Abstract The purpose of the present study was to find reliable non-invasive methods for imaging salivary glands and diagnosing primary Sj?gren's syndrome (SS) and to evaluate autonomic function and central nervous system (CNS) disorders in patients with primary SS. The patient population consisted of consecutive patients with primary SS, who fulfilled the International classification criteria for primary SS, from the Division of Rheumatology, Department of Internal Medicine in Oulu University Hospital. Magnetic resonance (MR) imaging and MR sialography of parotid glands were performed on 27 patients and 7 healthy controls and ultrasonography (US) of major salivary glands on 27 patients, 27 healthy controls, and 27 symptomatic controls with sicca symptoms or salivary gland swellings without SS. MR imaging and US showed heterogeneous parenchyma or adipose degeneration of the gland in 81% and 78% of patients, respectively. MR sialography showed ductal system changes, narrowings and dilatations, or cavities in 96% of patients. One healthy control and 2 symptomatic controls had abnormal findings of parotid or submandibular glands on US. Other controls had normal findings. The diagnostic specificity of US was 94%. Parenchymal structural changes on MR imaging and US were associated with anti-Ro/SSA positivity and weakly with the focus score index, but not with salivary or tear secretion, age, disease duration, or features of systemic activity of the disease, such as hypergammaglobulinemia or systemic complications. A comprehensive package of cardiovascular tests, including 24-hour heart rate variability, baroreflex sensitivity test with phenylephrine, Valsalva manoeuvre, deep breathing tests, and active orthostatic test, were conducted on 30 patients and 30 healthy, age and sex-matched, randomly selected population-based controls. No signs of autonomic dysfunction were found in patients compared to controls in any of the tests. The test results were not associated with saliva or tear secretion, age, disease duration, or clinical features of systemic activity of the disease. A case of severe inflammatory CNS disease associated with primary SS was described, and an investigation of the relevant literature was made. Though inflammatory CNS disease is a possible complication of primary SS, there is no consensus regarding its prevalence or significance in the literature. Diagnostics and treatment are empiric. In conclusion, MR imaging, MR sialography, and US yield such a definitive picture of the glandular changes in primary SS that they are promising alternatives for invasive examinations in the diagnostics of primary SS. Comprehensive cardiovascular tests revealed no signs of autonomic dysfunction in patients with primary SS compared to general population.

蠕動是一種能夠幫助食物通過胃腸道以及促進胃腸道產生能動性的類似波浪的收縮運動。它由一種叫做Cajal (ICC)間質細胞的起搏器細胞產生的慢波所控制。ICCs亦幫助由腸神經系統(ENS)到平滑肌的信息傳導。嚙齒動物和人類實驗表明，老化所導致的ICC細胞數量下降和腸神經退化與排便睏難和便秘有關。通過研究ICC和ENS在正常老化情況下和加速膽碱能神經元喪失的阿爾茲海默症(AD)老鼠模型(Tg2576)中的變化，我們對治療神經退化性疾病也許會有新的認識。本課題的目的在于，研究老化情況下正常老鼠模型及澱粉樣前體蛋白質(APP)過量表達下的AD老鼠模型的胃腸道在形態及功能上的變化。 / 六個月大的Tg2576和同齡野生型對照的全樣載片免疫組化實驗顯示， 十二指腸 (P < 0.05)和迴腸 (P < 0.01)中的腸神經細胞顯著降低，迴腸 (P < 0.001)中的GFAP陽性的腸神經膠質細胞也顯著消失。S100陽性的腸神經膠質細胞在胃竇（胃部中的起搏區域）(P < 0.05), 迴腸 (P < 0.05)和結腸 (P < 0.05)中顯著喪失。這些結果表明，在早期的AD階段，ENS已經出現變質。ICC細胞數量在六個月大的Tg2576和同齡野生型對照的所有腸胃部分並沒有顯著性差異 (P > 0.05)。同時，早期AD階段的基本蠕動節奏也並沒有發生改變。除此之外，結腸和十二指腸的GFAP/S100陽性的腸神經膠質細胞比例並沒有顯著增加，表明在早期AD階段，可能出現了炎症。 / 利用石蠟切片進行β澱粉樣蛋白免疫組化，天狼猩紅溶液化驗和硫代黃素T溶液化驗可以測試不溶的澱粉樣斑塊是否存在。結果指出在六個月大的Tg2576所有腸胃部分都觀察到澱粉樣斑塊聚集而在不同的腸胃部分聚集的程度都有所分別。除了結腸外，六個月大的野生型對照所有腸胃部分都觀察不到澱粉樣斑塊聚集。澱粉樣斑塊形成的增長可能和早期AD階段出現的腸神經細胞和腸神經膠質細胞喪失互相關聯。 / 應用電泳轉移酶標免疫印斑技術，測試六個月大的Tg2576和同齡野生型對照的迴腸和結腸中，膽碱乙酰轉移酶 (ChAT，出自興奮神經元)， 神經元型一氧化氮合酶(nNOS，出自抑制神經元)， 膠質細胞源性神經營養因子 (GDNF， 出自腸神經膠質細胞)和可溶解的β澱粉樣蛋白寡聚體的表達是否改變。和野生型對照相比，Tg2576的nNOS的表達在迴腸 (P < 0.05) 而不是結腸 (P > 0.05) 中顯著增加。而ChAT，GDNF和各β澱粉樣蛋白寡聚體 (十二聚物，九聚物和六聚物)在六個月大的Tg2576和同齡野生型對照之間並沒有顯著改變 (P > 0.05)。綜上結果表明，在早期AD階段，腸胃道中的抑制信號有所增加，但是β澱粉樣蛋白寡聚體可能不是引致腸胃道中的腸神經細胞和腸神經膠質細胞喪失的原因。 / 在腸胃道的組織學和生化實驗之後，我們利用了微電極陣列 (MEA) 系統來量度出自胃竇和迴腸的慢波信號。量度出來的主導頻率(DF)和功率分佈可以成為測量在老化的ICR老鼠和早期AD階段下腸胃道的功能有沒有變化的參數。在硝苯地平存在下，尼古丁顯著地刺激三個月大 (P < 0.05) 和 六個月大 (P < 0.05) 的ICR老鼠中胃竇和迴腸的慢波活動但未能引起十二個月大 (P > 0.05) 的ICR老鼠中的慢波活動，說明神經退化可能在十二個月的年齡開始。附加了河豚毒素的情況下，尼古丁不能再刺激三個年齡組中胃竇和迴腸的慢波活動 (P > 0.05)，由此證明了尼古丁是對腸神經細胞起作用再去激發ICC的活動。六個月大的Tg2576和同齡野生型對照之間的胃竇和迴腸的基准讀數沒有顯著分別 (P > 0.05)。然而，尼古丁顯著地增加野生型對照中胃竇和迴腸的DF和胃電過速範圍 (P < 0.05) 但是不能刺激Tg2576中胃竇和迴腸的電流活動 (P > 0.05)，示意在早期AD階段腸胃道中已經出現了腸神經細胞和/或腸神經膠質細胞喪失。 / 綜上所言，研究結果提出AD老鼠模型有形態學，生物化學和功能上的轉變。本課題提供了在研究神經退化疾病上的基礎，也支持ENS是中樞神經系統早期病變前的關口這個假設。 / Peristalsis is the wave-like contraction that moves food along the gastrointestinal (GI) tract and generates GI motility. Peristalsis is modulated by slow waves that originate from pacemaker cells called interstitial cell of Cajal (ICC). ICCs also modulate and transduce inputs from the enteric nervous system (ENS) to the smooth muscle. Recent studies in rodents and humans demonstrated that a decrease in ICC number and enteric neurodegeneration during ageing is associated with difficult bowel movements and constipation. By studying ICC and the ENS during normal aging and in a mouse model (Tg2576) of Alzheimer’s disease (AD) where cholinergic loss may be exaggerated, we may gain new perspectives on the treatment of degenerative diseases. The aim of the present study therefore, was to investigate the morphological and functional changes of the GI tract of mice during ageing and in an AD mouse model over-expressing amyloid precursor protein (APP) using an isolated tissue approach. / Whole mount immunohistochemistry of 6-month-old Tg2576 mice and their age-matched wild type (WT) controls revealed that there were significant losses of enteric neurons in the duodenum (P < 0.05) and ileum (P < 0.001), and of GFAP-positive enteric glial cells in the ileum (P < 0.001). There was also a loss of S100-positive glial cells in the antrum (pacemaker region in the stomach) (P < 0.05), ileum (P < 0.05) and colon (P < 0.05). These results indicated the alteration of the ENS during the early stages of AD. There were no differences in ICC arears of all GI regions between 6-month-old Tg2576 mice and their age-matched WT controls (P > 0.05), and there was no alteration of basal peristaltic rhythm during the early stages of AD. The non-significant increase of GFAP to S100 enteric glial cell ratio in the duodenum and colon might indicate an ongoing inflammatory process in these two GI regions during the early stages of AD. / The presence of insoluble amyloid plaques was studied using Aβ immunohistochemistry, Sirius red assay and Thioflavin-T assay on paraffin wax sections. The aggregation of amyloid plaques was observed in all the GI regions of 6-month-old Tg2576 mice and the levels of amyloid plaque varied in different regions. No amyloid plaques were found in the GI tract of 6-month-old WT animals excepting the colon. The increase in formation of amyloid plaques might be correlated to the losses of enteric neurons and enteric glial cells during the early stages of AD. / Western blot analysis was performed on frozen sections of tissues from the ileum and colon to investigate whether there were changes in choline acetyltransferase (ChAT, from excitatory neurons), neuronal nitric oxide synthase (nNOS, from inhibitory neurons), glial cell line-derived neurotrophic factor (GDNF, from enteric glia) and soluble Aβ oligomers between 6-month-old Tg2576 mice and WT controls. nNOS expression significantly increased in the ileum (P < 0.05) but not in the colon (P > 0.05) of Tg2576 mice compared with WT controls. There were no differences in the expressions of ChAT, GDNF and Aβ oligomers (docecamer, nonamer and hexamer) in the ileum and colon between Tg2576 mice and WT controls (P > 0.05). These results imply that there is an increase in the inhibitory signal in the GI tract during the early stages of AD but soluble Aβ oligomers might not be the cause of neuronal and glial losses in the GI tract. / Following histological and biochemical studies of different GI regions, slow wave signals from the antrum and ileum were measured using a microelectrode array (MEA) system. The dominant frequencies (DFs) and power distributions were measured and these served as parameters for measuring functional changes in the GI tract during ageing in ICR mice and the early stages of AD. In the presence of nifedipine, nicotine significantly stimulated the slow wave activities in the antrum and ileum of 3-month-old (P < 0.05) and 6-month-old (P < 0.05) ICR mice but failed to trigger the slow wave activities in 12-month-old (P > 0.05) ICR mice, suggesting the neurodegeneration might begin with the age between 6 and 12 months. With the addition of tetrodotoxin, nicotine failed to stimulate the slow wave activities in the antrum and ileum of three age groups (P > 0.05) and it showed that nicotine only acted on enteric neurons to trigger the ICC activities. There were no differences in the antral and ileal baseline recordings between 6-month-old Tg2576 mice and their age-matched WT controls (P > 0.05). However, nicotine significantly increased DFs and tachygastria ranges of the antrum and ileum in WT controls (P < 0.05) but failed to increase electrical activitiy of the antrum and ileum in Tg2576 mice (P > 0.05), thus suggesting a loss of neuronal and/or glial cells in the GI tract during the early stages of AD. / In conclusions, these findings suggest the mouse model for AD has morphological, biochemical and functional changes in the GI tract. The present studies provide a foundation for the investigation of degenerative diseases and support the hypothesis that the ENS may be the gateway for the early pathological changes in the central nervous system. / 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. / Detailed summary in vernacular field only. / Hui, Chin Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 180-200). / Abstracts also in Chinese. / PUBLICATIONS RELATED TO THE WORK IN THIS THESIS --- p.i / ABSTRACT --- p.ii / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / LIST OF ABBREVIATIONS --- p.vii / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Interstitial cells of Cajal (ICCs) as electrical pacemaker cells in GI tract --- p.1 / Chapter 1.2.1 --- ICC subtypes in GI tract --- p.2 / Chapter 1.3 --- Hypotheses of slow wave generation --- p.4 / Chapter 1.3.1 --- Mechanisms of the NSCC pacemaking hypothesis --- p.5 / Chapter 1.3.2 --- Mechanisms of the alternative hypothesis --- p.6 / Chapter 1.4 --- Involvement of ion channels in slow wave generation of ICC --- p.6 / Chapter 1.4.1 --- Calcium channels --- p.6 / Chapter 1.4.2 --- Sodium channels --- p.7 / Chapter 1.4.3 --- Potassium channels --- p.7 / Chapter 1.4.4 --- Chloride channels --- p.8 / Chapter 1.4.5 --- Non-selective cation channels --- p.8 / Chapter 1.5 --- Distribution of several types of receptors in ICC --- p.11 / Chapter 1.5.1 --- Purinergic receptors --- p.11 / Chapter 1.5.2 --- Muscarinic receptors --- p.11 / Chapter 1.5.3 --- Tachykinin receptors --- p.12 / Chapter 1.5.4 --- Vasoactive intestinal peptide receptors --- p.12 / Chapter 1.5.5 --- Serotonin receptors --- p.13 / Chapter 1.6 --- Introductions and functions of enteric nervous system --- p.15 / Chapter 1.6.1 --- Interaction amongst the central, peripheral and enteric nervous system: brain-gut axis --- p.15 / Chapter 1.6.2 --- Enteric neuronal subtypes in the GI tract --- p.15 / Chapter 1.6.2.1 --- Motor neurons --- p.16 / Chapter 1.6.2.2 --- Interneurons --- p.16 / Chapter 1.6.2.3 --- Intrinsic primary afferent neurons --- p.18 / Chapter 1.6.3 --- Enteric glial cells --- p.18 / Chapter 1.6.3.1 --- Enteric glial subtypes in the GI tract --- p.18 / Chapter 1.6.3.2 --- Communication between enteric neurons and glial cells --- p.19 / Chapter 1.6.3.3 --- Possible functions of enteric glial cells in the GI tract --- p.19 / Chapter 1.6.3.3.1 --- Secretion of neurotrophic factors --- p.20 / Chapter 1.6.3.3.2 --- Secretion of reduced glutathione --- p.20 / Chapter 1.6.3.3.3 --- Secretion of transforming growth factor-beta 1 --- p.21 / Chapter 1.7 --- Interactions amongst ICC, enteric neurons and enteric glial cells --- p.21 / Chapter 1.8 --- Gastrointestinal disorders --- p.22 / Chapter 1.8.1 --- Mechanisms for cell depletion --- p.22 / Chapter 1.8.1.1 --- Autoimmune attack --- p.22 / Chapter 1.8.1.2 --- Hyperglycaemia and diabetes mellitus --- p.24 / Chapter 1.8.1.3 --- Oxidative stress --- p.25 / Chapter 1.8.1.4 --- Ageing --- p.26 / Chapter 1.9 --- Alzheimer’s disease --- p.28 / Chapter 1.9.1 --- Genetics and pathogenesis of Alzheimer’s disease --- p.28 / Chapter 1.9.1.1 --- Aggregation of amyloid beta protein --- p.29 / Chapter 1.9.1.2 --- Genetic factors of AD --- p.29 / Chapter 1.9.1.3 --- Tau hyperphosphorylation and neurofibrillary tangles --- p.31 / Chapter 1.9.2 --- Current treatment for Alzheimer’s disease --- p.33 / Chapter 1.9.2.1 --- Symptomatic treatment --- p.33 / Chapter 1.9.2.2 --- Disease-modifying treatment --- p.34 / Chapter 1.9.2.3 --- Other potential drugs for AD treatment --- p.35 / Chapter 1.9.3 --- Possible animal models for AD investigation --- p.36 / Chapter 1.9.4 --- Possible correlations between Alzheimer’s disease and the enteric nervous system --- p.36 / Chapter 1.10 --- Aim of study --- p.37 / Chapter CHAPTER 2 --- Investigation into the morphologies of enteric nervous system and interstitial cell of Cajal in Tg2576 mice --- p.38 / Chapter 2.1 --- Introduction --- p.38 / Chapter 2.1.1 --- Molecular markers for ICC, ENC, and EGC --- p.38 / Chapter 2.1.2 --- Aims and objectives --- p.39 / Chapter 2.2 --- Materials and methods --- p.41 / Chapter 2.2.1 --- Animals --- p.41 / Chapter 2.2.2 --- Tissue preparation --- p.41 / Chapter 2.2.3 --- Immunohistochemistry --- p.42 / Chapter 2.2.4 --- Image acquisition and analysis --- p.43 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- General observations --- p.44 / Chapter 2.3.2 --- Area and pattern of ICCs and the ENS in the stomach --- p.46 / Chapter 2.3.3 --- Area and pattern of ICCs and the ENS in the duodenum --- p.52 / Chapter 2.3.4 --- Area and pattern of ICCs and the ENS in the jejunum --- p.56 / Chapter 2.3.5 --- Area and pattern of ICCs and the ENS in the ileum --- p.60 / Chapter 2.3.6 --- Area and pattern of ICCs and the ENS in the colon --- p.66 / Chapter 2.4 --- Discussion --- p.70 / Chapter 2.4.1 --- Major findings --- p.70 / Chapter 2.4.2 --- Possible alterations of the ENS during AD --- p.70 / Chapter 2.4.3 --- Morphological changes of the ENS in relation to genotype --- p.71 / Chapter 2.4.4 --- Morphological changes of ICCs in relation to genotype --- p.72 / Chapter 2.4.5 --- Morphological changes of the ENS and ICCs in relation to GI regions --- p.72 / Chapter 2.4.6 --- Inflammatory conditions in different GI regions --- p.73 / Chapter 2.5 --- Conclusion --- p.74 / Chapter CHAPTER 3 --- Formation of amyloid plaques in the brain and the GI tract of Tg2576 mice --- p.75 / Chapter 3.1 --- Introduction --- p.75 / Chapter 3.1.1 --- The absence of amyloid plaques in rodents --- p.75 / Chapter 3.1.2 --- Overexpression of human APP in transgenic mice --- p.76 / Chapter 3.1.3 --- Distribution of human APP and Aβ deposition in human and transgenic mice --- p.77 / Chapter 3.1.4 --- Transgene and promoter in Tg2576 mouse --- p.77 / Chapter 3.1.5 --- Methods for Aβ plaque detection --- p.78 / Chapter 3.1.6 --- Aim and objectives --- p.78 / Chapter 3.2 --- Materials and methods --- p.80 / Chapter 3.2.1 --- Animals --- p.80 / Chapter 3.2.2 --- Tissue processing --- p.80 / Chapter 3.2.3 --- Preparation of paraffin wax blocks and slide sections --- p.81 / Chapter 3.2.4 --- Aβ immunohistochemistry --- p.82 / Chapter 3.2.5 --- Sirius red assay --- p.83 / Chapter 3.2.6 --- Thioflavin-T assay --- p.84 / Chapter 3.2.7 --- Image acquisition --- p.84 / Chapter 3.3 --- Results --- p.85 / Chapter 3.3.1 --- Aβ immunohistochemistry --- p.85 / Chapter 3.3.1.1 --- The absence of positive immunoreactivity in the brain --- p.85 / Chapter 3.3.1.2 --- The presence of positive immunoreactivity in the GI tract of Tg2576 mice --- p.85 / Chapter 3.3.2 --- Sirius red assay --- p.92 / Chapter 3.3.2.1 --- The presence of positive immunoreactivity in the brain of Tg2576 mice --- p.92 / Chapter 3.3.2.2 --- Characteristics of Sirius red staining in the GI tract --- p.92 / Chapter 3.3.2.3 --- The presence of positive immunoreactivity in the GI tract of Tg2576 mice --- p.92 / Chapter 3.3.3 --- Thioflavin-T assay --- p.98 / Chapter 3.3.3.1 --- The presence of positive immunoreactivity in the brain of Tg2576 mice --- p.98 / Chapter 3.3.3.2 --- The presence of positive immunoreactivity in the GI tract of Tg2576 mice --- p.98 / Chapter 3.4 --- Discussion --- p.104 / Chapter 3.4.1 --- The presence of a small amount of amyloid plaques in the brain of young Tg2576 mice --- p.104 / Chapter 3.4.2 --- The presence of amyloid plaques in the GI tract --- p.104 / Chapter 3.4.3 --- Plaque formation in relation to genotype --- p.105 / Chapter 3.4.4 --- Possible effects of amyloid plaques in the brain and GI tract --- p.106 / Chapter 3.5 --- Conclusion --- p.108 / Chapter CHAPTER 4 --- Expression of Aβ oligomers, ChAT, nNOS and GDNF in the GI tract of Tg2576 mice --- p.109 / Chapter 4.1 --- Introduction --- p.109 / Chapter 4.1.1 --- Common and peripheral types of ChAT --- p.109 / Chapter 4.1.2 --- Three subtypes of NOS --- p.111 / Chapter 4.1.3 --- Functions of glial cell line-derived neurotrophic factor in the ENS --- p.112 / Chapter 4.1.4 --- Neurotoxicity of soluble Aβ peptides --- p.113 / Chapter 4.1.5 --- Aims and objectives --- p.113 / Chapter 4.2 --- Materials and methods --- p.115 / Chapter 4.2.1 --- Animals --- p.115 / Chapter 4.2.2 --- Preparation of materials --- p.115 / Chapter 4.2.3 --- Sample preparation --- p.117 / Chapter 4.2.4 --- Separating and stacking gels preparation --- p.118 / Chapter 4.2.5 --- Western blot --- p.119 / Chapter 4.2.6 --- Image acquisition and analysis --- p.120 / Chapter 4.3 --- Results --- p.122 / Chapter 4.3.1 --- Increase in nNOS expression in the ileum of Tg2576 mice --- p.122 / Chapter 4.3.2 --- No changes in the expressions of Aβ oligomers, ChAT, nNOS and GDNF in the colon of Tg2576 mice --- p.122 / Chapter 4.4 --- Discussion --- p.127 / Chapter 4.4.1 --- The absence of “cholinergic hypothesis of AD in the GI tract of Tg2576 mice --- p.127 / Chapter 4.4.2 --- Increased expression of nNOS in the ileum of Tg2576 mice --- p.128 / Chapter 4.4.3 --- Neuronal and glial losses may be related to the reduced GDNF expression --- p.129 / Chapter 4.4.4 --- No relationship between the Aβ oligomers and neuronal damages in the GI tract --- p.129 / Chapter 4.5 --- Conclusion --- p.129 / Chapter CHAPTER 5 --- Microelectrode array (MEA) study on slow wave activity in the GI tract --- p.131 / Chapter 5.1 --- Introduction --- p.131 / Chapter 5.1.1 --- Components in peristalsis-controlling unit --- p.131 / Chapter 5.1.2 --- Techniques in evaluating slow wave activity --- p.131 / Chapter 5.1.2.1 --- Patch clamp --- p.132 / Chapter 5.1.2.2 --- Calcium imaging --- p.132 / Chapter 5.1.3 --- Application of microelectrode array in evaluating slow wave activity --- p.134 / Chapter 5.1.4 --- Aims and objectives --- p.136 / Chapter 5.2 --- Methods and materials --- p.137 / Chapter 5.2.1 --- Animals --- p.137 / Chapter 5.2.2 --- Tissue preparation --- p.137 / Chapter 5.2.3 --- Electrical recordings --- p.138 / Chapter 5.2.4 --- Analysis and Statistics --- p.139 / Chapter 5.3 --- Results --- p.142 / Chapter 5.3.1 --- Experiments on ICR mice --- p.142 / Chapter 5.3.1.1 --- Nicotine stimulates the slow wave activity in the antrum in the presence of NIF but not in the presence of NIF and 500 nM TTX --- p.142 / Chapter 5.3.1.2 --- Nicotine stimulates the slow wave activity in the ileum in the presence of NIF but only partially stimulates activity in the presence of NIF and 500 nM TTX --- p.152 / Chapter 5.3.1.3 --- The use of 1 μM TTX completely blocked the nicotine stimulation in the ileum --- p.160 / Chapter 5.3.1.4 --- The dominant frequency of baseline increased in the ileum of 12-month-old ICR but not in the antrum in the presence of NIF --- p.162 / Chapter 5.3.2 --- Experiments on Tg2576 mice and their wild type controls --- p.164 / Chapter 5.3.2.1 --- No differences in both antral and ileal baseline DFs between 6- month-old non-transgenic and Tg2576 mice --- p.164 / Chapter 5.3.2.2 --- Nicotine stimulates slow wave activity in the antrum of 6-month-old wild type controls but not of Tg2576 mice --- p.164 / Chapter 5.3.2.3 --- Nicotine stimulates slow wave activity in the ileum of 6-month-old wild type controls but not of Tg2576 mice --- p.167 / Chapter 5.4 --- Discussion --- p.171 / Chapter 5.4.1 --- Pharmacological effects of nicotine in the GI tract --- p.171 / Chapter 5.4.2 --- Excitatory effects of nicotine in the slow wave activities of the stomach and ileum --- p.172 / Chapter 5.4.3 --- Changes of ICC functions and neuronal activities during ageing --- p.174 / Chapter 5.4.4 --- Enteric neurodegeneration leads to alteration in the ENS function in Tg2576 mice --- p.175 / Chapter 5.4.5 --- Conclusion --- p.176 / Chapter CHAPTER 6 --- Concluding discussion --- p.177 / REFERENCES --- p.180

It has been a widely accepted belief in scientific and public discourse at the end of the twentieth century that cannibalism was the principal means of transmission of the disease call 'Kuru'.The study argues that other explanations might have been excluded from consideration, in particular, iatrogenic transmission.Circumstantial evidence in support of this proposition is examined.The work begins with an examination of the relationship between a number of diseases including, X disease, poliomyelitis, louping ill, scrapie and kuru through the first half of the twentieth century. Major themes of the work revolve around the boundary between research on animal and human disease, the complexities of research in this area, and the different messages that exist simultaneously in three domains: scientific research and publications, government and institutional archives, and the public domain. The thesis argues that the circumstantial evidence presented needs to be considered seriously and that further research in the area is required before we can come to a reliable understanding of the factors involved in the transmission of kuru / Doctor of Philosophy (PhD)

Thesis (M.S.P.H.)--University of Michigan, 1938.

Thesis (M.S.P.H.)--University of Michigan, 1938.

Fused in sarcoma (FUS) is an RNA binding protein involved in DNA repair and RNA metabolism, including mRNA transcription, splicing, transport and translation. FUS is genetically and pathologically associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To explore the mechanisms by which pathogenic mutations in FUS cause neurodegeneration in ALS-FUS, we generated a series of FUS knock-in mouse lines that express the equivalent of the ALS-associated mutant proteins FUSP525L and FUSΔEX14 at physiological levels from the FUS locus. We demonstrate that heterozygous mutant FUS mice show progressive, age-dependent loss of vulnerable subpopulations of spinal motor neurons. While ALS-associated mutations in FUS lead to partial loss of function, we provide genetic evidence that the motor neuron phenotype observed is a consequence of a dose-dependent gain of function, associated with the insolubility of FUS and related RNA binding proteins (RBPs). Furthermore, we show that motor neuron degeneration is driven by cell autonomous mechanisms, associated with mutant FUS-independent inflammatory changes. In this faithful mouse model of ALS-FUS, we demonstrate that an antisense oligonucleotide (ASO) targeting the FUS transcript (ION363) results in the efficient silencing of both wild type and mutant FUS alleles, and that postnatal reduction of FUS protein levels in the brain and spinal cord delays disease onset in this mouse model of ALS-FUS. In a first-in-human trial of ION363, we demonstrate that repeated, intrathecal injections of this candidate therapeutic in an ALS patient with a FUSP525L mutation leads to the efficient silencing of both wild type and mutant FUS in the central nervous system, and a reduction in the burden of FUS aggregates that are a pathological hallmark of ALS-FUS. In mouse genetic and human clinical studies, we provide evidence in support of a therapeutic strategy by which silencing of the FUS gene may be used to prevent or delay disease onset in pre-symptomatic carriers of pathogenic FUS mutations, or to slow disease progression in symptomatic ALS- and FTD-FUS patients. In addition, we use this newly generated model to investigate the role of potential modifiers of FUS toxicity, including hnRNP U and UPF1, and study the role of chronic neuroinflammation in the disease progression that could lead to the development of novel therapeutics to provide immediate clinical benefit to patients with ALS-FUS.

Thesis (DPhil)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: This qualitative study comprised an in-depth investigation into the subjective - the explicitly human - experience of those suffering from Myalgic Encephalomyelitis (ME). I was, firstly, concerned with the nature and meaning of the social side of illness, that is, the sufferer's encounters with doctor, family member, friend and acquaintance alike. I was, secondly, set to develop greater insight into the essentially personal experience of being ill. I was able to conclude that, even amidst the inhumane presence of utter ignorance that permeate the very experience of ME, no ME sufferer is inevitably doomed a victim. They can always make a deliberate decision to have a say in their situation, in their own experience of their circumstances. They have the power of personal choice. Recommendations are, therefore, directed at expanding the potential extent and magnitude of this dynamic power. The substantive focus of my study was enriched by a deliberate concern with the methodological implications of my own intimate involvement within the research process. I was here primarily concerned with my personal contribution to the research process as well as the influence thereof on the research relationships developed and the research strategies chosen and applied. I could not but conclude that the understanding I explicate is, as all social science theory, essentially a human construction, developed by me, in my distinctly human capacity. Recommendations are, therefore, geared to sensitise all social researchers to their own contribution to the construction of that which is eventually presented (and taken) as truthful knowl~dge. In conclusion, I am thoroughly convinced that the experience of both illness and research is fundamentally human. This "humanness" cannot and should not be denied. Instead, I advocate a more deliberate focus on the human dimension of illness and research. Without such a focus, a more comprehensive understanding of either realm will continue to linger as but an elusive ideal. / AFRIKAANSE OPSOMMING: Hierdie kwalitatiewe studie is gebaseer op 'n in-diepte ondersoek gerig op die subjektiewe - die onteenseglik menslike - ervaring van diegene wat ly aan Myaligië Enkefalomiëlitis (ME). Ek was, eerstens, geïnteresseerd in die aard en betekenis van die sosiale dimensie van siekte, dit wil sê, die lyer se ervaring van sosiale kontak met dokters, gesinslede, vriende en kennisse. Ek was, tweedens, gerig op die ontwikkeling van 'n grondige insig in die uiters persoonlike ervaring van siek-wees. Ek het tot die gevolgtrekking gekom dat, selfs te midde van die onmenslike teenwoordigheid van blatante onkunde wat die ganse ervaring van ME kenmerk, geen ME lyer noodwendig tot 'n slagoffer-status gedoem is nie. Hulle kan altyd 'n doelbewuste besluit neem om 'n sê te hê in hul eie situasie, in hul eie ervaring van hul omstandighede. Hulle het die mag van persoonlike keuse. Aanbevelings is dus daarop gerig om die potentiële trefwydte en impak van hierdie dinamiese mag uit te brei. Die substantiewe dimensie van my studie is verryk deur 'n doelbewuste fokus op die metodologiese implikasies van my eie intieme betrokkenheid in die navorsingsproses. Ek was hoofsaaklik gemoeid met my persoonlike bydrae tot die navorsingsproses en die invloed daarvan op die ontwikkel van navorsingsverhoudings en die toepassing van gekose navorsingstrategieë. Hierdie fokus het gelei tot die besef dat die beskrywing wat ek aanbied, soos inderdaad alle sosiale teorie, essensieël 'n menslike konstruksie is, soos ontwikkel deur my, in my uitdruklik menslike kapasiteit. Aanbevelings is dus daarop gerig om alle sosiale navorsers te sensitiseer ten opsigte van hul eie bydrae tot die konstruksie van dit wat uiteindelik voorgestel (en geag) word as die waarheidsgetroue kennis. In slotsom, is ek oortuig dat die ervaring van beide siekte en navorsing fundamenteel menslik is. Hierdie "mensheid" kan en behoort nie ontken te word nie. Inteendeel, ek bepleit 'n doelbewuste fokus op die menslike dimensie van siekte en navorsing. Sonder só 'n fokus sal 'n meer diepgaande begrip van iedere area bloot 'n onbereikbare ideaal bly.

Compte-tenu de l’importance des populations exposées, les effets à long terme des pesticides sont un enjeu de santé publique majeur. Leur étude pose néanmoins des problèmes méthodologiques complexes. Notre objectif était de contribuer à la connaissance des effets chroniques des pesticides sur le système nerveux central en explorant par une approche épidémiologique innovante le rôle des insecticides organophosphorés dans l’apparition de troubles cognitifs. Nous avons développé et utilisé deux outils de mesure de l’exposition (une matrice culture-exposition : PESTIMAT, et des algorithmes basés sur des études de terrain : PESTEXPO) afin d’estimer l’exposition à 34 insecticides organophosphorés utilisés en vigne, en considérant pour chaque individu l’ensemble des tâches viticoles exposant aux pesticides (préparation, application, nettoyage, réentrée) réalisées au cours de sa vie. Ces outils ont été appliqués dans le cadre du premier suivi (2001-2003) de la cohorte PHYTONER constituée en 1997 par l’inclusion de 925 affiliés à la Mutualité Sociale Agricole de Gironde. L’exposition aux organophosphorés définie par des index cumulés à l’aide de nos deux outils était associée à une baisse des performances cognitives, de manière plus marquée pour les tests explorant la mémoire de travail visuelle et la vitesse de traitement de l’information. Les niveaux de risque variaient en fonction des organophosphorés et étaient plus marqués pour le mévinphos. Cette thèse a confirmé le risque de troubles cognitifs chez les utilisateurs professionnels de pesticides et pose la question d’une évolution ultérieure vers une démence. Elle a également démontré la faisabilité et la pertinence d’une approche s’attachant à établir des scores d’exposition à des matières actives pesticides spécifiques pour en analyser les effets de santé. / Given the number of exposed persons, long term effects of pesticides are a foremost Public Health concern. However their study raises complex methodological issues. Our objective was to contribute to the knowledge of the pesticide chronic effects on the central nervous system by exploring the role of organophosphate insecticides in occurence of cognitive disorders by an innovative epidemiological approach. Two exposure assessment tools were developed (a crop exposure matrix: PESTIMAT and algorithms based on field studies: PESTEXPO) to estimate the lifetime cumulated exposure to 34 organophosphate insecticides used in vineyards, taking into account pesticide exposure during tasks (mixing, spraying, cleaning, re-entry) performed by wine-growers. These tools were used in the framework of the first follow-up (2001-2003) of the PHYTONER cohort, initiated in 1997 by the enrollment of 925 workers affiliated to the farmer health insurance system in Gironde, France. Cumulative organophosphate exposure defined by an index using the two tools was associated with poor cognitive performances, particularly for tests exploring the visual working memory and the processing speed. Risk level varied depending on the organophosphate, and was more pronounced for mevinphos. This thesis supports the hypothesis that cognitive impairment may be associated with pesticide occupational use and raises the question of a further evolution towards dementia. It also demonstrated the feasibility and the relevance of an approach based on chemical specific exposure scores to analyze health effects.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 2, 2010). Includes bibliographical references.

The majority of the population in South Africa use traditional health care to treat various mental conditions. This thesis has two main objectives; to bring together a comprehensive and detailed record of psychotropic plants used in southern Africa by indigenous peoples for medicinal or cultural purposes. Secondly, this research attempts to investigate the validity and rationale of the use of these plants by screening them in various biological assays for psychotropic activity. Plants were selected, based on their traditional use and availability, and were screened in four assays, which detect biological activity of a useful nature. A number of in vitro enzymatic and neuronal signal transduction assays were employed in this thesis, the inhibition of the serotonin reuptake transporter protein (SERT); inhibition of catabolic enzymes (e.g. acetylcholinesterase, monoamine oxidase); GABAA- benzodiazepine receptor binding. The influence of legislation, past and present, on the state of traditional medicine is highlighted. Aspects of the philosophies and practises of the various practitioners of South African traditional medicine will be discussed. An annotated list compiled from available ethnobotanical literature of plants traditionally used for central nervous system-related purposes is provided. It contains more than 330 species, from 94 families, which are currently used or have been used for cultural, medicinal and recreational purposes related to the central nervous system (CNS). Where available, information pertaining to plant part used, preparation method, dosage, route of administration, known and potentially active constituents are included. Seventy five extracts from 34 indigenous plant species used in South African traditional medicine or taxonomically related to these were investigated for their affinity to the serotonin reuptake transport protein, making use of an in vitro [3H]-citalopram serotonin reuptake transport protein binding assay. Aqueous and 70% ethanolic extracts of various plant parts were screened and 45 extracts derived from 15 plant species showed affinity. The affinity of 12 extracts from four plants was characterized as high (more than 50% inhibition at 5, 1, and 0.5 mg/ml). Plant species with high affinity to the serotonin reuptake transport protein included Agapanthus campanulatus, Boophone disticha, Datura ferox and Xysmalobium undulatum. Agapanthus campanulatus yielded high activity in aqueous extracts from leaves and flowers. B. disticha showed high activity both in aqueous and ethanolic extracts of leaves and bulbs. D. ferox showed high activity in aqueous extracts from the seeds and X. undulatum showed high activity in the ethanolic extract of the whole plant. Two compounds, buphanadrine and buphanamine, were isolated by bioassay-guided fractionation on vacuum-liquid-chromatography (VLC) and preparative thin-layer-chromatography (TLC) from B. disticha. The structures of the compounds were determined by 1H and 13C NMR. Fractions were tested for affinity to the serotonin transporter in a binding assay using [3H]-citalopram as a ligand. The IC50 values of buphanidrine and buphanamine were 274 ìM (Ki = 132 ìM) and 1799 ìM (Ki = 868 ìM), respectively. The two alkaloids were also tested for affinity to the 5HT1A receptor, but only showed slight affinity. Aqueous and ethanol extracts of 43 plants that are traditionally used to treat against epilepsy and convulsions were initially tested in the GABAA-benzodiazepine receptor binding assay, where the binding of 3H-Ro 15-1788 (flumazenil) to the benzodiazepine site is measured. The GABAA-benzodiazepine receptor complex is involved in epilepsy and convulsions. Out of the 118 extracts tested, one aqueous and 18 ethanol extracts showed activity. The most active extracts were the ethanolic leaf extracts of Searsia tridentata, Searsia rehmanniana and Hoslundia opposita and the ethanolic corm extract of Hypoxis colchicifolia, which all showed good dose-dependent activity. A further forty-six ethanol extracts from another 35 species, both indigenous and exotic that are traditionally used predominantly as sedatives or to treat various CNS-related ailments were tested in the GABAA-benzodiazepine receptor-binding assay. Out of the 46 extracts tested, seven showed good activity and 10 showed moderate activity. The most active extracts were the ethanolic leaf extracts of Arctopus echinatus, Artemisa afra, four Helichrysum species and Mentha aquatica which all showed good dose-dependent activity. Two biflavonoids with activity in the 3H-Ro 15-1788 (flumazenil) binding assay were isolated by high pressure liquid chromatography (HPLC) fractionation of the ethanol extract of the leaves from Searsia pyroides. The structures of the two biflavonoids were elucidated by nuclear magnetic resonance spectroscopy (NMR) to be agathisflavone and amentoflavone. Agathisflavone and amentoflavone competitively inhibited the binding of 3H-Ro 15-1788 with a Ki of 28 and 37 nM, respectively. Extracts of Searsia dentata and Searsia pentheri were not as active as the extract from Searsia pyroides; both were found to contain apigenin and agathisflavone. The monomer apigenin, agathisflavone and amentoflavone were fitted into a pharmacophore model for ligands binding to the GABAA receptor benzodiazepine site. This reflected the affinities of the compounds in the [3H]-flumazenil binding assay. Mentha aquatica, a mint that is found in Europe and Africa, is used in Zulu traditional medicine for spiritual purposes. The ethanolic leaf extract showed a strong affinity to the GABA-benzodiazepine receptor. Viridiflorol from the essential oil and (S)-naringenin from an ethanolic extract was isolated by bioassay-guided fractionation using binding to the GABA-benzodiazepine site. Viridiflorol had an IC50 of 0.19 M and (S)-naringenin of 0.0026 M. Twenty plants used in Zulu traditional medicine for several CNS-related ailments were screened for MAO inhibition and specific MAO-B inhibition activity. MAO-B inhibitors are currently employed in the treatment of neurodegenerative related illnesses such as Parkinson's and Alzheimer's diseases. A photometric peroxidase linked assay was used to determine the inhibition of the oxidative deamination of tyramine by MAO isolated from rat liver. Ruta graveolens exhibited the best MAO inhibitory activity (ethyl acetate leaf extract = IC50 5 ± 1 ìg/ml, petroleum ether extract = 3 ± 1 ìg/ml) and specific MAO-B inhibition (ethyl acetate leaf extract = IC50 7 ± 6 ìg/ml petroleum ether extract = 3 ± 1 ìg/ml). Schotia brachypetala, Mentha aquatica and Gasteria croucheri also exhibited good MAO-B inhibition activity. Six extracts of varying polarity of Mentha aquatica were tested in a photometric peroxidase linked MAO bioassay. The 70% ethanol extract had highest inhibitory activity. (S)-Naringenin was isolated from the extract by bioassay guided fractionation on VLC and preparative TLC. The structure of the compound was determined by 1H, 13C and 13C-DEPT NMR and optical rotation. The IC50 values for MAO inhibition by naringenin were 342 ± 33 ìM for the rat liver mitochondrial fraction, 955 ± 129 ìM for MAO-A and 288 ± 18 ìM for MAO-B respectively. South African traditional medicine clearly utilizes many botanical species with CNS-related activity. Only a small number of the more than 330 southern African plant species reported to treat or alter the CNS have been scientifically evaluated. To date very few of the active compounds have been isolated and identified. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.