Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Yung, Him Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 133-146). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Publications based on work in this thesis --- p.vii / Abbreviations --- p.viii / Contents --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Objectives and overview of this study --- p.1 / Chapter 1.2 --- Rat pheochromocytoma (PC12) cells --- p.3 / Chapter 1.3 --- Prostanoids and their receptors --- p.4 / Chapter 1.4 --- Roles of prostanoids --- p.7 / Chapter 1.5 --- Nerve growth factor (NGF) and its receptors --- p.9 / Chapter 1.6 --- Change of gene expressions by NGF in PC12 cells --- p.10 / Chapter 1.7 --- Signaling pathways involved in NGF-induced differentiation of PC12 cells --- p.12 / Chapter 1.8 --- Classification of adenylyl cyclases --- p.14 / Chapter 1.9 --- Methods to study differentiation of PCI 2 cells --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.19 / Chapter 2.1 --- Materials --- p.19 / Chapter 2.2 --- Cell culture medium and buffers --- p.25 / Chapter 2.3 --- Buffers and solutions for assay of [3H]inositoI phosphates ([3H]IP) production --- p.25 / Chapter 2.4 --- Buffers and solutions for assay of [3H]cAMP production --- p.27 / Chapter 2.5 --- Buffers and solutions for Western blotting --- p.28 / Chapter 2.6 --- Methods --- p.30 / Chapter 2.6.1 --- Maintenance of PC12 cells --- p.30 / Chapter 2.6.2 --- General culture condition of PCI2 cells for NGF treatment --- p.31 / Chapter 2.6.3 --- Determination of phospholipase C activity in PC12 cells --- p.31 / Chapter 2.6.3.1 --- Principle of assay --- p.31 / Chapter 2.6.3.2 --- Column preparation --- p.32 / Chapter 2.6.3.3 --- Measurement of [3H]IP production --- p.33 / Chapter 2.6.3.4 --- Data analysis --- p.34 / Chapter 2.6.4 --- Determination of adenylyl cyclase activity in PC12 cells --- p.35 / Chapter 2.6.4.1 --- Principle of assay --- p.35 / Chapter 2.6.4.2 --- Column preparation --- p.35 / Chapter 2.6.4.3 --- Measurement of [3H]cAMP production --- p.36 / Chapter 2.6.4.4 --- Data analysis --- p.37 / Chapter 2.6.5 --- Determination of neurofilament protein expression in PC12 cells by Western blotting --- p.38 / Chapter 2.6.6 --- Determination of adenylyl cyclase isoform expression in PC12 cells by reverse transcriptase-polymerase chain reaction (RT-PCR) --- p.39 / Chapter 2.6.6.1 --- Isolation of total cellular RNA --- p.39 / Chapter 2.6.6.2 --- Synthesis of first strand cDNA by reverse transcription (RT) --- p.40 / Chapter 2.6.6.3 --- Polymerase Chain Reaction (PCR) --- p.41 / Chapter 2.6.6.4 --- Agarose gel electrophoresis --- p.41 / Chapter 2.6.7 --- Neurite quantification --- p.42 / Chapter 2.6.8 --- Trypan blue exclusion test --- p.42 / Chapter Chapter 3 --- Results --- p.45 / Chapter 3.1 --- Characterization of prostanoid receptor expression in PC12 cells . --- p.45 / Chapter 3.1.1 --- Study of the presence of Gq-coupled prostanoid receptors --- p.45 / Chapter 3.1.2 --- Study of the presence of Gs-co»pled prostanoid receptors --- p.47 / Chapter 3.1.3 --- Study of the presence of Gi-coupled prostanoid receptors --- p.48 / Chapter 3.1.4 --- Further proof of EP3 expression in PC12 cells --- p.50 / Chapter 3.1.5 --- Discussion --- p.51 / Chapter 3.2 --- Time course effect of NGF on PC12 cells --- p.65 / Chapter 3.2.1 --- Effect of NGF on PGE2-mediated inhibition of forskolin-stimulated [3H]cAMP production --- p.65 / Chapter 3.2.2 --- Effect of NGF on basal and forskolin-stimulated [3H]cAMP production --- p.67 / Chapter 3.2.3 --- Acute effect of NGF on [3H]cAMP production --- p.70 / Chapter 3.2.4 --- Effect of NGF withdrawal on basal and forskolin-stimulated [3H]cAMP production --- p.71 / Chapter 3.2.5 --- Effect of NGF on adenylyl cyclase gene expression --- p.72 / Chapter 3.2.6 --- Discussion --- p.74 / Chapter 3.3 --- Quantification of the degree of differentiation of PC12 cells --- p.89 / Chapter 3.3.1 --- Expression of neurofilament protein as a marker of differentiation --- p.89 / Chapter 3.3.2 --- Neurite assays --- p.90 / Chapter 3.3.2.1 --- Manual assessment of PC12 cells --- p.90 / Chapter 3.3.2.2 --- Quantification of images of PC1 2 cells --- p.91 / Chapter 3.3.3 --- Discussion --- p.93 / Chapter 3.4 --- Adenosine A2a receptor activity in PC12 cells --- p.106 / Chapter 3.4.1 --- Effect of NGF on A2Areceptor-mediated [3H]cAMP production --- p.106 / Chapter 3.4.2 --- Synergistic activation of adenylyl cyclase by A2A receptor and forskolin --- p.108 / Chapter 3.4.3 --- Chronic and acute effect of ADA and ZM241385 on [3H]cAMP production --- p.109 / Chapter 3.4.3.1 --- Chronic effect of ADA and ZM241385 --- p.110 / Chapter 3.4.3.2 --- Acute effect of ADA and ZM241385 --- p.111 / Chapter 3.4.4 --- Discussion --- p.112 / Chapter Chapter 4 --- Discussion and future perspectives --- p.121 / Chapter 4.1 --- Discussion --- p.121 / Chapter 4.2 --- Future perspectives --- p.131 / References --- p.133

Background Past studies from this laboratory have shown that quinpirole administration from postnatal day (P) 1–21 produces persistent supersensitization of the dopamine D2 receptor that persists throughout the animal's lifetime. Methods In Experiment 1, both male and female rats were treated with quinpirole or saline from P1–21 and tested on the place and match-to-place versions of the Morris water task (MWT) from P22–28. In Experiment 2, both male and female rats were administered either acute or chronic injections of quinpirole (1 mg/kg) or saline beginning on P1 until analysis for corticosterone (CORT) on P7, 14, or 21. Results Neonatal quinpirole treatment produced deficits on both versions of the MWT compared with saline control. One day after behavioral testing, brain tissue was harvested, and the hippocampus was analyzed for nerve growth factor (NGF) and brain-derived nerve growth factor (BDNF); NGF was found to be significantly decreased by neonatal quinpirole treatment. Acute or chronic quinpirole treatment on P14 produced a larger increase in CORT than controls and produced larger increases in CORT than control rats on P21. Conclusions These results demonstrate that neonatal quinpirole treatment produces cognitive deficits that could be related to decreases in hippocampal NGF and increases in CORT, resulting in abnormalities in hippocampal development.

INTRODUÇÃO: A degeneração do disco intervertebral (DIV) é um processo crônico e apontado como o maior causador de cervicalgia e lombalgia. Esse processo geralmente conta com a degradação da matriz extracelular, expressão de citocinas inflamatórias e fatores angiogênicos e axonogênicos. Entretanto, muito pouco se sabe sobre esse processo em DIVs assintomáticos durante o envelhecimento, principalmente no segmento cervical. O objetivo desse estudo foi de delinear o perfil de moléculas relacionadas à degeneração discal em DIVs cervicais e lombares. MÉTODOS: Discos intervertebrais humanos cervicais e lombares (C4-C6 e L4-S1) foram coletados em autópsia de 30 indivíduos presumivelmente assintomáticos e divididos em grupos jovem (GJ < 35 anos, n=60) e idoso (GI > 65 anos, n=60). O nível de degeneração foi constatado pela escala de Thompson, e foi correlacionado com a detecção imuno-histoquímica das moléculas de MMP-1, -2, -3, TIMP-1, IL-1beta, TNF-alfa, VEGF, NGF-beta e BDNF. RESULTADOS: Todos os DIVs mostraram algum grau de degeneração, embora mais acentuadas no GI. As moléculas empenhadas no estudo foram identificadas em ambos grupos. A detecção imuno-histoquímica foi prevalente no citoplasma das células nativas do DIV e na região de interseção entre a placa vertebral e o arranjo fibro-colágeno. O envelhecimento propiciou, no disco cervical, maior expressão de MMP-2, -3, VEGF, NGF-beta e BDNF, enquanto que no disco lombar, a maior expressão foi de MMP-1, -2 -3, TIMP-1, TNF-alfa, VEGF e NGF-beta. DISCUSSÃO: O envelhecimento de DIVs cervicais e lombares caracterizou-se por exibir um processo catabólico e extensivo remodelamento da matriz extracelular, os quais podem ser interpretados como eventos que antecipam a doença degenerativa discal. Esse processo é capaz de levar a angiogênese e axonogênese de modo a ampliar o metabolismo aeróbio do DIV e captar informação nociceptiva como forma de defesa, uma vez que até nos discos lombares de indivíduos jovens essa última característica pôde ser observada. Discos assintomáticos também exibem moléculas relacionadas à doença degenerativa discal e talvez a inibição de parte dessas possa resultar em terapia preventiva / INTRODUCTION: Degeneration of the intervertebral disc (DIV) is a chronic process that pointed as a major cause of neck and low back pain. This process generally includes an extracellular matrix degradation, expression of inflammatory cytokines, angiogenesis and axonogenesis factors. However, there is a little known about this process in asymptomatic DIVs during aging, especially in the cervical region. The aim of this study was to delineate the profile of molecules related to disc degeneration in the cervical and lumbar discs. METHODS: Human cervical and lumbar intervertebral discs (C4-C6 e L4-S1) were harvested at autopsy from 30 asymptomatic individuals, and divided according to age with young (GJ < 35 years old, n=60) and elderly (GI > 65 years old, n=60) groups. Gross degeneration was graded according to the Thompson scale and this was correlated to the immunohistochemical detection of molecules of MMP-1, -2, -3, TIMP-1, IL-1beta, TNF-alfa, VEGF, NGF-beta e BDNF. RESULTS: Discs from GJ were significantly less degenerated than those of GI. The molecules involved in the study were identified in both groups. The immunohistochemical detection was prevalent in the cytoplasm of native disc cells and the region between the vertebral plate and fibrous collagen arrangement (intersection). Aging provided in cervical disc, increased expression of MMP-2, -3, VEGF, NGF and BDNF-beta, whereas in the lumbar disc the highest expression of MMP-1, -2, -3, TIMP-1, TNF-alfa, VEGF and NGF-beta was seen. DISCUSSION: The aging of cervical and lumbar DIV was marked by catabolic process and a extensive remodeling on extracellular matrix which can be interpreted as a predict event of the degenerative disc disease. This process can lead to angiogenesis and axonogenesis in order to expand the aerobic metabolism of the DIV and get nociceptive information as a defense, since even in the lumbar discs of young individuals this last feature can be observed. Asymptomatic discs also exhibit molecules related to degenerative disc disease and perhaps the inhibition some of these can result in preventive therapy

O exercício físico pode atenuar os efeitos do envelhecimento sobre o sistema nervoso central, por meio do aumento da expressão de neurotrofinas, tais como fator neurotrófico derivado do cérebro (BDNF), o qual promove a ramificação dendrítica e melhora da maquinaria sináptica, pela interação com seu receptor TrkB. Receptores TrkB são produzidos no corpo da célula e transportados aos terminais axonais, por meio de SLP1, CRMP2, Rab27B e Sortilina onde são ancorados para realizar seu papel fisiológico. Sabendo que a relação entre o tráfego de receptores de neurotrofinas e o treinamento físico ainda é pouco conhecida, o objetivo do presente trabalho é analisar os níveis do receptor TrkB, bem como de seus transportadores anterógrados e retrógrados, no sistema nervoso central de ratos idosos, modelos de neurodegeneração, expostos a diferentes protocolos de treinamento físico moderado. Os ratos do primeiro grupo experimental foram expostos a 1mg/kg/dia de Rotenona ou DMSO durante 4 semanas, depois, juntamente com a exposição à rotenona, realizaram treinamento físico moderado em esteira, 5 vezes por semana, durante 40 minutos; ou permaneceram em repouso. Os ratos do segundo grupo experimental realizaram 6 semanas de treinamento, sendo em seguida expostos à rotenona por 4 semanas, e subdividos em dois grupos, um que continuou o exercício e outro que ficou sedentário. Os resultados encontrados sugerem que o treinamento físico parece reverter ou prevenir de maneira geral os danos presentes na neurodegeneração considerando as proteínas do tráfego de BDNF e seu receptor, e ainda, que a magnitude e direção destas alterações está diretamente relacionada ao protocolo de treinamento físico, bem como, a região do sistema nervoso central analisada / Physical exercise can attenuate the effects of aging on the central nervous system by increasing the expression of neurotrophins such as brain-derived neurotrophic factor (BDNF), which promotes dendritic branching and enhances synaptic machinery, through interaction with its receptor TrkB. TrkB receptors are synthesized in the cell body and are transported to the axonal terminals, through SLP1, CRMP2, Sortilin and Rab27B, to where receptors are anchored to perform its physiological role. However, the aspects of the neurotrophin receptors traffic after physical training is still a matter of investigation. Thus, the present study aims to analyze the expression levels of TrkB receptor and their anterograde carriers in aged Lewis rats, model of neurodegeneration, and its relationship with moderate exercise training. Rats from the first experimental group were exposed to 1mg/kg/day of Rotenone (ROT) or DMSO for 4 weeks, and then subjected or not to moderate exercise running on treadmill, five days a week, 40 minutes a day, combined with the drug. Rats from the second experimental group were trained for 6 weeks, followed by exposure to rotenone during 4 weeks, rats were then subdivided into two groups, one that continued the exercise and the other became sedentary. Results suggest that exercise training appears to reverse or prevent the impairment related to neurodegeneration considering the proteins involved in BDNF signaling, and also that the magnitude and direction of these changes in directly related to the physical training protocol, as well as the area of the central nervous system analyzed

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser.

Thesis (M.Sc.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 86-111).

This thesis examines the effects of nerve growth factor (NGF) and lithium on the regulation of neurotensin/neuromedin N (NT/N) gene expression in PC12 pheochromocytoma cells. In PC12 cells, the expression of the rat NT/N gene is strictly dependent on simultaneous exposure to combinations of NGF, glucocorticoids, activators of adenylate cyclase, and lithium. Transient transfection experiments indicated that a consensus AP-1 site located within the NT/N promoter is the principal target of NGF and lithium action. NGF rapidly, but transiently, induces the expression of several AP-1 genes in PC12 cells, suggesting that the effect of NGF on NT/N gene expression results from increased AP-1 activity. These results led to the prediction that the induction of NT/N gene expression should be rapid, transient and dependent on de novoprotein synthesis. These experiments also suggested that the NT/N gene is principally regulated through the initiation of transcription. However, post-transcriptional mechanisms may also be involved. Experiments in this thesis were designed to examine the regulatory mechanisms responsible for increased NT production in PC12 cells when treated with different inducer combinations and whether AP-1 factors could act as mediators in responses to NGF and lithium. Results described in this thesis indicate that the principal mechanism by which NGF and lithium regulate NT biosynthesis is by activating NT/N gene transcription. Comparison of NT/N mRNA, pro NT/N synthetic rates, proNT/N proteins and mature NT levels in induced PC12 cells, demonstrated that NGF and lithium had no effect on the translation of NT/N mRNA and had only a modest effect on post-translational processing. Nuclear run-on assays showed that NT/N transcription is transicntly activated in maximally induced cells. A rapid RNase protection assay was developed to examine both the kinetics of NT/N gene activation and whether activation requires newly synthesized proteins. Quantitation of nuclear NT/N precursor RNA. using a probe spanning the junction between exon onc and intron one, provides a sensitive measure of NT/N gene activity and by several criteria provides an accurate measure of NT/N transcription. When either NGF or lithium was combined with dexamethasone and forskolin, nuclear NT/N precursor RNA transiently accumulated, although each inducer displayed different kinetics, rapid and delayed, respectively. De novo protein synthesis was not required for activating NT/N transcription when NGF was used as the permissive agent, although newly synthesized proteins secm to be needed for subsequent down-regulation. The response to lithium displayed a marked requirement for new protein synthesis, consistent with the involvement of newly synthesized AP-1 factors. RNA blot analysis showed that lithium either alone or in combination with dexamethasone and forskolin induced c-jun and fra-1 gene expression with delayed kinetics, consistent with c-Jun/Fra-1 complexes mediating the effects of lithium on NT/N gene transcription. The pathway identified by lithium does not activate or require protein kinase C. This pathway is also active in neuronally-differentiated PC12 cells suggesting that it could be involved in the regulation of NT/N gcne exprcssion in the intact nervous system. These results and order of addition experiments demonstrate that NGF and lithium activate distinct pathways required for NT/N gene induction.

O exercício físico pode atenuar os efeitos do envelhecimento sobre o sistema nervoso central, por meio do aumento da expressão de neurotrofinas, tais como fator neurotrófico derivado do cérebro (BDNF), o qual promove a ramificação dendrítica e melhora da maquinaria sináptica, pela interação com seu receptor TrkB. Receptores TrkB são produzidos no corpo da célula e transportados aos terminais axonais, por meio de SLP1, CRMP2, Rab27B e Sortilina onde são ancorados para realizar seu papel fisiológico. Sabendo que a relação entre o tráfego de receptores de neurotrofinas e o treinamento físico ainda é pouco conhecida, o objetivo do presente trabalho é analisar os níveis do receptor TrkB, bem como de seus transportadores anterógrados e retrógrados, no sistema nervoso central de ratos idosos, modelos de neurodegeneração, expostos a diferentes protocolos de treinamento físico moderado. Os ratos do primeiro grupo experimental foram expostos a 1mg/kg/dia de Rotenona ou DMSO durante 4 semanas, depois, juntamente com a exposição à rotenona, realizaram treinamento físico moderado em esteira, 5 vezes por semana, durante 40 minutos; ou permaneceram em repouso. Os ratos do segundo grupo experimental realizaram 6 semanas de treinamento, sendo em seguida expostos à rotenona por 4 semanas, e subdividos em dois grupos, um que continuou o exercício e outro que ficou sedentário. Os resultados encontrados sugerem que o treinamento físico parece reverter ou prevenir de maneira geral os danos presentes na neurodegeneração considerando as proteínas do tráfego de BDNF e seu receptor, e ainda, que a magnitude e direção destas alterações está diretamente relacionada ao protocolo de treinamento físico, bem como, a região do sistema nervoso central analisada / Physical exercise can attenuate the effects of aging on the central nervous system by increasing the expression of neurotrophins such as brain-derived neurotrophic factor (BDNF), which promotes dendritic branching and enhances synaptic machinery, through interaction with its receptor TrkB. TrkB receptors are synthesized in the cell body and are transported to the axonal terminals, through SLP1, CRMP2, Sortilin and Rab27B, to where receptors are anchored to perform its physiological role. However, the aspects of the neurotrophin receptors traffic after physical training is still a matter of investigation. Thus, the present study aims to analyze the expression levels of TrkB receptor and their anterograde carriers in aged Lewis rats, model of neurodegeneration, and its relationship with moderate exercise training. Rats from the first experimental group were exposed to 1mg/kg/day of Rotenone (ROT) or DMSO for 4 weeks, and then subjected or not to moderate exercise running on treadmill, five days a week, 40 minutes a day, combined with the drug. Rats from the second experimental group were trained for 6 weeks, followed by exposure to rotenone during 4 weeks, rats were then subdivided into two groups, one that continued the exercise and the other became sedentary. Results suggest that exercise training appears to reverse or prevent the impairment related to neurodegeneration considering the proteins involved in BDNF signaling, and also that the magnitude and direction of these changes in directly related to the physical training protocol, as well as the area of the central nervous system analyzed