Systemic inflammation is a leading cause of mortality and morbidity in both human and equine intensive care patients. This systemic inflammatory response may be due to insult from bacterial, viral, fungal or parasitic invasion or from trauma or hypoxemia. Local and systemic release of a wide variety of endogenous pro-inflammatory mediators results in activation of the innate immune system in order to resolve the insult. In sepsis this initial appropriate host response becomes amplified and deregulated leading to refractory hypotension and multiple organ dysfunction. The exact incidence of sepsis (SIRS due to bacterial infection) has not been reported in the equine literature (Roy 2004). Since early recognition and treatment of sepsis are associated with improved outcome the search for markers to accurately predict presence of sepsis and likelihood of survival continues. The serum concentration of both procalcitonin and its related molecule CGRP have been documented to increase in humans with SIRS, yet no literature exists as to the production or role of CGRP in equine patients with SIRS. This study showed that equine CGRP was produced in detectable quantities by healthy adult horses and neonatal foals less than two weeks of age using a rat á-CGRP ELISA. The low percentage recovery of CGRP from samples and the high lower limit of detection for the assay prevented establishment of a normal concentration range of CGRP in healthy horses. In both adult horses and foals with documented SIRS, CGRP concentrations were significantly increased at time of presentation to the hospital (p<0.0002, p<0.003 respectively). A trend towards increased serum CGRP concentration was present in anaesethized horses exposed to endotoxin, but this was not statistically significant (p< 0.067). / Master of Science

我们的研究是基于发现镁金属的成骨现象。在我们组之前的工作中，我们发现大鼠股骨骨髓腔内植入镁棒后，在很短时间内（一周后）就会在股骨骨膜下部位形成新骨。这种镁导致的成骨现象是怎样发生的，以及我们能否利用镁金属的这种特性去促进骨质疏松骨折的愈合？对这两个问题的解答便构成了本篇论文的主要内容。 / 因为镁诱导的新生骨产生在骨膜下方，并且植入镁棒产生的成骨现象在骨膜剥除的部位消失，所以我们认为骨膜是镁成骨的关键点。骨膜是富含感觉神经纤维和干细胞的组织，而且动物骨的感觉神经分布主要集中在骨膜（约占总的神经数量的99%以上）。骨膜神经末端不仅仅感知痛觉触觉和温度觉，而且在外界刺激下释放感觉神经递质。神经递质包裹于处于神经末梢的囊泡当中，CGRP 是经典的也是分布最广的感觉神经递质。在动物体内，血液中CGRP 的含量随年龄的增加而减少，同时骨内的镁含量也随之流失。这也是老年龄动物骨折愈合较慢的原因之一。所以我们提出本课题的研究假设：镁金属降解产生的镁离子作用于骨膜部位的感觉神经末梢，刺激神经递质CGRP 的释放。骨膜内增多的CGRP 作用于骨膜内的干细胞进行成骨分化，最后形成新骨。我们进一步检测镁的这种成骨作用能否促进骨质疏松鼠骨折的愈合。 / 首先我们用过量的辣椒素破坏大鼠股骨的感觉神经末端之后，镁的成骨显著减少，这说明镁的成骨作用相当程度上依赖于通过骨膜的神经组织。我们通过免疫组化染色及蛋白定量测定发现，植入镁后的骨组织内CGRP（降钙素基因相关肽，一种感觉神经末端分泌的主要神经递质）含量增加了一倍多。我们用CGRP 受体拮抗剂同样发现可以部分抑制镁的成骨作用。我们推测镁降解过程中产生的镁离子在骨膜部位增加了感觉神经递质的释放，骨膜部位增多的神经递质作用于骨膜源性间充质干细胞以及骨髓源性间充质干细胞想成骨方向分化成骨。体外试验结果表明，CGRP 在高浓度下显著促进骨膜及骨髓源性干细胞的成骨分化。我们从大鼠的脊髓腰段L3-5 背根神经节分离出背根神经节神经元，在体外用荧光对神经元内的突触小泡进行染色，发现当培养液中的镁离子浓度升高时（1-2mM），这些富含神经递质CGRP 的突触小泡不但数目增加，而且从胞体中心向轴突末梢迁移。在这个过程中，我们同时记录到显著的镁离子内流。实验结果表明，镁离子可以促进神经元的复极化以及神经递质向轴突末端迁移聚集，从而在下一次刺激中释放出更多的神经递质。同时体外干细胞分化实验结果表明高浓度的镁离子（5-10mM）显著促进干细胞的成骨分化。对干细胞和神经元的胞内镁离子内流检测发现，在胞外镁离子浓度升高的情况下，胞内镁离子内流主要通过一种膜通道MagT1。至此，关于镁成骨的机制可以归纳为：镁金属在降解过程中产生的镁离子作用于骨膜感觉神经末梢，使之释放出更多的神经递质，增加释放的神经递质和镁离子共同促进分布在骨膜和骨髓的干细胞进行成骨分化，从而增加成骨。 / 镁的成骨效应使之有很大的潜力用于骨质疏松骨折的修复。由于镁金属强度不足以直接用来固定大鼠骨折，所以我们设计了一种中空的不锈钢针管作为髓内骨折固定针。针管中部与骨折线对应的部位开出一些小孔，细的镁棒可以插入针管，在体内镁降解产生的镁离子可以从中部的小孔释放出去发挥其成骨效应，进而促进骨折的愈合。我们用卵巢切除大鼠进行闭合性骨折造模，然后用我们设计的髓内针固定。X 射线结果表明，手术后第二、四周镁治疗组骨折愈合组织的面积和宽度显著大于对照组。Micro-CT 扫描结果也同样表明，镁治疗组骨折部位愈合组织的总体积和骨组织体积在术后第四周显著大于对照组。组织学染色表明，在术后第二周，镁治疗组的骨折部位的骨膜内成骨大量增加，并且有大量间充质细胞充塞与骨折部位。第术后第四周，更多的软骨组织形成于镁治疗组的骨折部位。荧光双染色结果也表明，镁治疗组的骨折部位在第四周有更多的新生骨形成。第八周和十二周的偏振光图像表明，镁治疗组的骨折愈合部位形成的胶原纤维比对照组更规则且更多更亮。这说明在骨折愈合后期软骨内成骨以及编制骨向层状骨转化的过程中，镁治疗组的骨重建更加规则。最后在第十二周的力学实验结果证明，镁治疗组的骨干所能承受的最大压力显著高于对照组（大约增强了27%）。这部分体内试验证明镁金属可以加速并优化大鼠骨质疏松骨折的愈合，而且我们设计的中空含镁髓内针可以作为将来临床新型骨折髓内固定针的原型。 / 结论：我们对镁成骨的作用和其机制进行了比较深入全面的研究，并初步证实镁金属可以用于动物骨质疏松骨折的修复。我们的研究结果为将来镁金属在临床尤其是骨科领域的实际应用提供了一些基本的理论依据。 / In the rodent femur, almost 99% of all sensory nerves are distributed densely in the periosteum. Neuropeptides encapsulated in the synaptic vesicles are located at the axon terminals and released through exocytosis after being stimulated at the sensory nerve endings. The neuropeptides released from nerve endings have an osteo-anabolic effect on osteoblasts. Among the many kinds of neuropeptides, which include α-calcitonin gene-related peptide (CGRP), substance P, and other amino molecules, CGRP is the classical and dominantly distributed peptide in sensory nerve endings. In aged animals, decreased serum CGRP and loss of bone Mg content may be the factors inhibiting fracture healing. / In this study, Mg was found to significantly promote new bone formation in the subperiosteal cortical region after it was intramedullarily implanted in the rat femur canal. Histomorphological analysis revealed that the newly formed bone grew from periosteum, a fibrous membrane constituted of blood vessels, sensory serves, and mesenchymal stem cells, and did not form any cartilage-like tissue, the latter of which is a feature of intramembranous ossification. Observation that Mg-induced new bone formation disappeared at the periosteum-stripped region revealed the existence of an interaction between the periosteum and Mg ions. / Based on previous findings, this study examined the following hypotheses: (1) Mg ions from Mg implanted in the rat femur canal act on sensory nerve endings in the periosteum and promote neuropeptide CGRP release, (2) mass CGRP release in the periosteum promotes periosteum-derived stem cells osteoblastogenesis and leads to new bone formation. Mg ions affect synaptic replasticity in dorsal root ganglia neurons, and (3) pure Mg metal affects fracture healing in ovariectomized (OVX) rats. / Neuropeptide CGRP plays a pivotal role in Mg-induced new bone formation. This hypothesis was supported by femur bone analysis showing that CGRP content significantly increased in Mg-implanted femur bone compared to control femur bone. When rat sensory nerves were destroyed by administration of high-dose capsaicin, induction of new bone formation by Mg implantation significantly decreased, proving that sensory nerves play an important role in Mg-induced osteogenesis. Because neuropeptide CGRP from sensory nerve endings may play a pivotal role in Mg’s osteogenic process, the effective CGRP antagonist BIBN4096bs was administered to Mg-implanted rats. Administration of the CGRP antagonist significantly reduced newly formed bone volume after Mg implantation. To examine whether this phenomenon is dependent on the interaction between neuropeptides and MSCs, which are richly distributed in the periosteum, periosteum-derived stem cells (PDSCs) and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from the periosteum and bone marrow, respectively. It was observed that high concentrations of CGRP significantly promoted osteogenic differentiation in both PDSCs and BMSCs while high concentrations of CGRP had an obvious chemotaxis effect on BMSCs. / Mg increases CGRP release by affecting DRG neurons. The results of immunochemical staining and ELISA CGRP quantification analysis of femur samples showed that femur CGRP content in Mg-implanted samples was almost twice that of controls. Previous studies reported that Mg ions could promote neural synaptic replasticity in hippocampus neurons in vitro. This study examined the hypothesis that Mg ions could promote synaptic replasticity in DRG neurons. The neural synaptic vesicles, which contain neuropeptides of DRG neurons, including CGRP, derived from the L3-5 dorsal root ganglion were stained in vitro. The synaptic vesicles were found to significantly increase in number when their medium was changed from Mg-free medium to Mg-rich medium of 1 mM and 2 mM and to migrate from the neuron body to its axon terminals. These results proved that Mg could facilitate neuron replasticity and prompt synaptic vesicle aggregation at axon terminals, indicating that much neuropeptide release occurs after stimulation. Real-time recording of the intracellular Mg signal revealed that DRG neuron Mg influx significantly increased after Mg medium had been added and that Mg influx into neurons was mainly through the membrane Mg ion channel MagT1. Implantation of Mg ions (MgCl₂) of high concentration was found to promote stem cell (PDSCs and BMSCs) osteogenic differentiation. Although the mechanism of Mg’s osteogenic effect on stem cells was not thoroughly studied, cellular Mg influx was found to increase in high-Mg medium through the membrane ion channel MagT1. / Mg accelerated bone fracture in ovariectomized rats. Mg metal is too soft to repair bone fracture in animal models. To overcome this challenge, we designed a novel intramedullary nail containing Mg to accelerate osteoporotic bone fracture healing in ovariectomized (OVX) rats. The novel nail is a hollow stainless steel needle with several interlacing arranged holes drilled midway through the needle. The Mg pin is inserted into the needle canal and Mg ions released through the holes on the needle reach the fracture line during degradation in vivo. Our findings indicate that use of this Mg-containing intramedullary nail could accelerate bone fracture healing in OVX rats. Review of post-surgery X-ray results showed that the fracture callus of the Mg-treated group was significantly larger than that of the control group at weeks 2 and 4. Review of micro-computed tomography (micro-CT) scanning images indicated that both the total volume and area of callus bone in the Mg-treated group exceeded those of the control group at week 4. However, no significant difference was found between the two groups regarding callus area and volume at week 12. / Histomorphological analysis showed a wider intramembranous ossification area and woven bone area in the Mg-treated group at weeks 2 and 4 and more cartilage tissue at the callus site in the Mg-treated group at week 4. Double fluorescence labeling staining revealed more densely stained newly formed bone in the Mg-treated group than the control group at week 4, indicating accelerated callus bone formation in the Mg-treated group. The callus was observed to be undergoing endochondral ossification and woven bone remodeling at weeks 8 and 12. Review of polarized light images showed brighter and more regularly arranged collagen fibers in the Mg-treated group compared to the control group. Biomechanical testing at week 12 revealed that the ultimate load of shaft bone in the Mg-treated group had increased 30% more than that of the control group. These results indicate that the novel Mg-containing intramedullary nail designed in this study could significantly accelerate and optimize osteoporotic fracture healing in OVX rat model. / Significance: The results of this study contribute to a thorough understanding of the osteogenic effect of Mg by explicating its bioeffect on neurons and stem cells. The novel Mg-containing intramedullary nail designed in this study appears promising in osteoporotic fracture healing and to have many potential clinical applications. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Yifeng. / Thesis (Ph.D.) Chinese University of Hong Kong, 2015. / Includes bibliographical references (leaves 170-180). / Abstracts also in Chinese.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.

by Chi Fung Yeung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 83-120). / Abstracts in English and Chinese. / abstract --- p.1 / table of contents --- p.3 / acknowledgments --- p.8 / declaration --- p.9 / list of figures and tables --- p.10 / list of abbreviations --- p.13 / Chapter chapter 1- --- general introduction --- p.15 / Chapter 1.1 --- VASOACTIVE PEPTIDES --- p.18 / Chapter 1.1.1 --- HISTORICAL BACKGROUND --- p.18 / Chapter 1.1.1.1 --- Adrenomedullin --- p.20 / Chapter 1.1.1.2 --- Calcitonin gene-related peptide --- p.21 / Chapter 1.1.1.3 --- Endothelin-3 --- p.22 / Chapter 1.1.2 --- "SYNTHESIS AND RELEASE OF ADRENOMEDULLIN, CALCITONIN GENE RELATED PEPTIDE AND ENDOTHELINS" / Chapter 1.1.2.1 --- SYNTHESIS AND RELEASE OF Adrenomeduulin --- p.24 / Chapter 1.1.2.2 --- SYNTHESIS AND RELEASE OF calcitonin gene related peptide --- p.25 / Chapter 1.1.2.3 --- SYNTHESIS AND RELEASE OF ENDOTHELINS --- p.25 / Chapter 1.1.3 --- "ADRENOMEDULLIN, CALCITONIN GENE RELATED PEPTIDE AND ENDOTHELINS IN THE CNS" --- p.26 / Chapter 1.1.4 --- "RECEPTORS OF ADRENOMEDULLIN, CALCITONIN GENE RELATED PEPTIDE AND ENDOTHELINS AND SIGNAL TRANSDUCTION" --- p.27 / Chapter 1.1.5 --- "BIOLOGICAL ACTIONS OF ADRENOMEDULLIN, CALCITONIN GENE RELATED PEPTIDE AND ENDOTHELINS" / Chapter 1.1.5.1 --- BIOLOGICAL ACTIONS OF ADRENOMEDULLIN --- p.30 / Chapter 1.1.5.1.1 --- haemodynamic effects of adrenomedullin --- p.30 / Chapter 1.1.5.1.2 --- Renal effects of Adrenomedullin --- p.31 / Chapter 1.1.5.1.3 --- Endocrine effects of Adrenomedullin --- p.31 / Chapter 1.1.5.1.4 --- Central effects of Adrenomedullin --- p.32 / Chapter 1.1.5.2 --- CALCITONIN GENE RELATED PEPTIDE / Chapter 1.1.5.2.1 --- haemodynamic effects of calcitonin gene related peptide --- p.33 / Chapter 1.1.5.2.2 --- Renal effects of calcitonin gene related peptide --- p.34 / Chapter 1.1.5.2.3 --- Endocrine effects of calcitonin gene related peptide --- p.34 / Chapter 1.1.5.2.4 --- Central effects of calcitonin gene related peptide --- p.35 / Chapter 1.1.5.3 --- ENDOTHELINS / Chapter 1.1.5.3.1 --- haemodyamic effects of endothelins --- p.36 / Chapter 1.1.5.3.2 --- Renal effects of Endothelins --- p.36 / Chapter 1.1.5.3.3 --- endocrine effects of endothelins --- p.37 / Chapter 1.1.5.3.4 --- Central effects of Endothelins --- p.37 / Chapter 1.1.6. --- PROLIFERATIVE OR ANTI-PROLIFERATIVE EFFECTS --- p.38 / Chapter 1.1.7 --- CLINICAL RELEVANCE OF VASOACTIVE PEPTIDES / Chapter 1.1.7.1 --- Clinical relevance of Adrenomedullin --- p.39 / Chapter 1.1.7.2 --- Clinical relevance of calcitonin gene related peptide --- p.40 / Chapter 1.1.7.3 --- Clinical relevance of Endothelins --- p.41 / Chapter 1.2 --- ASTROCYTES / Chapter 1.2.1 --- Historical background and astrocyte morphology --- p.42 / Chapter 1.2.2 --- Physiological roles of astrocytes --- p.44 / Chapter 1.2.3 --- Pathology of astrocytes --- p.45 / Chapter 1.3 --- NEUROPEPTIDE RECEPTORS AND ASTROCYTES / Chapter 1.3.1 --- Neuropeptide receptors on astrocytes --- p.47 / Chapter 1.3.2 --- Consequences of receptor activation --- p.49 / Chapter 1.4 --- AIMS OF THE THESIS --- p.50 / Chapter CHAPTER 2 --- "GENERAL MATERIALS AND METHODS, AND DATA ANALYSIS" / Chapter 2.1 --- MATERIALS / Chapter 2.1.1 --- ANIMALS --- p.54 / Chapter 2.1.2 --- PEPTIDE HORMONES --- p.54 / Chapter 2.1.3 --- ISOTOPES AND RADIOIMMUNOASSAY KITS --- p.54 / Chapter 2.1.4 --- CULTURE MATERIALS AND CHEMICALS --- p.55 / Chapter 2.1.5 --- PREPARATION OF MATERIALS / Chapter 2.1.5.1 --- Preparation of primary cultures of astrocytes --- p.55 / Chapter 2.1.5.2 --- Preparation of medium and binding buffer --- p.56 / Chapter 2.1.5.3 --- Preparation of 125I-labelled vasoactive peptides --- p.56 / Chapter 2.1.6 --- MEASUREMENT OF CELLULAR CYCLIC AMP --- p.57 / Chapter 2.1.7 --- DETERMINATION OF PROTEIN CONTENT OF CULTURED ASTROCYTES --- p.60 / Chapter 2.2 --- METHODS --- p.61 / Chapter 2.2.1 --- LIGAND BINDING: MEASUREMENT OF 125I-AM AND 125I-CGRP BINDING / Chapter 2.2.1.1 --- Binding kinetics --- p.61 / Chapter 2.2.1.2 --- Determination of specific binding of 125I-AM and 125I-CGRP --- p.61 / Chapter 2.2.1.3 --- Competition binding studies --- p.62 / Chapter 2.2.2 --- "BIOCHEMICAL INTERACTIONS BETWEEN ET3, AM, CGRP AND PKC-ANALOG" / Chapter 2.2.2.1 --- Determination of the production of cAMP in response to AM and CGRP --- p.62 / Chapter 2.2.2.2 --- Determination of the effect of PMA on AM and CGRP-dependent CAMP production --- p.63 / Chapter 2.2.2.3 --- Determination of the effect of the phorbol esters on AM and CGRP-dependent cAMP production --- p.63 / Chapter 2.2.2.4 --- Elucidation of antagonistic effect of staurosporine and Ro31-8220 --- p.64 / Chapter 2.2.2.5 --- Determination of the effects of ET-3 on AM and CGRP- dependent CAMP accumulation --- p.64 / Chapter 2.2.2.6 --- Determination of the effects of PKC inhIBition on ET-3 suppression of AM- and CGRP-induced CAMP responses --- p.64 / Chapter 2.2.2.7 --- Elucidation of antagonistic effect of cycloheximide --- p.65 / Chapter 2.3 --- STATISTIC ANALYSIS --- p.65 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- BINDING KINETICS OF 125I-AM AND 125I-CGRP --- p.68 / Chapter 3.2 --- SPECIFIC BINDING OF 125I-AM AND 125I-CGRP --- p.68 / Chapter 3.3 --- COMPETITION BINDING --- p.68 / Chapter 3.4 --- DOSE RESPONSE OF ADRENOMEDULLIN AND CGRP- STIMULATED CAMP PRODUCTION --- p.69 / Chapter 3.5 --- EFFECTS OF PHORBOL ESTERS ON AM AND CGRP DEPENDENT CAMP ACCUMULATION --- p.69 / Chapter 3.6 --- EFFECT OF PMA ON AM AND CGRP-DEPENDENT CAMP ACCUMULATION --- p.70 / Chapter 3.7 --- EFFECT OF ET-3 ON AM AND CGRP-DEPENDENT CAMP ACCUMULATION --- p.70 / Chapter 3.8 --- "THE EFFECTS OF STAUROSPORINE, RO 31-8220" --- p.71 / Chapter 3.9 --- EFFECT OF CYCLOHEXIMIDE ON THE SUPPRESSIVE ACTION OF ET3 --- p.72 / Chapter CHAPTER 4 --- DISCUSSION --- p.73 / Chapter CHAPTER 5 --- GENERAL CONCLUSION --- p.80 / REFERENCES --- p.83 / APPENDIX-PUBLISHED PAPER

Chan Hoi-Huen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 104-123). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Publications --- p.vi / Table of contents --- p.viii / List of Figures --- p.xii / List of Tables --- p.xiv / Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Blood vessels and blood pressure --- p.1 / Chapter 1.2 --- Smooth muscle --- p.2 / Chapter 1.3 --- Endothelium --- p.3 / Chapter 1.4 --- Vasodilation and Vasoconstriction --- p.5 / Chapter 1.5 --- Calcitonin gene-related peptide (CGRP) --- p.6 / Chapter 1.5.1 --- Discovery of CGRP --- p.6 / Chapter 1.5.2 --- Localization and distribution of CGRP --- p.7 / Chapter 1.5.3 --- Structure profile of CGRP --- p.8 / Chapter 1.5.4 --- CGRP and the vascular system --- p.10 / Chapter 1.6 --- Nitric oxide --- p.11 / Chapter 1.6.1 --- Production of NO by nitric oxide synthase (NOS) --- p.12 / Chapter 1.6.2 --- Actions of NO in smooth muscle --- p.14 / Chapter 1.6.3 --- Synergism with CGRP --- p.14 / Chapter 1.7 --- Other research of CGRP in the laboratory of Professor Ronald R. Fiscus --- p.15 / Chapter 1.8 --- "Aging, diabetes mellitus, sex hormones and cardiovascular system" --- p.16 / Chapter 1.9 --- Aim of study --- p.18 / Chapter Chapter 2 --- Methods and materials --- p.19 / Chapter 2.1 --- General experimental methods --- p.19 / Chapter 2.1.1 --- Measurement of blood pressure in anaesthetized rats --- p.19 / Chapter 2.1.2 --- Tissue bath experiments --- p.20 / Chapter 2.1.2.1 --- Preparation of isolated rat aortic rings --- p.20 / Chapter 2.1.2.2 --- Measurement of contractile and relaxant responses in the rat aortic rings --- p.21 / Chapter 2.1.3 --- Culture of aortic rat vascular smooth muscle cells --- p.22 / Chapter 2.1.4 --- Immunostaining for smooth muscle α-actin in cultured smooth muscle cells --- p.23 / Chapter 2.1.5 --- Determination of nitrite levels in smooth muscle cell culture media --- p.24 / Chapter 2.1.6 --- Measurement of protein contents --- p.25 / Chapter 2.1.7 --- Reversed Transcription- Polymerase Chain Reaction (RT-PCR) --- p.26 / Chapter 2.1.7.1 --- mRNA isolation --- p.26 / Chapter 2.1.7.2 --- Reverse transcription (RT) --- p.27 / Chapter 2.1.7.3 --- Polymerase chain reaction (PCR) --- p.27 / Chapter 2.1.7.4 --- Agarose slab gel electrophoresis --- p.29 / Chapter 2.1.7.5 --- Capillary electrophoresis --- p.29 / Chapter 2.2 --- Reagents --- p.30 / Chapter Chapter 3 --- Impairment of hypotension to calcitonin gene-related peptide in female rats with streptozotocin-induced diabetes mellitus or ovariectomy --- p.40 / Chapter 3.1 --- Introduction --- p.40 / Chapter 3.2 --- Methods --- p.45 / Chapter 3.2.1 --- Animal Preparation --- p.45 / Chapter 3.2.2 --- Statistical analysis --- p.46 / Chapter 3.3 --- Results --- p.47 / Chapter 3.3.1 --- "Body weight, blood glucose and initial blood pressure" --- p.47 / Chapter 3.3.2 --- Hypotensive responses to CGRP in ovariectomized rats --- p.48 / Chapter 3.3.3 --- Hypotensive responses to CGRP in diabetic rats --- p.49 / Chapter 3.3.4 --- Hypotensive responses to CGRP in rats with diabetes and ovariectomy --- p.50 / Chapter 3.4 --- Discussion --- p.50 / Chapter 3.5 --- Conclusions --- p.56 / Chapter Chapter 4 --- Severe impairment of CGRP-induced hypotension in vivo and vasorelaxation in vitro in elderly rats --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Methods --- p.64 / Chapter 4.2.1 --- Tissue preparation for vascular rings --- p.64 / Chapter 4.2.2 --- Vasorelaxation studies in vitro --- p.65 / Chapter 4.2.3 --- Animal preparation for in vivo studies --- p.65 / Chapter 4.2.4 --- Measurement of hypotensive responses to CGRP --- p.66 / Chapter 4.2.5 --- Statistical analysis --- p.66 / Chapter 4.3 --- Results --- p.67 / Chapter 4.3.1 --- Effect of age on CGRP-induced vasorelaxations in rings of thoracic aorta and caudal arteries --- p.67 / Chapter 4.3.2 --- Effect of age on acetylcholine-induced responses in aortic rings --- p.68 / Chapter 4.3.3 --- CGRP-induced hypotension in young female and male rats --- p.68 / Chapter 4.3.4 --- CGRP-induced hypotension in elderly female and male rats --- p.68 / Chapter 4.3.5 --- CGRP-induced hypotension in elderly female rats with ovariectomy --- p.69 / Chapter 4.4 --- Discussion --- p.69 / Chapter 4.5 --- Conclusions --- p.73 / Chapter Chapter 5 --- "Effects of CGRP on interleukin-Iβ-, lipopolysaccharides- and ginseng extract-induced production of nitrite oxide in vascular smooth muscle cells of elderly rats" --- p.82 / Chapter 5.1 --- Introduction --- p.82 / Chapter 5.2 --- Methods --- p.83 / Chapter 5.2.1. --- Animal model --- p.83 / Chapter 5.2.2. --- Culture of vascular smooth muscle cells --- p.84 / Chapter 5.2.3 --- Extraction of total RNA --- p.84 / Chapter 5.2.4 --- Reverse transcription and polymerase chain reaction (RT-PCR) --- p.35 / Chapter 5.2.5 --- Capillary electrophoresis with laser-induced fluorescence detector (CE-LIF) --- p.85 / Chapter 5.2.6 --- Determination of nitrite levels in smooth muscle cell culture media --- p.85 / Chapter 5.2.7 --- Measurement of protein contents --- p.86 / Chapter 5.3 --- Results --- p.86 / Chapter 5.3.1 --- "Effects of IL-Iβ, alone and in combination with CGRP, on NO production in young and elderly VSMCs" --- p.86 / Chapter 5.3.2 --- "Effects of LPS, alone and in combination with CGRP, on NO production in young and elderly VSMCs" --- p.89 / Chapter 5.3.3 --- "Effects of ginseng extract, alone and in combination with CGRP, on NO production in VSMCs" --- p.89 / Chapter 5.4 --- Discussion --- p.90 / Chapter 5.5 --- Conclusions --- p.93 / Chapter Chapter 6 --- General discussion and Conclusions --- p.100 / References --- p.104

Indiana University-Purdue University Indianapolis (IUPUI) / Nerve growth factor (NGF) is a neurotrophin upregulated with injury and inflammation. Peripheral administration of NGF causes hyperalgesia and allodynia in animals. Blocking NGF signaling reverses these effects. At the cellular level, chronic exposure of sensory neurons to NGF enhances expression the neurotransmitter, calcitonin gene-related peptide (CGRP). Acute exposure to NGF increases capsaicin-evoked CGRP release from sensory neurons in culture. Thus, NGF increases peptide release from neurons by: (1) increasing expression of peptides, and/or (2) altering their sensitivity. The increase in peptide outflow by either mechanism could contribute to development of hyperalgesia and allodynia. The signaling cascades mediating the actions of NGF in sensory neurons are unclear. Therefore, experiments were designed to determine which pathways regulate changes in iCGRP content and evoked release from primary sensory neurons in culture. The Ras/MEK/ERK cascade was identified as a possible regulator of iCGRP expression in response to NGF. To test this pathway, it was manipulated in neurons by (1) expression of dominant negative or constitutively active isoforms of Ras, (2) farnesyltransferase inhibition, (3) manipulation of the RasGAP, synGAP, and (4) blocking MEK activity. When the pathway was blocked, the NGF-induced increase in iCGRP expression was attenuated. When the Ras pathway was activated, iCGRP expression increased. These data indicate that Ras, and downstream signaling kinases, MEK and ERK, regulate the NGF-induced increases in CGRP in sensory neurons. To determine which pathway(s) regulate the increase in capsaicin-evoked iCGRP release upon brief exposure to NGF, the Ras/MEK/ERK pathway was manipulated as described above, and pharmacological inhibitors of the PI3 kinase, PLC, and Src kinase pathways were used. There were no differences observed in NGF-sensitization when the Ras and PI3 kinase pathways were inhibited, suggesting these two pathways were not involved. However, when the Src kinase inhibitor PP2 was used, the NGF-induced increase in release was completely blocked. Furthermore, the PKC inhibitor, BIM, also inhibited the sensitization by NGF. This data indicate Src and PKC regulate of sensitivity of sensory neurons in response to brief exposure to NGF. Thus, there is differential regulation of iCGRP content and evoked release from sensory neurons in response to NGF.

Lu Lifang. / "November 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. (192-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Peptidases regulate important physiological processes by controlling levels of bioactive peptides and occasionally through noncatalytic processes. This thesis presents a study of prolyl endopeptidase-like (PREPL), which is a peptidase involved in several human deletion syndromes, including hypotonia-cystinuria syndrome (HCS). Phenotypes tentatively attributed to PREPL deletion include hypotonia and decreased growth hormone (GH) levels. However, little is known about the mechanisms by which PREPL deletion causes these phenotypes. To better understand PREPL catalytic activity, we used an activity-based protein profiling fluorescence polarization screen to identify the first specific PREPL inhibitors. We proceeded to demonstrate the activity of these inhibitors in cells and discovered several classes of cell-active PREPL inhibitors. Further, one of these inhibitors, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile, was able to enter mouse brains. To characterize PREPL substrate specificity, we performed several substrate profiling experiments, but no substrates could be identified, in line with reports from other groups who used related approaches to attempt to identify PREPL substrates. To characterize any noncatalytic functions of PREPL, we used an affinity purification-mass spectrometry approach (AP-MS) to search for any protein-protein interactions of PREPL. We identified brain-expressed X-linked 2 (BEX2) as a novel interactor of PREPL, and confirmed this interaction by immunoblot. Several other proteins identified in the AP-MS experiment, including several members of the STRIPAK complex are being further investigated for possible PREPL interaction. To determine whether HCS phenotypes are in fact due to PREPL deletion and to delineate the molecular pathways involved, we generated a conditional PREPL knockout mouse. These mice were visibly smaller than wildtypes and growth curve analysis verified that from week three of life, there was a significant difference in weight between wildtype and knockout mice. Initial surface righting task experiments also indicate that PREPL knockout pups may have a hypotonia phenotype. In summary, we have developed several new tools for studying PREPL catalytic and noncatalytic function, demonstrated that PREPL deletion causes a GH-related growth deficiency and possible hypotonia and thus moved several steps closer to understanding the molecular mechanisms underlying PREPL deletion phenotypes. / Chemistry and Chemical Biology

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 12 uppsatser.