A lesão renal induzida pela I/R é a principal causa de IRA nos rins nativos e nos rins transplantados e estudos enfatizam a participação de células inflamatórias na sua patogênese, através da caracterização de lesão endotelial, infiltração leucocitária e a geração de mediadores inflamatórios pelas células epiteliais tubulares. Evidências recentes mostram que as células T CD4+ exercem um papel fundamental como mediadoras da agressão renal na I/R, ressaltando-se o envolvimento do paradigma Th1/Th2 como um possível mecanismo efetor. O presente estudo foi realizado com o objetivo de estudar a participação de algumas citocinas Th1 e Th2 no desenvolvimento da lesão de I/R renal. Para tanto, nós nos propusemos a desenvolver um modelo experimental de I/R renal em camundongos deficientes em IL-12, IFN-, e duplo deficientes em IFN- e IL-12 (representando defeito da via de ativação Th1), camundongos deficientes em IL-4 e IL-10 (representando defeito da via de ativação Th2) e duplo deficientes em IL-10 e IL-12, tendo como controles camundongos normais (selvagens). Todos os animais foram submetidos a uma lesão de I/R por ligadura reversível do pedículo renal por 45 minutos seguidos de 24 horas de reperfusão. Após a indução da isquemia, nós analisamos as alterações funcionais (creatinina e uréia por método bioquímico colorimétrico) e morfológicas renais (morfometria renal), além de investigar a expressão molecular de HO-1 (um gene de proteção tecidual), de t-bet (um transcrito envolvido na diferenciação Th1), de GATA-3 (um transcrito envolvido na diferenciação Th2), citocina pró-inflamatória IL-6 e de uma quimiocina pró-inflamatória MCP-1, visando caracterizar a influência da polarização Th1/Th2 da resposta imune na lesão renal induzida pela I/R. Nós mostramos que os camundongos deficientes em IL-4, IFN-, IL-10/IL-12 e IL-10 apresentaram uma disfunção renal importante, caracterizada por altos níveis séricos de creatinina e uréia e por um alto grau de agressão morfológica renal, avaliada pela percentagem de área de necrose tubular. Todos estes resultados foram significativamente mais acentuados que obtidos nos camundongos deficientes em IL-12 e IFN-/IL-12, sendo comparáveis aos animais selvagens. Por outro lado, a capacidade de reparação renal, medida pela percentagem de área de regeneração tubular, foi mais precoce nos camundongos deficientes em IFN-, quando comparada com os camundongos deficientes em IL-12 e IFN-/IL-12. A análise molecular quantitativa, utilizando-se o PCR em tempo real mostrou uma expressão significantimente maior de RNAm de HO-1, IL-6 MCP-1 e do fator transcricional pata Th2 (GATA-3) nos camundongos deficientes em IL-4, IFN-, IL-10/IL-12 e IL-10, em comparação com os camundongos deficientes em IL-12, IFN-/IL-12 que apresentaram baixa expressão do fator transcricional para Th1 (T-bet), em 24 horas após a I/R renal. Quando fomos fazer a transferência adotiva de médula óssea de animais deficientes em IL-12 e IL-4 para animais selvagens previamente com depleção subletal celular, esses animais vieram apresentar resultados semelhantes aos animais deficientes em IL-12 e IL-4, onde os animais selvagens reconstituídos com medula óssea dos animais IL-12 apresentaram menor níveis de uréia sérica e menor expressão de RNAm IL-6, enquanto que os animais selvagens recontituídos com medula óssea dos animais IL-4 apresentaram maior níveis de uréia sérica e maior expressão de RNAm IL-6, esses dados comprovam que realmente a citocina IL-12 parece ser a principal citocina envolvida no processo inflamatório desencadeado pela lesão de isquemia e reperfusão renal. Tais achados favorecem a hipótese dos efeitos deletérios das células com per_l Th1 e o papel protetor das células Th2 na lesão isquêmica renal. Estes novos episódios podem fornecer a base para uma melhor compreensão fisiopatológicos, bem como para o desenvolvimento de métodos preventivos e terapêuticos para a insuficiência renal aguda isquêmica. / Renal ischemia/reperfusion injury (I/R) is the major cause of acute renal failure (ARF) in the native as well as in the transplanted kidneys, with a complex pathogenesis that involves many components of inflammatory response such as leukocyte infiltration and generation of inflammatory mediators by tubular cells. Recent evidences show a critical role of the CD4+ T cell, with the Th1/Th2 paradigm as a possible ejectors mechanism. The present study has the objective to investigate the participation of some main Th1 and Th2 cytokines in the development of the renal of I/R. For that, we developed an experimental model of renal I/R in deficient in IL-12, IFN-, double deficient IFN- and IL-12 (representing defect of the pathway Th1), IL-4 and IL-10 (representing defect of the pathway Th2) and in double deficient IL-10 and IL-12 knockout mice, having wild-type mice as controls. All animals were submitted to renal I/R by reversible ligation of the renal pedicles for 45 minutes followed by 24 hours of reperfusion. After induction of the ischemia, we analyzed renal function and morphometric histological analyzes. Furthermore, we quantified the expression of HO-1 (cytoprotection gene), t-bet (transcription factor involved in the differentiation Th1), GATA-3 (transcription factor involved in the differentiation Th2), and cytokine pro-inflammatory IL-6 and chemokine pro-inflammatory MCP-1, aiming to characterize the influence of Th1/Th2 polarization in the immune response driven by the renal I/R. We showed that the IL-4, IFN-, IL-10 and IL-10/IL-12-de_cient mice presented an important kidney dysfunction, characterized by higher levels serum creatinine and tubular necrosis, being comparable to the wild-type animals. The molecular analyses, utilizing the real-time PCR, showed a significantly higher expression of HO-1, IL-6, MCP-1 and transcription factor involved in the differentiation Th2 (GATA-3) in the in IL-4, IFN-, IL-10 and IL-10/IL-12-defficient mice, in comparison with the in IL-12, IFN-/IL-12-de_cient mice showed lower expression transcription factor involved in the differentiation Th1 (T-bet) . Moreover, the renal dysfunction seen in Th2-de_cient mice was followed by a higher expression of IL-6. When we make the transfer adoptive of bone marrow of animals deficient in IL-12 and IL-4 to wild animals previously with sublethal cell depletion, these animals produce similar results to animals deficient in IL-12 and IL-4, where the animals reconstituted wild animals with bone marrow IL-12 showed lower levels of serum urea and lower expression mRNA of IL-6, while the wild animals reconstituted with bone marrow of animals IL-4 showed higher levels of serum urea and greater expression of mRNA IL-6. These data show that really the cytokine IL-12 appears to be the main cytokine involved in the inflammatory process triggered by the injury of ischemia and reperfusion kidney. Such _ndings favor the hypothesis of the deleterious effects of Th1 cells with prole and the protective role of Th2 cells in ischemic renal injury. These novel insights may provide basis for the better pathophysiological understanding, as well as for development of preventive and therapeutic methods for the ischemic acute renal failure.

"Ischemia is a condition of decreased tissue viability caused by a lack of perfusion, which prevents the delivery of oxygen and nutrients to biological tissue. Ischemia plays a major role in many clinical disorders, yet there are limited means by which tissue viability can be assessed. The long-term objective of this research is to develop a non-invasive or non-contact instrument for quantifying human tissue ischemia. Skeletal muscle ischemia is evaluated at this stage because skeletal muscle is easily accessible, its ischemia represents a clinical problem, and it can endure short periods of ischemia without suffering permanent injury. The ischemia monitor designed for this study is based on impedance spectroscopy, the measurement of tissue impedance at various frequencies. This study had three major goals. The first goal was to improve upon the design of the ischemia monitor to achieve optimal system performance in a clinical environment. Major considerations included electrode sterility, instrument mobility, and electrosurgical unit interference. The second goal was to collect both impedance and pH data from human subjects undergoing tourniquet surgeries, which induce skeletal muscle ischemia and result in changes of the tissue's pH and impedance. The average in recorded pH during ischemia was 0.0053 pH units/minute and the average change in Ro was -0.1481 Ohms/minute. The third goal was to develop a relationship between parameters of tissue impedance and pH utilizing neural networks. This goal was accomplished in three stages. First, the optimal neural network type for classifying impedance data and pH values was determined. Based on these results, the backpropagation neural network was utilized for all subsequent work. Then, the input parameters of the neural network were optimized using previously collected data. The number of inputs to the previously developed neural network were reduced by 35% (13/20) with a maximum of a 3% reduction in neural network performance. Finally, the neural network was trained and tested using human impedance and pH data. The network was able to correctly estimate tissue pH values with an average error of 0.0440 pH units. Through the course of this research the ischemia monitor based on impedance spectroscopy was improved, a methodology for the use of the instrument in the operating room was developed, and a preliminary relationship between parameters of impedance spectra and pH was established. The results of this research indicate the feasibility of the instrument to monitor both pH and impedance in a clinical setting. Additionally, it was demonstrated that impedance data collected non-invasively could be used to estimate the pH and level of ischemia in human skeletal muscle."

Chiu, Wui Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 107-128). / Abstracts in English and Chinese. / Abstract --- p.i / 中文摘要 --- p.iv / Acknowledgements --- p.vi / Publications --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xiv / List of Figures --- p.xv / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hypoxic-ischemic encephalopathy in human infants --- p.1 / Chapter 1.1.1 --- Incidence --- p.1 / Chapter 1.1.2 --- Biphasic development of HI brain damage --- p.2 / Chapter 1.1.2.1 --- Initiating mechanism: energy failure in immature brain --- p.3 / Chapter 1.1.2.2 --- Biochemical cascades --- p.4 / Chapter 1.1.2.2.1 --- Excitatory amino acid receptor activation by glutamate --- p.4 / Chapter 1.1.2.2.2 --- Intracellular calcium accumulation --- p.4 / Chapter 1.1.2.2.3 --- Formation of free radicals --- p.5 / Chapter 1.1.2.2.3.1 --- Reactive oxygen species (ROS) --- p.5 / Chapter 1.1.2.2.3.2 --- Nitric oxide (NO) --- p.6 / Chapter 1.1.2.3 --- Release of inflammatory mediators --- p.6 / Chapter 1.1.2.4 --- Mitochondrial dysfunction --- p.7 / Chapter 1.1.2.5 --- Final path to death: necrosis or apoptosis --- p.8 / Chapter 1.1.2.6 --- Ways to change: neuronal survival and proliferation signaling --- p.8 / Chapter 1.1.3 --- Interventions for neonatal hypoxia-ischemia --- p.9 / Chapter 1.2 --- Animal models mimicking hypoxia-ischemia brain injury --- p.12 / Chapter 1.2.1 --- Comparisons of animal models of hypoxia-ischemia --- p.12 / Chapter 1.2.2 --- Development of neonatal rat model with hypoxic-ischemic damage --- p.14 / Chapter 1.3 --- Neural stem/progenitor cells --- p.15 / Chapter 1.3.1 --- Effect of hypoxic-ischemia on neural stem/progenitor cells --- p.17 / Chapter 1.4 --- Thrombopoietin --- p.18 / Chapter Chapter 2 --- Objectives --- p.23 / Chapter Chapter 3 --- Materials and Methodology --- p.24 / Chapter 3.1 --- Establishment of neonatal rat model of HI brain damage and effects of TPO on neural protection --- p.24 / Chapter 3.1.1 --- Animal protocols --- p.24 / Chapter 3.1.2 --- Induction of HI brain damage in neonatal rats --- p.24 / Chapter 3.1.3 --- Treatment with TPO --- p.25 / Chapter 3.1.4 --- Sacrifice of rats --- p.25 / Chapter 3.1.5 --- Read-out measurements --- p.26 / Chapter 3.1.5.1 --- Brain weight --- p.26 / Chapter 3.1.5.2 --- Gross injury assessment of the right hemisphere --- p.26 / Chapter 3.1.5.3 --- Histology --- p.27 / Chapter 3.1.5.4 --- Blood cell count --- p.27 / Chapter 3.1.5.6 --- Functional assessments --- p.28 / Chapter 3.1.5.6.1 --- Grip traction test --- p.28 / Chapter 3.1.5.6.2 --- Elevated body swing test --- p.28 / Chapter 3.1.5.7 --- Statistical analysis --- p.28 / Chapter 3.2 --- Establishment of in vitro model of primary mouse NSPs and the effect of TPO on their proliferation --- p.29 / Chapter 3.2.1 --- Mouse embryo dissection for the extraction of NSP --- p.29 / Chapter 3.2.2 --- Culturing of NSP --- p.30 / Chapter 3.2.3 --- Immunofluorescence staining for stem cell markers --- p.31 / Chapter 3.2.4 --- Neurosphere assay with different combinations of mitogens --- p.31 / Chapter 3.2.5 --- Neurosphere assay with different concentrations of TPO --- p.32 / Chapter 3.2.6 --- Neurosphere assay under hypoxia --- p.32 / Chapter 3.2.7 --- Statistical analysis --- p.33 / Chapter Chapter 4 --- Effects of thrombopoietin on neonatal rat models of hypoxia-ischemia brain damage --- p.39 / Chapter 4.1 --- Summary of experimental settings --- p.39 / Chapter 4.2 --- Results --- p.39 / Chapter 4.2.1 --- Mortality --- p.39 / Chapter 4.2.2 --- Effects of TPO on p7 mild damage model 1 week post-surgery --- p.40 / Chapter 4.2.2.1 --- Body and brain weights --- p.40 / Chapter 4.2.2.2 --- Gross injury score --- p.41 / Chapter 4.2.2.3 --- Cortex and hippocampus area --- p.41 / Chapter 4.2.2.4 --- Blood cell counts --- p.42 / Chapter 4.2.3 --- Effects of TPO on p7 severe damage model 1 week post-surgery --- p.43 / Chapter 4.2.3.1 --- Body and brain weights --- p.43 / Chapter 4.2.3.2 --- Gross injury score --- p.43 / Chapter 4.2.3.3 --- Cortex area --- p.44 / Chapter 4.2.3.4 --- Blood cell counts --- p.44 / Chapter 4.2.4 --- Effects of TPO on p7 severe damage model 3 week post-surgery --- p.45 / Chapter 4.2.4.1 --- Body and brain weights --- p.45 / Chapter 4.2.4.2 --- Gross injury score --- p.46 / Chapter 4.2.4.3 --- Blood cell counts --- p.46 / Chapter 4.2.4.4 --- Functional outcomes --- p.46 / Chapter 4.2.5 --- Effects of TPO on pl4 severe damage model 1 week post-surgery --- p.47 / Chapter 4.2.5.1 --- Body and brain weights --- p.47 / Chapter 4.2.5.2 --- Gross injury score --- p.48 / Chapter 4.2.5.3 --- Cortex area --- p.48 / Chapter 4.2.5.4 --- Blood cell counts --- p.49 / Chapter 4.3 --- Discussion --- p.49 / Chapter Chapter 5 --- Effects of thrombopoietin on the proliferation of primary mouse neural stem/ progenitor cells in culture --- p.83 / Chapter 5.1 --- Summary of experimental settings --- p.83 / Chapter 5.2 --- Results --- p.83 / Chapter 5.2.1 --- Effect of EGF or bFGF withdrawal on NSP proliferation --- p.84 / Chapter 5.2.2 --- Dose effect of TPO treatment on NSP proliferation --- p.85 / Chapter 5.2.3 --- Effect of hypoxia --- p.85 / Chapter 5.2.4 --- Effect of TPO treatment in combination with hypoxia --- p.86 / Chapter 5.2.5 --- Detection of neural progenitor cell marker --- p.87 / Chapter 5.3 --- Discussion --- p.88 / Chapter Chapter 6 --- General discussion --- p.101 / Bibliography --- p.106

Ischaemic stroke has become one of the leading causes of death and disability in the world. Protease activated receptors (PARs, PAR-1 to PAR-4) belong to G protein coupled receptors that can be self-activated by tethered ligands (TL) revealed through proteolytic cleavage. Based on these TL, many activating peptides (APs) and antagonists have been synthesized to investigate PARs actions. / In the present study, the roles of PARs were examined in two models of ischaemic stroke. For the in vivo model, transient middle cerebral artery occlusion (tMCAO) was performed to establish cerebral ischaemia in rats. For the in vitro model, oxygen and glucose deprivation (OGD) was used to mimic an ischaemia insult in primary cultured rat embryonic cortical neurones. / Western blot studies showed that expressions of PAR-1 and PAR-2 were increased in the rat ischaemic brain cortex, whereas PAR-1 was reduced in the rat cortical neurones subjected to OGD. Pretreatments of PAR-1 AP (SFLLRN-NH₂) and PAR-2 AP (SLIGRL-NH₂) produced significant protection against ischaemia-induced damage. Pretreatment of PAR-3 AP (SFNGGP-NH₂) only improved ischaemic symptoms in in vivo but not in in vitro model. When treated after ischaemia, only PAR-1 AP produced significant reductions on ischaemia-induced damage. Protective actions of PAR-1 and PAR-2 APs were inhibited by PAR-1 antagonist (BMS-200261) and PAR-2 antagonist (ENMD-1068) respectively, but PAR-1 antagonist did not affect posttreatment effects of PAR-1 AP in in vitro model. Pre- and posttreatments of thrombin, and pretreatment of trypsin also protected ischaemia-induced damage in the two models. / PAR-1 AP produced marked increase in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and ratio of bcl-2/bax, but reduced contents of reactive oxygen species (ROS), nitric oxide (NO) and malondialdehyde (MDA) in both ipsilateral ischaemic brain cortices and in rat cortical neurones subjected to OGD. In the in vitro model, PAR-1 AP greatly decreased caspase-3 activity and TUNEL positive cells, while markedly increased mitochondrial membrane potential (MMP). All these protective actions were inhibited by its antagonist, which suggests it was mediated via activation of PAR-1. / In MCA isolated from normal and ischameic rats, PAR-2 AP and trypsin produced vasodilatation while PAR-3 AP elicited vasoconstriction. However, another PAR-3 AP had no effect in the two types of MCA. A high concentration of PAR-1 AP relaxed MCA isolated form ischaemic rats, and it was not inhibited by a PAR-1 antagonist. The vasodilator action of PAR-2 AP was inhibited by one of two PAR-2 antagonists tested. The vasodilator actions induced by PAR-1 and PAR-2 APs involved NO production since L-NAME was effective in inhibiting their actions. / In conclusion, PAR-1 AP was found to be the most efficacious in protecting the brain from ischaemia-induced damage when administered either before or after ischaemia insults. The protective actions were likely to be attributed to its anti-oxidant properties in the ischaemic brain that reduced apoptosis of brain cells. Therefore, PAR-1 was identified as a promising target for development of novel prophylactic and therapeutic treatments of ischaemic brain disease. / 缺血性腦中風已經成為全世界導致死亡和殘疾的最主要的疾病之一。蛋白酶激活受體（PARs, PAR-1 to PAR-4）屬於G蛋白偶聯受體並且可以通過蛋白水解生成系鎖配體（TL）從而作用於受體本身而激活信號通路。根據TL的序列已經合成了很多激活肽和拮抗劑，它們可以作為有價值的工具藥進行PAR的作用研究。 / 當前，PAR的作用在兩個缺血性腦中風模型中進行研究。體內模型是通過大鼠大腦中動脈阻塞手術而建立；體外模型是通過對大鼠胚胎大腦皮層神經元進行氧糖剝奪模擬缺血性損傷。 / 蛋白質印跡法的實驗表明PAR-1和PAR-2的表達在缺血側大腦皮層中有所增多，而PAR-1在氧糖剝奪的大鼠皮層神經元中表達卻有所降低。預處理PAR-1（SFLLRN-NH₂）和PAR-2（SLIGRL-NH₂）的激活肽顯著改善了缺血導致的損傷。預處理PAR-3激活肽（SFNGGP-NH₂）僅僅改善了體內缺血症狀，卻對體外缺血模型沒有效果。然而，當這些激活肽在缺血后給予的時候，只有PAR-1的激活肽顯著改善了缺血損傷。PAR-1的拮抗劑（BMS-200261）和PAR-2的拮抗劑（ENMD-1068）抑制了PAR-1和PAR-2激活肽的保護作用，但是體外實驗後處理PAR-1激活肽的保護作用卻未收影響。預處理及後處理凝血酶，預處理胰酶都在這兩個模型中顯示出保護缺血性損傷的作用。 / PAR-1激活肽在缺血同側大腦皮層以及經受氧糖剝奪的大鼠皮層神經元中，顯著提高了超氧化物歧化酶（SOD）、過氧化氫酶（CAT）、谷胱甘肽過氧化物酶（GSH-Px）的活力以及bcl-2/bax的比例，同時顯著降低了活性氧自由基（ROS）、一氧化氮（NO）以及丙二醛（MDA）的含量。在體外模型中，PAR-1激活肽還顯著降低了caspase-3的活力以及TUNEL陽性細胞的比例，同時顯著提高了線粒體膜電位（MMP）。所有這些作用都可以被拮抗劑抑制，說明PAR-1激活肽的保護作用是通過激活PAR-1介導的。 / 不管是從正常還是缺血的大鼠中分離出來的大腦中動脈，PAR-2激活肽和胰酶都可以使之舒張，PAR-3激活肽卻對其有收縮作用。然而，另外一種PAR-3激活肽卻未顯現出對血管活性的影響。高劑量的PAR-1激活肽只可以在分離于缺血大鼠的大腦中動脈中引起舒張，但此作用不能被其拮抗劑所抑制。PAR-2激活肽導致的血管舒張只可以被檢測的兩個拮抗劑中的其中一個所抑制。PAR-1和PAR-2激活肽引起的血管舒張與NO的產生有關，因為L-NAME可以有效抑制它們的作用。 / 總之，不管是預處理還是後處理的給藥方式，PAR-1的激活肽在保護大腦的缺血性損傷中都是最有效果的。保護作用可能可以歸因于其抗氧化以及抗凋亡的特性。所以，PAR-1是研究防治缺血性腦疾病的發展中富有希望的一個靶點。 / Zhen, Xia. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 194-206). / Abstracts also in Chinese. / Title from PDF title page (viewed on 11, October, 2016). / 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.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 119-147) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 119-147) Also available online.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (Ph.D.)--Memorial University of Newfoundland, Faculty of Medicine, 2000. / Typescript. Bibliography: p. 137-164.

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Center for Neuroscience. Title from pdf file main screen (viewed on October 18, 2009). Includes bibliographical references.