=0.009) with fewer grade 1 and more grade 3 tumours in the second cohort. Further study showed the grade difference to be exerted by the tumours in screened women in the second cohort with there being no difference in grade between symptomatic patients in the two groups. 64.2% of the tumours in cohort 1 and 71.5% of the tumours in cohort 2 were ER positive (p=0.042); this is also likely to be a clinically significant difference. The difference between the cohorts appeared to be exerted by high percentage of screen-detected tumours in cohort 2 being ER positive; however this finding still supports a theory of changing biology. 44.9% of the tumours in cohort 1 and 49.9% of tumours in cohort 2 were PR positive (p=0.181). 21.5% of tumours in cohort 1 and 20.6% of tumours in cohort 2 were Her-2 positive; this was not a significant difference. An increase in ER positivity was seen in all age groups in the study, though multivariate analysis did suggest a contribution from a higher number of women over 60 in the more recent cohort. Kaplan-Meier analysis showed survival to be higher in the second cohort than the first. There was a significant difference in survival between ER positive and negative patients. Cox’s regression was performed; as expected this showed a multifactorial contribution to increases in survival in these cohorts rather than it being entirely due to changes in ER status. However the changes in ER status shown in a population of Glasgow patients over time may mean that the results of clinical trials carried out in many years ago need to be interpreted with caution when applying them to the women of today. The results of this project were published in the British Journal of Cancer. Overall, the epidemiological studies within this thesis shed an important new light on the factors contributing to breast cancer incidence in Scotland, with a major finding being a significant association between birth cohort and breast cancer incidence suggesting a significant impact being made by reproductive risk factors. This hypothesis is supported by the analysis of risk factor trends in Scotland undertaken within the thesis. The laboratory study has shown a significant lowering in grade and increase in ER positive status of tumours in a cohort of Glasgow women over time; while the changes are statistically explainable by known effects of a breast screening programme on tumour detection they could still represent a true change in biology. The results of all the studies contained in the thesis could have significant implications for future health service planning.

Abstract Background: Pulmonary hypertension (PH) is a rare condition that can occur as a primary disease process, Idiopathic Pulmonary Hypertension (IPH) or secondary to other disorders. In Familial IPH mutations have been identified in the bone morphogenetic protein receptor II gene (BMPRII) (chromosome 2q32-31) a member of the Transforming Growth Factor  (TGF) (Lane et al, 2000). Despite the mutation being present in all cells, vascular wall remodelling is only seen in the pulmonary circulation with marked thickening of the intima and neointimal formation, muscularisation of small-generation resistance vessels and thickening of the adventitial layer together with increased ECM deposition. Similar appearances are noted in the pulmonary circulation’s response to hypoxia. for this projectProlonged exposure of the pulmonary circulation to hypoxia results in vasoconstriction and subsequent vascular wall remodelling. The hypothesis of this work is that the pulmonary circulation’s response to hypoxia may be partially explained by the existence of differences exist in cell signalling pathways in between adventitial fibroblasts from pulmonary and systemic arteries in HPAF. Studies from the Scottish Pulmonary Vascular (SPVU) Laboratory have shown that pulmonary arterial fibroblasts (PAFB) in bovine and rat models of acute hypoxic exposure preferentially proliferate to hypoxia, whereas systemic arterial fibroblasts (SAFB) do not , that the stress mitogen activated protein kinase p38 MAPK is consistently activated in PAFB exposed to acute hypoxia, and is constitutively upregulated in PAFB cultured from rats exposed to chronic hypoxia (Welsh et al, 1998; Welsh et al; 2001). This response to hypoxic exposure has been shown to be dependent on p38 MAPK activity, as use of SB203580 can block the hypoxia-mediated proliferative response to acute hypoxia (Scott et al, 1998; Welsh et al, 2001). Aims and methods: We wished to establish whether the pro-proliferative response of PAFB to acute hypoxic exposure previously noted in bovine and rat models could also be demonstrated in a human model. We wished to establish a role for both classic MAPK and stress MAPKs in hypoxia-mediated PAFB proliferation. We also wished to examine the role of hypoxia inducible factor 1 (HIF1) in human arterial fibroblast responses to acute hypoxia. There is a body of literature that documents cross talk between p38 MAPK and the Bone Morphogenetic Protein (BMPR) signalling pathways. We wished to establish whether Smad proteins (involved in the downstream signalling cascade from BMPR) might play a role in human pulmonary and systemic arterial fibroblast proliferation to acute hypoxia. Following approval from the local Ethics Committee, PAFB were harvested from patients undergoing lobectomy for the treatment of lung cancer. Left internal mammary arteries (SAFB) were harvested from patients undergoing coronary artery bypass grafting. Cells from systemic and pulmonary arterial fibroblasts were grown in conditions of normoxia or acute hypoxia (PO2 35 mmHg ~ 5% O2). Cellular proliferation was assessed using [3H]Thymidine uptake as a surrogate. p38, p44/p42 - ERK1/2 and JNK MAPKs and Smad protein activity was assessed using Western Blotting Techniques with the use of appropriate primary and secondary antibodies and Chemiluminescence to detect the presence of protein. p38 MAPK isoform activity was assessed using Catch and Release® immunophoresis techniques. Findings and conclusions: We demonstrated that acute hypoxic exposure results in human PAFB proliferation, associated with increased p44/p42 – ERK 1/2 MAPK activity, but dependent on p38 MAPK  activity. We also found that the p38 MAPK  isoform was expressed in human PAFB following hypoxic exposure but this did not appear to be involved in the hypoxia-mediated proliferative response. p38 MAPK  activity appeared to occur in a bi-phasic pattern with peaks of activity at t = 6 and 16 hours, the second peak was found to be responsible for the hypoxia-mediated proliferation seen in these cells in agreement with previous work from the SPVU laboratory (Scott et al, 1998; Welsh et al., 2001). The second peak in p38 MAPK  activity was synchronous with peak HIF1 activity (between t = 8 –16 hours). We demonstrated that HIF1 activity can be abrogated by pre-incubation of human PAFB with SB203580 suggesting a mechanistic link between p38 MAPK  activation and HIF1 in a human model of acute hypoxic exposure. We have also demonstrated that that BMPR2-associated Smad 1, 5 and 8 activation is increased in hypoxic human SAFB, suggestive of the activation of an anti-proliferative pathway in these cells that is not associated with p38 MAPK activity. To our knowledge this is the first demonstration of an active response in SAFB to acute hypoxic exposure that involves the active upregulation of an anti-proliferative pathway in these cells. In addition we have demonstrated that in hypoxic pulmonary arterial fibroblasts phospho Smad 1, 5 and 8 expression is reduced (suggestive of the down-regulation of an anti-proliferative pathway) and can be further abrogated by pre-incubation with SB203580. This suggests that in SAFB Smad 1, 5 and 8 activation occurs independent of p38 MAPK activation while in PAFB, p38 MAPK activity augments Smad 1, 5 and 8 activation.

Feline immunodeficiency virus (FIV) is an important veterinary pathogen with comparative significance because of its similarities to its human counterpart HIV. Since FIV is the only non-primate lentivirus which induces AIDS-like symptoms in its natural host, it serves as a valuable animal model for both prophylactic and therapeutic studies of HIV. It is accepted that the induction of neutralising antibodies (NAbs) is a key element in the control of lentiviral infection, since T-cell based vaccines alone failed to prevent infection in most experimental animal model systems. In this project a robust and reproducible in vitro neutralisation assay was developed and optimised, permitting the assessment of the NAb response in naturally infected cats and with the potential to evaluate candidate vaccines. It was demonstrated that, in general, primary FIV strains in the UK belong to subtype A, and therefore the development of a regional, subtype A-specific, FIV vaccine could be considered for use in the UK. The identification of a neutralisation resistant isolate of FIV led to the finding that a linear neutralisation determinant was located within the V5 region of Env and mutations in this region may lead to immune evasion in vivo. In addition, a second neutralisation determinant was identified in the C3/V4 region of Env. Finally, it was observed that a small proportion of naturally infected cats generated NAbs against FIV. Of these, only a very small proportion of the cats had antibodies with the potential to cross neutralise strains within the same subtype as the homologous isolate. Nonetheless, a plasma sample from a single cat was identified that neutralised all strains tested, including strains from different subtypes and geographical regions. It is likely that studies of the homologous isolate that induced the broad NAb response may be capable of inducing a similar broad response in vaccinated cats. Such a finding would have important implications for the design of potential novel lentiviral immunogens.