Prostaglandins (PG) play a major role in many endogenous processes including inflammation, labor, reproduction, and blood clotting. In the last two decades, these lipid signaling molecules have shown great potential as ocular hypotensive agents. Intraocular pressure (IOP) is a major risk factor in primary open-angled glaucoma (POAG), the second leading cause of blindness world-wide. Currently, prostaglandin F(2α) analogues are the most widely prescribed medications used to treat ocular hypertension. Studies have identified that almost all prostaglandin analogues exhibit anti-hypertensive effects in the eye, although they are not clinically available. Initial studies attributed the decrease in IOP observed to changes in hydraulic conductivity across the pressure-independent or uveoscleral pathway. More recent studies have shown that prostaglandin F(2α) analogues also lower IOP by affecting the pressure-dependent or trabecular pathway--the diseased tissue in POAG. Little is currently known about PG endogenous function, or the etiology of POAG. However, these studies suggest prostaglandin involvement in the maintenance of IOP in humans and identify the potential of PG analogues to treatment ocular hypertension. The research and findings presented in this dissertation address three specific aims designed to test the hypothesis that Endogenous prostaglandins, prostaglandin enzymes and prostaglandin receptors are involved in regulating conventional outflow facility. Specific aim 1 characterizes the distribution and activity of prostamide/prostaglandin F synthase (PM/PGFS) in the mouse and human eye using immunohistochemistry, western blot analysis and thin layer chromatography. Using techniques in biochemistry, molecular biology and physiology, specific aim 2, identifies the presence of the PG-EP₄ receptor within the outflow pathways, and the efficacy of a selective PG-EP₄ agonist, 3,7-dithiPGE₁, is also determined. Finally, specific aim 3 identifies PG-EP4 receptor coupling and downstream signaling using in vitro assays of transfected and primary cell lines to measure cAMP accumulation after treatment with a PG-EP₄ agonist. Collectively, these studies reveal the importance of PGE₂ synthesis and signaling to the conventional outflow pathway. They identify the PG-EP₄ receptor as a regulator of aqueous outflow and provide more specific therapeutic targets for the treatment of POAG.

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Metabolic homeostasis is orchestrated by the hypothalamus through the neuroendocrine and the autonomic nervous systems. The hypothalamic nuclei respond to the peptide leptin secreted from adipose tissue to suppress feeding and increase energy expenditure by promoting fat metabolism via sympathetic activity. Another important, but perhaps less appreciated function of central leptin signaling is to elevate peripheral insulin sensitivity. Environmental and genetic risk factors that affect hypothalamic leptin signaling can lead to obesity and type 2 diabetes mellitus (T2DM). Here, we discovered that LIM domain only 4, LMO4, is a novel protein participating in central leptin signaling. In a process strikingly similar to T2DM in humans, CaMKIIα-Cre;LMO4flox/flox mice, which have LMO4 knocked out in the postnatal brain including the hypothalamus, develop visceral adiposity, reduced insulin sensitivity, obesity and diabetes when fed with regular chow. Central leptin signaling was significantly lost in key hypothalamic nuclei of mutant mice. Caloric restriction prevents obesity but not insulin resistance in these mice. Taken together, our results suggest that LMO4 function in the brain is required for central leptin signaling to control fat metabolism and peripheral insulin sensitivity.

Metabolic homeostasis is orchestrated by the hypothalamus through the neuroendocrine and the autonomic nervous systems. The hypothalamic nuclei respond to the peptide leptin secreted from adipose tissue to suppress feeding and increase energy expenditure by promoting fat metabolism via sympathetic activity. Another important, but perhaps less appreciated function of central leptin signaling is to elevate peripheral insulin sensitivity. Environmental and genetic risk factors that affect hypothalamic leptin signaling can lead to obesity and type 2 diabetes mellitus (T2DM). Here, we discovered that LIM domain only 4, LMO4, is a novel protein participating in central leptin signaling. In a process strikingly similar to T2DM in humans, CaMKIIα-Cre;LMO4flox/flox mice, which have LMO4 knocked out in the postnatal brain including the hypothalamus, develop visceral adiposity, reduced insulin sensitivity, obesity and diabetes when fed with regular chow. Central leptin signaling was significantly lost in key hypothalamic nuclei of mutant mice. Caloric restriction prevents obesity but not insulin resistance in these mice. Taken together, our results suggest that LMO4 function in the brain is required for central leptin signaling to control fat metabolism and peripheral insulin sensitivity.

Metabolic homeostasis is orchestrated by the hypothalamus through the neuroendocrine and the autonomic nervous systems. The hypothalamic nuclei respond to the peptide leptin secreted from adipose tissue to suppress feeding and increase energy expenditure by promoting fat metabolism via sympathetic activity. Another important, but perhaps less appreciated function of central leptin signaling is to elevate peripheral insulin sensitivity. Environmental and genetic risk factors that affect hypothalamic leptin signaling can lead to obesity and type 2 diabetes mellitus (T2DM). Here, we discovered that LIM domain only 4, LMO4, is a novel protein participating in central leptin signaling. In a process strikingly similar to T2DM in humans, CaMKIIα-Cre;LMO4flox/flox mice, which have LMO4 knocked out in the postnatal brain including the hypothalamus, develop visceral adiposity, reduced insulin sensitivity, obesity and diabetes when fed with regular chow. Central leptin signaling was significantly lost in key hypothalamic nuclei of mutant mice. Caloric restriction prevents obesity but not insulin resistance in these mice. Taken together, our results suggest that LMO4 function in the brain is required for central leptin signaling to control fat metabolism and peripheral insulin sensitivity.

Metabolic homeostasis is orchestrated by the hypothalamus through the neuroendocrine and the autonomic nervous systems. The hypothalamic nuclei respond to the peptide leptin secreted from adipose tissue to suppress feeding and increase energy expenditure by promoting fat metabolism via sympathetic activity. Another important, but perhaps less appreciated function of central leptin signaling is to elevate peripheral insulin sensitivity. Environmental and genetic risk factors that affect hypothalamic leptin signaling can lead to obesity and type 2 diabetes mellitus (T2DM). Here, we discovered that LIM domain only 4, LMO4, is a novel protein participating in central leptin signaling. In a process strikingly similar to T2DM in humans, CaMKIIα-Cre;LMO4flox/flox mice, which have LMO4 knocked out in the postnatal brain including the hypothalamus, develop visceral adiposity, reduced insulin sensitivity, obesity and diabetes when fed with regular chow. Central leptin signaling was significantly lost in key hypothalamic nuclei of mutant mice. Caloric restriction prevents obesity but not insulin resistance in these mice. Taken together, our results suggest that LMO4 function in the brain is required for central leptin signaling to control fat metabolism and peripheral insulin sensitivity.

Orientadores: Antonio Carlos Bannwart, Vanessa Cristina Bizotto Guersoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica e Instituto de Geociências / Made available in DSpace on 2018-08-27T08:44:42Z (GMT). No. of bitstreams: 1 Geest_CharlieVanDer_M.pdf: 5712468 bytes, checksum: 765eb52c38dad02a122dbc8c5387f363 (MD5) Previous issue date: 2015 / Resumo: Os procedimentos de manutenção de equipamentos ou desligamento de emergência de uma unidade estacionária de produção de petróleo no mar ocorrem com freqüência na produção de petróleo. No caso de produção de petróleo parafínico escoando em uma linha de produção no fundo do mar, onde a temperatura é em torno de 4 ° C, a perda de calor podel conduzir o óleo a TIAC (Temperatura Inicial de Aparecimento de Cristais) do óleo. Isso leva à cristalização de parafinas. Dependendo do tempo que o óleo permaneça estático, neste ambiênte, perdendo calor, é possivel que se forme uma estrutura parafínica que bloqueie completamente a linha de produção. Linhas de produção com óleos parafínicos são um problema grave para a garantia de escoamento. É importante saber a pressão mínima necessária para reiniciar o escoamento para projetar as instalações de bombas na superfície. Óleos parafínicos são fluidos complexos e tem sido estudados há muito tempo. O comportamento reológico é um dos principais fatores que aumenta a complexidade do problema, no entando, para se prever o comportamento do óleo, o estudo de outros fenomênos é necessários, tais como compressibilidade e encolhimento térmico. Estudos experimentais foram feitos para estudar a reologia, compressibilidade e o encolhimento térmico, individualmente, em um reômetro Hakke Mars 3 e em uma celula PVT. Depois de todos os parâmetros estudados individualmente uma linha em escala reduzida foi construída para estudar o comportamento do óleo numa tubulação com a influência de todos os fenômenos físicos juntos. Todos os experimentos foram realizados com dois óleos reais parafínicos para melhor generalizar os resultados. O óleo apresenta o comportamento reológico denominado tixotrópia. Tixotropia é um fenômeno complexo e não é completamente compreendido pela comunidade científica, e foi definida pelo Dicionário de física de Oxford: "Certos materiais se comportam como sólidos sob pequenas tensões aplicadas, mas sob tensões maiores se tornam líquidos. Quando as tensões são removidas do material este volta para sua consistência inicial. Esta propriedade é particularmente associada com determinadas colóides que formam géis quando deixados em repouso, mas que se tornam líquidos quando mexido ou agitado, devido a uma redistribuição da fase sólida ". Neste estudo, o obetivo era simular o comportamento de dois óleos comerciais parafínico diferentes, com base nos resultados experimentais. A partir da revisão da literatura, o modelo de de Souza Mendez and Thompson (2013) foi selecionado para a reologia. O modelo não foi capaz de prever os dados perfeitamente, então, uma alteração foi feita para melhorar suas previsões, sem alterar o significado físico das equações. A abordagem matemática da linha foi feita com base no balanço de massa e quantidade de movimento, para simular os resultados da linha de produção foi feito um algoritmo baseado no método númerico das diferenças finitas. Um dos principais objetivos deste trabalho era simular a linha, baseado nas equações de conservação de massa e movimento, implementando o modelo elastico viscoplástico tixotrópico. Porém, como os resultados experimentais não mostraram um resultado onde a reologia era realmente relevante, a fase final do algoritmo considerou somente um fluido newtoniano fracamente compressível / Abstract: The Procedure of shutdown of an oil production unit for equipment maintenance or emergency occurs with high frequency during its operational lifetime. In the case of waxy crude oil production, when, the crude stays at the bottom of the sea at 4°C, the loss of heat leads to the WAT (Wax Appearance Temperature) of the oil, which leads to the crystallization of wax. If the oil stays static for sufficient time, it might form a structure that completely blocks the pipeline. Flow lines filled with gelled waxy crudes oil is a severe problem to flow assurance. It is important to know the minimum pressure necessary to restart the subsea flowline to design the surface pump facilities. Gelled waxy crudes are complex fluids and have been studied for a long time. One of the main factors that explain the complexity of the problems is the rheological behavior, though, in order to predict the minimum pressure, others physical parameters are required, such as compressibility and shrinkage. An experimental study was done with a rheometer to study the rheology, then, with the PVT cell to study the compressibility and the shrinkage. These parameters were studied individually and afterwards an apparatus was built to study the behavior of the oil in a pipeline were their influence were evalueted. All the experiments were performed with two waxy crudes oils to better generalize the results. The oil has a temporal rheological behavior defined as thixotropic. Thixotropy is a complex flow phenomenon and is not completely understood by the scientific community, and was defined by the Oxford Encyclopedic Dictionary of Physics as: "Certain materials behave as solids under very small applied stresses but under greater stresses become liquids. When the stresses are removed the material settles back into its original consistency. This property is particularly associated with certain colloids which form gels when left to stand but which become sols when stirred or shaken, due to a redistribution of the solid phase." In this study, the idea was to simulate the behavior of two different commercial waxy crude oils, based on the experimental results. From the literature survey, the model of Souza-Mendez-Thompson (2013) was chosen to evaluate the rheological behavior. The model was not able to predict the data perfectly, then, a modification was made to improve its predictions without changing the physical meaning of the equations. A mathematical approach was done based on mass and momentum balance to simulate the results of the pipeline with a difference finite method. The main goals of this study was to simulate the pipeline with an elasto viscoplastic thixotropic model within the algorithm based on mass and momentum equations. But, as the experimental results were showing that the rheological behavior was not a relevant phenomena in the restart process, the final phase of the algorithm was reconsider, since did not apply for our experimental data. The results considering the oil as a Newtonian fluid with high viscosity had good accuracy / Mestrado / Explotação / Mestre em Ciências e Engenharia de Petróleo

Cerebrospinal fluid (CSF) infusion tests can be used to estimate the dynamic properties of the CSF system. Idiopathic normal pressure hydrocephalus (INPH) is a syndrome signified by a disturbance to the CSF system, where the cause is unknown and the diagnosis is difficult to determine. As an aid in identifying patients with INPH who will improve after shunt surgery, infusion tests are commonly used to determine the outflow conductance (Cout), or outflow resistance (Rout=1/Cout), of the CSF system. The tests are also used to determine shunt function in vivo. The general aim of this thesis was to develop and validate CSF infusion methods, to investigate the dynamics of the CSF system. The methods should be applicable to patients with INPH, to aid in the quest to further improve the diagnosis and management of this syndrome. An existing mathematical model describing the dynamics of the CSF system was further developed. The characteristics of the model were verified and the effect of expanding intracranial air on the intracranial pressure (ICP) was simulated. The simulations supported the recommendation to maintain sea-level pressure during air ambulance transportation of patients with suspected intracranial air. A recently developed infusion apparatus was evaluated, on an experimental model as well as on a patient material. The repetitiveness in estimating Cout was found to be good. A statistically significant difference was found between the repeated Cout estimations in the patient group, indicating that there might have been a small physiological change introduced during the infusion test. A parameter, ∆Cout, was proposed and evaluated. It proved to reflect the reliability of individual Cout investigations in a clinically useful way, as well as to provide easily interpreted information. An adaptive algorithm for assessment of Cout was developed and evaluated on a patient group. The new algorithm was shown to reduce the investigation time, from 60 minutes, by 14.3 ± 5.9 minutes (mean ± SD), p<0.01, without reducing the reliability of the estimated Cout below clinically relevant levels. The relationship between ICP and CSF outflow was studied in a group of patients investigated for INPH. It was found that in the range of moderate increase from baseline pressure, the assumption of a pressure independent Rout was confirmed (p=0.5). However, at larger pressure increments, the relationship had a non-linear tendency (p<0.05). This indicates that the traditional view of a pressure independent Rout might have to be questioned in the region where ICP exceeds baseline pressure too much. Infusion tests can be performed in different ways, where three main categories may be distinguished. The bolus infusion method was compared to the constant pressure and constant flow infusion methods, on an experimental model as well as on a patient material. When physiological pressure fluctuations were added to the model, significant differences were found in the determination of Cout in the range of clinical importance, i.e. low Cout (p<0.05). The finding was supported by the patient investigations, the difference was however not significant. With the application of the new methods developed in this thesis, and the increased knowledge concerning relationships between CSF dynamic parameters, the CSF infusion test was further improved with the ability to increase measurement reliability in a reduced time. This constitutes a good basis to perform a large multi-centre study with the main goal to determine the predictive value of the parameter Cout.

Primary open-angle glaucoma (POAG) is one of the leading causes of blindness worldwide. The only modifiable risk factor for POAG is elevated intraocular pressure, resulting from increased aqueous humor production or decreased drainage. Resistance to drainage in the aqueous outflow pathway is believed to reside in the juxtacanalicular connective tissue (JCT) and to be modulated by the inner wall (IW) endothelium of Schlemm’s canal (SC); however, the mechanisms that increase resistance in POAG remain unclear. To cross the IW, aqueous humor passes through I-pores on giant vacuoles (GVs) or B-pores between adjacent endothelial cells. Additionally, outflow around the circumference of the eye is segmental, or non-uniform, and fluorescent tracers can be used to label areas of high-flow and non-flow. The morphological differences in the endothelial cells of SC and their GVs in high- vs. non-flow areas have not been fully elucidated. In this project, we investigated the role of GVs and pores in the IW endothelial cells of SC in regulating segmental outflow in human eyes. We used serial block-face scanning electron microscopy to generate thousands of serial images and visualize these structures in 3D at the ultrastructural level. First, we 3D-reconstructed 45 individual IW cells and their GVs and quantified the number of connections each cell makes with the underlying JCT matrix/cells. We found that cells in high-flow areas made significantly fewer connections to JCT matrix/cells compared to cells in non-flow areas. Secondly, we analyzed 3,302 GVs for I-pores and basal openings and found a significantly greater percentage of GVs with both basal openings and I-pores in high-flow area compared to non-flow area, suggesting this type of GVs form a channel through which aqueous humor passes from JCT to SC. We also found that GVs with I-pores were significantly larger than those without I-pores. Our results suggest that decreasing number of cellular connections and increasing number of GVs with pores may be potential strategies to increase the amount of high-flow area and aqueous outflow for glaucoma treatment. Together, these studies add to our understanding of the role of GVs and pores in regulating segmental flow around the eye.