Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 4 uppsatser.

Thesis (M.Sc.) Uppsala : Sveriges lantbruksuniv.

Thesis (M.Sc.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2009/06/18). Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references (leaves 181-233). Also available online through Digital Dissertations.

Η φυσιολογική ανάπτυξη του εμβρύου των θηλαστικών επιτυγχάνεται μέσα από τη συντονισμένη ρύθμιση της γονιδιακής έκφρασης των κυττάρων που προκύπτουν από τις διαιρέσεις του ζυγωτού. Έτσι λαμβάνει χώρα ο σταδιακός καθορισμός της τύχης των πολυδύναμων αυτών κυττάρων, τα οποία προσανατολίζονται κατάλληλα κατά μήκος του πρόσθιο-οπίσθιου και του ραχιαίο-κοιλιακού άξονα του εμβρύου. Ο κατάλληλος προσανατολισμός εξασφαλίζει την έκθεση των κυττάρων σε τοποειδικά εκφραζόμενους μεταγραφικούς παράγοντες που ελέγχουν τη μεταγραφή συγκεκριμένων γονιδίων. Μέσα λοιπόν από σύνθετα γονιδιακά ρυθμιστικά δίκτυα συμβαίνει η διαφοροποίηση των πρόδρομων κυττάρων προς ειδικούς τύπους κυττάρων με συγκεκριμένη μορφή και λειτουργία. Συχνά, μάλιστα, σχεδιάζονται πειραματικά πρωτόκολλα που μιμούνται τις διαδικασίες της διαφοροποίησης συγκεκριμένων κυτταρικών τύπων ξεκινώντας από βλαστοκύτταρα. Ιδιαίτερο ενδιαφέρον μεταξύ αυτών εμφανίζει η διαφοροποίηση κύτταρων που παράγουν ινσουλίνη, τα οποία θα μπορούσαν να αξιοποιηθούν για τη θεραπεία ευρέως διαδεδομένων ασθενειών όπως ο ινσουλινοεξαρτώμενος διαβήτης. Η αποκάλυψη, επομένως, των ρυθμιστικών δικτύων που κατευθύνουν τη διαφοροποίηση αυτών των κυττάρων υπόσχεται να βελτιώσει τα σχετικά πρωτόκολλα διαφοροποίησης αλλά και να συμβάλλει ενδεχομένως στην κατανόηση των μοριακών μηχανισμών της ασθένειας. Με τον όρο ρυθμιστικό δίκτυο υποδηλώνεται ένα σύνολο αλληλεπιδράσεων μεταξύ μεταγραφικών παραγόντων (trans-trans) αλλά και μεταξύ γονιδιακών περιοχών και πρωτεϊνών που ελέγχουν τη μεταγραφή (cis-trans). Στην παρούσα διατριβή, αντικείμενο εξέτασης αποτέλεσαν οι ρυθμιστικές αλληλουχίες ενός συνόλου γονιδίων τα οποία, με βάση πειραματικά δεδομένα διερεύνησης της γονιδιακής έκφρασης με μικροσυστοιχίες DNA, φαίνονταν να σχετίζονται με τη διαφοροποίηση των β-κυττάρων του παγκρέατος των θηλαστικών. Oι ρυθμιστικές αλληλουχίες συνήθως προσδένουν μεταγραφικούς παράγοντες και λόγω του σημαντικού τους ρόλου δέχονται έντονη εξελικτική πίεση. Δεδομένης λοιπόν της εξελικτικής συντήρησης των ρυθμιστικών στοιχείων, χρησιμοποιήθηκε η μέθοδος της συγκριτικής γονιδιωματικής με στόχο την ανεύρεση συντηρημένων ρυθμιστικών αλληλουχιών. Με σύγκριση ορθόλογων γονιδιωματικών περιοχών σε ένα σύνολο οργανισμών που περιλάμβανε ακόμη και τους πιο απομακρυσμένους φυλογενετικά οργανισμούς που διαθέτουν πάγκρεας, ταυτοποιήθηκαν συντηρημένες θέσεις πρόσδεσης μεταγραφικών παραγόντων. Πιο αναλυτικά, τα γονίδια που μελετήθηκαν επιλέχθηκαν με κριτήριο τη γνωστή ή πιθανή ρύθμισή τους από το μεταγραφικό παράγοντα NGN3, τον κύριο καθοδηγητή της διαφοροποίησης των ενδοκρινών κυττάρων του παγκρέατος. Με βάση πειραματικά δεδομένα ανάλυσης της γονιδιακής έκφρασης και με τη βοήθεια ισχυρών και ευέλικτων αλγορίθμων αναζητήθηκαν κοινά cis ρυθμιστικά στοιχεία στα ορθόλογα επαγόμενων και καταστελλόμενων από τον NGN3 γονιδίων. Παράλληλα αναζητήθηκαν διαφορές ανάμεσα στις ρυθμιστικές περιοχές των αντίστοιχων ομάδων γονιδίων. Αποκαλύφθηκε, έτσι, η ύπαρξη μιας συντηρημένης ρυθμιστικής περιοχής σε όλα τα ορθόλογα των οργανισμών που διαθέτουν πάγκρεας, η οποία περιλάμβανε θέσεις πρόσδεσης για μεταγραφικούς παράγοντες που εμπλέκονται στις διαδικασίες διαφοροποίησης των κυττάρων της ενδοκρινούς μοίρας του παγκρέατος. Η συγκεκριμένη περιοχή δεν εντοπίστηκε σε γονίδια των οποίων η έκφραση δεν σχετίζεται με την προηγούμενη διαδικασία, είτε αυτά εκφράζονται συνεχώς (B-ACTIN), είτε δεν εκφράζονται καθόλου στο πρώιμο έμβρυο (B-GLOBIN). Επιπλέον, η διερεύνηση εντοπισμού νουκλεοτιδικών προτύπων στις αλληλουχίες των ρυθμιστικών στοιχείων αποκάλυψε, επιπροσθέτως την παρουσία αλληλουχιών πρόσδεσης για τον παράγοντα AP4 μέσα στα ρυθμιστικά στοιχεία των καταστελλόμενων από τον NGN3 γονιδιών. Έγινε, έτσι, διάκριση των προαναφερθέντων ρυθμιστικών στοιχείων σε αυτά που φέρουν θέση πρόσδεσης για τον AP4 και σε αυτά που δεν φέρουν. Παράλληλα η ανάλυση ολόκληρου του γονιδιώματος του ποντικού και η στατιστική επεξεργασία των αποτελεσμάτων κατέδειξαν πως και τα δύο στοιχεία δεν εντοπίστηκαν τυχαία στα γονίδια που ελέγχονταν μεταγραφικά από τον NGN3. Δεδομένου ότι στην πλειοψηφία των γονιδίων που εξετάστηκαν οι ρυθμιστικές αυτές περιοχές εντοπίστηκαν μακριά από το σημείο έναρξης της μεταγραφής εκτιμήθηκε πως πρόκειται για ακολουθίες με ρόλο ενισχυτή, οι οποίες σε περίπτωση που μπορούν να προσδέσουν επιπροσθέτως και τον παράγοντα AP4 μετατρέπονται σε επιλεκτικούς καταστολείς των αντιστοίχων γονιδίων. Το τελευταίο συμπέρασμα υποστηρίχτηκε και από την ανάλυση της περιεκτικότητας των ρυθμιστικών στοιχείων σε GC που έδειξε ότι, όπως και οι περισσότερες αλληλουχίες με ρυθμιστικό ρόλο κατά την εμβρυογένεση, έτσι και οι συγκεκριμένες ήταν πτωχές σε GC, κάτι όμως που γενικότερα δεν ισχύει για τους υποκινητές. Η διερεύνηση των αλληλεπιδράσεων των πρωτεϊνών-μεταγραφικών παραγόντων (trans-trans) που κατά πρόβλεψη προσδένονται στα συντηρημένα ρυθμιστικά στοιχεία αποκάλυψε την ύπαρξη ενός συμπλόκου από γενικούς και ειδικούς μεταγραφικούς παράγοντες. Το σύμπλοκο αυτό συνδεόμενο με ειδικούς μεταγραφικούς ρυθμιστές μπορεί να λειτουργεί άλλοτε ως επαγωγέας και άλλοτε ως καταστολέας της μεταγραφής συγκεκριμένων γονιδίων. Σημαντικό ρόλο στη διαφορική λειτουργία του συγκεκριμένου συμπλόκου θεωρήθηκε ότι διαδραματίζει η αλλαγή των επιπέδων ακετυλίωσης της χρωματίνης λόγω της παρουσίας ακετυλασών και αποακετυλασών στο σύμπλοκο. Οι αλληλεπιδράσεις μεταξύ των πρωτεϊνών (trans-trans), μαζί με τις αλληλεπιδράσεις μεταξύ των γονιδίων που αναλύθηκαν (cis-cis) αλλά και οι συνδυασμοί αυτών ενσωματώθηκαν σε ένα ευρύτερο ρυθμιστικό δίκτυο με κεντρικό ρυθμιστή τον NGN3. Προέκυψε, λοιπόν, ένα ρυθμιστικό δίκτυο, από το οποίο υποδεικνύεται ότι με επιλεκτική επαγωγή συγκεκριμένων γονιδίων και με καταστολή άλλων επιτυγχάνεται τελικά η διαφοροποίηση κυττάρων ικανών να παράγουν ινσουλίνη. / Mammalian development occurs by the progressive determination of cells from a pluripotent undifferentiated state through successive states of gradually restricted developmental potential, until the full complement of mature terminally differentiated cells has been specified. Embryonic development is a complex and highly orchestrated process during which multiple cell movements and changes in gene expression must be spatially and temporally coordinated to ensure that embryogenesis proceeds correctly. Complex genetic regulatory networks receive input in the form of extracellular signals and output instructions on the regulated expression of specific genes. The linchpins of the regulatory networks are the cis-regulatory elements that directly control gene expression through interpretation of the tissue-specific transcription factors (trans-elements). Embryonic stem cells are orientated across the dorso-ventral and the anterior-posterior axis of the early embryo. The orientation of progenitor cells along these two axes is thought to influence their fate by defining the identity and concentration of inductive signals to which they are exposed. In an effort to develop cell-based therapies, (i.e. for diabetes) experimental protocols aim to mimic the biological procedures that take place during embryonic development in order to differentiate embryonic stem cells towards specific cell types. One of the foremost challenges towards the development of cell therapies for diabetic people is to achieve the directed differentiation of cells capable of producing insulin. Elucidation of the genetic networks involved in the endocrine pancreas specification are thought to be essential for devising rational protocols to efficiently differentiate embryonic stem cells or pancreas progenitor cells into fully differentiated endocrine subtypes. Computational approaches allow the unravelling of complex regulatory networks including genomic (cis-cis) or proteomic (trans-trans) interactions or a combination (cis-trans) of both. In this study the genomic regulatory regions (cis elements) of several genes known and putative targets of the transcription factor NGN3 were analyzed. The NGN3 transcription factor is the major regulator of “insulin-producing cell” formation. Taking into account data from microarray experiments from pancreas progenitor cells, in which NGN3 has been induced, genes shown to be co-regulated (upregulated or downregulated) by this transcription factor were selected for analysis. Using a combination of sophisticated computational tools for exploiting and analyzing genomic data and developing the suitable algorithms, an extensive in silico analysis of the regulatory regions of these genes was performed. Evolutionarily conserved regions are linked with experimentally identified regulatory elements. Comparative genomics are commonly used in order to identify transcription factor binding sites, which are functionally important regions that are thought to be well-conserved. Analysis of genomic regulatory regions included not only genes corregulated by NGN3, but also their orthologs in several species including the most phylogenetically distant species (fish), which have pancreas. In parallel, housekeeping genes, like B-ACTIN, and those not expressed in embryos and stem cells, like B-GLOBIN, were used as negative controls. Regulatory region analysis revealed the presence of a highly conserved regulatory element, where many transcription factors with established involvement in pancreas development bind, in all the orthologs of several genes co-regulated by NGN3. Furthermore, motif identification in separate clusters of the regulatory elements of either upregulated or downregulated genes revealed the presence of additional binding motifs for the factor AP4 only in downregulated genes. In parallel, the regulatory region analysis of the entire mouse genome and the statistical analysis of the upcoming results showed that both types of regulatory elements (with and without AP4) were non-randomly identified inside the regulatory regions of genes whose transcription is controlled by NGN3. Moreover the selective presence of the AP4 binding sequence into this region renders it a highly specific suppressor found in only a small number of genes downregulated by NGN3. Taking into account that both these regulatory elements were identified at considerable distances from each gene’s transcription start site, it was assumed that they represent enhancers, and those capable of binding AP4 were considered silencers. This conclusion was enforced by the compositional analysis of these regions showing low GC levels, similarly to the majority of the regulatory regions implicated in embryonic development, something that has not been reported for promoter sequences. Moreover, analysis of protein-protein interactions showed that some of the transcription factors, predicted to bind onto these elements, together with other non-specific transcription factors, constitute a core transcription control complex. This protein complex interacts with the remaining members of the predicted cluster of transcription regulators and works either as an inducer or a suppressor of transcription. This is determined by the presence of a HAT and/or an HDAC in this protein complex assumed to locally control chromatin acetylation. Based on these data, we constructed a model of the complex regulatory network that describes how through the transcriptional regulation of the analyzed genes mainly guided by ΝGN3 the gradual differentiation of cells capable of producing insulin takes place.

The thesis aims to optimize the methods of laboratory culture and to fill in the gaps in knowledge of D. virgifera virgifera ecophysiology. The experiments on embryology and development proved that the diapause of D. virgifera virgifera is of obligatory type and can be terminated by exposure to long-day conditions (20:4; photo : scotophase) and constant temperature of 25 °C. Approximately 10% of eggs completed the development to adults within 4 months. Besides numerous other environmental conditions that are discussed, temperature seems to be a key factor influencing longevity in this pest. Higher temperature of 25 °C prolonged survival by more than 20 days in 2015, compared to 22 °C in 2014.

No description available.

Made available in DSpace on 2014-06-11T19:26:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-10-13Bitstream added on 2014-06-13T20:33:41Z : No. of bitstreams: 1 silva_bm_me_jabo.pdf: 548750 bytes, checksum: 36f590119119f6fe622e3615252bd6ac (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A acetilação das histonas é um dos principais mecanismos de reprogramação epigenética do genoma dos gametas a fim de estabelecer um estado totipotente para o desenvolvimento normal. O objetivo deste trabalho foi avaliar o efeito da utilização da Tricostatina A (TSA) sobre os níveis de acetilação das histonas e o desenvolvimento embrionário, avaliando-se a contagem do número de células da massa celular interna (MCI), trofectoderma (TE), e células totais, e os níveis da reação de imunocitoquímica para H3K9ac. Para isso, três experimentos foram delineados. No primeiro experimento, verificou-se que o grupo tratado com 5nM de TSA por 144h durante o cultivo in vitro (CIV) aumentou (p<0,01) o nível de H3K9ac em relação ao grupo controle. A taxa de clivagem e o desenvolvimento embrionário foram avaliados em um segundo experimento, e o grupo tratado com 5nM de TSA não apresentou diferença na produção de embriões comparado ao grupo controle. No experimento 3, o tratamento com a TSA não apresentou efeitos significativos em relação ao número de células da MCI, porém houve redução (p<0,01) tanto do número de células da TE quanto de células totais comparado ao grupo controle. Portanto, conclui-se que o tratamento com TSA não altera o desenvolvimento embrionário pré-implantacional de embriões fêmeas de bovinos, mostrando efeito negativo sobre o desenvolvimento dos embriões tratados / Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genome to establish a totipotent state for normal development. The aim of this work was to evaluate the use of Trichostatin A (TSA) on the levels of histone acetylation and embryonic development, the assessment of counting the cell number of inner cell mass (ICM), trophectoderm (TE), and total cells, and the levels of H3K9ac for immunocytochemical reaction. Therefore, three experiments were designed. In the first experiment, it was verified that the group treated with TSA for 5nM for 144h during in vitro culture (IVC) increased (p <0.01) the level of H3K9ac in the control group. Cleavage rate and embryo development were evaluated in a second experiment, and the group treated with 5nM TSA showed no difference in embryo production compared to control. In Experiment 3, treatment with TSA had no significant effect on the cell number of ICM, but decreased (p <0.01) both the cell number of TE and total cells as compared to control. Therefore, it is concluded that treatment with TSA does not alter the preimplantation embryonic development female bovine embryos, showing a negative effect on the development of embryos treated

Made available in DSpace on 2014-06-11T19:29:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-02-13Bitstream added on 2014-06-13T20:59:33Z : No. of bitstreams: 1 goncalves_fs_me_jabo.pdf: 514303 bytes, checksum: 2ff947581f5831ab66e019d8c9ec7990 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Recentemente, tem-se dado atenção crescente aos efeitos deletérios dos radicais livres sobre os oócitos e embriões cultivados in vitro em mamíferos. No entanto, poucos estudos foram conduzidos sobre a ação desses agentes sobre a interação entre o espermatozóide-oócito durante a fertilização in vitro (FIV). Com o intuito de melhorar os resultados da produção in vitro de embriões bovinos, este trabalho teve por objetivo avaliar os efeitos da suplementação do meio de fecundação in vitro com antioxidantes cisteamina (Cist) e 2-Mercaptoetanol (2-ME) sobre a capacitação espermática, fecundação, qualidade dos zigotos e competência no desenvolvimento embrionário. Os espermatozóides apresentaram o DNA íntegro quase na totalidade entre os diferentes tratamentos e durante o tempo no processo da FIV. / Recently, deleterious effects of free radicals over oocytes and embryos cultivated in vitro in mammals have received increasing attention. Nonetheless, few studies were conducted about the action of those agents over the interaction between the sperm-oocyte during in vitro fertilization (IVF). Aimed at improving results of in vitro production of bovine embryos, this work had as objective to evaluate the effects of supplementation of the in vitro fertilization environment with antioxidants cisteamine (Cist) and 2-Mercaptoethanol (2-ME) over the spermatic capacitation, fertilization, quality of zygotes and competency in the embryonic development. Spermatozoas exhibited an integral DNA almost in the totality of the different treatments and along the time in the IVF process.

Orientador: Gisele Zoccal Mingoti / Banca: Joaquim Mansano Garcia / Banca: Rubens Paes de Arruda / Resumo: Recentemente, tem-se dado atenção crescente aos efeitos deletérios dos radicais livres sobre os oócitos e embriões cultivados in vitro em mamíferos. No entanto, poucos estudos foram conduzidos sobre a ação desses agentes sobre a interação entre o espermatozóide-oócito durante a fertilização in vitro (FIV). Com o intuito de melhorar os resultados da produção in vitro de embriões bovinos, este trabalho teve por objetivo avaliar os efeitos da suplementação do meio de fecundação in vitro com antioxidantes cisteamina (Cist) e 2-Mercaptoetanol (2-ME) sobre a capacitação espermática, fecundação, qualidade dos zigotos e competência no desenvolvimento embrionário. Os espermatozóides apresentaram o DNA íntegro quase na totalidade entre os diferentes tratamentos e durante o tempo no processo da FIV. / Abstract: Recently, deleterious effects of free radicals over oocytes and embryos cultivated in vitro in mammals have received increasing attention. Nonetheless, few studies were conducted about the action of those agents over the interaction between the sperm-oocyte during in vitro fertilization (IVF). Aimed at improving results of in vitro production of bovine embryos, this work had as objective to evaluate the effects of supplementation of the in vitro fertilization environment with antioxidants cisteamine (Cist) and 2-Mercaptoethanol (2-ME) over the spermatic capacitation, fertilization, quality of zygotes and competency in the embryonic development. Spermatozoas exhibited an integral DNA almost in the totality of the different treatments and along the time in the IVF process. / Mestre