Orientador: Licio Augusto Velloso / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T16:23:48Z (GMT). No. of bitstreams: 1 Razolli_DanielaSoares_M.pdf: 1686452 bytes, checksum: 30a86d391dc3f2eada8b43a2a1e30b03 (MD5) Previous issue date: 2011 / Resumo: O receptor ionotrópico NMDA (N-metil-D-aspartato) é um receptor de glutamato envolvido em uma série de processos essenciais para o funcionamento adequado do sistema nervoso central. A ativação ou expressão anômala deste receptor pode contribuir para o desenvolvimento de doenças como Parkinson e Alzheimer. Um estudo utilizando um microarranjo de DNA realizado em nosso laboratório revelou que apenas dois dentre 1.176 genes hipotalâmicos avaliados, estão simultaneamente envolvidos na resposta a estímulos termogênicos induzidos pela ingestão de dieta hipercalórica e pela exposição à baixa temperatura; sendo eles, a subunidade 2B do receptor NMDA, e a proteína-G ligadora de GTP. Tal fato sugere que os processos envolvidos com o controle do gasto energético são complexos e finamente regulados. Ainda, dados prévios mostraram a co-localização entre receptores NMDA e neurônios MCH (Hormônio Concentrador de Melanina), o qual é um dos principais neurotransmissores hipotalâmicos envolvidos no controle do gasto energético. Dessa forma, o objetivo do presente estudo foi avaliar a expressão e a funcionalidade de receptores NMDA em hipotálamo de ratos alimentados com dieta padrão ou rica em lipídeos. Como o hipotálamo responde aos hormônios leptina e insulina no processo de controle de ingestão alimentar e gasto energético, decidimos avaliar se o receptor NMDA pode modular a expressão de neurotransmissores hipotalâmicos por meio da via da leptina. Para tal, ratos foram tratados por via intracerebroventricular (icv) com glutamato (agonista de NMDA) e AG490 seguido de glutamato. O AG490 é um inibidor químico da JAK2 - proteína inicial da via de sinalização da leptina. Nossos resultados mostraram que a administração icv de glutamato reduz a ingestão alimentar e o peso dos animais, modulando a expressão de neurotransmissores orexigênicos e anorexigênicos de maneira parcialmente dependente da via da leptina / Abstract: The ionotropic NMDA receptor is a glutamate receptor involved in a number of essential functions in the central nervous system. The anomalous expression or activation of this receptor can contribute to development of diseases such as Parkinson and Alzheimer. A previous study performed in our lab revealed that only two out of 1.176 genes were simultaneously modulated in the hypothalamus of rats exposed to two distinct thermogenic stimuli, i.e., exposition to low temperature and feeding a high-fat diet; the 2B NMDA receptor subunit and the GTP protein G. This fact suggests that energy expenditure is a complex and finely controlled process. In addition, previous data showed co-localization between NMDA receptor and MCH neurons, which is one of the most important hypothalamic neurotransmitters involved in the energy expenditure control. Thus, this study aimed at evaluating the expression and function of NMDA receptor in the hypothalamus of rats fed on control or high-fat diets. Since the hypothalamus is responsive to leptin and insulin, we evaluated if glutamate can modulate hypothalamic neurotransmitter expression through the leptin pathway. Rats were treated via intracerebroventricular (icv) with glutamate or glutamate followed of AG490 (a chemical inhibitor of JAK2 - inicial leptin pathway protein). Our results showed that icv glutamate reduce food intake and body weight, modulating the orexigenic and anorexigenic neurotransmitters through a mechanism, at least in part, dependent on leptin pathway / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

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Improving crop yield by generating stress tolerant plants is the enduring objective of this research. A small class of bioactive fatty acid derivatives, N-acylethanolamines (NAEs), including anandamide (NAE 20:4), an endocannabinoid receptor ligand, affects a wide range of physiological and behavioral functions in animals. In plants, NAEs to the exclusion of anandamide are found to be ubiquitous and abundant in seed tissues and are shown to be involved in mediating abscisic acid (ABA) -dependent or -independent stress responses. Early land plants such as Physcomitrella patens (moss) have been shown to tolerate abiotic stresses. We hypothesized that NAEs are involved in mediating stress responses in moss. Gas chromatography-mass spectrometry was employed in NAE detection and quantification in moss. Selective lipidomic approach revealed novel NAE metabolites. The endocannabinoid receptor ligand anandamide and its precursor molecules were detected and quantified. Exogenous treatment of NAE 12:0, NAE 20:4 and ABA showed a growth inhibitory effect for all three metabolites. NAE 20:4 was more potent than NAE 12:0 to degrees similar to the plant hormone ABA. In silico analyses of NAE catabolizing enzyme fatty acid amide hydrolase from Arabidopsis showed eight putative FAAH candidates in this moss. Candidates showed high similarities with plants as well as animal FAAH proteins. Primers specific to NAE pathway genes have been designed for expression analysis. Our recent identification of the ligand NAE 20:4 in this moss, provides us with a unique opportunity to address if 1) early land plants, such as mosses, retained the endocannabinoid signaling mechanism that is akin to animals but not to plants and 2) if such distinctive NAE profile and mechanism by which it may function in moss plant is responsible, in part, for their natural ability to resist high temperatures, dehydration, osmotic and salt stresses. Insights into unique lipids composition and signaling pathways that mosses acquire naturally, during their successful transition from water to land, may lead to development of tools necessary to enhance abiotic stress tolerance in vegetative tissues of higher plants and thus contribute to improvement of crop productivity.

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<p dir="ltr">The Pekin duck, a major poultry product, is an important livestock species that requires further study to understand their physiological needs and welfare. Welfare improvements can be obtainable by measuring a duck’s response to its environment and developing management practices that allow ducks to explore positive natural behaviors and minimize negative affective states. Assessing welfare includes measuring physical and mental states of an animal and how it copes with its environment. This thesis sets out to use established indicators for mental state, such as serotonin (5-HT) and dopamine (DA), to validate methods of measuring duck affective state applied to environment changes. Affective states can be assessed using neurotransmitter concentrations inserted into turnover equations to understand serotonergic and dopaminergic activity within the synapse. Similarly, gene expression can be linked to affective state by investigating the rate-limiting enzyme in 5-HT and DA synthesis. Developing neuroendocrine profiles for the Pekin duck can elucidate how environmental changes affect physiology and welfare. </p><p dir="ltr">Preening cups are a semi open water source placed within duck barns to improve welfare and allow for positive natural behaviors. Since ducks are waterfowl, many believe these birds need access to open water to develop naturally and have positive welfare. Our lab designed an experiment, referenced in Chapter 3, to compare ducks raised with preening cups to ducks raised with only water nipple lines. At Purdue Animal Science Research and Education Center (ASREC), 260 grow-out Pekin ducks were raised to the industry standard. There were 2 pens that had access to preening cups on day 18 and 2 pens had access to only nipple lines. We collected duck brains on day 18 before preening cup placement (PRE, n = 6). On day 43, we collected duck brains from pens with preening cups (PC, n = 6) and from pens without preening cups (CON, n = 6). Then, brains were hemisected and further dissected into caudal mesencephalon (CM), rostral mesencephalon (RM), diencephalon (DI), and forebrain (FB). The right portions of each brain were used to investigate neurotransmitter concentrations and turnover using mass spectrometry. While the left portions were used to investigate gene expression of tryptophan hydroxylase (TPH1, TPH2) and tyrosine hydroxylase (TH). Our results found no significant differences in 5-HT turnover or 5-HT static levels associated with preening cups across collection days. In the CM, we found a decrease in DA turnovers for PC and CON (p = 0.0067) when compared to PRE. Also, we found a decrease in DA turnover for PC when compared to PRE and CON (p = 0.003) in the RM. In the CM, we found increases in TPH1 expression (p = 0.022) for PC and CON when compared to PRE and in TH expression (p = 0.022) for CON when compared to PRE. There were no significant differences found in the RM and DI brain areas for gene expression. In the FB, we found a decrease in TH gene expression (p = 0.031). Overall, our data highlights an increase in dopaminergic activity within the midbrain. This increase in DA can be correlated with aggressive behavior witnessed from ducks housed with preening cups. Elevated DA is associated with addiction and resource guarding. In this experimental study, we concluded that preening cups placed with a small number of ducks (n = 65 ducks per preening cup) may elicit aggressive behavior and decrease welfare. </p><p dir="ltr">The results of this initial study led us to the question of how a duck affective state is altered by preening cups in a large commercial setting. A commercial Pekin duck barn will place one preening cup for approximately 1,000-1,500 ducks, which means a barn will carry about 3-5 preening cups. Similar methodology was used in Chapter 4 to investigate the ducks affective state using mass spectrometry and qRT-PCR to assess 5-HT and DA activity within the brain. We visited four commercial duck barns with an average of 7,500 ducks on day (d)21 prior to preening cup placement, d28 one week after preening cup placement, and d35 one day prior to processing. We collected litter samples to test moisture content (n = 3/barn/day) and found no differences before or after preening cup placement. We performed a transect walk by moving systematically through each barn and recording the frequency of welfare concerns. All barns showed low percentages of welfare concerns while differences were likely due to natural aging of ducks in a production system. Brains were collected from two locations in the barn including ducks actively using the preening cup (PC) and ducks across the barn not actively performing any behaviors other than standing or sitting (CON). Brains were divided into CM, RM, and DI brain areas and each half was assessed with 5-HT and DA turnover along with TPH1, TPH2, and TH gene expression. No differences were found for 5-HT turnover or static levels. This suggests that preening cups are not altering affective state. DA turnover was decreased in the CM (p < 0.05) and DI (p < 0.001) due to age, but no differences were found between collection location (PC vs CON). We link this increase in dopaminergic activity to natural aging and preparation for puberty as no aggressive behavior was witnessed in commercial barns. Aggression and resource guarding of commercial preening cups is unlikely due to the increased number of ducks per water source and the large space the ducks can access. There was increased TPH1 expression within the RM brain area for d35 ducks when compared to d28 and d21. We conclude that affective state is not variable within the barn based on collection locations. Overall, 5-HT was not affected by preening cups and DA was affected by age. This means that preening cups may not improve affective state and welfare, but they also do not cause determinantal effects either. </p><p dir="ltr">Another major welfare concern associated with environment is transportation for Pekin ducks. To assess how affective state is altered by crating and transportation of hens and drakes, Chapter 5 used similar methodology as the previous studies. Transportation may cause an acutely stressful event while activating a physiological stress response. Chapter 5 measured several central and peripheral physiological parameters to assess responses to transportation. Thirty-six, 23-week-old breeder ducks from a commercial facility were randomly assigned to one of three treatment groups. The control group (CON) was caught and immediate euthanized in the pen, the crate group (CRA) was caught and crated for 90 minutes before euthanasia, and the transport group (TRA) was caught, crated, and loaded into the back of a truck to be driven on country roads at 55 mph for 90 minutes before immediate euthanasia. Blood was collected for corticosterone ELISA analysis and blood smear heterophil to lymphocyte ratio (HLR) analysis. We found that hens showed an increase in HLR (p = 0.035) and serum corticosterone (p = 0.01) due to CRA. Drakes and hens showed an increase in HLR (p = 0.035) and serum corticosterone (p = 0.0084) for TRA. These results suggest that transportation is a stressor that elicits a sex dependent response where hens increase corticosterone and HLR under CRA unlike drakes who only showed a stress response under TRA. Likewise, a sex difference was shown for 5-HT turnover where hens show a stepwise increase from CON to CRA (p = 0.01) and from CRA to TRA (p = 0.016) in the CM and RM. There were no differences for DA turnover while TPH1 gene expression was decreased (p = 0.03) for TRA hens when compared to CON and CRA hens. Our results suggest that transportation negatively shifts affective state by increasing stress responses in ducks and reducing serotonergic activity in hens. In the future, care should be taken to evaluate stress between sexes and minimize transportation time. </p><p dir="ltr">In conclusion, preening cups and transportation are important aspects of a Pekin ducks’ life that alter affective state and physiology. We recommend proper management of preening cups to ensure positive welfare by placing water sources over open pits to prevent excess accumulation of water in the barn. We recommend that while evaluating stressors, researchers need to assess different sexes and collection time from the onset of the stressor. Future studies can include determining affective state for other enrichments such as environmental enrichment devices or investigating commercial barns without preening cups during the production cycle. Continued research could measure affective state following shorter transportation stress or chronic stress to resolve what timeframes alter 5-HT and DA turnover. Transportation may be minimized by installing on-site processing facilities to stun and process animals without transportation to reduce negative affective states. In an academic setting, pharmaceutical intervention may be interesting to investigate to improve affective state during stressful events.</p>

<p>This thesis investigates the potential of using positron emission tomography (PET) to study the biosynthesis and release of serotonin (5HT) at the brain serotonergic neuron. As PET requires probe compounds with specific attributes to enable imaging and quantification of biological processes, emphasis was placed on the evaluation of these attributes. </p><p>The experiments established that the 5HT transporter radioligand [¹¹C]-3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile, [¹¹C]DASB, is suitable for imaging and quantification of transporters in rats and rhesus monkeys. In addition, the binding of [¹¹C]DASB in brain tissue is decreased when 5HT concentrations are increased by tranylcypromine administration. The sensitivity of [¹¹C]DASB binding, under these experimental conditions, to increased endogenous 5HT concentrations demonstrates the potential of in vivo monitoring of 5HT release in rat and monkey models.</p><p>The irreversible binding of 5-hydroxy-L-[β-¹¹C]tryptophan, [¹¹C]HTP, in the monkey brain was lower in the presence of NSD1015, which was used to inhibit the decarboxylase step in 5HT synthesis. [¹¹C]HTP seems thus to have potential for tracking changes in the activity of this biosynthesis enzyme. In contrast, the accumulation of [¹¹C]HTP was unaffected by clorgyline, which was used to inhibit metabolism of the probe in the brain. This appears to indicate that elimination of the main metabolite from the brain could be negligible and thus will not alter [¹¹C]HTP quantification. The extent and distribution of the irreversible binding of a substrate for the first enzyme in 5HT formation, α-[¹¹C]methyl-L-tryptophan, [¹¹C]AMT, was different from those for [¹¹C]HTP. This suggests that the two studied probe compounds provide estimates related to the enzyme activity of different steps in the 5HT biosynthesis pathway. </p><p>A reference tissue version of the Patlak method for the analysis of data obtained by PET was also developed. This approach takes into account irreversible binding in the reference region and appears, therefore, to yield more reliable parameter estimates than the conventional reference Patlak analysis. The method is recommended for parameter estimation of [¹¹C]HTP data when no metabolite-corrected plasma curve is available. </p><p>Knowledge of altered 5HT synthesis and release in disease states and the consequences for effective pharmacotherapy can improve our knowledge of the aetiology of certain psychiatric and neurological diseases and enhance our ability to design more effective drugs.</p>

Mechanistische und kinetische Untersuchungen von komplexen zellulären Prozessen in situ sind in den vergangenen Jahren durch den Einsatz photoaktivierbarer Biomoleküle, sogenannter caged Verbindungen, möglich geworden. Bei den caged Verbindungen handelt es sich um photolabile inaktive Derivate von biologisch aktiven Molekülen, aus denen durch ultraviolettes Licht mit Hilfe einer photochemischen Reaktion die natürliche, biologisch aktive Substanz schnell freigesetzt werden kann. Im Rahmen der vorliegenden Arbeit wurden caged Verbindungen von den Neurotransmittern Octopamin und Dopamin, dem Octopamin-Antagonist Epinastin, den Proteinsyntheseinhibitoren Emetin und Anisomycin, dem Protonophor CCCP und dem Riechstoff Bourgeonal hergestellt. Zur Synthese dieser caged Verbindungen wurden sowohl bekannte als auch verschiedene im Rahmen dieser Arbeit neu entwickelte photolabile Schutzgruppen mit einem (Cumarin-4-yl)methyl- bzw. einem 2-Nitrobenzyl-Gerüst eingesetzt. Entsprechende Syntheseverfahren wurden erarbeitet. Anschließend erfolgte eine umfassende physikalisch-chemische sowie photochemische Charakterisierung der erhaltenen caged Verbindungen. Dabei wurde besonders auf gute Löslichkeit in Wasser bei physiologischer Ionenstärke, schnelle und effiziente Photoreaktivität, hohe Extinktion bei Wellenlängen von 350-430 nm und gute solvolytische Stabilität bei geringer Toxizität der freigesetzten Schutzgruppe geachtet. Ein Schwerpunkt bei der photochemischen Charakterisierung bildeten die Untersuchungen zur Quantifizierung der 2-Photonen-Anregung, uncaging action cross-sections, der Cumarinylmethyl-caged Verbindungen, aufgrund ihrer Bedeutung für die Photofreisetzung von Biomolekülen, da die gleichzeitige Absorption von 2 IR-Photonen eine höhere dreidimensionale Auflösung und eine wesentlich tiefere Gewebepenetration erlaubt. Mit Hilfe von Kooperationspartnern wurden zeitaufgelösten Fluoreszenz- und IR-Messungen an verschiedenen (Cumarin-4-yl)methoxycarbonyl-caged Modellverbindungen durchgeführt, mit denen die Geschwindigkeitskonstanten k1 und kdecarb des Photolysemechanismus ermittelt wurde. Am Ende folgten die Anwendungserprobungen ausgewählter caged Verbindungen in einem Translationsassay bzw. in Zelluntersuchungen. / In the last years mechanistic and kinetic in situ studies of complex cellular processes become possible by employing photoactivate Biomolecules, also called caged compounds, as a tool for these studies. Caged compounds are photolabile inactive derivates of biologic active molecules which are fast laid off the nature biologic active molecule by a photochemical reaction which was triggered by UV-light. In the present dissertation caged compounds of the neurotransmitters octopamine and dopamine, of the octopamine antagonist epinastine, of the proteine synthesis inhibitors emetine and anisomycine, of the ionophore CCCP and of the odorus substance Bourgeonal are synthesized. As precursors for the synthesis of that caged compounds some reported and several in these work newly developed photolabile protecting groups with (coumarin-4-yl)methyl- or 2-nitrobenzyl-scaffold were used. Corresponding Synthesis were designed. Afterwards the received caged compounds were global physical-chemical and photochemical characterised. In favour it was specifically valued for highly water solubility at pH 7,2, fast and efficient photo reactivity, high extinctions at wavelength 350-430 nm, well solvolytic stability and less toxicity of the redundant protecting groups. One key aspect of photochemical characterisation were the studies of uncaging action cross-sections of the coumarinylmethyl-caged molecules, because of their relevance for the photorelease of biomolecules in tissues. The simultaneous absorption of 2 IR-photons allowed highly three-dimensional release and a essentially deeper penetration in tissues. With the aid of co-operation partners were time-released fluorescence- and IR- measurements with several (coumarin-4-yl)methoxycarbonyl-caged molecules realised and therefore the rate constant k1 und kdecarb of the photolyse mechanismus were determined. At the end of the dissertation the achieved caged compounds were testet in translation assays and several cell cultures.

This thesis investigates the potential of using positron emission tomography (PET) to study the biosynthesis and release of serotonin (5HT) at the brain serotonergic neuron. As PET requires probe compounds with specific attributes to enable imaging and quantification of biological processes, emphasis was placed on the evaluation of these attributes. The experiments established that the 5HT transporter radioligand [11C]-3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile, [11C]DASB, is suitable for imaging and quantification of transporters in rats and rhesus monkeys. In addition, the binding of [11C]DASB in brain tissue is decreased when 5HT concentrations are increased by tranylcypromine administration. The sensitivity of [11C]DASB binding, under these experimental conditions, to increased endogenous 5HT concentrations demonstrates the potential of in vivo monitoring of 5HT release in rat and monkey models. The irreversible binding of 5-hydroxy-L-[β-11C]tryptophan, [11C]HTP, in the monkey brain was lower in the presence of NSD1015, which was used to inhibit the decarboxylase step in 5HT synthesis. [11C]HTP seems thus to have potential for tracking changes in the activity of this biosynthesis enzyme. In contrast, the accumulation of [11C]HTP was unaffected by clorgyline, which was used to inhibit metabolism of the probe in the brain. This appears to indicate that elimination of the main metabolite from the brain could be negligible and thus will not alter [11C]HTP quantification. The extent and distribution of the irreversible binding of a substrate for the first enzyme in 5HT formation, α-[11C]methyl-L-tryptophan, [11C]AMT, was different from those for [11C]HTP. This suggests that the two studied probe compounds provide estimates related to the enzyme activity of different steps in the 5HT biosynthesis pathway. A reference tissue version of the Patlak method for the analysis of data obtained by PET was also developed. This approach takes into account irreversible binding in the reference region and appears, therefore, to yield more reliable parameter estimates than the conventional reference Patlak analysis. The method is recommended for parameter estimation of [11C]HTP data when no metabolite-corrected plasma curve is available. Knowledge of altered 5HT synthesis and release in disease states and the consequences for effective pharmacotherapy can improve our knowledge of the aetiology of certain psychiatric and neurological diseases and enhance our ability to design more effective drugs.