As sÃndromes mielodisplÃsicas (SMD) representam um grupo heterogÃneo de doenÃas clonais que acometem a cÃlula precursora hematopoÃtica pluripotente, caracterizando-se por baixa contagem de cÃlulas no sangue perifÃrico, displasia em uma ou mais linhagens celulares, hematopoese ineficiente, alÃm do risco aumentado de progressÃo para leucemia mielÃide aguda. Embora a doenÃa possa acometer pacientes de outras faixas etÃrias, Ã mais frequente naqueles com idade avanÃada, com mÃdia ao diagnÃstico de 60 a 75 anos. As anormalidades cromossÃmicas sÃo observadas em aproximadamente 50% de todos os casos de SMD, estando, em alguns casos, relacionadas com achados clÃnicos e morfolÃgicos. O objetivo deste trabalho foi determinar, atravÃs da tÃcnica de FISH (hibridizaÃÃo in situ por fluorescÃncia), a frequÃncia de alteraÃÃes envolvendo os genes MLL, RB e TP53 em pacientes com SMD e associar estas alteraÃÃes com os achados citogenÃticos. Os casos inseridos no estudo foram oriundos do ambulatÃrio de SMD do Hospital UniversitÃrio Walter CantÃdio. Dos 33 pacientes selecionados, 17 pertenciam ao grupo com idade acima de 60 anos. 52% dos pacientes foram classificados, segundo a OMS, como citopenia refratÃria com displasia em mÃltiplas linhagens (CRDM) e 61% estratificados, segundo o IPSS, como de risco intermediÃrio 1 (INT-1). Um total de 78% dos pacientes apresentaram alteraÃÃes citogenÃticas, dentre eles 31% possuÃam cariÃtipos complexos (mais de 3 alteraÃÃes por metÃfase). A tÃcnica de FISH permitiu identificar em 18% dos pacientes alteraÃÃes envolvendo um dos trÃs genes avaliados. TrÃs pacientes apresentaram alteraÃÃo do gene TP53, sendo detectada em dois deles (registros 31 e 6) a deleÃÃo de um Ãnico alelo ou de ambos os alelos do gene, respectivamente, e no terceiro (registro 2), detectou-se a amplificaÃÃo do gene TP53, sendo estas alteraÃÃes nÃo visualizadas atravÃs da citogenÃtica clÃssica, por se tratar de um tÃcnica menos sensÃvel. Detectou-se em 6% dos pacientes (registros 7 e 22) rearranjo do gene MLL, no primeiro a FISH descartou a suposta deleÃÃo do gene alegada pela citogenÃtica, provando que o mesmo estava presente no genoma do paciente, porÃm de forma rearranjada e no segundo a citogenÃtica nÃo conseguiu demonstrar o rearranjo do gene. Quanto ao gene RB, a FISH permitiu identificar apenas um paciente (3%) com deleÃÃo de um dos alelos do gene, sendo esta alteraÃÃo tambÃm nÃo detectada pela citogenÃtica clÃssica. A FISH possibilitou identificar, durante a avaliaÃÃo do gene TP53, dois pacientes (registros 5 e 10) apresentando pelo menos 6 cÃpias extras do cromossomo 17, devendo essa alteraÃÃo se tratar de um pequeno clone hiperdiplÃide detectado parcialmente no primeiro paciente e nÃo detectado no segundo. Nos seis pacientes que apresentaram alteraÃÃo dos genes avaliados, a FISH proveu informaÃÃes que adicionaram, confirmaram ou alteraram o resultado previamente emitido pela citogenÃtica clÃssica, sendo estas uma das principais aplicaÃÃes desta tÃcnica devido sua alta sensibilidade quando comparada ao mÃtodo clÃssico. / Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal disorders affecting the hematopoietic pluripotent cell, characterized by low cell counts in peripheral blood, dysplasia in one or more cell lines, inefficient hematopoiesis and increased risk of progression to acute myeloid leukemia. Although the disease can affect patients of other age groups, they are more frequent in those with advanced age with an average 60 to 75 years at diagnosis. Chromosomal abnormalities are observed in approximately 50% of all cases of MDS and are related with clinical and morphological findings. The aim of this study was to determine, through the technique of FISH (fluorescence in situ hybridization), the frequency of changes involving the MLL, RB, and TP53 genes in patients with MDS and associate these changes with cytogenetic findings. The cases included in the study were selected in the ambulatory of SMD from University Hospital Walter CantÃdio. Thirty three patients were selected, 17 had aged over 60 years. 52% of patients were classified, according to WHO criteria, as refractory cytopenia with dysplasia in multiple lineages (RCDM) and 61% stratified, according to IPSS, as intermediate risk 1 (INT-1). 78% of patients had abnormalities detected by cytogenetics, among them 31% had complex karyotypes (more than 3 changes per metaphase). 18% of patients had changes at least in one of the three genes valued in this study by FISH. Three patients showed alterations of TP53 gene, being detected in two patients (records 31 and 6) the deletion of a single allele or both alleles of the gene, respectively, and in the third (record 2), we detected amplification of TP53 gene, all this changes were not detected by classical cytogenetics, because it is a less sensitive technique. 6% of patients (records 7 and 22) had rearrangement of MLL gene. In the first case, FISH discarded the gene deletion alleged by cytogenetic, proving that it was present in the genome of the patient, but in a rearranged form, and in the second case cytogenetics failed to demonstrate rearrangement of the gene. For the RB gene, FISH identified only one patient (3%) with deletion of one allele of the gene, and this change was also not detected by classical cytogenetics. During evaluating the TP53 gene, FISH allowed identification of two patients (records 5 and 10) presenting at least six extra copies of chromosome 17, probably representing a small hyperdiploid clone partially detected in the first patient and not detected in the second . In the six patients who showed abnormalities of the genes analyzed, FISH has provided information that added, changed or confirmed the result previously given by classical cytogenetics, which are a major application of this technique due to its high sensitivity compared to the traditional method.

Prostatic diseases are common; benign prostate hyperplasia (BPH) is almost ubiquitous in elderly men and 899,000 men were diagnosed with prostate cancer worldwide in 2008. The incidence of both is increasing and expected to continue to rise. Therefore, prostatic diseases represent a considerable economic burden, but there are currently no reliable markers available to accurately differentiate indolent from aggressive disease nor to predict who will benefit from treatment for either BPH or prostate cancer. This results in over and under-treatment of both diseases with consequent patient related morbidity and mortality. The molecular mechanisms underlying the natural history of prostatic diseases remain elusive. It is accepted that prostate cell growth and survival are exquisitely dependent upon activation of the androgen receptor (AR) by androgens. Following ligand binding, AR undergoes further phosphorylation at serine residues, which inhibit proteolytic degradation, stabilise AR and influence AR transactivation. It is therefore plausible that alterations in AR phosphorylation may drive prostatic disease progression. However, few studies have explored the significance of AR phosphorylation, or the kinases driving AR serine phosphorylation in the clinical setting. The over-riding objective of this study was to establish the clinical relevance of AR serine phosphorylation status in prostate tissue in both BPH and prostate cancer. The specific aims of the current study were: • To firstly establish and validate a panel of AR phosphospecific antibodies. • To evaluate site specific AR serine phosphorylation expression levels in prostate cancer and BPH patient cohorts, with full clinical data and follow-up. • To investigate the expression of candidate kinases mediating such phosphorylation. This involved establishing tissue banks with linked comprehensive clinical databases, and utilising this tissue to establish AR phosphorylation expression profiles for each patient. Six AR phosphospecific antibodies (pARS81, pARS94, pARS213, pARS515, pARS578, pARS650) were verified using peptide competition assays and western blotting. Cdk1, ERK1/2, Akt and PKC were identified as putative kinases mediating AR phosphorylation using the online kinase search tool Scansite 2.0. Immunohistochemistry was performed on hormone naïve diagnostic prostate cancer tissue relating to 90 patients. High expression levels of AR phosphorylation at serine sites 81, 515 and 578 were each associated with a poorer clinical outcome. Following cox regression analysis, cytoplasmic pARS515 expression (p=0.038, HR 4.5 (95% CI 1.1–20.6)) and pARS81 nuclear expression (p=0.030, HR 0.033 95% CI 0.002-0.721) were independently associated with shorter time to biochemical relapse and shorter disease specific survival respectively. Cdk1 and/or pCdk1161 were significantly associated with pARS81 and pARS515 as predicted by Scansite 2.0. Similarly, nuclear PKC expression was significantly associated with pARS578 expression both in the cytoplasm and the nucleus. In patients with PSA at diagnosis ≤20ng/ml, high cytoplasmic pARS515 expression was associated with significantly shorter time to biochemical relapse (p=0.019). This translated into significantly shorter disease-specific survival (p<0.001, 10y survival 38.1% vs 100%). Prostate cancer patients with a low serum PSA level at diagnosis may be suitable for delayed radical treatment via active surveillance. An investigation was therefore undertaken in 51 prostate cancer patients treated by active surveillance. Active surveillance is a deferred radical treatment approach which provides a potential solution to the problem of over treatment as a result of over-diagnosis. However some patients harbour occult aggressive disease and delay in treatment may result in disease progression and failure of radical therapy. Although none of the individual AR serine phosphorylation sites were associated with clinical outcome measures on univariate analysis, high expression of total AR in the cytoplasm (p=0.021, HR 4.6 (95% CI 1.3-16.8)) and presence of perineural invasion in the tumour specimen (p=0.003, HR 8.6 (95% CI 2.1-35.7)) were deemed independent with regards to shorter time to treatment intervention in a cox regression analysis. Validation of the results seen in the first active surveillance prostate cancer cohort was undertaken in a second prospectively collected cohort consisting of 84 active surveillance patients. The results in the first cohort were not replicated in the second. Although cytoplasmic pARS81 was associated with time to intervention (p=0.032) and pARS515 expression trended towards an association (p=0.072), an increase in patient numbers in both cohorts may have provided more reliable results. However even with the numbers available in contrast to the first active surveillance cohort, but in line with the pilot prostate cancer cohort, Cdk1 was associated with pARS515 expression, and pCdk1161 trended towards an association. BPH is also an androgen driven disease dependent upon the AR. Previous research into predictive and prognostic markers in BPH is scant. Therefore a comprehensive analysis of clinical and novel pathological factors, including markers of inflammation, was performed in 336 BPH patients. Following this a complete panel of AR serine phosphorylation sites, and associated kinases, was analysed with reference to clinical outcome measures in the BPH cohort. Low expression levels of total AR and AR phosphorylated at Ser-81, 515 and 650 were associated with poorer clinical outcomes. Low expression of smooth muscle pARS515 (p=0.029, HR 0.31 (95% CI 0.10-0.94)) and older age (p=0.004, HR 5.13 (95% CI 1.43-18.41)) were deemed independent on cox regression analysis with regards to shorter time to postoperative acute urinary retention (AUR). Furthermore, low expression of pARS515 in the smooth muscle was associated with increased incidence of postoperative AUR in patients over 70 years old (25.1% vs 2.8% at 10 years following transurethral resection of prostate (TUR)), (p=0.002, HR 0.20 (95% CI 0.06-0.62)). This may have important clinical implications in postoperative counselling of these patients. In addition it may influence the decision to commence early postoperative medical treatment (with 5-alpha-reductase inhibitors and/or alpha blockers) on a prophylactic basis in these patients. Cytoplasmic pARS650 expression (p=0.010, HR 0.50 (95% CI 0.29-0.86)) and PSA at diagnosis (p=0.018, HR 1.89 (95% CI 1.11-3.16)) were independently associated with time to failure of surgical intervention. Furthermore, low expression of pARS650 in the cytoplasm was associated with increased failure of surgical intervention in patients with PSA ≥4ng/ml at diagnosis (45.5% vs 13% at 5 years post TUR), (p=0.026, HR 0.52 (95% CI 0.29-0.93)). This comprehensive study on immunohistochemical expression of site specific AR serine phosphorylation and associated kinases fills a gap in the current literature. It has demonstrated the clinical significance of AR serine phosphorylation in prostate cancer and BPH and uncovered potentially exciting new avenues for future investigation. Site specific serine phosphorylation of the AR may serve as a prognostic and predictive biomarker in prostatic disease and has potential as a future target for therapeutic intervention.

Predators can have both direct and indirect effects on how their prey interact with parasites. This thesis explores these effects using observational and experimental approaches. A behavioural experiment suggested that the direct effects of predators are size- and sex-biased, with small and male guppies, Poecilia reticulata Peters 1859, more prone to Gyrodactylus turnbulli Harris 1986 parasite-induced vulnerability to predation (Chapter 2). Trait-mediated indirect effects of predators also appear important to this host-parasite interaction, as revealed by surveys of natural Trinidadian populations under different predation regimes. First, predator-driven life history evolution predicts an apparent population divergence in parasite tolerance (Chapter 3). Similar divergence in a second trait, social behaviour, may drive sex- and age-biased parasitism: the guppies most liable to shoal have the highest infection probability (Chapter 4). Social behaviour is thus an important driver of parasite transmission, but how parasites affect social interactions remains poorly understood. The second part of this thesis investigates how guppies may use sensory information to mitigate this cost of sociality. Many fishes rely on chemical and visual information and the interaction between sensory modalities to behave appropriately; for example, chemical cues change how guppies respond to visual cues (Chapter 5). In a social context, guppies use both chemical and visual cues to detect infection in conspecifics, but only avoid those in the later stages of infection (Chapter 6). Infection avoidance behaviour is not innate, but likely results from juvenile guppies imprinting on cues of conspecifics, and associating with these cues in adulthood (Chapter 7). This imprinting-mediated avoidance appears to be adaptive: a transmission experiment showed that the onset of avoidance behaviour coincides with the stage of infection at which conspecifics are most infectious (Chapter 8). The sensory ecology of the host and the community in which it lives therefore have important implications for disease dynamics.

The growing problem of insecticide resistance is jeopardising current pest control strategies and current insecticide development pipelines are failing to provide new alternatives quickly enough. Metabolomics offers a potential solution to the bottleneck in insecticide target discovery. As a proof of concept, metabolomics data for permethrin exposed Drosophila melanogaster was analysed and interpreted. Changes in the metabolism of amino acids, glycogen, glycolysis, energy, nitrogen, NAD+, purine, pyrimidine, lipids and carnitine were observed along with markers for acidosis, ammonia stress, oxidative stress and detoxification responses. Many of the changed metabolites and pathways had never been linked to permethrin exposure before. A model for the interaction of the observed changes in metabolites was proposed. From the metabolic pathways with the largest changes, candidate genes from tryptophan catabolism were selected to determine if the perturbed pathways had an effect on survival when exposed to permethrin. Using QPCR it was found that all genes in the entire pathway were downregulated by permethrin exposure with the exception of vermilion suggesting an active response to try and limit flux through tryptophan catabolism during permethrin exposure. Knockdown of the tryptophan catabolising genes vermilion, cinnabar and CG6950 in Drosophila using whole fly RNAi resulted in changes in susceptibility to permethrin for both topical and oral routes of exposure. Knockdown of the candidate genes also caused changes in susceptibility when the insecticides fenvalerate, DDT, chlorpyriphos and hydramethylnon were orally administered. These results show that tryptophan catabolism knockdown has an effect on surviving insecticides with a broad range in mode of action. Symptoms that occur in Drosophila during exposure to the different insecticides were also noted. To gain further understanding into the mechanisms affecting survival, tissue specific knockdown was performed revealing tissue and gender specific changes in survival when vermilion, cinnabar and CG6950 are knocked down. Metabolomics was performed on the knockdown strains to determine the efficacy of the knockdowns on tryptophan catabolism and to identify any knock-on effects. The results indicate that tryptophan metabolite induced perturbations to energy metabolism and glycosylation also occur in Drosophila along with apparent changes in the absorption of ectometabolites. As the knockdown of vermilion, cinnabar and CG6950 tended to result in reduced susceptibility to insecticides, they would make poor targets for insecticidal compounds, however, they may be the first examples of genes that are not directly involved in insecticide metabolism or cuticle synthesis that increase insecticide tolerance in Drosophila. As the first metabolomics data set showed evidence for oxidative stress during permethrin exposure, preliminary work was begun for identifying the tissue specificity and timing of oxidative stress in both Dipterans and Lepidopterans using Drosophila and Bombyx mori as models. In Drosophila oxidative stress did not begin immediately suggesting that the insecticide itself is not a cause, however, a rapid increase in oxidative stress occured over a six hour period after a day of oral exposure implicating catabolites of permethrin. Bombyx were highly susceptible to permethrin showing oxidative stress in the Malpighian tubule and silk gland when exposed. This study has shown that metabolomics is highly effective at identifying pathways which modulate survival to insecticide exposure. It has also brought insight into how insecticide induced pathology may cause death. Data has also been generated which could help characterize the putative transaminase CG6950.

Soft tissue sarcomas (STS) comprise a heterogenenous group of greater than 50 malignancies of putative mesenchymal cell origin and as such they may arise in diverse tissue types in various anatomical locations throughout the whole body. Collectively they account for approximately 1% of all human malignancies yet have a spectrum of aggressive behaviours amongst their subtypes. They thus pose a particular challenge to manage and remain an under investigated group of cancers with no generally applicable new therapies in the past 40 years and an overall 5-year survival rate that remains stagnant at around 50%. From September 2000 to July 2006 I undertook a full time post-doctoral level research fellowship at the MD Anderson Cancer Center, Houston, Texas, USA in the department of Surgical Oncology to investigate the biology of soft tissue sarcoma and test novel anti- sarcoma adenovirus-based therapy in the preclinical nude rat model of isolated limb perfusion against human sarcoma xenografts. This work, in collaboration with colleagues as indicated herein, led to a number of publications in the scientific literature furthering our understanding of the malignant phenotype of sarcoma and reported preclinical studies with wild-type p53, in a replication deficient adenovirus vector, and oncolytic adenoviruses administered by isolated limb perfusion. Additional collaborative and pioneering preclinical studies reported the molecular imaging of sarcoma response to systemically delivered therapeutic phage RGD-4c AAVP. Doxorubicin chemotherapy is the single most active broadly applicable anti-sarcoma chemotherapeutic yet only has an approximate 30% overall response rate with additional breakthrough tumour progression and recurrence after initial chemo-responsiveness further problematic features in STS management. Doxorubicin is a substrate for the multi- drug resistance (mdr) gene product p-glycoprotein drug efflux pump and exerts its main mode of action by induction of DNA double-strand breaks during the S-phase of the cell cycle. Two papers in my thesis characterise different aspects of chemoresistance in sarcoma. The first shows that wild-type p53 suppresses Protein Kinase Calpha (PKCα) phosphorylation (and activation) of p-glycoprotein by transcriptional repression of PKCα through a Sp-1 transcription factor binding site in its -244/-234 promoter region. The second paper demonstrates that Rad51 (a central mediator of homologous recombination repair of double strand breaks) has elevated levels in sarcoma and particularly in the S- G2 phase of the cell cycle. Suppression of Rad51 with small interfering RNA in sarcoma cell culture led to doxorubicin chemosensitisation. Reintroduction of wild-type p53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression via transcriptional repression of the Rad51 promoter through increased AP-2 binding. In light of poor response rates to chemotherapy, escape from local control portends a poor prognosis for patients with sarcoma. Two papers in my thesis characterise aspects of sarcoma angiogenesis, invasion and metastasis. Human sarcoma samples were found to have high levels of matrix metalloproteinase-9 (MMP-9) with expression levels that correlated with p53 mutational status. MMP-9 is known to degrade extracellular collagen, contribute to the control of the angiogenic switch necessary in primary tumour progression and facilitate invasion and metastasis. Reconstitution of wild-type p53 function led to decreased levels of MMP-9 protein and mRNA as well as zymography-assessed MMP-9 proteolytic activity and decreased tumour cell invasiveness. Reintroduction of wild-type p53 into human sarcoma xenografts in-vivo decreased tumour growth and MMP-9 protein expression. Wild-type p53 was found to suppress mmp-9 transcription via decreased binding of NF-κB to its -607/-595 mmp-9 promoter element. Studies on the role of the VEGF165 in sarcoma found that sarcoma cells stably transfected with VEGF165 formed more aggressive xenografted tumours with increased vascularity, growth rate, metastasis, and resistance to chemotherapy. Use of the anti-VEGFR2 antibody DC101 enhanced doxorubicin sensitivity at sub-conventional dosing, inhibited tumour growth, decreased development of metastases, and reduced tumour micro-vessel density while increasing the vessel maturation index. These effects were explained primarily through effects on endothelial cells (e.c.s), rather than the tumour cells per se, where DC101 induced e.c. sensitivity to doxorubicin and suppressed e.c. production of MMPs. The p53 tumour suppressor pathway is the most frequently mutated pathway in sarcoma. Recapitulation of wild-type p53 function in sarcoma exerts a number of anti-cancer outcomes such as growth arrest, resensitisation to chemotherapy, suppression of invasion, and attenuation of angiogenesis. Using a modified nude rat-human sarcoma xenograft model for isolated limb perfusion (ILP) delivery of wild-type p53 in a replication deficient adenovirus vector I showed that functionally competent wild-type p53 could be delivered to and detected in human leiomyosarcoma xenografts confirming preclinical feasibility - although not efficacious due to low transgene expression. Viral fibre modification to express the RGD tripeptide motif led to greater viral uptake by sarcoma cells in vitro (transductional targeting) and changing the transgene promoter to a response element active in cells with active telomerase expression restricted the transgene expression to the tumour intracellular environment (transcriptional targeting). Delivery of the fibre-modified, selectively replication proficient oncolytic adenovirus Ad.hTC.GFP/ E1a.RGD by ILP demonstrated a more robust, and tumour-restricted, transgene expression with evidence of anti-sarcoma effect confirmed microscopically. Collaborative studies using the fibre modified phage RGD-4C AAVP confirmed that systemic delivery specifically, efficiently, and repeatedly targets human sarcoma xenografts, binds to αv integrins in tumours, and demonstrates a durable, though heterogeneous, transgene expression of 1-4 weeks. Incorporation of the Herpes Simplex Virus thymidine kinase (HSVtk) transgene into RGD-4C AAVP permitted CT-PET spatial and temporal molecular imaging in vivo of transgene expression and allowed quantification of tumour metabolic activity both before and after interval administration of a systemic cytotoxic with predictable and measurable response to treatment before becoming apparent clinically. These papers further the medical and scientific community’s understanding of the biology of soft tissue sarcoma and report preclinical studies with novel and promising anti- sarcoma therapeutics.

This thesis describes work undertaken by the author at the University of Oxford. It begins by providing an introduction to malaria infection and pathophysiology, and a review of the latest attempts to produce an effective malaria vaccine. It goes on to describe the rationale behind the vaccines developed by the University of Oxford and others. A brief introduction to the process of planning and carrying out clinical trials of vaccines is then provided, and is followed by chapters describing two clinical trials, designed to test the safety, immunogenicity and then efficacy of candidate malaria vaccines. These trials were performed in Oxford, to examine two different vaccination approaches. The first intended to broaden the specificity of the vaccine induced immune response, by providing multiple antigens in one vaccine, aiming thereby to improve protection from malaria infection. The second regimen used a combination vaccine intending to induce both humoral and cellular immunity simultaneously, thereby providing enhanced efficacy against malaria infection. Neither approach was sufficient to provide protection from infection in the challenge studies described; however, some impact on the disease was detected in the second study. This is examined in detail. The laboratory work described studies background immune responses (both cellular and humoral) to vaccine antigens in a malaria exposed population, intended to support the inclusion of these antigens in the multi-antigen vaccine. The remaining chapters describe work in parasite life cycle modelling, undertaken to aid interpretation of results of these clinical trials, and finally an examination of the clinical course of malaria in the control volunteers who have taken part in the many challenge studies conducted in Oxford.

>Magister Scientiae - MSc / This thesis describes the cloning and recombinant expression of domains from the human RBBP6 protein for future in vitro binding studies with pRb and p53. RBBP6 is a splicing-associated protein that is known to interact with both p53 and the Retinoblastoma gene product (pRb), and has recently been shown to be highly upregulated in oesophageal cancer. The pRb binding domain (RbBD) and the p53 binding domain (p53BD) were each expressed using the glutathione-S-transferase (GST) tag affinity system, and affinity purified using a glutathione-linked agarose column. Purified fusion proteins were cleaved to separate the target protein from GST using PreScission ™ Protease, for which there is a recognition sequence located immediately upstream of the multiple cloning site on the pGEX-6P series of plasmids. The pRb binding and p53 binding domains were further purified using cation exchange chromatography. Mass spectrometry confirmed that the RbBD was expressed as a single species of the expected molecular weight. However preliminary NMR analysis suggested that the domain was not fully folded. A total yield of 8 mg of protein was achieved from 11 of culture, which make it feasible to express 15Nand 12Clabelled samples for NMR. The p53BD was found to be expressed at lower levels and subject to C-terminal degradation, which suggest that the C-terminus is unstructured most likely due to the presence of polylysine tail. Human pRb protein was also successfully expressed and purified using the GST affinity system. Human p53 protein was expressed but was found to be insoluble and attempts to purify it were not pursued. Attempts to confirm the interactions between human RBBP6 and p53 and pRb proteins are on-going but fall outside the scope of this thesis. Expression constructs for the RING and zinc finger domains from human RBBP6 were also cloned into the pGEX system for future structural studies using NMR. Both domains were found to be expressed as soluble fusion proteins in preliminary expression studies.

Inputs of the major element potassium into the ocean from rivers and on-axis high temperature hydrothermal systems have likely varied on geological timescales. Variable uptake of potassium into lavas altered in low-temperature, off-axis, hydrothermal systems could keep the potassium concentration in seawater within the narrow range (~9.5 to 11 mmol L-1) observed in the Phanerozoic. To test this hypothesis a better understanding of the timing of alteration, and of the role of changing environmental conditions on seawater/basalt reactions is required. The age of 69 samples of the secondary, potassium-rich, phyllosilicate mineral celadonite from lavas in the Troodos ophiolite were determined using Rb-Sr radiometric dating to test whether potassium uptake occurs within a specific time interval. Measurements used tandem quadrupole ICP-MS/MS. Combined with published radiometric ages the dataset revealed regional differences in the duration of celadonite formation in the Troodos ophiolite lavas. In one area, where significant hydrothermal sediments were deposited on the lavas, celadonite formed as much as ~40 Myr after the crust accreted, whereas in an area with rare hydrothermal sediments celadonite formation was largely limited to the first ~20 Myr after crustal accretion. These differences in duration of celadonite formation in the upper oceanic crust are interpreted as reflecting differences in distribution of hydrothermal sediments that act as a source of labile Fe that is needed for celadonite formation. To test if there are significant variations of duration and timing of celadonite formation on various scales in the upper oceanic crust I measured the first in-situ Rb-Sr ages of celadonite in lavas from DSDP and ODP drill cores. These ages show that ~80% of celadonite formed from pervasive fluid flow within the first ~20 Myr after the oceanic crust accreted. All celadonite ages roughly correlate with the cumulative heat flow removed from the oceanic lithosphere in the same time interval. In combination the >100 new celadonite ages presented here provide strong evidence that most celadonite forms in the first ~20 Myr after crustal accretion and environmental conditions could be significant in controlling potassium uptake. To determine whether the potassium sink from seawater into altered seafloor lavas varied over time I compile a dataset of the potassium content of lavas from DSDP and ODP drill cores (0 to 180 Myr age range). Estimates of the average potassium content of individual holes reveal that this varies with age. However, holes of similar age show a similar magnitude of variability to that which occurs over this time. To investigate the source of the variability of potassium in altered lavas I modelled the effects of bottom seawater temperature and pH using PHREEQC. The models indicate that if the fluid is in equilibrium with K-feldspar, Na-beidellite and calcite, an increase in bottom seawater temperature and/or decrease in pH would lead to the potassium concentration in the off-axis fluid to increase significantly. This emphasizes the need for future studies to investigate feedback mechanisms between low-temperature hydrothermal alteration in response to changing environmental conditions. / Graduate / 2022-07-12

Denna avhandling går ut på att undersöka verkningsgradens temperaturberoende för SHG (second harmonic generation) från 1030nm till 515nm, i en kvasifasmatchad KTP-kristall, för både pulsad och CW (continuous wave)-laser. Detta undersöktes då det har tidigare noteras att verkningsgraden hos en KTP-kristall avtar när effekten på lasern ökar. Det är på grund av ökad värmeutveckling från absorption (se Figur 1). I denna rapport undersöktes detta genom att variera lasereffekten, kyla KTP-kristallen med en temperaturgradient och mäta dess effekter på verkningsgraden. För att undersöka fallet med CW skapades ett simuleringsprogram i Python, då det inte fanns en tillräckligt stark CW-laser tillgänglig. En laser med femtosekundspulser användes för att mäta verkningsgraden för pulsad laser. Resultaten visade att CW-lasern som förväntat var känslig för temperaturförändringar och att det orsakade att verkningsgraden minskade med effekten. Dessutom visade simuleringen att en temperaturgradient var ett effektivt sätt att höja verkningsgraden om den applicerades korrekt. Den pulsade lasern uppvisade ett annat beteende. Den var mycket stabil för stora temperaturspan, 7% skiljde mellan högsta och lägst effekt på ett temperaturspan på 70°C.

No description available.