The development of the diencephalon can be summarised as a process in which cells that initially appear similar give rise to a complex structure that contains a variety of cell groups called nuclei. It involves two stages: the early patterning of the diencephalic prosomeres and the later formation of the individual nuclei. It has been shown that transcription factors and morphogens regulate the first stage but their further effects on the second stage remain unclear. The ventral lateral geniculate nucleus (vLGN) is involved in the visual system and is shown to have complex origins from the thalamus, the zona limitans intrathalamica (ZLI) and the prethalamus. The transcription factor Pax6 is involved in the development of brain structures including the cortex, the diencephalon and the major axonal tracts in the forebrain by playing a multifaceted role in patterning, proliferation, differentiation, migration and axon guidance. It is known that Pax6 is essential in setting up the prosomeric boundaries in the developing diencephalon but its role in the formation of individual nuclei has not yet been explored. By using a conditional Pax6 knock-out mouse driven by Zic4Cre with a green fluorescent protein (GFP) reporter showing the Cre activity, the formation of the thalamic nuclei vLGN, dorsal lateral geniculate nucleus (dLGN) and VP (ventral posterior nuclei) was examined in postnatal day 0 (P0) Pax6+/+, Pax6fl/+ and Pax6fl/fl pups. Using this mouse model, I found an increase in nuclear volume at the rostral level and a global decrease in cell density in the P0 Pax6fl/fl vLGN, whereas in the dLGN an increase of GFP+ve cell proportion was observed. In Pax6fl/+, I found an increase in GFP+ve cell proportion in the caudal part of the vLGN and across the dLGN. No significant change was observed in the VP in either the Pax6fl/+ or the Pax6fl/fl. The defects in the vLGN and dLGN could be caused by: 1. disruption of the expression of patterning factors such as Shh and Nkx2.2; 2. cell proliferation defcts and abnormal apoptosis; 3. ocular developmental defects; 4. failure in cell sorting/migration; 5. cell fate change. During my PhD, I tested the first three theories and explored the fourth but was not able to pursue the last due to the time limit of the project. To test the hypothesized mechanisms underlying those defects seen in the vLGN and dLGN, I performed BrdU labelling to study the time origin of cells that contribute to these two nuclei and discovered that E11.5 and E12.5 are the main ages when these cells and the GFP+ve subpopulation are born. Then I carried out experiments to examine the cell proliferation and cell apoptosis in the thalamus (pTH-R, rostral part of the progenitor zone of the thalamus, and pTH-C, caudal part of the progenitor zone of the thalamus) and the prethalamus (Pth) from E11.5 to E13.5 and found: 1. the proliferation rate decreased in the pTH-R in Pax6fl/+ at E11.5; 2. the growth fraction decreased in both pTH-C and pTH-R in E12.5 Pax6fl/fl; 3. there is no change in cell proliferation in the GFP+ve subpopulation; 4. no abnormal apoptosis is observed in either the whole cell population or the GFP+ve subpopulation. Judging by the amplitude of the change in proliferation in the pTH-R and pTH-C at E11.5 and E12.5, it is unlikely that these changes alone are responsible for the phenotypes seen in P0 vLGN and dLGN. Then I examined the expression patterns of Shh and Nkx2.2 and the expansion of both was observed in Pax6fl/fl at both E12.5 and E13.5, which could explain the volume change of the vLGN but not the change in the proportion of GFP+ve subpopulation in both the vLGN and dLGN. Then I continued to examine if the ocular input from the retinal ganglionic cells are severely affected by the deletion of Pax6 and found no gross change in the conditional mutants, which rejected the ocular developmental defects theory. At the end of my PhD, I performed a BrdU short-term survival experiment and a brain slice culture combined with live imaging experiment to explore the possibility of abnormal cell migration causing the vLGN and dLGN phenotypes and found that the cells moving along the border of the thalamus and prethalamus move faster in the Pax6fl/fl than in the Pax6fl/+, but rather than moving directly toward the lateral surface of the diencephalon, they take a detour. These findings indicate that the deletion of Pax6 causes minor changes in the proliferation of E11.5 to E13.5 diencephalon and expansion of regional marker expression such as Shh and Nkx2.2, which could potenially affect the volume and change the proportion of GFP+ve cells in P0 vLGN and dLGN. Migration defects caused by Pax6 could also contribute to the phenotype observed in those two nuclei. Potential cell fate change caused by Pax6 deletion could be another factor that contributes to the defects in the conditional mutants. More work needs to be done to test the migration defect and cell fate change hypotheses in future.

La compréhension usuelle de l'appariement proton-neutron, dont les traces sont actuellement étudiées en noyaux N = Z, s'appuie sur le mécanisme de paires de Cooper et modèles BCS type. Dans cette thèse, nous présentons une approche alternative qui, contrairement aux modèles BCS, conserve exactement le nombre de particules et l'isospin. Dans cette approche, l'état fondamental de noyaux N = Z est décrit comme un condensé de quartets alpha-similaires faits de deux neutrons et deux protons couplés à l'isospin total T = 0 et de spin J total = 0. La comparaison avec des calculs exacts du modèle en couches montre que le modèle de condensation quartet (QCM) donne une description très précise de l'appariement des corrélations à noyaux N = Z, bien mieux que les modèles BCS. Il est également montré que l'appariement proton-neutron et alpha de type condensation sont importantes non seulement pour les noyaux N = Z, mais aussi pour les noyaux avec des neutrons excédentaires. Dans ce dernier cas, le condensat de quartets d'alpha coexiste avec le condensat des paires de neutrons en excès par rapport au isotopes N = Z. En utilisant le cadre de QCM, nous avons également étudié la compétition entre l’appariement proton-neutron isovectoriel et isoscalaire dans les noyaux N = Z. Nos résultats indiquent que la contribution de l'appariement isoscalaire aux corrélations d'appariement du fondamental est très faible par rapport à l'appariement isovectoriel. / The common understanding of proton-neutron pairing, whose fingerprints are currently investigated in N = Z nuclei, relies on Cooper pair mechanism and BCS-type models. In the present thesis we present an alternative approach which, contrary to BCS models, conserves exactly the particle number and the isospin. In this approach the ground state of N=Z nuclei is described as a condensate of alpha-like quartets built by two neutrons and two protons coupled to the total isospin T=0 and total spin J=0. The comparison with exact shell model calculations shows that the quartet condensation model (QCM) gives a very accurate description of pairing correlations in N=Z nuclei, much better than the BCS models. It is also shown that proton-neutron pairing and alpha-type condensation are important not only for N=Z nuclei but also for nuclei with excess neutrons. In the latter case the condensate of alpha-like quartets coexist with the condensate of the neutron pairs in excess relative to the N=Z isotope. Using the framework of QCM we have also studied the competition between the isovector and the isoscalar proton-neutron pairing in nuclei with N=Z. Our results indicate that the contribution of isoscalar pairing to the ground state pairing correlations is very small compared to the isovector pairing.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

The renin-angiotensin system (RAS) is critical for the control of blood pressure (BP) and salt balance in mammals. Studies reveal that local RAS are present in the rat brain and renin is the first effector of the brain RAS for generating angiotensin II (Ang II) which exerts diverse physiological actions in both peripheral and central nervous system. The existence of renin within the brain has now been demonstrated by numerous studies. Previous studies suggest that renin may go through angiotensin-dependent and independent pathway to influence vascular tone, by Ang II type 1 receptor (AT1R) and renin specific (pro)renin receptor (PRR), respectively. Studies also indicate that AT1R and PRR are highly expressed in the nucleus tractus solitarii (NTS), which is important for central feedback regulation of BP. Further studies have shown that Ang II contributes to the release of NO, which plays an important role in cardiovascular regulation in the NTS. These results indicate that renin plays cardiovascular modulatory role in the NTS. However, the mechanisms how renin modulate cardiovascular functions in the NTS remained unclear. In the present study, I investigated the molecular mechanisms of renin-induced cardiovascular effects in the NTS. Unilateral microinjection of renin into the NTS of WKY rats produced prominent depressor and bradycardic effects. Pretreatment with a non-selective NOS inhibitor L-NAME, eNOS specific inhibitor L-NIO, Akt inhibitor IV, and PI3K inhibitor LY294002 significantly attenuated the cardiovascular response evoked by renin, whereas nNOS specific inhibitor Vinyl-L-NIO and MEK inhibitor PD98059 did not cause significant changes. Western blot studies showed renin increased eNOSS1177 and AktS473 phosphorylation instead of nNOSS1416 and ERK1/2T202/Y204 phosphorylation, and pretreatment with LY294002 blocked renin-induced eNOSS1177 and AktS473 phosphorylation. These results indicated that renin might go through PI3K-Akt-eNOS pathway to increase eNOS activity and ultimately result in NO release. The cardiovascular effects of renin were also attenuated by renin specific inhibitor aliskiren, angiotensin converting enzyme inhibitor lisinopril, AT1R antagonist losartan, and intracellular Ca2+ chelator, BAPTA-AM instead of G protein £]£^ subunit inhibitor gallein, PLC inhibitor U73122, calmodulin inhibitor (W-7) and (pro)renin receptor blocker, handle region peptide. These results indicated that renin mainly through AT1R to regulate BP. Therefore, my results indicated that the modulation of cardiovascular effects of renin in the NTS involves AT1R-PI3K-Akt pathway to activate eNOS activation.

Autophagy is an important cellular process in maintenance of protein homeostasis. Emerging evidence indicates differential roles of autophagy in cellular function under different pathophysiologic conditions. In some circumstance, autophagy results in cell survival, wheras in other situations it results in cell death. Endotoxin affects neurons in the nucleus tractus solitarii (NTS), baroreceptor afferent terminal site in the brain stem, resulting in cardiovascular depression. The aim of this study was to examine whether modulation of autophagic activity in NTS and other brain regions subserving cardiovascular regulation are associated with cardiovascular depression during experimental endotoxemia. Adult male Sprague-Dawley rats received continuously intraperitoneal infusion via osmotic minipump of lipopolysaccharide (LPS, 2.5 mg/kg/day) or normal saline (NS). Body weight (BW) and systolic blood pressure (SBP) were recorded in animals on days 1, 2, 3, 5, 7, 10, and 14 after LPS treatment. Western bolotting was used to assess the expression of autophagic activity marker, microtubule-associated protein 1 light chain 3 (LC3). Rapamycin (0.55 mg/Kg/day), chemical reported to activate autophagy, was infused continuously into the lateral ventricle of the endotoxemic rats for 7 days via osmotic minipump. Both BW and SBP of rats were decreased in the initial 5 days, followed by a gradual return to baseline after LPS treatment. There was a trend in the decrease in autophagic activity (using the ratio of LC3-¢º/LC3-I as an experimental index) at NTS. However, there is no apparent association between the change in autophagic activity at NTS and the LPS-induced cardiovascular depression. In addition, there was no obvious change in the autophagic activity at RVLM, hypothalamus and hippocampus. Intracranial infusion of rapamycin, a mTOR inhibitor that maintains cellular autophagic activity, resulted in a further enhancement of cardiovascular depression induced by LPS. These results suggest that continuously intraperitoneal infusion via osmotic minipump of LPS result in decreases of body weight and systolic blood pressure. However, the present study provides no direct evidence to support for a cause-and-effect role of autophage at NTS, RVLM, hypothalamus as well as hippocampus in the LPS-induced cardiovascular depression during experimental endotoxemia.

The mammalian brain contains a regulatory center in the diencephalic region known as the hypothalamus that plays a critical role in physiological homeostasis, and contains a variety of centers for behavioral drives, such as hunger and thirst. Located deep within the hypothalamus is the suprachiasmatic nucleus (SCN), or the master biological clock, that organizes rhythmic physiology and behavior, such that critical events take place at the most appropriate time of the day or night and in the most appropriate temporal, phase relationships. Cell-to-cell communication is essential for conveying inputs to and outputs from the SCN. The goal of the present study was to use an immortalized neural cell line (SCN2.2), derived from the presumptive anlage of the rat suprachiasmatic nucleus, as an in vitro model system to study intercellular communication among SCN cells. I tested whether the pineal neurohormone melatonin could modulate cell-to-cell signaling, via both dye coupling (gap junctional communication) and calcium waves (ATP-dependent gliotransmission). I also tested whether extracellular ATP could influence the spread of calcium waves in SCN2.2 cells. Lastly, the ability of extracellular ATP to modulate SCN physiological responses to melatonin in SCN2.2 cells was examined. I show that melatonin at a physiological concentration (nM) reduced dye coupling (gap junctional communication) in SCN2.2 cells, as determined by a scrape loading procedure employing the fluorescent dye lucifer yellow. Melatonin caused a significant reduction in the spread of calcium waves in cycling SCN2.2 cultures as determined by ratiometric calcium imaging with Fura-2 AM, a calcium sensitive indicator dye. This reduction was greatest when an endogenous circadian rhythm in extracellular ATP accumulation, determined by luciferase assay, was at its trough or lowest extracellular concentration. In addition, melatonin and ATP interacted in the regulation of gliotransmission (calcium waves), and this interaction was also specific to particular phases of the endogenous SCN physiological rhythmicity. Thus, I have established that a complex interaction exists between established melatonin signaling pathways and this newly discovered ATP accumulation rhythm, with the mechanisms underlying this relationship linked to endogenous cycling of SCN cellular physiology.

Developmental changes within the hypothalamus are necessary for maturation of the reproductive neuroendocrine axis. Recent reports have implicated several neuronal networks in this process, but genes involved in their regulation have not been elucidated. Using a well-established model for nutritional induction of precocious puberty, objectives were to 1) use microarray technology to examine changes in gene expression within the arcuate nucleus (ARC) of the hypothalamus in pre-pubertal heifers fed high or low-concentrate diets, and 2) determine if high-concentrate diets are required for nutritional induction of precocious puberty. In Experiment 1, early-weaned, cross-bred heifers were fed either a high-forage/low-gain (HF/LG; 0.45 kg/d) or a highconcentrate/ high-gain (HC/HG; 0.91 kg/d) diet for 91 d. Analysis of microarray data indicated that 346 genes were differentially expressed (P < 0.05) between HC/HG and HF/LG heifers. Expression of three key metabolic genes [neuropeptide Y (NPY), agoutirelated protein (AGRP), and growth hormone receptor (GHR)] observed to be differentially expressed in the microarray analysis was investigated further by quantitative PCR. Real-time RT-PCR indicated that expression of NPY, AGRP and GHR was lower (P < 0.05) in HC/HG compared to HF/LG heifers. In contrast, concentrations of insulin (P < 0.05), IGF-1 (P < 0.002) and leptin (P = 0.1) were greater in HC/HG compared to HF/LG. For Experiment 2, 48 heifers were used in 2 replicates (24 heifers/replicate) in a 2 x 2 factorial design to examine the roles of diet type (HF vs HC) and rate of gain (LG, 0.45 kg/d vs HG, 0.91 kg/d) on age at puberty. Heifers were fed HC/HG, HC/LG, HF/HG or HF/LG (n = 12/group) for 14 wk, and then switched to a common growth diet (0.68 kg/d) until puberty. Heifers in both HG groups reached puberty at a younger age (54.5 ± 1.8 wk) than heifers in both LG groups (60.2 ± 1.9 wk; P < 0.04). A marked increase (P < 0.01) in serum concentrations of leptin occurred in HC/HG heifers between 24 and 30 wk of age. This increase in circulating leptin was not observed in other groups. Overall, results indicate that nutritional regulation of reproductive neuroendocrine development involves the control of NPY, AGRP and GHR expression. The abrupt increase noted for circulating leptin in heifers fed HC/HG diets, if timed and sustained appropriately, could represent an important temporal cue for activation of the neuroendocrine system and the onset of puberty.

Insulin resistance was thought as the major etiology of hypertension of the metabolic syndrome. Both human and animal studies revealed sympathetic overactivity were present in the metabolic syndrome. Nowadays, most of the studies that examined the etiologies of hypertension of metabolic syndrome were focused on the pathophysiologic effects of insulin resistance on the peripheral vessels. However, there was no study ever examined the insulin resistance in cardiovascular regulatory centers of central nervous system or the pathogenesis of sympathetic overactivity in metabolic syndrome. Our previous study demonstrated that insulin plays a cardiovascular regulatory role in the nucleus tractus solitarii (NTS), one of the cardiovascular regulatory centers in the brain stem. We also demonstrated that the cardiovascular regulatory effects of insulin in the NTS were accomplished through activating PI3K-PKB/Akt-NO signaling pathways. Recently, increases in oxidative stress could raise the incidence rate of diabetes mellitus and cardiovascular diseases had been reported. Besides, it has been reported that there were marked increases in reactive oxidative species (ROS) in various hypertension animal models. It was also reported that elevation of ROS in various tissues may activate the mitogen-activated protein kinase (MAPK) superfamily. Activated MAPKs may phosphorylate insulin receptor substrate 1 (IRS1) on the serine 307 residue. It has been reported that IRS1S307 phosphorylation would inhibit normal insulin signal transduction. The aims of this thesis were to investigate whether the neuronal cells in the NTS would develop insulin resistance in the metabolic syndrome rats, whether development of insulin resistance in the NTS cause hypertension in the metabolic syndrome rats, which signaling molecule in insulin signaling pathway is the key molecule that cause insulin resistance in the NTS, and what the pathogenesis of insulin resistance is in the NTS of metabolic syndrome rats. In the pioneer study, Wistar-Kyoto (WKY) rats were fed with 10% fructose water as their drinking water for 8 weeks. Another group of fructose-fed WKY rats were fed with insulin sensitizer, rosiglitazone, since the 5th week. Blood pressure was measured by tail vein sphygmomanometer every week and venous blood were draw to measure blood sugar and insulin level every other week. Thereafter, all the rats enrolled in this study were fed with 10% fructose water with/without rosiglitazone for 2-3 weeks. My results demonstrated the blood pressure of fructose-fed WKY rats was significantly elevated after 2-week fructose feeding. But at the same time, HOMA-IR did not elevated, which indicated the insulin resistance in the peripheral did not develop yet. Interestingly, at the same time, endogenous insulin in the NTS was significantly elevated in the fructose-fed group. The cardiovascular responses of insulin in the NTS were diminished in the fructose-fed group. While in the rosiglitazone-treated group, the blood pressure and endogenous insulin in the NTS were decreased the baseline level. The cardiovascular responses of insulin in the NTS were restored in the rosiglitazone-treated group. These results indicated insulin resistance do develop in the NTS of fructose-fed rats, and the neuronal insulin resistance in the NTS can induce hypertension. The immunoblotting results demonstrated the phosphorylation of IRS1S307 was significantly elevated in the fructose-fed rats. While the phosphorylation of its downstream molecules, such as AktS473 and eNOSS1177, were significantly decreased as compared with the control group. In the NTS of rosiglitazone-treated group, the phosphorylation of IRS1S307 was decreased, and the phosphorylation of AktS473 and eNOSS1177 were restored. These results indicated that the underline pathogenesis of insulin resistance in the NTS was phosphorylation on the inhibitory serine residue of IRS1, which interfered with the normal insulin signal transduction in the NTS. Increases in ROS in the NTS of fructose-fed rats were demonstrated in the DHE histostaining. Phosphorylation of p38MAPK in the NTS of fructose-fed rats was also detected by immunoblotting. In the NTS of Tempol-treated fructose-fed rats, the phosphorylation of p38MAPK reduced and the nitric oxide production elevated to the basal level. Blood pressure decreased significantly when p38MAPK inhibitor, SB203680, was microinjected into the NTS of fructose-fed rats. These results indicated the pathogenesis of insulin resistance in the NTS is increases in ROS in the NTS, which activate p38MAPK and then phosphorylate IRS1S307. In conclusion, the neuronal cells in the NTS may develop insulin resistance in fructose-fed rats, and the neuronal insulin resistance in the NTS contributes to the hypertension of metabolic syndrome. The mechanism of insulin resistance in the NTS is phosphorylation on the serine 307 residue of IRS1, which interfere with insulin signaling and subsequent NO production in the NTS. The pathogenesis of IRS1S307 phosphorylation is activated p38MAPK which in turn is activated by ROS in the NTS.

Carbon monoxide (CO) has been identified as an endogenous biological messenger in the brain. Heme oxygenase (HO) catalyzes the metabolism of heme to CO and biliverdin. CO has been shown to act as a neurotransmitter and neuronal messenger in the brain. We reported recently that CO was involved in central cardiovascular regulation, modulated the baroreflex, may affect glutamatergic neurotransmission, and metabotropic glutamate receptors (mGluRs) may be coupled to the activation of HO in the nucleus tractus solitarii (NTS) of rats. We also reported previously that adenosine can increase the release of glutamate in the NTS. The present study was designed to investigate the possible interaction of CO, adenosine, and mGluRs groups in the NTS. Male Sprague-Dawley rats were anesthetized with urethane, and blood pressure were monitored intra-arterially. Unilateral microinjection of ascending doses of hemin (0.01 to 3.3 nmol), a heme molecule cleaved by HO to yield CO, produced decreases in blood pressure and heart rate dose-dependently. In addition, similar cardiovascular effects were observed in adenosine (2.3 nmol) and several agonists for mGluRs groups such as DHPG (group ¢¹) (0.03 nmol), APDC (group ¢º) (0.3 nmol)and L-AP4 (group ¢») (0.3 nmol). These cardiovascular effects of hemin were attenuatd by prior administration of the adenosine receptor antagonist DPSPX (0.92 nmol). Similarly, pre-treatment of HO inhibitor ZnPP¢Á or ZnDPBG (1 nmol) also attenuated the depressor and bradycadic effects of adenosine. Among the mGluRs agonists, prior administration of ZnPP¢Á (1 nmol), an inhibitor of HO activity, significantly attenuated the cardiovascular effects of APDC and L-AP4, and failed to prevent the cardiovascular responses of DHPG. These results indicated an interaction between CO and adenosine, and group ¢º and ¢» mGluRs may be coupled to the activation of HO in central cardiovascular regulation.

Insulin resistance plays an intricate role in the development of cardiovascular diseases, hypertension is associated with insulin-resistant states such as diabetes and obesity. However, the underlying mechanism to explain the associations between hypertension and insulin resistance is unknown. The insulin exerts various biological effects in different type of cells. Clinical studies have reported that insulin has been shown to stimulate the protein kinase Akt via activation of PI3K in endothelial cells. Furthermore, insulin stimulated production of nitric oxide (NO) is inhibited by wortmannin in human umbilical vein endothelial cells (HUVECs). We also reported previously that NO contributes to the regulation of blood pressure in central nervous system. The aim of this study was to elucidate the potential mechanisms linking hypertension with insulin resistance and whether insulin signaling may involved in cardiovascular regulation in rat NTS neurons, we investigated the cardiovascular effects of insulin in the nucleus tractus solitarii (NTS) of urethane-anesthetized male spontaneous hypertensive rat (SHR) and normotensive Wistar-kyoto rats (WKY). Unilateral microinjection of insulin (100 IU/ml) into the NTS produced prominent depressor and bradycardic effects in 8/16 week-old WKY. However, no significant cardiovascular effects were found in adult SHR (16 week-old) after insulin injection. Furthermore, young SHR (8 week-old) with normal blood pressure showed depressor and bradycardic effects after insulin injection. Pretreatment with PI3K inhibitor LY294002 and NO synthase inhibitor L-NAME into the NTS attenuated the cardiovascular response evoked by insulin in WKY and young SHR but not in adult SHR. Furthermore, insulin induced Akt phosphorylation in-situ in WKY and SHR rats. Thus, these results indicated that insulin-PI3K-Akt-NOS sensitive mechanisms were involved in WKY and young SHR (normotensive) but not in adult SHR (hypertensive). The results also suggested the possible defective insulin signaling may resulted in the development of hypertension in adult SHR.