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Ovarian cysts in dairy cattle importance of serum LH concentrations in maintenance of cysts and expression of mRNAs for steroidogenic enzymes and gonadotropin receptors /Calder, Michele D. January 1998 (has links)
Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 110-136). Also available on the Internet.
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Regulation of expression of 5 alpha-reductase type 1 in mouse leydig cellsKrishnan, Shruti. January 2005 (has links) (PDF)
Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: 33-36.
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Σχεδιασμός και ανάπτυξη νέων αναλόγων της εκλυτικής ορμόνης της ωχρινοτρόπου ορμόνης (GnRH)Παππά, Ελένη Β. 27 January 2012 (has links)
Η Εκλυτική ορμόνη της Ωχρινοτρόπου Ορμόνης των θηλαστικών (GnRH-I ή GnRH) είναι ένα δεκαπεπτίδιο το οποίο εκκρίνεται από τον υποθάλαμο κατά ώσεις. Η GnRH είναι δυνατό να δρα σαν ρυθμιστής διαφόρων συστημάτων τα οποία, για τη λειτουργία τους, απαιτούν την έκκριση της LH και της FSH. Πράγματι, η GnRH και τα ανάλογα της χρησιμοποιούνται εκτεταμένα για τη θεραπεία ενός μεγάλου αριθμού ασθενειών οι οποίες σχετίζονται με τις φυλετικές ορμόνες. Η χρόνια χορήγηση αναλόγων της GnRH προκαλεί απευαισθητοποίηση των υποδοχέων το οποίο οδηγεί στην παύση της έκκρισης γοναδοτροπινών και κατά συνέπεια στην καταστολή της λειτουργίας των ωοθηκών και των όρχεων (chemical castration).
Εκτός από την υπόφυση οι υποδοχείς της GnRH εκφράζονται σε αρκετούς φυσιολογικούς και διάφορους καρκινικούς ιστούς, συμπεριλαμβανομένων του μαστού, του ωοθηκών, του ενδομητρίου και του προστάτη. Ο ρόλος της GnRH και των υποδοχέων της σε αυτούς τους εξωϋποθαλαμικούς ιστούς δεν έχει ξεκάθαρος. Ωστόσο, έχει δειχθεί ότι σε διάφορα είδη καρκινικών κυττάρων συμπεριλαμβανομένων και των PC3 και LNCaP τα ανάλογα της GnRH αναστέλλουν τον πολλαπλασιασμό ή οδηγούν σε προγραμματισμένο κυτταρικό θάνατο (απόπτωση).
Η lamprey Εκλυτική Ορμόνη της Ωχρινοτρόπου Ορμόνης (lGnRH-III) είναι η τρίτη μορφή της GnRH η οποία έχει απομονωθεί από τα ψάρια του είδους lamprey (το γένος Petromyzon marinus). Αυτό το δεκαπεπτίδιο το οποίο διαφέρει από τη GnRH των ανθρώπων (GnRH-I) στα αμινοξέα των θέσεων 5-8, διεγείρει την απελευθέρωση οιστραδιόλης και της προγεστερόνης στα ενήλικα θηλυκά lamprey αλλά έχει αμελητέα ενδοκρινή δράση στα συστήματα των θηλαστικών. Την τελευταία δεκαετία έχουν δημοσιευτεί στοιχεία τα οποία υποστηρίζουν την απευθείας αντικαρκινική δράση της lGnRH-III. Τα παραπάνω οδηγούν στο χαρακτηρισμό της lGnRH-III ως ένα εξαιρετικό μόριο-οδηγό για την ανάπτυξη αναλόγων της GnRH με αυξημένη και πιθανώς εκλεκτική αντικαρκινική δράση.
Οι πιο πρόσφατες αναφορές για απευθείας δράση των αναλόγων της GnRH στην αναστολή του πολλαπλασιασμού καρκινικών κυττάρων τα οποία παράγουν τη φυσική ορμόνη και εκφράζουν τους υποδοχείς της εγκαινιάζουν ένα νέο πεδίο έρευνας και προσφέρουν νέα ώθηση στο σχεδιασμό και τη σύνθεση νέων αναλόγων της GnRH. Μελέτες οι οποίες διερευνούν τη σχέση δομής-δραστικότητας της GnRH-Ι και ιδιαιτέρως της lGnRH-III και των αναλόγων τους στο εξωϋποθαλαμικό σύστημα είναι περιορισμένες. Η ανάγκη διερεύνησης του πεδίου αυτού θεωρείται μείζονος σημασίας καθώς ο πληθυσμός των καρκινοπαθών αυξάνει συνεχώς και η πάθηση κατατάσσεται μεταξύ των σημαντικότερων πεδίων ερευνητικής μελέτης. Ανάλογα της GnRH χορηγούνται σε ασθενείς με ορμονοεξαρτώμενους όγκους με στόχο την αναστολή των γοναδοτροπινών και την παύση της έκκρισης των φυλετικών ορμονών. Η προσέγγιση του πεδίου με χρήση αναλόγων της GnRH με ενδεχόμενη κυτταροστατική δράση αποτελεί μια σύγχρονη και πολλά υποσχόμενη προοπτική. Από τη άλλη μεριά, ένα από τα μεγαλύτερα προβλήματα το οποίο εμφανίζεται στα φάρμακα πεπτιδικής φύσης είναι η γρήγορη και εκτεταμένη αποικοδόμηση τους από διάφορα ένζυμα. Προκειμένου να βελτιωθεί η βιοδιαθεσιμότητα των αναλόγων της GnRH έχουν χρησιμοποιηθεί διάφορες προσεγγίσεις. Η εισαγωγή N-μεθυλιωμένων-αμινοξέων περιορίζει διαμορφωτικά τον πεπτιδικό σκελετό και χρησιμοποιείται συχνά για την αύξηση της πρωτεολυτικής σταθερότητας των φαρμάκων πεπτιδικής φύσης. Μια πιο ολοκληρωμένη προσέγγιση είναι αυτή η οποία συνδυάζει την σταθερότητα έναντι της ενζυμικής αποικοδόμησης με τη διατήρηση της βιολογικής δράσης.
Στόχος της ∆ιατριβής είναι η συμβολή στο πεδίο των σχέσεων δομής-δραστικότητας της GnRH μέσω του ορθολογικού σχεδιασμού, της ανάπτυξης και του ελέγχου νέων συνθετικών αναλόγων της. Συγκεκριμένα, συνθέσαμε δώδεκα νέα ανάλογα τα οποία περιλαμβάνουν διαμορφωτικά περιορισμένα αμινοξέα στις θέσεις 4 και 6 και μελετήσαμε τις δομικές αλλαγές οι οποίες προκύπτουν από τις αλλαγές αυτές με φασματοσκοπία NMR, τη σταθερότητα έναντι της ενζυμικής αποικοδόμησης από την α-χυμοθρυψίνη και τη σουμπτιλισίνη και την δράση τους επί του πολλαπλασιασμού των καρκινικών κυττάρων PC3 και LNCaP. Η NMeSer εισήχθη στη θέση 4 του μορίου αντικαθιστώντας τη Ser και η Gly6 αντικαταστάθηκε από διάφορα D- αμινοξέα (DLys, Nε- τροποποιημένη DLys, Glu, DGlu). Το Ν-τελικό γλυκιναμίδιο έχει επαλειφθεί και στη θέση του έχει γίνει εισαγωγή της αιθυλ-ομάδας (Fujino modification), όπως στο μόριο του Λεουπρολιδίου. Συνθέσαμε επίσης είκοσι δύο νέα ανάλογα της lGnRH-III με τροποποιήσεις σε διάφορες θέσεις του μορίου και μελετήσαμε τη δράση τους επί του πολλαπλασιασμού των PC3 και LNCaP κυττάρων. Η Asp6 της lGnRH-III αντικαταστάθηκε από Asn, Asn(OMe), Glu ή Gln, η Trp3,7 από DTrp ή L/D-Tic, το pGlu1 αντικαταστάθηκε από Glu ή Ac-Glu και η His5 με την Lys8 άλλαξαν μεταξύ τους θέσεις. Επιπλέον, συνθέσαμε και τέσσερα κυκλικά ανάλογα της lGnRH-III καθώς τα κυκλικά πεπτίδια συχνά χαρακτηρίζονται από μεγαλύτερη μεταβολική σταθερότητα και δραστικότητα σε σχέση με τα ευθύγραμμα. Τέλος, μελετήσαμε τη δομή της lGnRH-III με τη χρήση ΝΜR φασματοσκοπίας και τα χαρακτηριστικά της παρουσιάζονται σε σύγκριση με αυτά της GnRH-I. Για τη σύνθεση των αναλόγων της GnRH-I σαν στερεό υπόστρωμα χρησιμοποιήθηκε η [3-(αιθυλ-Ν-Fmoc-αμινομέθυλ)ινδολ-1- υλ] ακετυλ ΑΜ ρητίνη για την παραλαβή αμιδίων ενώ, για τη σύνθεση της GnRH-I, της lGnRH-III και των αναλόγων της lGnRH-III χρησιμοποιήθηκε η ρητίνη του Sieber χρησιμοποιώντας την Fmoc/tBu μεθοδολογία. Η ενζυμική σταθερότητα των αναλόγων της GnRH-I προσδιορίστηκε έπειτα από επώαση των πεπτιδίων με την α-Chymotrypsin και τη Subtilisin ενώ, η δράση έναντι του κυτταρικού πολλαπλασιασμού της GnRH-I, της lGnRH-III και των αναλόγων τους μελετήθηκε με ανθρώπινα καρκινικά κύτταρα προστάτη (PC3 και LNCaP).
Το πρωτόκολλο σύνθεσης το οποίο ακολουθήθηκε οδήγησε στην παραλαβή πεπτιδίων σε μεγάλες αποδόσεις και υψηλή καθαρότητα. Η μελέτη της επίδρασης των νέων αναλόγων της GnRH στην αναστολή του πολλαπλασιασμού των καρκινικών κυττάρων του προστάτη εγκαινιάζει το πεδίο σε επίπεδο σχέσεων δομής-δραστικότητας και συνεισφέρει στην αδιάκοπη έρευνα των δομικών απαιτήσεων των αναλόγων της GnRH προσφέροντας νέες προοπτικές στον σχεδιασμό δραστικών αναλόγων της GnRH. Tέλος, προέκυψαν πεπτιδικά ανάλογα της ορμόνης τα οποία σε σχέση με το Λεουπρολίδιο, το οποίο αποτελεί δραστικό συστατικό φαρμάκων για την αντιμετώπιση των ανωτέρω ασθενειών και με τη lGnRH-III, εμφανίζονται στις βιολογικές δοκιμές στις οποίες εξετάστηκαν δραστικότερα και με αυξημένη πρωτεολυτική σταθερότητα και συνεπώς αποτελούν καλές, υποψήφιες για περαιτέρω βιολογική/κλινική μελέτη, ενώσεις. Σε επίπεδο σχέσεων δομής-δραστικότητας οι τελευταίες είναι δυνατόν να αποτελέσουν τη βάση για τον μελλοντικό σχεδιασμό νέων αναλόγων της GnRH με υψηλή δραστικότητα και ενδεχόμενη φαρμακευτική εφαρμογή. / Mammalian Gonadotropin-releasing hormone (GnRH-I or GnRH) is a neuroendocrine decapeptide secreted from the hypothalamus in a pulsatile manner. Due to its hypophysiotropic actions, GnRH may act as a modulator of the activity of diverse systems that require LH and FSH secretion for their function. Indeed, GnRH and its analogs are used extensively for the treatment of a wide range of hormone-dependent diseases, including steroid-dependent tumors. Chronic administration of GnRH analogs desensitizes GnRH pituitary receptors, results in arrest of gonadotropin secretion, and thereby suppresses ovarian and testicular function (chemical castration).
In addition to its pituitary expression, the GnRHR is expressed in several normal tissues and in a number of malignant tumors, including cancers of the breast, ovary, endometrium and prostate. The specific function of GnRH and its receptor in these extrapituitary sites is not entirely clear. However, it was demonstrated that in many cancer cells including PC3 and LNCaP GnRH analogs inhibit cell proliferation or lead to a programmed cell death (apoptosis). Lamprey gonadotropin-releasing hormone III (lGnRH-III) is the third isoform of GnRH isolated from the sea lamprey (Petromyzon marinus). This decapeptide that differs in residues 5–8 from human GnRH (GnRH-I), stimulates the release of estradiol and progesterone in the adult female sea lamprey but has negligible endocrine activity in mammalian systems. In the last decade data concerning the superior direct antitumor activity of sea lamprey lGnRH-III have been published. These observations make lGnRH-III an excellent starting compound for the development for the development of constrained peptide analogs with increased and potentially selective anticancer activity.
The direct antiproliferative effect of the GnRH analogs in many malignant tissues inaugurates the field and leads to the design of effective GnRH analogs with new perspectives. Information concerning the impact of systematic single amino acid modifications of GnRH-I and especially of lGnRH-III on malignant cells is still lacking. The structural investigation of this field seems to be a very important issue. On the other hand, one of the main problems of peptide drugs is their fast and extensive degradation by several peptidases. In order to improve the availability of GnRH analogs, various approaches have been adopted. Incorporation of N-methylated amino acids in peptides results in conformational constrained peptide backbones and is commonly used for increasing proteolytic stability of peptide drugs. In the latter approach, stability against proteolysis should be achieved along with preservation or enhancement of biological activity.
The aim of this study was the contribution in the field of the structure-activity relationships based in the rational design, development (modern synthesis) and biological characterization of the new analogs of GnRH. Particularly, we synthesized twelve new GnRH-I analogs containing conformationally restricted amino acids in positions 4 and 6, and studied the structural changes imposed by these modifications through NMR spectroscopy, the stability against enzymatic degradation by α-chymotrypsin and subtilisin and their direct antiproliferative effect on prostate cancer cells PC3 and LNCaP. NMeSer was inserted in position 4 of the peptide sequence, replacing Ser4 and Gly6 was substituted by several D- amino acids (DLys, Nε- modified DLys, Glu, DGlu). The synthesized GnRH-I analogs lack the carboxy-terminal Gly10-amide of GnRH-I and an ethylamide residue has been added to Pro9 (Fujino modification), as in Leuprolide structure. We also synthesized twenty two lGnRH-III analogs with modifications in several positions of lGnRH-III and studied their effect on PC3 and LNCaP prostate cancer cell proliferation. Asp6 of lGnRH-III was substituted by Asn, Asn(OMe), Glu or Gln, Trp3,7 by DTrp or L/D-Tic, pGlu1 was replaced by Glu or Ac-Glu and His5 and Lys8 switched places. Cyclopeptides are of great importance both in pharmaceutical and chemical respect. These molecules often exhibit increased biological activity and selectivity. Furthermore, they are more stable in metabolism than the parent linear molecules. Taking into consideration of these potentials we synthesized four cyclic analogs of lGnRH-III as well. The new analogs of GnRH-I were assembled on a [3-((Ethyl-Fmoc-amino)- methyl)-1-indol-1-yl]-acetyl AM resin to provide the peptide amide while, GnRH-I, lGnRH-III and lGnRH-III analogs were assembled on a Sieber Amide resin using Fmoc/tBu methodology. The enzymatic stability of GnRH-I analogs were determined after incubation with α-Chymotrypsin and Subtilisin while the antiproliferative activity of GnRH-I, lGnRH-III and their analogs was performed with human prostate cancer cell lines (PC3 and LNCaP). Modern peptide synthesis afforded us peptides in high yields which could be easily purified. This study provides information which can be used in the design of new GnRH analogs with improved enzymatic stability and high antiproliferative activity on PC3 and LNCaP prostate cancer cell proliferation.
This structure-activity relationship study of GnRH analogs contributes to the on-going research for more potent GnRH analogs with prolonged and selective activity.
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GnRH/GnIH e seus receptores no sistema olfato-retinal de zebrafishCorchuelo Chavarro, Sheryll Yohana [UNESP] 29 May 2015 (has links) (PDF)
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000854973.pdf: 2773395 bytes, checksum: daf1de70009034e8b87b75a4cc4b7610 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O hormônio liberador de gonadotropina (GnRH) é um dos fatores chaves na regulação neuroendócrina da reprodução dos vertebrados. Alguns peixes apresentam três variantes do GnRH: o GnRH1 envolvido na secreção de gonadotropinas, o GnRH2 que regula o comportamento alimentar e sexual e o GnRH3 expresso no bulbo olfatório e o nervo terminal cujas fibras nervosas inervam a retina e o epitélio olfatório. O zebrafish possui duas variantes do GnRH (GnRH2 e GnRH3), sendo o GnRH3 a variante hipofisiotrófica. Estudos mostram possível envolvimento do GnRH no sistema olfato-retinal. No sistema olfatório o GnRH regula a sensibilidade na detecção de alimento, o reconhecimento intra e interespecífico, entre outros. Na retina, o GnRH3 pode estar envolvido na acuidade visual e do processamento de informação da retina. Existem estudos que reportam a presença de receptores de GnRH em diferentes camadas da retina, no entanto ainda não é clara a presença de receptores no epitélio olfatório. Neste contexto, no presente estudo analisamos a localização do gnrh2, gnrh3 e seus receptores (gnrhr1,2,3 e 4) e do gnih (hormônio inibidor de gonadotropinas) no epitélio olfatório, a retina e o bulbo olfatório de machos e fêmeas adultos e comparamos a expressão destes genes em fêmeas em diferentes estágios de maturação gonadal. Para tanto, o RNA total do epitélio olfatório, retina, bulbo olfatório, cérebro e gônadas foi extraído. Com base na sequência dos genes gnrh2, gnrh3, gnrhr1, gnrhr2, gnrhr3 e gnrhr4, primers forward e reverse foram desenhados para RT-PCR e qPCR. Sondas para a hibridização in situ também foram construídas para verificar os sítios de expressão destas moléculas no epitélio olfatório, retina e gônadas. Imunohistoquímica com os anticorpos anti-GnRH3 (BB8 e GF6) foram realizadas para localizar a proteína do GnRH3 nos tecidos analisados. O presente estudo apresenta um panorama da expressão do sistema... / The gonadotropin releasing hormone (GnRH) is one of the key factors involved in the neuroendocrine regulation of vertebrate reproduction. Some fish species have three GnRH variants: GnRH1 involved in gonadotropin secretion, GnRH2 regulating food and sexual behaviors and the GnRH3 which is expressed in the olfactory bulb and terminal nerve whose fibers innervate the retina and the olfactory epithelium. Two GnRH variants (GnRH2 and GnRH3) are present in the zebrafish, in which GnRH3 acts as the hypophisiotrophic variant. Recent studies have been showing the role of GnRH in the olfactory-retinal system. In the olfactory system, GnRH regulates food detection, and intra and interspecific recognition. In retina, GnRH3 may be involved in visual acuity modulation and retinal processing information. Moreover, studies have reported the presence of GnRH receptors in the retina, but not yet in the zebrafish olfactory epithelium. Therefore, the current study analyzed the presence of GnRH2, GnRH3 and its receptors (GnRH-R1,2,3 and 4) and GnIH (gonadotropin inhibitory hormone) in the olfactory epithelium, olfactory bulb, retina and in gonads of adult zebrafish. We also compared the expression of these genes during the different stages of ovarian maturation in zebrafish. For that, total RNA of the olfactory epithelium, olfactory bulb, retina and gonads was extracted with the PureLink® RNA Mini Kit(Ambion®). RT-PCR and qPCR analysis were performed using forward and reverse primers for gnrh2, gnrh3, gnrhr1, gnrhr2, gnrhr3, gnrhr4 for . Probes for in situ hybridization were constructed to verify the expression sites of these molecules in the olfactory epithelium, retina, and gonads. Immunohistochemistry usinganti-GnRH3 antibodies (BB8 and GF6) were performed to identify the GnRH3 protein in these tissues. The current study presents a general expression view of GnRH/GnIH and their receptors in the olfactory epithelium-olfactory bulb-retinal axis during ... / FAPESP: 2014/02481-9
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GnRH/GnIH e seus receptores no sistema olfato-retinal de zebrafish /Corchuelo Chavarro, Sheryll Yohana. January 2015 (has links)
Orientador: Laura Satiko Okada Nakaghi / Coorientador: Rafael Henrique Nóbrega / Banca: Elisabeth Criscuolo Urbinati / Banca: Matias Pandolfi / Resumo: O hormônio liberador de gonadotropina (GnRH) é um dos fatores chaves na regulação neuroendócrina da reprodução dos vertebrados. Alguns peixes apresentam três variantes do GnRH: o GnRH1 envolvido na secreção de gonadotropinas, o GnRH2 que regula o comportamento alimentar e sexual e o GnRH3 expresso no bulbo olfatório e o nervo terminal cujas fibras nervosas inervam a retina e o epitélio olfatório. O zebrafish possui duas variantes do GnRH (GnRH2 e GnRH3), sendo o GnRH3 a variante hipofisiotrófica. Estudos mostram possível envolvimento do GnRH no sistema olfato-retinal. No sistema olfatório o GnRH regula a sensibilidade na detecção de alimento, o reconhecimento intra e interespecífico, entre outros. Na retina, o GnRH3 pode estar envolvido na acuidade visual e do processamento de informação da retina. Existem estudos que reportam a presença de receptores de GnRH em diferentes camadas da retina, no entanto ainda não é clara a presença de receptores no epitélio olfatório. Neste contexto, no presente estudo analisamos a localização do gnrh2, gnrh3 e seus receptores (gnrhr1,2,3 e 4) e do gnih (hormônio inibidor de gonadotropinas) no epitélio olfatório, a retina e o bulbo olfatório de machos e fêmeas adultos e comparamos a expressão destes genes em fêmeas em diferentes estágios de maturação gonadal. Para tanto, o RNA total do epitélio olfatório, retina, bulbo olfatório, cérebro e gônadas foi extraído. Com base na sequência dos genes gnrh2, gnrh3, gnrhr1, gnrhr2, gnrhr3 e gnrhr4, primers forward e reverse foram desenhados para RT-PCR e qPCR. Sondas para a hibridização in situ também foram construídas para verificar os sítios de expressão destas moléculas no epitélio olfatório, retina e gônadas. Imunohistoquímica com os anticorpos anti-GnRH3 (BB8 e GF6) foram realizadas para localizar a proteína do GnRH3 nos tecidos analisados. O presente estudo apresenta um panorama da expressão do sistema... / Abstract: The gonadotropin releasing hormone (GnRH) is one of the key factors involved in the neuroendocrine regulation of vertebrate reproduction. Some fish species have three GnRH variants: GnRH1 involved in gonadotropin secretion, GnRH2 regulating food and sexual behaviors and the GnRH3 which is expressed in the olfactory bulb and terminal nerve whose fibers innervate the retina and the olfactory epithelium. Two GnRH variants (GnRH2 and GnRH3) are present in the zebrafish, in which GnRH3 acts as the hypophisiotrophic variant. Recent studies have been showing the role of GnRH in the olfactory-retinal system. In the olfactory system, GnRH regulates food detection, and intra and interspecific recognition. In retina, GnRH3 may be involved in visual acuity modulation and retinal processing information. Moreover, studies have reported the presence of GnRH receptors in the retina, but not yet in the zebrafish olfactory epithelium. Therefore, the current study analyzed the presence of GnRH2, GnRH3 and its receptors (GnRH-R1,2,3 and 4) and GnIH (gonadotropin inhibitory hormone) in the olfactory epithelium, olfactory bulb, retina and in gonads of adult zebrafish. We also compared the expression of these genes during the different stages of ovarian maturation in zebrafish. For that, total RNA of the olfactory epithelium, olfactory bulb, retina and gonads was extracted with the PureLink® RNA Mini Kit(Ambion®). RT-PCR and qPCR analysis were performed using forward and reverse primers for gnrh2, gnrh3, gnrhr1, gnrhr2, gnrhr3, gnrhr4 for . Probes for in situ hybridization were constructed to verify the expression sites of these molecules in the olfactory epithelium, retina, and gonads. Immunohistochemistry usinganti-GnRH3 antibodies (BB8 and GF6) were performed to identify the GnRH3 protein in these tissues. The current study presents a general expression view of GnRH/GnIH and their receptors in the olfactory epithelium-olfactory bulb-retinal axis during ... / Mestre
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Indução da ovulação e funcionalidade do corpo lúteo em novilhas Nelore pré-púberes /Vrisman, Dayane Priscila. January 2017 (has links)
Orientador: Maria Emilia Franco Oliveira / Coorientador: Pedro Paulo Maia Teixeira / Coorientador: Fábio Morato Monteiro / Banca: Pietro Sampaio Baruselli / Banca: Lindsay Unno Gimenes / Resumo: Devido a comum ocorrência de regressão prematura (RP) do corpo lúteo (CL) em novilhas após primeira ovulação (OV), os objetivos do estudo foram: 1) acompanhar a dinâmica lútea após indução da OV em novilhas Nelore pré-púberes e 2) determinar diferenças relacionáveis a funcionalidade dessa estrutura. Cinquenta e sete fêmeas (289,61±32,28 kg, ECC de 5,66±0,65 e 17,47±0,81 meses de idade) foram divididas em dois grupos de tratamento para indução da OV. No grupo GP4+GnRH foi utilizado dispositivo intravaginal de progesterona (P4) de 3º uso por 10 dias e, 48 horas após remoção, aplicado 0,02mg de acetato de buserelina (GnRH), e no grupo GGnRH foi utilizado somente o GnRH. Os CLs formados foram acompanhados pela ultrassonografia a cada dois dias até a sua regressão funcional (diminuição do sinal vascular do Doppler colorido e concentrações de P4 abaixo de 1 ng/mL), sendo determinado para cada dia o diâmetro, área, valores numéricos (VPN) e heterogeneidade dos pixels e percentual (%) de vascularização. A velocidade do pico sistólico, velocidade diastólica final, índice de resistência e o índice de pulsatilidade (IP) da artéria ovariana também foram determinados para cada avaliação, além da concentração sérica de P4. Essas características foram comparadas entre os tratamentos, funções dos CLs (duração normal ou regredido prematuramente), dias das avaliações e suas interações, utilizando o procedimento MIXED do programa SAS (p≤0,05). Três animais de cada tratamento não responderam ao ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Due to the common occurrence of premature regression (PR) of the corpus luteum (CL) in heifers after the first ovulation (OV), the aims of this study were to: 1) monitor the luteal dynamics after OV induction in prepubertal Nellore heifers, and 2) determine differences related to the functionality of this structure. Fifty-seven females (BW 289.61±32.28 kg, BCS 5.66±0.65 and 17.47±0.81 months old) were divided into two treatment groups for OV induction. In the group GP4+GnRH, an intravaginal progesterone (P4) device of 3rd use was used for 10 days and, 48 hours after its removal 0.02 mg of buserelin acetate (GnRH) was applied, and in the GGnRH group only GnRH was used. Formed CLs were monitored via ultrasonography every two days until functional regression (decrease of the vascular signal of color Doppler and serum P4 concentrations below 1 ng/mL), being determined for each day the diameter, area, numerical values (NV) and heterogeneity of the pixels, and vascularization percentage (%). The systolic and diastolic peak velocity, resistance and pulsatility index (PI) of the ovarian artery were also determined for each day in addition to the serum P4 concentration. These characteristics were compared between treatments, CLs functions (normal duration or prematurely regressed), days of evaluations and their interactions, using the MIXED procedure of SAS program (p≤0.05). Three animals from each treatment did not respond to the OV inductor (6/57=11%), which determined an ovulation ... (Complete abstract click electronic access below) / Mestre
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Clonagem, caracterização e análise filogenética das subunidades alfa e beta do hormônio folículo estimulante de Pirarucu (Arapaima gigas) visando sua síntese em células CHO / Cloning, characterization and phylogenetic analisys of the alpha and beta subunits of the follicle stimulating hormone of Pirarucu (Arapaima gigas) in view of its synthesis in cho cellsRoberto Feitosa de Carvalho 16 June 2014 (has links)
O Pirarucu (Arapaima gigas) é um peixe gigante da família Arapaimidae, nativo das bacias amazônicas que pode chegar a dois metros de comprimento e pesar mais de 200 Kg. Está presente no Equador, na Colômbia, no Peru, na Bolívia e no Brasil. Atualmente a espécie está ameaçada de extinção devido à pesca predatória e ao aumento da presença humana em seus viveiros naturais. No presente trabalho, os cDNAs da subunidade (ag-GTHα) e da subunidade β do hormônio folículo estimulante de A. gigas (ag-FSH) foram isolados e clonados pela primeira vez, possibilitando uma melhor compreensão da diversidade e evolução desta glicoproteína em peixes e a futura síntese biotecnológica deste hormônio para fins reprodutivos e alimentícios. Tanto o cDNA do ag-GTHα quanto aquele do ag-FSHβ foram sintetizados pela reação de transcriptase reversa (RT) e pela reação em cadeia da polimerase (PCR) utilizando como molde RNA total proveniente das glândulas hipofisárias de A. gigas. O cDNA da subunidade ag-GTHα possui uma sequência codificadora (Open Reading Frame, ORF) de 348 pb, o que corresponde a uma proteína de 115 aminoácidos com um suposto peptídeo sinal de 24 aminoácidos e com um peptídeo maduro de 91 aminoácidos. Dez resíduos de cisteínas responsáveis pela formação de cinco pontes dissulfeto, dois sítios de N-glicosilação e três resíduos de prolinas apresentaram-se altamente conservados quando comparados com outras espécies de peixes. A comparação baseada em sequências de aminoácidos de GTHα de 38 espécies de peixes revelou alta identidade do A. gigas com membros das seguintes ordens: Acipenseriformes, Anguiliformes, Siluriformes e Cypriniformes (87,1-89,5%), e, a menor identidade com os Gadiformes e Cyprinodontiformes (55%). A identidade com a análoga sequência de GTHα de Homo sapiens foi de 67%. Para a subunidade ag-FSHβ, a ORF correspondente foi de 381 pb, produzindo uma proteína de 126 aminoácidos com um peptídeo sinal de 18 e um peptídeo maduro de 108 aminoácidos. Quando comparado com as sequências de Anguilla marmorata, Acipenser gueldenstaedtii e Homo sapiens, o peptídeo maduro de ag-FSHβ mostrou conter 12 resíduos de cisteínas responsáveis pela formação de seis pontes dissulfeto, duas prolinas e um sítio de glicosilação perfeitamente conservados, apresentando identidades de 63, 50 e 45% respectivamente em suas sequências de aminoácidos. As árvores filogenéticas construídas mostraram, em geral, que o A. gigas, da ordem dos Osteoglossiformes, se apresenta como grupo irmão dos Clupeocefala, enquanto os Elopomorpha (Anguiliformes) formam o grupo mais basal de todos os teleósteos aqui analisados. / Pirarucu (Arapaima gigas) is a giant fish of the Arapaimidae family native to the Amazon river basin, that can reach 3 meters in length, weighing up to 250 Kg. It is present in Equador, Colombia, Peru, Bolivia and Brazil. This species is in danger of disappearing due to exploitation by the fishing industry and increasing human presence in its natural habitat. In the present work the cDNAs of the gonadotropin -subunit (ag-GTH) and of follicle-stimulating hormone β subunit (ag-FSHβ) were isolated and cloned for the first time. As a consequence, a better understanding of the diversity and evolution of this glycoprotein in fish and its future biotechnological synthesis for reproductive and alimentary purposes will be possible. Both cDNAs of ag-GTHα and ag-FSHβ have been synthesized via reverse transcriptase reaction (RT-PCR) and polymerase chain reaction (PCR) using as a template total RNA extracted from A. gigas pituitary glands. Ag-GTHα- subunit has a coding sequence (open reading frame, ORF) of 348 bp, corresponding to a 115 amino acid protein, with a putative signal peptide of 24 aminoacids and a mature peptide of 91 amino acids. Ten cysteine residues, responsible for the formation of five disulfide linkages, two N-glycosylation sites and three proline residues were found highly conserved when compared to other fish species. A comparison based on the amino acid sequence of the GTHα- subunit from 38 different species of fish showed high identity of A. gigas with members of the following orders: Acipenseriformes, Siluriformes and Cypriniformes (87.1-89.5%), while the lowest identity was found with Gadiformes and Cyprinodontiformes (55%). In comparison with the analogous sequence of Homo sapiens an identity of 67% was found. For the ag-FSHβ subunit an ORF of 381 bp, coding for a 126 amino acid protein, with a signal peptide of 18 and a mature peptide of 108 amino acids, was found. When compared with the Anguilla marmorata, Acipenser gueldenstaedtii and Homo sapiens, the mature peptide of ag-FSHβ showed the presence of the twelve cysteine residues responsible for the formation of six disulfide linkages, two proline residues and one glycosylation site, all of them perfectly conserved. The identity with the mentioned species were 63, 50 and 45% respectively. The obtained phylogenetic trees have shown, in general, that A. gigas of the order of Osteoglossiformes appears as sister group of Clupeocephala, while Elopomorpha (Anguilliformes) forms the most basal group of all analyzed teleosts.
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Influência de diferentes isoformas de fosfodiesterases no controle da maturação de oócitos bovinos / Influence of different phosphodiesterase isoforms on the control of bovine oocyte maturationFabiane Gilli Zaffalon 31 July 2014 (has links)
A maturação in vitro do oócito é um dos fatores limitantes na produção in vitro de embriões. In vivo, esta maturação é um processo altamente orquestrado no qual a meiose é retomada pela onda de gonadotrofina que antecede a ovulação e que induz à queda dos níveis de AMPc no oócito. No entanto, os oócitos aspirados ao serem retirados dos folículos ovarianos retomam espontaneamente a maturação comprometendo a competência de seu desenvolvimento. O AMPc é sintetizado pela adenilato ciclase (AC) e degradado pelas fosfodiesterases (PDE), existindo algumas relacionadas à degradação do AMPc e outras do GMPc. Sendo assim, a proposição deste trabalho foi averiguar a contribuição de diferentes isoformas de fosfodiesterases na retomada da meiose e nos níveis de GMPc, AMPc e ainda, determinar quando há manutenção de AMPc em níveis elevados observando sua influência na competência oocitária e ativação da MAPK. Para isso, os complexos cumulus-oócito (CCOs) foram maturados in vitro na ausência, presença ou associação de inibidores de PDEs-AMPc e GMPc específicas e FSHr. As amostras foram avaliadas em relação a: 1) taxa de maturação; 2) níveis intracelulares de AMPc e GMPc nos CCOs; 3) taxa de desenvolvimento de blastocistos ; 4) ativação da MAPK em oócitos e células do cumulus. Os resultados obtidos no primeiro experimento indiaram que o inibidor da PDE3 foi o mais eficaz (p<0,05) em atrasar a retomada da meiose, às nove horas de maturação, porém, isolado ou em associação com o inibidor da PDE8, não foi capaz de alterar (p>0,05) os níveis de AMPc. No experimento dois, o inibidor da PDE5 isolado não influenciou a retomada da meiose (p>0,05), porém, quando associado aos inibidores da PDE3 e 8 houve atraso na retomada (p<0,05) e ainda alteraram os níveis de GMPc e AMPc (p<0,05) nas primeiras horas de maturação. O experimento três mostrou a influencia do FSHr durante a MIV, o qual estimulou a retomada da meiose, mas em associação com inibidores da PDE5 e 8 atrasa a retomada (p<0,05). Além disso, o FSHr provoca aumento do nível de AMPc e sua associação com inibidores de PDE5 e PDE8 ocasionou elevação adicional (p<0,05). As condições de cultivo estudadas no experimento quatro mostraram que a maturação induzida (pré-MIV de duas horas com agentes para elevar AMPc seguindo de 22 horas de MIV com FSH associado a inibidores de PDEs) atrasaram a retomada da meiose às nove horas de maturação, mas não afetaram progressão da meiose às 24, 28 e 30 horas. Os tratamentos, porém, não melhoraram a competência oocitária após a fertilização in vitro e ocasionaram pequenas variações na ativação da MAPK em oócitos e células do cumulus. / In vitro maturation of oocytes is a limiting factor in the in vitro production of bovine embryos. In vivo, this maturation is a highly orchestrated process in which meiosis resumption by the gonadotropin surge that precedes ovulation induces the decrease in cAMP levels in the oocyte. However, when oocytes are removed from follicles, they spontaneously resume maturation compromising the competence for its development. cAMP is synthesized by adenylyl cyclase (AC) and degraded by phosphodiesterases (PDE), and there are some PDEs related to degradation of cAMP and/or cGMP. Thus, the purpose of this work was to investigate the contribution of different isoforms of phosphodiesterases in the resumption of meiosis and levels of cAMP and, also, to determine differences in signaling pathways when maintaining high levels of cAMP and its influence on oocyte competence. For this purpose, cumulus-oocyte complexes (COC) were matured in vitro in the presence, absence or combination of inhibitors of cAMP- and cGMP-specific PDEs and FSH. Samples be were evaluated in relation to: 1) maturation rate, 2) intracellular levels of cAMP and cGMP in COCs, 3) rate of blastocyst development and 4) activation of MAPK in oocytes and cumulus cells. The results of the first experiment showed that the PDE3 inhibitor is more effective (p <0.05) in delaying meiosis resumption, at nine hours of maturation, but was not capable of altering cAMP levels (p> 0.05) either alone or in combination with the PDE8 inhibitor. In experiment two, the PDE5 inhibitor alone did not affect the meiosis resumption (p> 0.05), however, when associated with PDE3 and PDE8 inhibitors it delayed their resumption (p <0.05) and also altered cGMP and cAMP levels of (p <0.05) in the early hours of maturation. The third experiments showed the influence of FSHr during IVM, which stimulated the resumption of meiosis, but in combination with PDE5 and PDE8 inhibitors meiosis was delayed (p <0.05). Furthermore, FSHr causes increased levels of cAMP and its association with PDE5 and PDE8 inhibitors caused an additional increase (p <0.05). Culture conditions studied in experiment four showed that induced maturation (pre-IVM for two hours with agents to elevate cAMP followed by 22 hours IVM with FSH associated with PDE inhibitors) delayed the resumption of meiotic maturation at nine hours, but has no effect on meiosis progression at 24, 28 and 30 hours. The treatments, however, did not improve oocyte competence after in vitro fertilization and caused minor variations in the activation of MAPK in oocytes and cumulus cells.
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Modeling electrical spiking, bursting and calcium dynamics in gonadotropin releasing hormone (GnRH) secreting neuronsFletcher, Patrick Allen 11 1900 (has links)
The plasma membrane electrical activities of neurons that secrete gonadotropin releasing
hormone (GnRH), referred to as GnRH neurons hereafter, have been studied extensively.
A couple of mathematical models have been developed previously to explain different
aspects of these activities including spontaneous spiking and responses to stimuli such as current injections, GnRH, thapsigargin (Tg) and apamin. The goal of this paper is to
develop one single, minimal model that accounts for the experimental results reproduced
by previously existing models and results that were not accounted for by these models.
The latter includes two types of membrane potential bursting mechanisms and the
associated calcium oscillations in the cytosol. One of them has not been reported in
experimental literatures on GnRH neurons and is thus regarded as a model prediction.
Other improvements achieved in this model include the incorporation of a more detailed
description of calcium dynamics in a three dimensional cell body with the ion channels
evenly distributed on the cell surface. Although the model is mainly based on data
collected in cultured GnRH cell lines, we show that it is capable of explaining some
properties of GnRH neurons observed in several of other preparations including mature
GnRH neurons in hypothalamic slices. One potential explanation is suggested. A
phenomenological reduction of this model into a simplified form is presented. The
simplified model will facilitate the study of the roles of plasma membrane electrical
activities on the pulsatile release of GnRH by these neurons when it is coupled with a
model of pulsatile GnRH release based on the autoregulation mechanism. / Science, Faculty of / Mathematics, Department of / Graduate
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Post-transcriptional Regulation Of Gene Expression : Role Of 3' Untranslated Region Of FSHBeta mRNAManjithaya, Ravi R. 08 1900 (has links) (PDF)
No description available.
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