Aortic valve disease (AVD) affects millions of people of all ages around the world. Current treatment for AVD consists of valvular replacement with a non-living prosthetic valve, which is incapable of growth, self-repair, or remodeling. While tissue engineering has great promise to develop a living heart valve alternative, success in animal models has been limited. This may be attributed to the fact that understanding of valvular cell biology has not kept pace with advances in biomaterial development. Aortic valve leaflets are exposed to a complex and dynamic mechanical environment unlike any in the vasculature, and it is likely that native endothelial and interstitial cells respond to mechanical forces differently from other vascular cells. The objective of this thesis was to compare valvular cell phenotype to vascular cell phenotype, and assess the influence of steady shear stress on valvular cell biology. This thesis demonstrates that valvular endothelial cells respond differently to shear than vascular endothelial cells, by aligning perpendicular to the direction of steady shear stress, and by the differential regulation of hundreds of genes in both static and fluid flow environments. Valvular interstitial cells expressed a combination of contractile and synthetic phenotypes not mimicked by vascular smooth muscle cells. Two three-dimensional leaflet models were developed to assess cellular interactions and the influences of steady laminar shear stress. Valvular co-culture models exhibited a physiological response profile, while interstitial cell-only constructs behaved more pathologically. Steady shear stress enhanced physiological functions of valvular co-cultures, but increased pathological response of interstitial cell-only constructs. These results showed that valvular cells, whether cultured separately or together, behaved distinctly different from vascular cells. It was also determined that shear stress alone cannot induce tissue remodeling to more resemble native valve leaflets. The leaflet models developed in this thesis can be used in future experiments to explore valvular cell biology, assess the progression of certain forms AVD, and develop targeted diagnostic and therapeutic strategies to hopefully eliminate the need for valvular replacement entirely.

The lower esophageal sphincter (LES) is characterized by basal tone and appropriately timed neurogenic relaxation. The physiological mechanisms underlying these crucial LES functions remain poorly understood. The current studies were designed to characterize the electrophysiological properties and neural regulation of LES circular smooth muscle (CSM), and to determine whether interstitial cells of Cajal (ICC) play a role in neurotransmission. Conventional intracellular recordings were performed in CD1, nNOS knock-out, eNOS knock-out and W/Wv mutant mice. Mouse LES consists of “sling” and “clasp” smooth muscle, which were studied separately in CD1 mice. In subsequent studies of mutant mice and respective controls, only the clasp muscle was examined, Immunohistochemical c-Kit staining of ICC was performed in wild-type and W/Wv mutant mice that were first characterized electrophysiologically. The smooth muscle of the LES clasp and sling displayed unitary membrane potentials with a resting membrane potential (RMP) of ~ -43 mV. Spontaneous nifedipine-sensitive action potentials superimposed on the unitary potentials were usually recorded in the LES clasp, but not sling muscle. A monophasic inhibitory junction potential (IJP) was recorded in sling CSM, whereas a biphasic IJP consisting of an initial IJP, followed by long-lasting slow IJP (LSIJP) was recorded in clasp. Further pharmacological studies using control and various knockout mice suggest that: 1. the CSM of the mouse LES is innervated by cholinergic, nitrergic and purinergic nerves; 2. the LSIJP is mediated entirely by nitrergic nerves, whereas purinergic and nitrergic nerves produce the monophasic IJP in the LES sling and initial phase of biphasic IJP in the LES clasp; 3. Ca2+/CaM-kinase II is involved in the regulation of the nitrergic IJPs; 4. TREK-1 K+ channels are not involved in the nitrergic IJP; 5. purinergic and cholinergic neurotransmission is intact in LES CSM of W/Wv mutant mice, whereas nitrergic neurotransmission is impaired in about half of the animals. In animals in which nitrergic neurotransmission was intact, ICC-IM were markedly deficient immunohistologically, suggesting that ICC are not required for nitrergic neurotransmission; 6. impaired nitrergic neurotransmission in W/Wv mutant mice is associated with dysfunction of a Ca2+-dependent signaling cascade primed by spontaneous Ca2+ release from the sarcoplasmic reticulum. / Thesis (Ph.D, Physiology) -- Queen's University, 2008-01-24 15:54:52.175

Hypertension, a risk factor for aortic valve stenosis, increases transvalvular load and can elicit extracellular matrix (ECM) remodeling. Elevated cyclic pressure and the vasoactive agent angiotensin II (Ang II) both promote collagen synthesis, an early hallmark of aortic sclerosis. It was hypothesized that increased collagen production induced by elevated pressure conditions or the presence of Ang II would affect the mechanical properties of leaflet tissue by decreasing extensibility. Porcine aortic valve leaflets were exposed to pressure conditions of increasing magnitude with and without Ang II. Biaxial mechanical testing was performed to determine peak stretch. Collagen content was determined using a quantitative dye-binding method. The results demonstrated Ang II and elevated pressure decrease the extensibility of leaflet tissue and increase the collagen content in the ECM. In conclusion, the results demonstrated that both elevated pressure and Ang II play a role in altering the biomechanical properties of aortic valve leaflets.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24458号 / 理博第4957号 / 新制||理||1708(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 高橋 淑子, 准教授 佐藤 ゆたか, 教授 中務 真人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

In der kontroversen Diskussion um die unklare Ätiologie der Appendizits wird oft eine Motilitätsstörungen angeführt. Die Interstitial cells of Cajal sind bedeutend für die Motilität und die Entstehung der Peristaltik im menschlichen Kolon. Bei einigen Motilitätsstörungen des Darmes wurde eine Rarifizierung dieser Zellen beobachtet. Die ICC wuden noch nie in der Appendix vermiformis beschrieben.In dieser Arbeit wurden erstmals die ICC in der Appendix mittels einer immunhistochemischen Färbung durch einen maus-monoklonalen Antikörper ( NCL- cKit) nachgewiesen sowie analysiert. Es konnten keine Subgruppen IC-SMP und IC-MP in der Appendix nachgewiesen werden. Die IC-LM zeigten sich reduziert im Vergleich zum Kolon. Die IC-CM konnten zahlreich und regelmäßig dargestellt werden. Eine unterschiedliche Verteilung bzw. Dichte der ICC in der normalen Appendix, der akut und chronisch entzündeten Appendizits konnte nicht nachgewiesen werden.Schlussfolgernd und in Übereinstimmung mit den Beobachtungen anderer Autoren besitzt die Appendix eine reduzierte Motilität und eine physiologische Koprosthase, die aber allein nicht zu einer Entzündung führt. Erst unter dem Einfluss der aus der Literatur bekannten Kofaktoren wird die Koprosthase verstärkt. Erst dies führt zu einer Alteration der Schleimhaut und zur Appendizitis. / The aetiology of the childlike appendicitis is not generally known, but a motility disorder is discussed. The Interstitial cells of Cajal (ICC) are important for the motility and the development of the peristalsis of the colon. In some motility disorders the ICC are abnormaly distributed. The ICC of the human vermiform appendix has never been examined before. We proved and analysed the ICC of the appendix with the mous-monoclonal antibody against c-kit, (NCL-cKit). We could not identify subtypes of ICC, as IC-SMP or IC-MP in the appendix. The IC-LM were reduced compared to the colon. The IC-CM were numerously and regularly distributed. There were no differences in the reduced incidence of ICC between normal vermiform appendix, acute inflamed and chronic inflamed appendicitis.In conclusion and conformance with other observations the appendix has a physiological motility disorder and koprosthasis. Only if some influences of presumably additional cofactors (e.g. inflamation, lymphoid hyperplasia, obstruction), the koprosthasis is intensified, the mucosa is irritated and the appendicitis is developed.

Hypertension is a critical public health issue worldwide, and in the United States, it is the leading cause of heart disease, stroke, and kidney failure, contributing to more than 1,100 deaths per day. It is proposed that the renal medulla combats increased blood pressure by releasing a neutral lipid from the lipid droplets of medullary interstitial cells, termed medullipin, which induces diuresis- natriuresis and vasodepression. The renal medulla is enriched with fatty acid lipid ethanolamides including the endocannabinoid anandamide (AEA), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA), along with their primary hydrolyzing enzyme fatty acid amide hydrolase (FAAH). Our lab is investigating the relationship of these lipid ethanolamides and their metabolites to medullipin. We have shown that intramedullary infusion of AEA stimulated diuresis-natriuresis without changing mean arterial pressure (MAP) in an acute surgical model using anesthetized normotensive C57BL/6J mice. The hypothesis that infusion of a FAAH-selective inhibitor, PF-3845, would produce similar responses as exogenous AEA was tested. Intramedullary infusion of PF-3845 stimulated diuresis-natriuresis, decreased MAP, and increased lipid ethanolamide concentrations in kidney tissue in C57BL/6J mice. Since the decrease in MAP observed with PF-3845 was not consistent with the results of exogenous AEA, this study hypothesized that increased PEA concentrations in the renal medulla observed with PF-3845 produced the decrease in MAP. Therefore, the effects of PEA administration into the renal medulla were investigated. Intramedullary infusion of PEA stimulated diuresis and natriuresis without changing MAP in normotensive C57BL/6J mice. However, intramedullary PEA administration to mice made hypertensive using L-NAME, an inhibitor of nitric oxide synthase, was assessed. Intramedullary infusion of PEA stimulated diuresis, but also decreased MAP in L-NAME-induced hypertensive mice. The mechanism of PEA-induced diuresis was evaluated for the contributions of its FAAH-mediated hydrolysis and the CB1 receptor. Intramedullary infusion of PEA stimulated diuresis in FAAH knockout mice and CB1 knockout mice. The possible source of PEA in the renal medulla was investigated using renal medullary interstitial cells cultured from mice. In cultured mouse medullary interstitial cells (MMICs), treatment with PF-3845 increased cytoplasmic lipid droplets detected by Sudan Black B (SBB) staining and increased PEA in the culture medium. Physiologic stimuli that may regulate PEA production and release from MMICs were also evaluated. Increased osmolarity increased NAPE-PLD protein levels, increased SBB stained droplets in MMICs, and increased PEA concentrations in the culture medium. Overall, it is concluded that the PEA-induced diuretic and natriuretic effect is independent of FAAH-mediated hydrolysis and the CB1 receptor, and that PEA can serve as an antihypertensive regulator in the renal medulla that may be regulated by medullary interstitial cells.

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Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-10-13T19:41:46Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTACAO GIOVANNA GUSMÃO.pdf: 640329 bytes, checksum: eb48b6d00fc9a30b18bc1c3414978fc5 (MD5) / Made available in DSpace on 2016-10-13T19:41:46Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTACAO GIOVANNA GUSMÃO.pdf: 640329 bytes, checksum: eb48b6d00fc9a30b18bc1c3414978fc5 (MD5) Previous issue date: 1998-11-13 / Os hormônios tireoidianos são essenciais para um crescimento e desenvolvimento normal pós natal. Várias investigações têm tentado estabilizar o papel específico dos hormônios tireoidianos nas funções e desenvolvimento do trato reprodutor masculino. Os hormônios tireoidianos possivelmente exercem uma ação recíproca entre o esteróide testicular e as células de Sertoli durante o período prematuro. O presente estudo tentou avaliar o efeito da tiroxina sobre os níveis de testosterona no soro e no fluido intersticial testicular (FIT) em ratos machos. O hipertireoidismo foi induzido com a tiroxina (20 g\kg) em ratos Wistar machos com 22 dias de idade, pesando 80g por um período de 5,10, 15 e 20 dias de tratamento. Os grupos controles receberam solução salina 0,1 ml/100g do peso corporal, como veículo. Após cada período de tratamento, os animais foram pesados e sacrificados, para obtenção do sangue, e os testículos foram pesados, para coleta do fluido intersticial testicular (FIT). Os nossos resultados mostram que a tiroxina em ratos sexualmente prematuros, promove um aumento de testosterona, tempdependente. Observa-se também, que a nível testicular, no FIT, os níveis de testosterona encontram-se elevados. A tiroxina promoveu um aumento do peso corporal e testicular, modificando a função metabólica. Estudos evidenciaram que os receptores de LH e hCG é regulado em homogeneizado celular de testículo em ratos tratados com tiroxina por 20 dias. A capacidade de ligação específica foi obtida pela motodologia envolvendo a saturação de receptores do LH e hCG, aumentando a concentração de LH marcado e hCG não marcado. Outros resultados mostram que a tiroxina é capaz de alterar os níveis de receptores de LH de alta afinidade presentes em homogeneizado testicular, promovendo uma redução da capacidade de ligação específica (Bmax), obtido através da Análise de Scatchard. Conclui-se que a tiroxina pode acelerar a formação de testosterona no lumen intersticial no processo de maturação sexual, modificando possivelmente o mecanismo intracelular das células testiculares, e que a capacidade de ligação do LH e hCG é alterada após o tratamento com tiroxina. / Thyroid hormones are essential for normal pos-natal growth and development. Several investigators have attempted to establish a specific role of thyroid hormones in reproductive tract growth and function. The thyroid hormones possibly exert a reciprocal action between the testicular steroid and the Sertoli cells during the early period. The present study tried to evaluate the effect of the thyroxine over the serum levels of the testosterone and in the interstitial fluid of the testis in male rats. The hyperthyroidism was induced with the thyroxine (20 g\kg), In Wistar male rats 22 days of age, weighing 80kg for a period of 5, 10, 15 and 20 days. The control group received 0,9% saline solution as vehicle. After each treatment period, the animals were weighty and killed to obtainment of blood, and the testis was weighty to collect of testicular interstitial fluid (TIF). These results show that the thyroxin in premature rats, promote an increase of testosterone, dependent time. It is observed either an increase on testosterone level in the TIF. The thyroxin promoted an increase the corporeal and testicular weighty, capable in modify a metabolic function. In the previos study the regulation of LH and hCG receptors by homogenized testicular cells of the 20 days treatment. The specific binding capacity was also obtained by LH and hCG receptors with increasing concentration LH labelled and hCG no labelled. The Scatchard analysis of the saturad curve has show that a affinity constant (ka) is around 108 M-1. This result demonstred that the control present more binding specific that thyroxine wich is a maximum induction of hCG receptor. These results indicate that the thyroxine is able to accelerate the formation the interstitial lumen in the process of sexual maturation, altering possibly the intra-cell mechanism of testicular cells, and the LH and hCG binding capacity is changed after the thyroxine treatment.

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