Thesis (M.S.)--The University of North Carolina at Greensboro, 2008. / Directed by Paul Davis; submitted to the Dept. of Kinesiology. Title from PDF t.p. (viewed Jul. 20, 2010). Includes bibliographical references (p. 104-112).

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 156 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 45-50).

Ο Λείος Μυϊκός Φαινότυπος (ΛΜΦ) χαρακτηρίζεται από σημαντική πλαστικότητα και παίζει κομβικό λειτουργικό ρόλο τόσο σε φυσιολογικές όσο και σε παθολογικές καταστάσεις. Στις αρτηρίες, η μεταβολή του ΛΜΦ στο φάσμα από φυσιολογικός-συσταλτός μέχρι συνθετικός-παθολογικός εμπλέκεται αιτιολογικά σε διάφορες ανθρώπινες ασθένειες όπως στην αθηροσκλήρωση, στην υπέρταση και στην επαναστένωση των αγγείων μετά από εγχείρηση. Η έκφραση γονιδίων ΛΜΦ έιναι επίσης μεγάλης σημασίας σε ασθένειες άλλων οργάνων, όπως η ίνωση των νεφρών και του ήπατος και η μετάσταση του καρκίνου. Συνεπώς, η μελέτη των μοριακών μηχανισμών μέσω των οποίων επηρεάζεται ο ΛΜΦ είναι πολύ σημαντική για την ανάπτυξη νέων τεχνικών περιορισμού της εξέλιξης αυτών των νόσων. Η λειτουργία πολλών ιστών όπως είναι τα αγγεία ελέγχεται σε σημαντικό βαθμό από το συμπαθητικό νευρικό σύστημα. Σκοπός λοιπόν της παρούσας εργασίας ήταν πρώτα-πρώτα να εξετάσουμε εάν in vitro η διέγερση των α και β αδρενεργικών υποδοχέων πιθανώς ρυθμίζει την έκφραση γονιδίων ΛΜΦ και προκαταρκτικά να διακριβώσουμε μέσω ποιών μοριακών μηχανισμών οι α1 (υπότυποι α1Α και α1Β) και οι β-ΑΥς επηρεάζουν και ελέγχουν το ΛΜΦ. Κατά δεύτερο λόγο θελήσαμε να εξετάσουμε εάν η εξωγενής έκφραση του μεταγραφικού παράγοντα Μυοκαρδίνη προκαλεί επιθηλιο-μεσεγχυματική μετάβαση (ΕΜΤ-Epithelial to Mesenchymal Transition) σε ενδοθηλιακά κύτταρα in vitro. Για τα πρώτα πειράματά μας χρησιμοποιήσαμε δύο διαφορετικούς κυτταρικούς πληθυσμούς: α) διαφοροποιημένα λεία μυϊκά κύτταρα αορτής αρουραίου της κυτταρικής σειράς A7r5, και β) κύτταρα ινοβλαστών της κυτταρικής σειράς ΝΙΗ3Τ3 προερχόμενα απο ποντικό, προκειμένου να δούμε αντίστοιχα πως η δράση των ΑΥ ελέγχει την έκφραση των γονιδίων αυτών σε διαφοροποιημένα ΛΜΚ (A7r5) και σε αδιαφοροποίητα κύτταρα που όμως μπορούν να μετατραπούν σε «ΛΜΚ» (ΝΙΗ3Τ3). Ως δείκτη για την έκφραση του ΛΜΦ, εξετάσαμε την έκφραση κυτταροσκελετικών, δομικών πρωτεϊνών-δεικτών, όπως η λείου μυϊκού τύπου α-Ακτίνη (SM-α-Αctin),, η λείου μυϊκού τύπου βαριά αλυσίδα της Μυοσίνης (SM-MHC), η λείου μυϊκού τύπου Καλπονίνη (SM-Calponin) και η λείου μυϊκού τύπου πρωτεΐνη 22α (τρανσγελίνη) (SM22α), σε δύο επίπεδα: α) είτε χρησιμοποιώντας αντισώματα, είτε β) με την χρήση πλασμιδίων αναφοράς όπου minimal υποκινητές των παραπάνω γονιδίων ελέγχουν την προσμετρούμενη έκφραση λουσιφεράσης. Η ενεργοποίηση της μεταγραφής αυτών των γονιδίων ρυθμίζεται, σε μεγάλο μέρος, από τις αλληλουχίες CArG (CArG boxes) στους υποκινητές τους, στις οποίες προσδένεται ο παράγοντας Serum Response Factor (SRF). Στα ΛΜΚ, ο μεταγραφικός παράγοντας SRF ενεργοποιεί τη μεταγραφή των γονιδίων-δεικτών μέσω δημιουργίας ενός απαραίτητου συμπλόκου με έναν μεταγραφικό παράγοντα της οικογένειας των Μυοκαρδινών, η οποία αποτελείται από 3 μέλη : τη Μυοκαρδίνη και τις συγγενείς πρωτεΐνες MRTF-A και MRTF-B. Στη μελέτη που πραγματοποιήσαμε, αρχικά παρατηρήσαμε ότι οι δύο αυτοί κυτταρικοί πληθυσμοί δεν εκφράζουν τους α1-ΑΥς (α1Α και α1Β υπότυποι) ενδογενώς, αλλά, με εισαγωγή του αντίστοιχου πλασμιδίου των α1Α ή α1Β ΑΥς, το σύστημα καθίσταται λειτουργικό. Επιπλέον ανακαλύψαμε ότι τα κύτταρα A7r5 εκφράζουν ενδογενώς τους β-ΑΥς. Από τα πειράματα που πραγματοποιήσαμε, καταλήξαμε στο συμπέρασμα ότι οι υποδοχείς αυτοί επηρεάζουν και καθορίζουν με διαφορετικό τρόπο το ΛΜΦ, ανάλογα με τον υπότυπο των ΑΥς και ανάλογα με τον τύπο των κυττάρων. Πιο αναλυτικά, όσον αφορά στα κύτταρα A7r5 παρατηρήσαμε ότι: α) η ενεργοποίηση των α1Α-ΑΥ επάγει την έκφραση και των τεσσάρων γονιδίων-δεικτών που καθορίζουν το ΛΜΦ τόσο σε μεταγραφικό όσο και σε πρωτεϊνικό επίπεδο, β) η ενεργοποίηση του υποκινητή της SM22α από τους α1Α-ΑΥς εξαρτάται από τα στοιχεία CΑrG ενώ η ενεργοποίηση του υποκινητή της SM-Calponin δεν εξαρτάται από τα στοιχεία αυτά, γ) η ενεργοποίηση των α1Β-ΑΥ επάγει τη μεταγραφική ενεργότητα των υποκινητών των γονιδίων SM22α και SM-Calponin ενώ δεν επηρεάζει την μεταγραφική ενεργότητα των υποκινητών των γονιδίων SM-α-Αctin και της SM-MHC, δ) η ενεργοποίηση των υποκινητών της SM22α και της SM-Calponin από τους α1Β-ΑΥς εξαρτάται από τα στοιχεία CΑrG, ε) οι β-ΑΥς, σε αντίθεση με τους α1-ΑΥς, επηρεάζουν με διαφορετικό τρόπο τη μεταγραφή των γονιδίων-δεικτών που καθορίζουν το ΛΜΦ, κυρίως ελαττώνοντας τη μεταγραφική ενεργότητα των υποκινητών των γονιδίων SM-Calponin, SM-α-Αctin και SM-MHC ενώ δεν επηρεάζουν τη μεταγραφική ενεργότητα του SM22α. Επίσης, όταν παρόμοια πειράματα έγιναν στα κύτταρα ΝΙΗ3Τ3 παρατηρήσαμε ότι: α) οι α1Α-ΑΥs επάγουν τη μεταγραφή των υποκινητών και των τεσσάρων γονιδίων-δεικτών που καθορίζουν το ΛΜΦ, β) στα κύτταρα αυτά η ενεργοποίηση των υποκινητών της SM22α και της SM-Calponin από τους α1Α-ΑΥς δεν εξαρτάται από τα στοιχεία CΑrG, γ) η ενεργοποίηση των α1Β-ΑΥs επάγει τη μεταγραφική ενεργότητα των υποκινητών των γονιδίων SM22α, SM-Calponin και SM-MHC ενώ δεν επηρεάζει την ενεργότητα του υποκινητή της SM-α-Αctin. Από τα παραπάνω λοιπόν, καταλήξαμε στα εξής συμπεράσματα: 1) Η διέγερση των α1-ΑΥ οδηγεί σε άυξηση της έκφρασης των γονιδίων-δεικτών του ΛΜΦ, και άρα οι α1-ΑΥς λειτουργούν, τουλάχιστον in vitro, ως παράγοντες προώθησης του ΛΜΦ. 2) Η δράση των α1-ΑΥ εξαρτάται μόνο μερικώς απο την ύπαρξη στοιχείων CArG (SRE), και διαφέρει ανάλογα με τον υποκινητή, άρα οι διάφοροι υποκινητές χρησιμοποιούν διαφορετικά τα στοιχεία CArG που περιέχουν, πράγμα που ενισχύει την υπόθεση συνδυαστικής χρήσης μεταφραφικών παραγόντων για την «πλαστική» έκφραση του λειτουργικού ΛΜΦ. 3) Οι δύο υπότυποι των α1-ΑΥ που εξετάστηκαν, α1Α-ΑΥς και α1Β-ΑΥς, έχουν προφανείς διαφορές ως πρός την διέγερση της έκφρασης των ΛΜ γονιδίων, και άρα πιθανώς θα παίζουν διαφορετικό λειτουργικό ρόλο στα αγγεία και στους άλλους ιστούς όπου εκφράζονται. 4) Αντίθετα με τους α1-ΑΥς, οι β-ΑΥς λειτουργούν ανασταλτικά στην έκφραση των γονιδίων του ΛΜΦ, και άρα το τελικό προϊόν της συμπαθητικής διέγερσης στα αγγεία θα είναι η συνισταμένη των δράσεων α και β ΑΥ, που θα διαφέρει στα διάφορα αγγεία ανάλογα με την σχετική έκφραση των υποδοχέων αυτών. Παράλληλα, εξετάζοντας ενδοθηλιακά κύτταρα φλέβας ανθρώπινου ομφάλιου λώρου (HUVEC), είδαμε ότι η εξωγενής έκφραση της Μυοκαρδίνης προκαλεί αλλαγές στο φαινότυπό τους, τόσο σε μορφολογικό επίπεδο όσο και μέσω de novo έκφρασης της SM-α-Ακτίνης και της SM-Καλπονίνης σε πρωτεϊνικό επίπεδο. Παρατηρήσαμε δηλαδή αλλαγές που είναι συμβατές με την επιθηλιο-μεσεγχυματική μετάβαση (ΕΜΤ-Epithelial to Mesenchymal Transition), διαδικασία με πολύ σπουδαίο ρόλο στην εμβρυογένεση, στη διαμόρφωση ιστών και οργάνων, αλλά επίσης και στην ίνωση, στη μετάσταση του καρκίνου και στην παθολογική αγγειογένεση. Συμπερασματικά, τα ενδοθηλιακά κύτταρα των αγγείων συμμετέχουν στη μετάβαση αυτή και μπορεί να συμβάλλουν σε διεργασίες κυτταρικής διαφοροποίησης και ιστικής δόμησης. Συμπεραίνουμε ότι η έκφραση της Μυοκαρδίνης επαρκεί για να προκαλέσει ΕΜΤ σε ανθρώπινα ενδοθηλιακά κύτταρα, με πιθανές συνέπειες για τη δυνατότητα trans-διαφοροποίησης και συνεισφοράς αυτών των κυττάρων, σε ανακατασκευή και αναδιοργάνωση ιστών, όπως πχ. στην αθηροσκλήρωση και την καρδιακή ανεπάρκεια. / The Smooth Muscle Cell (SMC) Phenotype is characterized by important plasticity and it plays crucial functional role in physiological and pathological conditions. Modulation of SMC Phenotype in arteries is a key etiological feature of some major human pathologies, including atherosclerosis, hypertension and vessel restenosis. Expression of SMCs marker-genes is also important in chronic diseases of other organs, such as in kidney or hepatic fibrosis and in cancer metastasis. So, study of the molecular mechanisms which affect SMC phenotype is necessary in order to develop new therapeutic approaches to combat to these diseases. Function of many tissues such as the vasculature is regulated by the sympathetic nervous system. The aim of this study was first, to examine in vitro whether the stimulation of alpha (α) and beta (β) adrenergic receptors (ARs) can control the expression of SMCs marker-genes in vitro and in case it did, to probe the molecular mechanisms via which α1-ARs (subtypes α1A and α1B) and β-ARs affect and regulate SMC Phenotype. Secondly, we wanted to investigate if the exogenous expression of Myocardin can cause Epithelial to Mesenchymal Transition (ΕΜΤ)-like changes in endothelial cells in vitro. For our experiments, we used two different cell populations : a) A7r5, which are differentiated Smooth Muscle Cells isolated from rat embryonic aorta, and b) ΝΙΗ3Τ3, mouse fibroblasts, in order to examine how stimulation of ARs modulates the expression of these marker-genes in differentiated SMCs (A7r5) and in mesenchymal cells which can convert to SMCs (ΝΙΗ3Τ3), respectively. As a marker for the expression of SMC Phenotype, we monitored the expression of cytoskeletal, structural protein-markers, such as Smooth Muscle-α-Actin (SM-α-Actin), SM-Myosin Heavy Chain (SM-MHC), h1-Calponin (SM-Calponin) and SM22α (transgelin) at two levels: a) using specific antibodies or b) using reporter plasmids in which the minimal promoters of the above genes drive luciferase gene transcription and hence activity. The coordinate transcriptional activation of these genes is, in major part, regulated by the function of CArG boxes in their promoters, which bind Serum Response Factor (SRF). In SMCs, SRF mediates its transcriptional effects via essential complex formation with members of the Myocardin family, which includes Myocardin (Myocd), Myocardin-Related Transcription Factor-A (MRTF-A) and MRTF-B. In our study, we initially noticed that these two cell populations do not express α1-ARs (subtypes α1Α and α1Β) endogenously, but when we transfect them with the plasmids expressing α1Α and α1Β ARs, the cells respond to α1-ARs agonist stimulation. In addition, we discovered that A7r5 cells express endogenous β-ARs. From our experiments, we concluded that these receptors can modulate SMC Phenotype in distinct way. This depends on both the specific subtype of receptor as well as on the cellular background (cell type). More specific, we observed that in A7r5 cells: a) activation of α1A-ARs by phenylephrine induces the expression of all four marker-genes at a transcriptional and at a protein level, b) activation of the SM22α minimal promoter by α1A-ARs depends on CΑrG boxes, while activation of the SM-Calponin minimal promoter does not depend on the presence of CΑrG boxes, c) activation of α1B-ARs induces the transcriptional activity of the minimal promoters of SM22α and SM-Calponin but does not affect the transcriptional activity of the minimal promoter of SM-α-Αctin and SM-MHC, d) activation of both the SM22α and the SM-Calponin minimal promoters by α1B-ARs depends on the presence of CΑrG boxes, e) on the contrary, β-ARs affect the transcription of SMCs marker-genes in an opposite way to α1-ARs reducing the transcriptional activity of the minimal promoters of SM-Calponin, SM-α-Αctin and SM-MHC genes, without affecting the transcriptional activity of the SM22α promoter. Additionally, we noticed that in ΝΙΗ3Τ3 cells: a) α1Α-ARs induce transcriptional activity of minimal promoters of SMC marker-genes, b) activation of minimal promoters of SM22α and SM-Calponin by α1A-ARs does not depend on CΑrG boxes, c) activation of α1B-ARs induce the transcriptional activity of the minimal promoters of SM22α, SM-Calponin και SM-MHC but does not affect the transcriptional activity of the minimal promoter of SM-α-Αctin. Based on the above findings, we conclude that: 1) Stimulation of α1-ARs drives an increased expression of SMC marker genes and consequently α1-ARs function, at least in vitro, as factors which have the ability to induce/maintain the SMC Phenotype. 2) The activity of α1-ARs depends variably on the presence of CArG boxes (SREs) and differs between these minimal promoters. In essence, different promoters use their CArG boxes in a different way. This is in support of the hypothesis that combinational use of transcriptional factors is essential for «plastic» expression of the SMC Phenotype. 3) The two subtypes of α1-ARs examined, α1Α and α1Β, display obvious differences in stimulating SM-specific gene expression and consequently these subtypes may play different functional role in vessels and in other tissues in which they are expressed. 4) On the contrary, β-ARs inhibit the expression of SMC marker-genes. Therefore, the final result of vascular sympathetic stimulation would depend on the combined action of α και β ARs. This action will differ in different vessels, depending on the relative expression of these receptors and their subtypes. In addition, adenoviral expression of Myocardin in human umbilical vein endothelial Cells (HUVECs) induced phenotypic alterations, evidenced by morphological changes and by de novo expression of SM-α-Αctin and SM-Calponin at the protein level. These observations are compatible with an Epithelial to Mesenchymal Transition (EMT), a process which plays an important role in embryogenesis, tissue and organs formation and angiogenesis, but also participates, in fibrosis and cancer metastasis. Consequently, vascular endothelial cells can undergo in EMT and may contribute in cellular differentiation and in tissue formation. We conclude that the expression of Myocardin is sufficient to cause EMT-like changes in human endothelial cells. This may lead to cellular trans-differentiation and contribution of these cells in active tissue remodeling such as in atherosclerosis and in cardiac failure.

Orientador: Maria Cristina Thomaz / Banca: Fábio Enrique Lemos Budiño / Banca: Jacinta Diva Ferrugem Gomes / Banca: Hirasilva Borba Alves de Souza / Banca: Jorge de Lucas Júnior / Resumo: Objetivou-se avaliar os efeitos de concentrações crescentes de ractopamina em dietas para fêmeas suínas, abatidas com 110 kg de peso, quanto ao desempenho, características de carcaça, rendimento de cortes comerciais, composição e cortes cárneos do pernil, qualidades física, química, sensorial e perfil de ácidos graxos da carne, potencial para impacto ambiental e avaliação econômica. Foram utilizadas 468 fêmeas suínas, distribuídas entre quatro dietas: DC - dieta controle, composta principalmente por milho e farelo de soja; DC5 - dieta controle contendo 5 mg de ractopamina/kg; DC10 - dieta controle contendo 10 mg de ractopamina/kg e DC15 - dieta controle contendo 15 mg de ractopamina/kg. O peso inicial foi de 84,77±7,20 kg e final de 110,59±7,70 kg. O período experimental foi de 28 dias, quando então os animais foram destinados ao abate. Utilizou-se o delineamento em blocos ao acaso, para controlar diferenças de peso entre os animais. Encontrou-se reduções lineares para o consumo diário de ração e conversão alimentar, e aumentos lineares para área de olho de lombo, pesos de cortes inteiros do pernil e paleta, assim como para os pesos do coxão mole (Semitendinosus) e da alcatra (Gluteus medius), havendo efeito inverso para a quantidade de gordura no pernil, conforme o aumento das concentrações de ractopamina nas dietas. Não foi observado efeito sobre a qualidade e o perfil de ácidos graxos da carne. Houve diminuição da produção de fezes+urina por animal nas duas primeiras semanas de fornecimento, e alteração na composição das fezes. Notou-se aumentos nos custos da alimentação, e redução linear na receita líquida parcial sem bonificação. Para a receita líquida parcial com bonificação, observou-se efeito quadrático, sendo a concentração estimada de 4,05 mg de ractopamina/kg de dieta, a que determinou a melhor receita ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the effects of increasing ractopamine concentration in diets for gilts, slaughtered at 110 kg of body weight, on performance, carcass characteristics, whosale cuts, composition and retail cuts of ham, physical, chemical and sensorial quality and fat acids profile of meat, potential environmental impact and economical evaluation of using this additive. It was used 468 gilts, allotted into four diets: DC - control diet, based on corn and soybean meal; DC5 - control diet containing 5 mg of ractopamine/kg; DC10 - control diet containing 10 mg of ractopamine/kg; DC15 - control diet containing 15 mg of ractopamine/kg. The initial weight of animals was 84.77±7.20 kg and the final 110.59±7.70 kg. The assay was carried out by 28 days and the animals were conducted to slaughter. It was used a randomized block design, to control the differences of body weight. Linear reductions were observed for daily feed intake and feed:gain ratio, and linear increases in loin eye area, wholesale cuts of ham and shoulder and retail cuts of Semimembranosus and Gluteus medius and an inverse effect for fat of ham, by increasing the ractopamine concentration in diets. There was no effect on meat quality and fat acids profile of meat. A reduction on feces+urine production by animal was observed in the two weeks of ractopamine administration and a change on feces composition was noted. An increasing trend on feeding cost and a linear reduction in the net income without bonification was noted. For net income with bonification, there was a quadratic trend and better results were obtained with the estimated concentration of 4.05 mg of ractopamine/kg of diet. These results indicate that ractopamine inclusion in diets for gilts slaughtered at 110 kg of body weight is justified with bonification for lean carcass, and in this scenario, it was recomended to use 4 mg of ractopamine/kg of diet / Doutor

Beta blockers are commonly prescribed for the chronic treatment of hypertension, one of the most prolific disease states worldwide. The beta blockers selected for this study include acebutolol hydrochloride, labetalol hydrochloride, metoprolol tartrate oxprenolol hydrochloride and propranolol hydrochloride. All of these compounds have a short elimination half-life, necessitating multiple dose per day regimens and therefore the development of sustained release dosage forms incorporating these agents was considered beneficial in terms of extending the dosing interval, with the aim of improving patient compliance and subsequent therapeutic outcomes. Preformulation studies that were conducted included moisture content analysis by Karl Fischer titration, and DSC, a method used to predict potential interactions between the drugs and tablet excipients. Tablets were manufactured by both wet granulation and direct compression techniques, and the resultant drug release characteristics were evaluated using the USP Apparatus 3(BIO.DIS). A validated isocratic HPLC method, capable of separating the five drug candidates simultaneously, was developed and used for the analysis of drug samples. Tablet quality was assessed using analyses that included the physical assessment of weight, diameter, thickness, hardness and friability, as well as content uniformity of tablets, before and after dissolution testing. Direct compression tablet formulations containing each of the five beta blockers were successfully adapted from a prototype wet granulation matrix tablet containing metoprolol tartrate, and various formulation variables were investigated to establish,their effect on the rate and extent of drug release from these tablets. The grade and quantity of ethylcellulose used in the wet granulation and direct compression formulae influenced the release rate of some drug candidates. In addition, an alternative formulation method, involving freeze-drying of the drug with an ethylcellulose dispersion, was shown to have potential for altering release rates further. Anti-frictional agents, talc and colloidal silicon dioxide, did not affect drug release from these matrices,however, they affected the physical character:istics such as tablet weight and thickness, of the resultant tablets. All of the matrix tablets formulated were shown to release drug according to square root of time kinetics, in a sustained manner over a 22 hour period.

É importante caracterizar os múltiplos sistemas neurais que compõem a memória de diferentes tarefas comportamentais, visto que eles possuem diversas nuances que podem ser específicas de uma tarefa ou compartilhadas, dependendo do caso. Ressalta-se ainda que dentro da mesma tarefa estes sistemas podem agir de diferentes maneiras, dependendo de qual fase e/ou divisão da memória que estivermos nos referindo. Este trabalho teve como objetivo verificar se as sinapses dopaminérgicas D1 e noradrenérgicas presentes no hipocampo participam da consolidação da memória de dois paradigmas da tarefa labirinto radial de oito braços. Foram usados ratos Wistar machos adultos (2 a 3 meses), com peso médio de 220g, mantidos com água a vontade. Os animais receberam cânulas na região CA1 do hipocampo e foram treinados em um dos tipos de protocolo: com ou sem intervalo, sendo que receberam infusões intrahipocampais de SKF 38393, agonista dopaminérgico D1, ou NE, 0 ou 3h pós-treino no primeiro e segundo dias de treinamento. Como resultados obtivemos que NE obteve um efeito amnésico quando dada 0 h pós-treino no modelo sem intervalo e quando dada 3 h pós-treino no modelo com intervalo. O SKF não teve nenhum efeito independente do modelo ou tempo de administração. Estes achados sugerem que as sinapses noradrenérgicas hipocampais são importantes na consolidação da memória de ambos paradigmas nos primeiros dias de treinamento e que o sistema noradrenérgico atua diferentemente em cada paradigma. / Learning and memory can be studied in a variety of tasks. Tasks that require different structures, and different memory processes.It is important to characterize the neurotransmitter systems are involved time-dependently in memory consolidation of different tasks and depending on the case the systems can act in different ways inside of the same task , depending of which phase and/or division of the memory that we are referring. This work had as aim to determine wheter dopaminergic type 1 (D1) and β- noradrenergic receptors in the hippocampus participate in memory consolidation of two paradigms of the task eight arms radial maze. Male Wistar rats (2 to 3 months) were used, with medium weight of 220g, maintained with water "ad-libitum". The animals received stems in the area CA1 of the hippocampus and they were trained in one of the protocol types: win-stay or win-shift, and they received infusions intrahipocampais of D1 dopaminergic receptor agonist SKF 38393 or NE, 0 or 3 h post-training in the first and second days of training. Results showed NE had an amnestic effect when given 0 h post-training in the model win-stay and when given 3 h post-training in the model win-shift. SKF didn't have any independent effect of the model or time of administration. These discoveries suggest that β- noradrenergic receptors in the hippocampus are important in the consolidation of the memory of both paradigms in the first days of training and that the noradrenergic system acts differently in each paradigm.

A dispersive liquid-liquid microextraction (DLLME) method has been developed, optimized and validated for the extraction of seven beta-agonists (Cimaterol, Cimbuterol, Clenproperol, Clenbuterol, Ractopamine, Isoxsuprine and Ritodrine) from bovine muscle. The homogenized tissue samples were hydrolyzed enzymaticaly by beta-glucuronidase and extracted with DLLME. The extraction parameters (pH, extraction solvent, extraction solvent volume, disperser solvent) were accurately optimized. Separation of the beta-agonists was by gradient elution on C18 LC column using acetonitrile and formic acid aqueous solutions as mobile phases, multiple reaction monitoring (MRM) scan mode was used. The seven beta-agonists were then simultaneous determined and identified in single analysis using 4000 Qtrap LC-MS/MS system. The DLLME method was validated using ISO 17025 and the EU criteria (Commission Decision 2002/657/EC) for validation of analytical method, good precision, repeatability and spiked recoveries were obtained. The limits of detection and quantification for the residues were between 0.0728 – 0.0922 μg/kg and 0.243 – 0.307 μg/kg respectively for beta-agonists. The overall recoveries were between 85% and 100% with the relative standard deviation of (RSDs) between 3.0% and 10%. The recoveries from the developed DLLME method were compared with those obtained from dSPE. DLLME proved to be comparable to SPE. The real samples test showed that the DLLME method developed is accurate and sensitive for the determination of beta-agonist residues in bovine muscle. / Chemistry / M. Sc. (Analytical Chemistry)

É importante caracterizar os múltiplos sistemas neurais que compõem a memória de diferentes tarefas comportamentais, visto que eles possuem diversas nuances que podem ser específicas de uma tarefa ou compartilhadas, dependendo do caso. Ressalta-se ainda que dentro da mesma tarefa estes sistemas podem agir de diferentes maneiras, dependendo de qual fase e/ou divisão da memória que estivermos nos referindo. Este trabalho teve como objetivo verificar se as sinapses dopaminérgicas D1 e noradrenérgicas presentes no hipocampo participam da consolidação da memória de dois paradigmas da tarefa labirinto radial de oito braços. Foram usados ratos Wistar machos adultos (2 a 3 meses), com peso médio de 220g, mantidos com água a vontade. Os animais receberam cânulas na região CA1 do hipocampo e foram treinados em um dos tipos de protocolo: com ou sem intervalo, sendo que receberam infusões intrahipocampais de SKF 38393, agonista dopaminérgico D1, ou NE, 0 ou 3h pós-treino no primeiro e segundo dias de treinamento. Como resultados obtivemos que NE obteve um efeito amnésico quando dada 0 h pós-treino no modelo sem intervalo e quando dada 3 h pós-treino no modelo com intervalo. O SKF não teve nenhum efeito independente do modelo ou tempo de administração. Estes achados sugerem que as sinapses noradrenérgicas hipocampais são importantes na consolidação da memória de ambos paradigmas nos primeiros dias de treinamento e que o sistema noradrenérgico atua diferentemente em cada paradigma. / Learning and memory can be studied in a variety of tasks. Tasks that require different structures, and different memory processes.It is important to characterize the neurotransmitter systems are involved time-dependently in memory consolidation of different tasks and depending on the case the systems can act in different ways inside of the same task , depending of which phase and/or division of the memory that we are referring. This work had as aim to determine wheter dopaminergic type 1 (D1) and β- noradrenergic receptors in the hippocampus participate in memory consolidation of two paradigms of the task eight arms radial maze. Male Wistar rats (2 to 3 months) were used, with medium weight of 220g, maintained with water "ad-libitum". The animals received stems in the area CA1 of the hippocampus and they were trained in one of the protocol types: win-stay or win-shift, and they received infusions intrahipocampais of D1 dopaminergic receptor agonist SKF 38393 or NE, 0 or 3 h post-training in the first and second days of training. Results showed NE had an amnestic effect when given 0 h post-training in the model win-stay and when given 3 h post-training in the model win-shift. SKF didn't have any independent effect of the model or time of administration. These discoveries suggest that β- noradrenergic receptors in the hippocampus are important in the consolidation of the memory of both paradigms in the first days of training and that the noradrenergic system acts differently in each paradigm.

Master of Science / Department of Animal Sciences and Industry / John Michael Gonzalez / The objective of this body of work was to examine effects of growth-promoting technologies (GP) on Longissimus lumborum meat tenderness, focusing on alterations of muscle fiber cross-sectional area (CSA) and collagen solubility. Two studies were conducted and analyzed as randomized complete block designs with repeated measures with GP and day of postmortem aging (DOA) as main effects. Treatments consisted of: a control (CON), implant only (IMP), and implant and [beta]-adrenergic agonist (COMBO). The [beta]-adrenergic agonist utilized for the first was zilpaterol hydrochloride, while the second study examined ractopamine hydrochloride. Objective tenderness of strip loin steaks was measured through Warner-Bratzler shear force (WBSF) after 2 (study 2) or 3 (study 1), 7, 14, 21, or 35 d of postmortem aging. Muscle fiber CSA and collagen solubility were analyzed via immunohistochemistry and hydroxyproline content, respectively. For the first study there was a treatment × DOA interaction (P < 0.01) for WBSF. Compared to CON steaks, IMP steaks had greater (P = 0.01) WBSF on d 3, but were similar (P = 0.21) by d 14. The COMBO steaks remained less tender at all-time points (P < 0.04) except d 21 (P = 0.13) when compared to the CON. Growth-promoting treatment increased the CSA of all three muscle fiber types (P < 0.01), but had no effect on collagen solubility measures (P > 0.21). The second study observed no treatment × DOA interaction (P = 0.54) for WBSF, but GP increased (P < 0.01) WBSF across all DOA. Growth-promoting treatment tended to increase the CSA of type I and IIX fibers (P < 0.10), and increased (P < 0.01) type IIA fiber CSA. In agreement with the first study, there was no treatment × DOA interaction or treatment effect on collagen solubility (P > 0.75). The addition of GP to feedlot heifer production increased WBSF of strip loin steaks and fiber CSA, but did not impact collagen characteristics.