The aim of this thesis was to validate the use of infrared thermography (IRT) to non-invasively measure stress and/or pain in cattle. The main approach was to measure changes in heat emitted from superficial capillaries around the eye (referred to as eye temperature) in response to various aversive husbandry procedures used routinely on farms. In addition, various exogenous challenges were given to investigate the role of the hypothalamic-pituitary-adrenal (HPA) axis and the autonomic nervous system (ANS) in regulating the eye temperature response. No evidence was found to support the hypothesis that an increase in eye temperature was due to HPA activity in cattle. A rapid drop in eye temperature occurred immediately after disbudding, an electric prod, startling and shouting. It is suggested that this was caused by the redirection of blood from the capillary beds via sympathetically-mediated vasoconstriction. Therefore, the role of the ANS was tested by measuring eye temperature, heart rate variability (HRV) and plasma catecholamine responses simultaneously. Somatic pain from disbudding and initial responses to surgical castration included a synchronised drop in eye temperature, increases in catecholamines and changes in HRV indicative of increased sympathetic activity. The role of the sympathetic nervous system was further confirmed by a drop in eye temperature that occurred following an epinephrine challenge. In contrast, deeper visceral pain from castration caused a more marked increase in eye temperature and changes in HRV indicative of increased parasympathetic tone. The underlying mechanism driving the increase in eye temperature is unknown; however, it is possible that it may be caused by vasodilation due to increased parasympathetic activity. These differences in ANS responses to different procedures, detected by IRT and HRV, may be due to the nature of the pain and the relative fear associated with the procedure. In summary, this research showed that during stress or pain, the heat emitted from superficial capillaries around the eye changes as blood flow is regulated under ANS control and these changes can be quantified using IRT. A combination of IRT and HRV is a non-invasive way to measure ANS activity and assess acute welfare impacts of husbandry practices in cattle. Further research using pharmacological inhibition and stimulation of the ANS activity would be beneficial to fully understand the underlying regulatory mechanisms of the eye temperature and HRV responses in cattle and other species during stress and/or pain. The full capability of IRT and HRV for detection of disease and emotional states and the effects of different intensities of pain, individual traits and previous experience also deserve attention.

兒茶酚胺類神經傳遞系統被認為與包括記憶學習等行為功能有很重要的關連，在記憶多元化理論的假設下，該神經系統與其它者對於特定記憶學習行為應有再確認之必要。過去對於空間性記憶的研究，其相關支持證據所依據之實驗操弄泰半集中在海馬迴系統上。但仍有部份研究指出大腦中的其他區域可能同樣與一般記憶的運作有密切的關連。特別是兒茶酚胺系統所在之紋狀總體組織部位（包括尾狀核與阿控博核），vP A僅與感覺接受、運動反應及增強作用等機制有關外，同時可能也扮演影響記憶表現的重要因子。本研究使用慾求性的八角迷津為工具，藉其地點學習與反應學習這兩種不同的迷津作業，及利用兒茶酚胺類的藥物或神經毒素，探討相關的記憶習得與記憶保持歷程所造成的影響。實驗的操弄包括：(1) 迷津作業之地點學習與反應學習以探討這兩種記憶之行為機制。(2) 記憶習得與記憶保持階段以瞭解這兩種迷津作業所引發記憶之全部歷程。(3) 中樞（阿控博核或尾狀核）神經毒素之破壞以及周邊藥物注射以確認兒茶酚胺類藥物對於記憶之神經藥理機制。本研究分為兩大實驗進行，實驗一以地點學習為主，實驗二以反應學習為主。實驗結果可以簡單歸納如下：(1) 兩 種學習作業的記憶策略有不同的習得歷程及需要不同的處理訊息。(2) 在迷津學習前用神經毒素 6-OHDA 破壞尾狀核或阿控博核，皆會影響地點記憶的習得，但對於反應記憶的習得，則需要同時破壞尾狀核及阿控博核才有類似的干擾效果。(3) 相對於神經毒素 6-OHDA的干擾效果，DSP-4皆不影響地點學習與反應學習的習得歷程。(4) 在記憶保持階段中，周邊注射兒茶酚胺類藥物 d-amphetamine、haloperidol 與 propranolol均會干擾地點記憶的提取，但卻不影響反應記憶的提取表現。(5) 於地點記憶與反應記憶習得後，給予尾狀核加阿控博核的雙側 6-OHDA 注射均不影響這兩種記憶的提取表現。實驗結果顯示兒茶酚胺類神經傳導系統對於記憶功能具有明顯的影響，其中紋狀體扮演了相當重要的角色。相對於不影響記憶提取之歷程，紋狀體的破壞對記憶習得歷程有阻滯之效果，其內部之尾狀核與阿控博核分別依不同之迷津作業具有相異之效果，且多巴胺系統較正腎上腺素系統明顯的參與了影響效果，這些結果顯示兒茶酚胺類神經傳導系統與記憶表現有密切的關連。 / Catecholamine (CA) neurotransmission systems are critically involved in the control of many behavioral functions including learning and memory. The role of CA in mediating learning and memory is recently focused on the basis of multiple memory hypothesis. In addition to the previous finding of spatial memory relevant to the hippocampal areas, the striatum containing the caudate nucleus and the nucleus of accumbens is thought to be important for executing the learning and memory function. By the use of radial arm maze (RAM), the present study examined the effects of CA related neurotoxins and drugs on the acquisition and retention stages of both place and response tasks. Two major parts of experiments were designed to reveal the neurobehavioral mechanisms for the place and response tasks of RAM. Food-deprivated rats were trained to enter the arms baited with chocolate in the eight-arm maze. Specific four arms were baited for each rat in the place task, while randomly selected four arms each cued with a piece of sand paper on the arm entrance were baited for the rat in the response task.The results can be summarized as followings. (1) Differen behavioral processes were shown in performing the place and response tasks. (2) The acquisition deficits were significantly produced by 6-hydroxydopamine (6-OHDA) lesion on either caudate or accumbens for the place task, whereas the acquisition of response task was only impaired by 6-OHDA lesions of both caudate and accumbens together. (3) In contrast to 6-OHDA, adrenergic neurotoxin DSP-4 did not significantly affect subjects to acquire either task. (4) During the retention stage, the performance of place task was significantly disrupted by d-amphetamine, haloperidol, or propranolol. However, this was not the case for the retrieval of response task. (5) Once acquired, neither place nor response task performamce could be influenced by 6-OHDA simultaneously administered on the caudate and accumbens areas.Taken together, these data collected from RAM support the idea that the striatal CA is essential for the leraning and memory. Shift of the CA neurotransmission function induced by either 6-OHDA lesions or relevant drugs can disrupt the RAM behavior, which impairment to be detectable is depended on the learning task itself as well as the time of a specific task being leraned.

<p>The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated.</p><p>This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.</p>

The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated. This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.

The primary goal of my thesis research was to characterize the basis for the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in primary culture diabetic cardiomyocytes. Toward this goal, I have investigated several key regulatory sites in this signaling pathway which could promote the hypersensitive activation of phosphorylase. Specifically, I investigated (1) which adrenergic receptors are involved in mediating the hypersensitive response of glycogen phosphorylase to epinephrine stimulation; (2) whether the presence of fatty acid metabolites affects phosphorylase activation; (3) whether the hypersensitive response of phosphorylase results from altered signal transduction through the β-adrenergic receptor system or from a post-receptor defect; and (4) the potential role for phosphorylase kinase in mediating the hypersensitive response of phosphorylase to catecholamine stimulation. The basis for adrenergic receptor mediation of the catecholamine-induced activation of glycogen phosphorylase was investigated in adult rat cardiomyocytes isolated from normal and alloxan-diabetic animals. Cells derived from diabetic animals exhibited a hypersensitive response to epinephrine stimulation which was apparent 3 hours after cell isolation and was further enhanced upon maintenance of the myocytes in culture for 24 hours. Normal cells initially lacked the hypersensitive response to epinephrine stimulation although upon maintenance of these cells in culture for 24 hours, the hypersensitive response was acquired in vitro. To assess alpha- and beta- adrenergic mediation of the response, normal and diabetic cardiomyocytes were incubated with propranolol, a β-receptor antagonist, prior to direct α1receptor stimulation with phenylephrine. Both normal and diabetic myocytes failed to undergo activation of phosphorylase in 3 or 24 hour cell cultures. In addition, the effects of epinephrine on phosphorylase activation were completely inhibited by propranolol whereas prazosin, an α-receptor antagonist, was unsuccessful. This data suggests that the hypersensitive response of glycogen phosphorylase in normal and diabetic cardiomyocytes is solely mediated through β-adrenergic receptor activation. Since the accumulation of various fatty acid metabolites can affect certain enzymes and signal transduction pathways within the cell, the potential effect of various fatty acid metabolites on phosphorylase activation was investigated. To determine the potential effects of fatty acid metabolites on phosphorylase activation in cultured cardiomyocytes, normal and alloxan-diabetic cells were incubated with either carnitine or palmitoylcarnitine prior to stimulation with epinephrine. Pretreatment of cardiomyocytes with or without carnitine or palmitoylcarnitine for 3 or 24 hours before epinephrine stimulation failed to alter phosphorylase activation. The addition of exogenous carnitine in the absence and presence of insulin was also unsuccessful in attenuating the hypersensitive phosphorylase activation response in 3 and 24 hour, normal and alloxan-diabetic derived cardiomyocytes. To determine if carnitine palmitoyltransferase 1 (CPT-1) activity was responsible for the hypersensitive response of phosphorylase in the diabetic myocytes, both normal and diabetic myocytes were maintained for 3 and 24 hours in the absence and presence of etomoxir, a CPT-1 inhibitor. Subsequent activation of phosphorylase by epinephrine in normal and diabetic myocytes was unaltered in the presence of etomoxir. Collectively, these data fail to support a critical role for fatty acid metabolite involvement in the hypersensitive activation of glycogen phosphorylase in acute, alloxan-diabetic cardiomyocytes. To assess potential G-protein involvement in the response, normal and diabetic-derived myocytes were incubated with either cholera or pertussis toxin prior to hormonal stimulation. Pretreatment of cardiomyocytes with cholera toxin resulted in a potentiated response to epinephrine stimulation whereas pertussis toxin did not affect the activation of this signaling pathway. To determine if the enhanced response of phosphorylase activation resulted from an alteration in adenylyl cyclase activation, the cells were challenged with forskolin. After 3 hours in primary culture, diabetic cardiomyocytes exhibited a hypersensitive response to forskolin stimulation relative to normal cells. However, after 24 hours in culture, both normal and diabetic myocytes responded identically to forskolin challenge. The present data suggest that a cholera toxin sensitive G-protein mediates the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in diabetic cardiomyocytes. This response, which is present in alloxan-diabetic cells, and is induced in vitroin normal cardiomyocytes, is primarily due to a defect at a post-receptor site. To assess the role of phosphorylase kinase in the hypersensitive activation of glycogen phosphorylase in the diabetic heart, phosphorylase kinase activity was measured initially in perfused hearts (to optimize the assay parameters) and subsequently in primary culture cardiomyocytes. Results from these experiments demonstrate that the present method for measuring phosphorylase kinase activity is a reliable indicator of the enzyme's activity in the heart, although the assay conditions must be further optimized before this system can be applied to the measurement of phosphorylase kinase activity in primary cultured cardiomyocytes.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-17T11:03:10Z No. of bitstreams: 1 lilianesenapinheiro.pdf: 8108520 bytes, checksum: b03200480798ddb2cf88a9276e4c9d8d (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T13:09:54Z (GMT) No. of bitstreams: 1 lilianesenapinheiro.pdf: 8108520 bytes, checksum: b03200480798ddb2cf88a9276e4c9d8d (MD5) / Made available in DSpace on 2016-10-22T13:09:54Z (GMT). No. of bitstreams: 1 lilianesenapinheiro.pdf: 8108520 bytes, checksum: b03200480798ddb2cf88a9276e4c9d8d (MD5) Previous issue date: 2008-06-25 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O diabetes mellitus (DM) reduz a secreção de catecolaminas (CAs) das células cromafins adrenais, sendo esse um evento patofisiológico crítico por favorecer a ocorrência de episódios de hipoglicemia grave decorrentes do próprio tratamento da doença. Vários trabalhos relatam a participação de proteínas quinase C (PKCs) nas vias de síntese e secreção de CAs nas células cromafins. Os objetivos desse trabalho foram analisar o efeito do DM sobre a expressão das isoformas α, ε e ζ de PKC em células cromafins de ratos e avaliar se o controle glicêmico reverte os efeitos da doença. Foram utilizados ratos Wistar com DM induzido por estreptozotocina. Foram estabelecidos três grupos experimentais, ratos controles (C), diabéticos tratados com salina (DTS) ou com insulina (DTI). As análises foram feitas 15 dias após a indução. Utilizamos as técnicas de imunohistoquímica e Western Blot. A insulinoterapia foi estabelecida após estudos do comportamento alimentar e da variação dos níveis glicêmicos de ratos controles e doentes durante 24h consecutivas. Foi testada a eficácia de diferentes esquemas de tratamento com insulina. O tratamento estabelecido consistiu em injeções de insulina NPH, sendo 1U aplicada às 13h e 4U às 19h. Após os 15 dias de tratamento, o ganho médio de massa corporal dos ratos C (+37±3g) e DTI (+43±3g) foram similares enquanto os DTS emagreceram (-9±6g). A média da glicemia de jejum dos ratos C (74±1mg/dl) e dos DTI (93±6mg/dl) foram similares e dentro dos níveis normais, enquanto que a dos ratos DTS foi elevada (471±23mg/dl). A insulinoterapia restabeleceu os níveis plasmáticos do colesterol total, c-LDL e c-VLDL nos ratos DTI. O DM não alterou os níveis de c-HDL, triglicerídos e frutosamina. As análises da expressão de PKCs mostraram que a PKCα é a mais expressada seguida de ζ e depois de ε. O DM reduziu em 39,5% a expressão da PKCα, enquanto a de ζ foi aumentada em 74,2%. A expressão da PKCε não foi afetada pelo DM. O tratamento com insulina reverteu o efeito do DM sobre a expressão de PKCα, a expressão da PKCε continuou inalterada e a expressão da PKCζ permaneceu elevada (+32,6%) quando comparada aos ratos C. Concluímos que em células cromafins adrenais, o diabetes afeta a expressão de isoformas de PKCs de maneira diferenciada. Trabalhos realizados em nosso laboratório mostraram que o DM reduz o conteúdo total (21,1%), a secreção basal (-24,3%) e a estimulada por carbacol (-28,9%) e K+ (42,2%) de CAs. Como observado para PKCα, a insulinoterapia reverteu o efeito do DM sobre o conteúdo total. Já foi demonstrado que PKCα participa de uma via de sinalização que estimula a atividade de tirosina hidroxilase. Por outro lado, o tratamento não restabeleceu os processos secretórios, sugerindo que PKCζ possa estar envolvida nessa alteração. Há fortes evidências de que PKCζ regula canais de K+ retificadores, o que pode explicar o efeito da doença sobre o processo de secreção via despolarização da membrana. / The diabetes mellitus (DM) reduces the catecholamine (CAs) secretion of adrenal chromaffin cells, a critical pathophysiologic event that promotes the occurrence of serious hypoglycemia episodes, consequence of the disease treatment. Several papers report the participation of protein kinase C (PKC) on catecholamine synthesis signal pathways of adrenal chromaffin cells. The objectives of this work were to study the effect of DM on expression of PKC isoforms α, ε and ζ in rat chromaffin cells and to evaluate if the glicemic control revert the effect of the illness. Male Wistar rats with diabetes induced by streptozotocin were used. Three experimental groups were determined: Control (C), diabetic rats receiving saline solution (DS) and diabetic rats receiving insulin (DI). The analyses were made after 15 days of DM induction. Immunohistochemistry and western blotting techniques were done. The insulin therapy protocol was established after studying the feeding behavior and glycemic level variations during the whole 24h. The information made possible to establish the time of insulin applications. Several schemes of insulin treatments were tested to keep the diabetic rat as close as possible to normoglycemia path. The best results were found by using 1U at 1:00 PM and 4U at 7:00 PM of NPH insulin. After 15 days of treatment the acquired body weight was similar between C and DI rats, 37±3g and 43±3g, respectively. The DS rats emaciated 9±6g. The fasting glycemic levels were 74±1mg/dl, 93±6mg/dl and 471±23mg/dl to C, DI and DS rats, respectively. The insulin therapy reestablishes the plasmic levels of total cholesterol, c-LDL and c-VLDL on DI rats. The DM did not change the levels of c-HDL, triglycerides and frutosamine. The PKCα is the more expressed isoform in adrenal chromaffin cells, followed by ζ and ε. The DM reduced 39,5% the PKCα expression and, unlike, increased 74,2% the expression of PKCζ. The expression of PKCε was not affected by DM. The insulin treatment reverted the effect of DM on PKCα, the expression of PKCε remained unchanged and the expression of PKCζ remained higher than the control group (+32,6%). Studies of our laboratory show that the DM causes reduction on adrenal catecholamine content (21,1%), basal secretion (-24,3%) and catecholamine secretion stimulated by carbachol (-28,9%) and high K+ (-42,2%). The insulin therapy, in like manner as observed on PKCα, reverted the DM effect on adrenal catecholamine content. It was shown that PKCα participates on signal transduction pathway that stimulates the activity of tyrosine hydroxylase. Otherwise, the insulin treatment did not restore the secretory processes, suggesting that PKCζ could be involved in this process. There are strong evidences showing that PKCζ regulates the voltage-dependent delayed rectifier K (Kv) and its expression was not normalized by insulin therapy.