Cardiovascular disease remains one of the most prevalent diseases in the United States and has remained as the leading cause of death. Large Mendelian randomization studies have found significant correlations between high blood pressure and cardiovascular disease (CVD). In fact, high blood pressure is the single most important independent risk factor for CVD. The purpose of this study was to determine the effect of L-citrulline on blood pressure to determine whether it could be advised as an effective treatment for high blood pressure. L-citrulline is a naturally occurring amino acid that readily converts to L-arginine within the human body. L-arginine has shown promise in decreasing both systolic blood pressure (SBP) and diastolic blood pressure (DBP) significantly by potently increasing levels of nitric oxide (NO) in the body. L-arginine, however, displays poor oral bioavailability compared to L-citrulline. Thus, L-citrulline may be a more effective method in raising plasma arginine levels, increasing NO, and decreasing SBP and DBP. A thorough systematic review and meta-analysis was conducted to extrapolate this effect. Using online databases, hundreds of articles were screened, and ultimately 11 studies were chosen, encompassing 224 total participants. Results showed an overall significant effect of L-citrulline on both resting SBP (MD: -3.74; 95% CI [-6.74, -0.74]; p=0.01) and DBP (MD: -2.00; 95% CI [-3.93, -0.06]; p=0.04). Further analysis of funnel plots was used to determine publication biases and subgroup analysis was performed to determine specific trial moderators that could have affected the overall outcome. In most cases, L-citrulline displayed a significant effect on blood pressure, and more research is warranted to investigate its potential therapeutic effect on cardiovascular health.

Plasmodium falciparum is the protozoan parasite that is the causative agent of malaria in humans. The World Health Organization estimates there were 219 million cases of malaria resulting in 435,000 deaths in 2017. Despite recent progress in control and treatment of the disease, malaria remains a major threat due to the emergence of resistance to all known antimalarial drugs. As a result, there is pressing need for the discovery of drugs with novel mechanisms of action to circumvent the current mechanisms of resistance. Protein kinases in Plasmodium serve critical functions in its life cycle and are implicated in multiple stages of cellular progression. These qualities make protein kinases attractive antimalarial drug targets. In addition, a large portion of the Plasmodium kinases have no orthologs in the mammalian kinome, which increases the potential for selective targeting. For these reasons, we hypothesized that targeting the plasmodial kinome may give rise to effective antimalarials. The goal of this study was to identify and characterize plasmodial inhibitors from a kinase-focused library of 2,480 compounds. An initial phenotypic screen was performed at a fixed one micromolar concentration to identify compounds with antiplasmodial activity. From this library, we have identified multiple scaffolds with submicromolar potency and ample selectivity. Further characterization was sought for hits including elucidating the cellular mechanism of action, physiochemical profiling, and structure-activity relationship profiling (SAR). Efforts are currently underway to identify the molecular targets of these compounds. As part of this effort, a probe-based chemoproteomics method was used. This method involved using soluble ATP probes to label kinases in lysates of inhibitor-treated cells. Samples from this experiment are currently undergoing mass spectrometric analysis. These results will help elucidate which kinases are targeted by the compounds of interest. Ultimately, this work will provide a starting point for the development of new therapeutics to treat drug-resistant malaria.

Osteoarthritis (OA) is a debilitating disease caused by the deterioration of articular cartilage and is a leading cause of disability in the United States and worldwide. Much current research into improved treatment for this disease is focused on tissue engineering through the growth of cartilage sheets made by articular chondrocytes. However, as chondrocytes proliferate in vitro, they also lose their ability to produce dense extracellular matrix, a necessary component of articular cartilage conferring mechanical strength. SOX9, a transcriptional activator, increases type II collagen expression, a key articular cartilage extracellular matrix component. Thus, SOX9 promotes an articular cartilage phenotype. Therefore, increasing SOX9 expression and activity as a transcriptional activator in culture has the potential to improve tissue engineering outcomes. This project serves to generate SOX9 promotor-driven secreted luciferase reporter chondrocytes to monitor chondrogenic properties temporally and non-destructively for use in high-throughput analysis of culturing conditions and drug screening.

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A number of aspects of the melano-macrophage centres of fish were investigated. In teleosts these centres consist of aggregates of pigment-laden macrophages and various leucocytes. They are usually embedded in haemopoietic tissue (mainly within the spleen and kidney) in association with the blood-supplying vessels and in a few species a distinct lymphoid cuff surrounds the entire structure. Salmonids are exceptional in that their melano-macrophages are scattered throughout the haemopoietic tissue and do not form distinct aggregates. At least four types of pigments have been described to occur in these phagocytes, viz. melanin, the lipogenic pigments ceroid and lipofuscin, and the haematogenous pigment haemosiderin. In light microscopy the centres appear in varying hues of yellow, brown and black. It has been suggested that these centres could well represent the primitive analogues of germinal centres of the lymph nodes of birds and mammals. As all previously available information came exclusively from a restricted number of teleost species it was considered of primary importance to carry out a study of the distribution and cytological organization of these centres in living representatives of Agnatha, Chondrichthyes and Osteichthyes. Seventy-two species of fish had their haemopoietic tissues examined by light microscopy for pigment-containing macrophages. Except for the lamprey Lampetra fluviatilis, all fish species were observed to possess these pigment cells. An evolutionary pattern was evident in both the distribution and the degree of organization of melano-macrophages; three major evolutionary trends were discernible, viz: i. a progressive increase in the abundance of pigment cells; ii. a structural evolution from a random distribution of individual pigmented macrophages observed in Agnatha and Chondrichthyes to organized centres characteristic of all Osteichthyes except the salmonids; iii. a change in organ location of these pigment cells from the liver in Agnatha, Chondrichthyes and the primitive bony fishes to the spleen and kidney in the advanced bony fishes. The increasing sophistication in cytological organization of the melano-macrophage centres is concomitant with the increasing levels of complexity of the cytoarchitecture of the lymphoid system. The increasing proclivity of the centres for the main lymphoid organs follows closely upon the evolution of the lymphoid system and represents a major advance in the evolution of lympho-reticular relationships. These analogies provide additional evidence that these centres are of a lymphatic nature and may well represent the primitive analogues of germinal centres of birds and mammals. Ontogenically pigment-bearing macrophages appear following upon first feeding. Immunological maturity appears to be attained at first feeding and the fact that it is shortly afterwards that melano-macrophages appear within the lymphoid tissues (eventually leading to melano-macrophage centre formation within them), seems to add weight to the evidence of a structural and functional relationship between melano-macrophage centres and lymphoid tissues. The very marked changes observed in the melano-macrophage centres during cachexia provided a convenient tool for studying these pigmented macrophages. In adult rainbow trout Salmo gairdneri and plaice Pleuronectes platessa kept at 12°C, the density of melano-macrophages and melano-macrophage centres respectively had increased considerably after 6 weeks of complete deprivation of food and by 10 weeks very high densities were observable. At higher temperatures (25°C), employing Tilapia zillii and swordtails Xiphophorus helleri, a very marked increase in the density of these centres was already evident after 3 weeks of complete starvation. With first feeding rainbow trout fry kept at 12°C, high densities of melano-macrophages were observed within the spleen and kidney after 3 weeks of complete starvation. No other treatment employed in this study was observed to induce any significant changes in the melano-macrophages of either fingerlings or adult fish. These results suggest that tissue atrophy is a major factor contributing to the formation of the pigments observed within the melano-macrophages. Electron microscopic observations employing normal and cachectic plaice indicated the following possible modes of origin for the pigments within melano-macrophages: i. melanin granules seem to be derived from their being simply phagocytosed from the classical melanin-containing cells that have been ruptured or otherwise damaged; ii. lipogenic pigments appear to derive from damaged cellular components such as effete mitochondria through the process of peroxidation of their unsaturated lipids; iii. haemosiderin is almost certainly derived from the breakdown of haemoglobin from effete erythrocytes. Since lipid peroxidation and recycling of iron compounds lead to the formation of free radicals and cations, these potentially toxic entities are bound to arise spontaneously within melano-macrophage centres. In view of this there is raised the possibility that the melanin within the centres could be playing a very important role through its well-recognized ability to absorb free radicals and its strong affinity for cations. While this would account for the presence of all these types of pigments within melano-macrophages, it could also explain why pigment cells are so often observed at sites of infection or tissue injury. All ingested cellular debris appears to be subjected to lysosomal enzyme activity and it is the indigestible residues (indigestible unsaturated lipids mainly) that give rise to the pigments which gradually accumulate. The absence of pigment in young larval fish, its steady accumulation with age in clinically normal fish and its presence without exception in older fish indicate that the pigments being studied seem to satisfy the criteria set forth for a basic biological aging process. The role of melano-macrophage centres in iron storage in normal and diseased fish was studied. The spleen, kidney and liver centres of fourteen species of clinically normal teleost fish were examined histochemically for haemosiderin. This was found to be present in varying amounts within the splenic centres of most specimens, but in contrast it was rarely found in the centres of the kidney and the liver. Linder conditions of starvation and in diseased fish, a markedly increased deposition of ferric iron occurred in the splenic centres of nearly all fish examined. By comparison, the iron content in the kidney and liver centres was generally still very low. These results suggest that although the centres in the various haemopoietic organs resemble each other morphologically and in their relation with the associated tissues, there could well be important functional differences between the centres of different organs. When rainbow trout that had been splenectomised were starved, accumulation of haemosiderin was diverted to the kidney melano- macrophages; the liver was still virtually devoid of iron. The possible implications of these findings are discussed. In conclusion, all the available evidence seem to indicate that the melano-macrophage centres of fish pnd the germinal centres of birds and mammals are similar in many ways. A major difference is the high levels of pigments in the centres of fish. This seems to be related to the inability of the latter to control their body temperature. Fatty acids of living organisms shift towards greater unsaturation under lower environmental temperatures as a means of maintaining protoplasmic viscosity within the range necessary for normal metabolic processes. Thus fish, because of their poikilothermic nature, have high levels of unsaturated fatty acids in their bodies and are thus more prone to lipofuscin formation. It has also been suggested that intracellular digestive processes of fish macrophages may not be well developed on the evolutionary scale. This could also lead to increased accumulation of indigestible materials within the melano-macrophage centres. Finally these results are discussed with special emphasis being placed on the following three points: i. that the melano-macrophage centres should be regarded as sites where a large variety of materials are aggregated, processed, sifted and disposed of in a variety of ways rather than regarding them as static areas passively accepting and storing any materials that come their way. Of special significance are those materials that are required for recycling such as iron-containing compounds; ii. that there are important functional differences between the melano-macrophage centres of different organs; iii. that as more information becomes available the melano-macrophage centres may well become useful as sensitive indicators of the state of health of the fish. A technique for bleaching the pigments within melano- macrophage centres in ultra-thin sections was developed. Treatment of the sections with permanganate for five minutes followed by fifteen minutes in metabisulphite resulted in complete bleaching of almost all the pigments.

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Bibliography: leaves 158-189. / x, 189 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1994