The design and construction of a state-of-the-art ultra-high vacuum spectrometer for the performance of angle-resolved inverse photoemission spectroscopy is presented. Detailed descriptions of its most important components are included, especially the Geiger-Muller ultraviolet photodetectors. By building on recent developments in the literature, we expect our spectrometer to achieve resolution comparable or superior to that of other prominent groups, and in general be one of the foremost apparatus for studying the momentum dependence of the unoccupied states in strongly correlated materials. Summaries of the theory of angle-resolved inverse photoemission spectroscopy and the basics of ultra-high vacuum science are also included.

The design and construction of a state-of-the-art ultra-high vacuum spectrometer for the performance of angle-resolved inverse photoemission spectroscopy is presented. Detailed descriptions of its most important components are included, especially the Geiger-Muller ultraviolet photodetectors. By building on recent developments in the literature, we expect our spectrometer to achieve resolution comparable or superior to that of other prominent groups, and in general be one of the foremost apparatus for studying the momentum dependence of the unoccupied states in strongly correlated materials. Summaries of the theory of angle-resolved inverse photoemission spectroscopy and the basics of ultra-high vacuum science are also included.

Doctor of Philosophy (Medicine) / Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true ‘barrier characteristics’ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Estudos sobre evolução têm sido desenvolvidos desde a publicação dos trabalhos de Charles Darwin sobre a origem das espécies pela seleção natural em 1859. Durante o século XX grandes avanços foram obtidos com a utilização de modelagens matemáticas e computacionais, pois com exceção de algumas espécies que podem ter sua evolução analisada in vivo, o tempo necessário para aquisição de dados é enorme e por este motivo o enfoque computacional passou a representar uma ferramenta essencial. Nesta tese são apresentados os conceitos básicos para se entender o processo evolutivo de populações assexuadas como mutação, seleção e relevos adaptativos, bem como os resultados numéricos sobre sua evolução através do processo conhecido como catraca de Muller, que baseia-se na perda estocástica da classe de indivíduos mais adaptados da população através das mutações adquiridas ao longo de sua linhagem. Neste trabalho foram estudadas diversas dinâmicas, como a de populações que estão sujeitas à passagens seriais com gargalo, onde observamos que a velocidade da catraca na não pára devido aos altos valores de epistase, enquanto que para populações com tamanho variável (crescimento e decrescimento exponencial) a catraca pára durante o período de crescimento até a população atingir o limite permitido pelo meio-ambiente, sendo que a partir deste ponto ela se comporta como no modelo de infinitos sítios tradicional. Por último, são apresentados os resultados de populações que interagem entre si em uma dinâmica presa-predador, onde o comportamento da catraca pode ser entendido com base nas dinâmicas das populações descritas anteriormente. Um outro problema abordado nesta tese é o da utilização de medidas da topologia de árvores genealógicas para verificar a presença da seleção na evolução de uma população. Apesar dos comprimentos dos ramos das árvores apresentarem alterações quando comparados ao caso neutro, observamos que os testes estatísticos utilizados não são suficientes para inferir o efeito da seleção em populações reais. / Studies about evolution have been developed since Charles Darwin\'s publications about the Origin of species and Natural Selection in 1859. During the XX century major developments were achieved through mathematical and computational modeling, since only few number of species that their evolution can be studied in vivo, once that the time scale involed for data acquisition procedure is considerable, and for this reason the computational approach become an important tool in this study. In this thesis are presented the basic concepts to understand the process of evolution in a population as mutation, selection and adaptive landscapes, in addition some numerical results about the evolution of an asexual population using the process known as Muller\'s ratchet, that can be characterized by the stochastic loss of the most fitted class of individuals through mutations that are acquired in their lineages. During this work several dynamics were studied, likewise the populations under serial bottleneck passages, where we observed that the velocity of the ratchet never stops for high epistatic coefficients, while in population whose size can varies (increasing or decreasing exponentially) the ratchet halts during population\'s increasing until these individuals do not reach the maximum number permitted, and after this point this population behaves like the traditional infinite genome size model. At last, we show the results of populations that can interact between themselves in a predator-prey dynamics, where the behaviour of the ratchet can be understood in the previous dynamics. Another problem that was studied in this thesis is related with several topology measures of genealogical trees in order to verify the selection in a population evolution. Despite branch\'s length of the trees changed due to the selection, we could see that the statistical tests used do not be sufficient to infer the effect of selection under real populations.

Esta tese aborda diversos temas em evolução molecular, usando extensivamente o formalismo de funções geratrizes para obter resultados analíticos sempre que possível. Em primeiro lugar, apresenta-se a solução exata para o comportamento dinâmico de uma população infinita de seqüências infinitamente longas (não há mutações reversas) evoluindo sob a ação de mutações deletérias em um relevo adaptativo multiplicativo ou truncado. Além disso, foi estudado o comportamento de uma população submetida a sucessivas diluições de intensidades arbitrárias, como ocorre em alguns protocolos de evolução experimental. Foram obtidas expressões matemáticas que, em princípio, podem ser úteis na caracterização de populações reais de microorganismos. Demonstrou-se também que um processo estocástico de ramificação multidimensional generalizado é uma excelente ferramenta para analisar numericamente os efeitos da degeneração mutacional (especificamente, de um fenômeno denominado catraca de Muller) em populações sob variadas condições de crescimento exponencial. Finalmente, simulações foram extensivamente utilizadas para analisar a história evolutiva de populações finitas e averiguar a possibilidade de certas grandezas, como certas medidas da topologia de árvores genealógicas, serem empregadas na elaboração de testes estatísticos capazes de detectar as marcas deixadas pela seleção natural. / This thesis discusses some topics of molecular evolution, extensively using generating function methods to find analytical results whenever possible. In first place, it gives the exact solution for the dynamics of an infinite population of infinitely long sequences (no back mutations) evolving under the action of deleterious mutations on either multiplicative or truncated fitness landscapes. In addition, the behavior of a population subject to successive dilutions of arbitrary intensity, just like some experimental evolution protocols, is found. The mathematical expressions, in principle, may prove useful in characterizing real populations of microor¬ganisms. It was also demonstrated that a generalized multidimensional branching process is a nice tool in numerically studying mutational degeneration effects (specifically a pheno¬menon called Muller\'s ratchet) in populations under a wide variety of exponential growth settings. Finally, the evolutionary history of finite populations was studied by simulations to probe the viability of certain statistic, like some topological measures in genealogical trees, being incorporated in statistical tests to detect the fingerprints of natural selection.

Indiana University-Purdue University Indianapolis (IUPUI) / Chromosomal mechanisms of sex determination vary greatly in phylogenetically closely related species, indicative of rapid evolutionary rates. Sex chromosome karyotypes are generally conserved within families; however, many species have derived sex chromosome configurations. Insects display a plethora of sex chromosome systems due to rapid diversification caused by changes in evolutionary processes within and between species. A good example of such a system are insects in the blow fly family Calliphoridae. While cytogenetic studies observe that the karyotype in blow flies is highly conserved (five pairs of autosomal chromosomes and one pair sex chromosome), there is variation in sex determining mechanisms and sex chromosome structure within closely related species in blow flies. The evolutionary history of sex chromosomes in blow fly species have not been fully explored. Therefore, the objective of this research was to characterize the sex chromosome structures in four species of blow flies and investigate the selective forces which have played a role in shaping the diverse sex chromosome system observed in blow flies. The blow fly species used in this study are Phormia regina, Lucilia cuprina, Chrysomya rufifacies and Chrysomya albiceps. Phormia regina,and Lucilia cuprina have a heteromorphic sex chromosome system and are amphogenic (females produce both male and female offspring in equal ratio). In contrast, Chrysomya rufifacies and Chrysomya albiceps, have a homomorphic sex chromosome system, are monogenic (females produce unisexual progeny), have two types of females (arrhenogenic females – male producers and thelygenic females – female producers), and sex of the offspring is determined by the maternal genotype. To accomplish these tasks, a total of nine male and female individual draft genomes for each of the four species (including three individual draft genomes of Chrysomya rufifacies – male, and the two females) were sequenced and assembled providing genomic data to explore sex chromosome evolution in blow flies. Whole genome analysis was utilized to characterize and identify putative sex chromosomal sequences of the four blow fly species. Genomic evidence confirmed the presence of genetically differentiated sex chromosomes in P. regina and L. cuprina; and genetically undifferentiated sex chromosomes in C. rufifacies and C. albiceps. Furthermore, comparative analysis of the ancestral Dipteran sex chromosome (Muller element F in Drosophila) was determined to be X-linked in P. regina and L. cuprina contributing to sex chromosome differentiation but not sex-linked in C. rufifacies and C. albiceps. Evolutionary pressures are often quantified by the ratio of substitution rates at non-synonymous (dN) and synonymous (dS) sites. Substitution rate ratio analysis (dN/dS) of homologous genes indicated a weaker purifying selection may have contributed to the loss of sex-linked genes in Muller element F genes of the undifferentiated sex chromosome as compared to the differentiated sex chromosome system. Overall, the results presented herein greatly expands our knowledge in sex chromosome evolution within blow flies and will reinforce the study of sex chromosome evolution in other species with diverse sex chromosome systems.

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