Erlangen-Nürnberg, Univ., Diss., 2009.

University, Diss., 2001--Köln.

Orientador: Irani Quagio-Grassiotto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-26T00:26:51Z (GMT). No. of bitstreams: 1 Guimaraes_ArieneCristinaDias_M.pdf: 29601114 bytes, checksum: 7068d413d8a83c3eb916a37e2aa5119b (MD5) Previous issue date: 2000 / Resumo: A ultraestrutura do desenvolvimento oocitário em 5errasalmus spilopleura (Teleostei, Characiformes, Serrasalminae), foi estudada em gônadas de fêmeas sexualmente maduras, coletadas mensalmente de março de 1998 a fevereiro de 1999 no reservatório de Jurumirim, Alto do Rio Paranapanema, SP. Nos ovários de 5. spi!opleura, as oogônias são encontradas em ninhos, inseridos nas lamelas ovígeras. São células pequenas com núcleo grande e nucléolo único. Entram em oogênese, dando origem aos oócitos primários iniciais que se desenvolvem progressivamente em oócitos pré vitelogênicos, oócitos com alvéolos corticais em formação, oócitos em vitelogênese e oócitos em vitelogênese final. No núcleo dos oócitos primários iniciais se organizam os complexos sinaptonêmicos. Terminada a troca gênica, os oócitos diplotênicos são envolvidos por células pré foliculares, deixam os ninhos, e entram em crescimento primário. No interior dos folículos, os oócitos pré-vitelogênicos crescem em volume devido ao aumento progressivo das organelas citoplasmáticas. Neles surgem vesículas não elétron-densas na região citoplasmática cortical e tem início a formação da zona radiata. O envoltório folicular é constituído por células foliculares pavimentosas, uma fina lâmina basal e a teca, cujas células são alongadas. Entrando em crescimento secundário, os oócitos com alvéolos corticais em formação apresentam estruturas vesiculares, de conteúdo filamentoso, na região citoplasmática cortical, as quais fundem se entre si e com as vesículas não elétron-densas formadas na etapa anterior. Tais vesículas são precursoras dos alvéolos corticais. A zona radiata torna-se mais espessa. O envoltório folicular mantém o padrão descrito, porém, a lâmina basal se desenvolve. Nos oócitos em vitelogênese, além dos precursores dos alvéolos corticais, surgem grânulos de conteúdo elétron-denso, os quais fundem-se formando os grânulos de vitelo. Nestes, a zona radiata encontra-se totalmente formada e organiza se em três camadas de diferentes elétron-densidades. As células foliculares tornam-se menos alongadas e com espaços intercelulares dilatados, enquanto a teca e a lâmina basal mantêm o padrão anterior. Nos oócitos em vitelogênese final, os grânulos de vitelo espalham-se por todo o citoplasma e deslocam os alvéolos corticais para a periferia da célula. A zona radiata conserva a estrutura tripartida. As células foliculares apresentam-se cúbicas, as da teca permanecem alongadas e recobertas por um epitélio pavimentoso / Abstract: The ultrastructure of oocyte growth in the gonads of sexually mature Serrasalmus spilopleura (Teleostei, Characiformes, Serrasalminae) was studied. These were collected monthly from the Jurumirim reservoir, Alto Paranapanema River, SP, between March 1998 and February 1999. The oogonia in S. spilopleura are found in nests in the ovaries, inside the ovigerous lamellae. The oogonia are small cells with large nuclei and a single nucleolus. They enter oogenesis, giving rise to primary oocytes, which progressively evolve into pre-vitellogenic oocytes, oocytes with cortical alveoli formation, oocytes undergoing vitellogenesis, and oocytes in final vitellogenesis. Synaptonemal complexes are formed in the nucleus of early primary oocytes. At the end of crossing-over, the diplotenic oocytes are surrounded by pre-follicular cells, leave the nests, and begin primary growth. The pre-vitellogenic oocytes inside the follicles increase in volume due to the progressive increase of cytoplasmic organelles. In these oocytes, clear vesicles appear in the cortical cytoplasmic region, and the zona radiata begins to formo The follicular envelope consists of squamous follicular cells, a thin basal lamina, and the theca, which has elongated cells. The secondary growth corresponds to the period in which the oocytes with growing cortical alveoli show vesicular structures with a filamentous content in the cortical cytoplasmic region. These vesicular structures fuse with each other and with the clear vesicles. These vesicular structures are the precursors of the cortical alveoli. The zona radiata becomes thicker, the follicular envelops maintains its pattern, but the basal lamina develops. As well as the precursors of the cortical alveoli, electron-dense granules form in oocytes undergoing vitellogenesis. These fuse and form yolk granules. The zona radiata is completely formed and arranged in three layers of different electron-densities. The follicular cells become less elongated, with dilated intercellular spaces. The theca and basal lamina show the same pattern. In the oocytes undergoing final vitellogenesis, the yolk granules are scattered throughout the cytoplasm, displacing the growing cortical alveoli toward cell periphery. The zona radiata maintains its tripartite structure. The follicular cells are cubical; the theca cells are elongated and covered with a squamous epithelium / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Orientador: Irani Quagio-Grassiotto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T00:23:14Z (GMT). No. of bitstreams: 1 Guimaraes_ArieneCristinaDias_D.pdf: 8092294 bytes, checksum: 89d4c2955b40132b1d27eea816e79977 (MD5) Previous issue date: 2004 / Resumo: Durante o desenvolvimento oocitário o metabolismo celular é intenso. Várias vias biossintéticas interagem na formação dos componentes e estruturas celulares, destinadas a viabilizar a fertilização e o desenvolvimento inicial do embrião e da larva. Nos oócitos dos teleósteos aparecem e se desenvolvem com esse objetivo, o envelope oocitário, os alvéolos corticais, as inclusões lipídicas e os grânulos de vitelo. Estudos morfológicos, fisiológicos e bioquímicos apontam diferentes sítios e organelas celulares como participantes nesse processo. As modificações morfo-fisiológicas que têm início durante o crescimento primário e completam-se ao longo do crescimento secundário foram acompanhadas nos oócitos de Serrasalmus spilopleura mediante a utilização de técnicas citoquímicas (estruturais e ultra-estruturais). Durante o crescimento primário, as células foliculares mostram corpos densos sob ação do ósmioimidazol (Im), enquanto no envelope oocitário, a substância amorfa é positiva ao método do vermelho de rutênio (VR). No citoplasma desses oócitos, corpos positivos ao Im estão associados aos grupamentos mitocondriais e ao retículo endoplasmático, grande quantidade de ribossomos é evidenciada pelo EDTA ou método de Bernhard e o corpúsculo de Balbiani responde à técnica da prata amoniacal (AgA). Além disto, elementos do retículo endoplasmático, cisternas e vesículas do complexo de Golgi, lisossomos, corpos multivesiculares e algumas vesículas elétron-densas reagem positivamente à detecção de fosfatases ácidas (AcPase). Elementos do retículo endoplasmático, vesículas do complexo de Golgi e algumas vesículas elétron-densas também respondem positivamente à impregnação com tetróxido de ósmio e iodeto de zinco (ZIO). Elementos do retículo endoplasmático, algumas cisternas e vesículas do complexo de Golgi, algumas microvesículas dos corpos multivesiculares e outras vesículas respondem positivamente à impregnação pelo tetróxido de ósmio e iodeto de potássio (KI). Durante o crescimento secundário, no envelope oócitário a substância amorfa é positiva à AgA e as microvilosidades respondem ao Im e ao VR. No citoplasma dos oócitos, depósitos lipídicos esparsos respondem ao Im, os 2 alvéolos corticais são positivos ao VR, ao Im e à AgA, enquanto os grânulos de vitelo reagem à AgA. Ainda nos oócitos em crescimento secundário, elementos do retículo endoplasmático incluindo espaço intermembranoso do envoltório nuclear, alguns dictiossomos, lisossomos, grânulos de vitelo, regiões do envelope oocitário e sítios nas células foliculares respondem à AcPase. Alvéolos corticais, estruturas citoplasmáticas heterogêneas, regiões do envelope oocitário e sítios nas células foliculares respondem ao ZIO. Ainda, elementos do retículo endoplasmático, outras vesículas e sítios nas células foliculares respondem positivamente ao KI. As respostas às técnicas utilizadas são discutidas em relação à biologia dos oócitos / Abstract: During oocyte development metabolic activity is intense. Many biosynthetic pathways interact during the formation of the cell structrural components, destined to promote the fertilization and the initial embryo and larval development. Therefore, the egg envelope, the cortical alveoli, lipidic inclusions and yolk granules appear and develop in the Teleostei oocytes. Morphological, physiological and biochemical studies show that different cell sites and organelles are at work in these processes. The morphophysiological changes that occur during oocyte primary and secondary growths in Serrasalmus spilopleura were studied under cytochemistry techniques (ultrastructural and structural). During oocyte primary growth, follicular cells show dense bodies when submitted to osmium-imidazole (Im), while in the egg envelope, the amorphous material react to ruthenium red technique (VR). In the oocyte cytoplasm, dense bodies, positive to Im, are associated to mitochondrial clusters and to endoplasmic reticulum, submitted to EDTA or Bernhard method large amounts of ribosomes are evident, and the Balbiani body reacts to amoniacal silver technique (AgA). Also in the previtellogenic oocytes, endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react positively to AcPase detection. These endoplasmic reticulum components, Golgi complex cisternae and vesicles also react positively to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are already strongly contrasted in response to osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are stained with ZIO, while microvesicles of multivesicular bodies and other large vesicles, which are isolated in the cytoplasm, are contrasted by KI. During oocyte secondary growth, in the egg envelope, the amorphous material react to AgA and the microvilli react positively to Im and to VR. In the oocyte cytoplasm, scattered lipidic depositions react to Im, the cortical alveoli are positive to VR, to Im and to AgA, while the yolk granules react to AgA. Endoplasmic reticulum components including the nuclear envelope intermembrane space, some dictiosomes, lysosomes, yolk granules, egg envelope regions and follicular cell sites are reactive to AcPase. The cortical alveoli, heterogeneous cytoplasmic structures, egg envelope regions and follicular cell sites are reactive to ZIO. Still, endoplasmic reticulum components, other vesicles and follicular cell sites react to KI. The reactions to the applied techniques are discussed in relation to the oocytes biology / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

The evolutionary success of insects is believed to be at least partially facilitated by symbioses between insects and prokaryotes. Bacterial endosymbionts confer various fitness advantages to their hosts, for example by providing nutrients lacking from the insects’ diet thereby enabling the inhabitation of new ecological niches. The Florida carpenter ant Camponotus floridanus harbours endosymbiotic bacteria of the genus Blochmannia. These primary endosymbionts mainly reside in the cytoplasm of bacteriocytes, specialised cells interspersed into the midgut tissue, but they were also found in oocytes which allows their vertical transmission. The social lifestyle of C. floridanus may facilitate the rapid spread of infections amongst genetically closely related animals living in huge colonies. Therefore, the ants require an immune system to efficiently combat infections while maintaining a “chronic” infection with their endosymbionts. In order to investigate the immune repertoire of the ants, the Illumina sequencing method was used. The previously published genome sequence of C. floridanus was functionally re-annotated and 0.53% of C. floridanus proteins were assigned to the gene ontology (GO) term subcategory “immune system process”. Based on homology analyses, genes encoding 510 proteins with possible immune function were identified. These genes are involved in microbial recognition and immune signalling pathways but also in cellular defence mechanisms, such as phagocytosis and melanisation. The components of the major signalling pathways appear to be highly conserved and the analysis revealed an overall broad immune repertoire of the ants though the number of identified genes encoding pattern recognition receptors (PRRs) and antimicrobial peptides (AMPs) is comparatively low. Besides three genes coding for homologs of thioester-containing proteins (TEPs), which have been shown to act as opsonins promoting phagocytosis in other insects, six genes encoding the AMPs defesin-1 and defensin-2, hymenoptaecin, two tachystatin-like peptides and one crustin-like peptide are present in the ant genome. Although the low number of known AMPs in comparison to 13 AMPs in the honey bee Apis mellifera and 46 AMPs in the wasp Nasonia vitripennis may indicate a less potent immune system, measures summarised as external or social immunity may enhance the immune repertoire of C. floridanus, as it was discussed for other social insects. Also, the hymenoptaecin multipeptide precursor protein may be processed to yield seven possibly bioactive peptides. In this work, two hymenoptaecin derived peptides were heterologously expressed and purified. The preliminary antimicrobial activity assays indicate varying bacteriostatic effects of different hymenoptaecin derived peptides against Escherichia coli D31 and Staphylococcus aureus which suggests a functional amplification of the immune response further increasing the antimicrobial potency of the ants. Furthermore, 257 genes were differentially expressed upon immune challenge of C. floridanus and most of the immune genes showing differential expression are involved in recognition of microbes or encode immune effectors rather than signalling components. Additionally, genes coding for proteins involved in storage and metabolism were downregulated upon immune challenge suggesting a trade-off between two energy-intensive processes in order to enhance effectiveness of the immune response. The analysis of gene expression via qRT-PCR was used for validation of the transcriptome data and revealed stage-specific immune gene regulation. Though the same tendencies of regulation were observed in larvae and adults, expression of several immune-related genes was generally more strongly induced in larvae. Immune gene expression levels depending on the developmental stage of C. floridanus are in agreement with observations in other insects and might suggest that animals from different stages revert to individual combinations of external and internal immunity upon infection. The haemolymph proteome of immune-challenged ants further established the immune-relevance of several proteins involved in classical immune signalling pathways, e.g. PRRs, extracellularly active proteases of the Toll signalling pathway and effector molecules such as AMPs, lysozymes and TEPs. Additionally, non-canonical proteins with putative immune function were enriched in immune-challenged haemolymph, e.g. Vitellogenins, NPC2-like proteins and Hemocytin. As known from previous studies, septic wounding also leads to the upregulation of genes involved in stress responses. In the haemolymph, proteins implicated in protein stabilisation and in the protection against oxidative stress and insecticides were enriched upon immune challenge. In order to identify additional putative immune effectors, haemolymph peptide samples from immune-challenged larvae and adults were analysed. The analysis in this work focussed on the identification of putative peptides produced via the secretory pathway as previously described for neuropeptides of C. floridanus. 567 regulated peptides derived from 39 proteins were identified in the larval haemolymph, whereas 342 regulated peptides derived from 13 proteins were found in the adult haemolymph. Most of the peptides are derived from hymenoptaecin or from putative uncharacterised proteins. One haemolymph peptide of immune-challenged larvae comprises the complete amino acid sequence of a predicted peptide derived from a Vitellogenin. Though the identified peptide lacks similarities to any known immune-related peptide, it is a suitable candidate for further functional analysis. To establish a stable infection with the endosymbionts, the bacteria have to be transmitted to the next generation of the ants. The vertical transmission of B. floridanus is guaranteed by bacterial infestation of oocytes. This work presents the first comprehensive and detailed description of the localisation of the bacterial endosymbionts in C. floridanus ovaries during oogenesis. Whereas the most apical part of the germarium, which contains the germ-line stem cells, is not infected by the bacteria, small somatic cells in the outer layers of each ovariole were found to be infected in the lower germarium. Only with the beginning of cystocyte differentiation, endosymbionts are exclusively transported from follicle cells into the growing oocytes, while nurse cells were never infected with B. floridanus. This infestation of the oocytes by bacteria very likely involves exocytosis-endocytosis processes between follicle cells and the oocytes. A previous study suggested a down-modulation of the immune response in the midgut tissue which may promote endosymbiont tolerance. Therefore, the expression of several potentially relevant immune genes was analysed in the ovarial tissue by qRT-PCR. The relatively low expression of genes involved in Toll and IMD signalling, and the high expression of genes encoding negative immune regulators, such as PGRP-LB, PGRP-SC2, and tollip, strongly suggest that a down-modulation of the immune response may also facilitate endosymbiont tolerance in the ovaries and thereby contribute to their vertical transmission. Overall, the present thesis improves the knowledge about the immune repertoire of C. floridanus and provides new candidates for further functional analyses. Moreover, the involvement of the host immune system in maintaining a “chronic” infection with symbiotic bacteria was confirmed and extended to the ovaries. / Der evolutionäre Erfolg von Insekten wird zumindest teilweise Symbiosen zwischen Insekten und Prokaryonten zugeschrieben. Dabei übertragen bakterielle Symbionten verschiedenste Fitnessvorteile an ihre Wirte. Beispielsweise ermöglicht die Bereitstellung von Nährstoffen, welche in der Nahrung des Insekts fehlen, die Erschließung neuer ökologischer Nischen. Die Florida Rossameise Camponotus floridanus trägt endosymbiontische Bakterien der Gattung Blochmannia. Diese primären Endosymbionten kommen hauptsächlich im Zytoplasma von spezialisierten Zellen des Mitteldarms, den sogenannten Bakteriozyten, vor. Blochmannien wurden aber auch in Oozyten und Eiern gefunden, was ihre vertikale Übertragung an Individuen der nächsten Generation ermöglicht. Als soziale Insekten leben C. floridanus in großen Kolonien von nah verwandten Individuen. Ihre Lebensweise begünstigt möglicherweise die schnelle Ausbreitung von Infektionen, weshalb erwartet werden müsste, dass die Ameisen ein effizientes Immunsystem besitzen. Gleichzeitig muss jedoch die „chronische“ Infektion mit den bakteriellen Symbionten aufrechterhalten werden. In der vorliegenden Arbeit wurde das Immunrepertoire der Ameisen mittels Illumina Sequenzierung charakterisiert. Zunächst wurde das vor kurzem publizierte Genom von C. floridanus funktionell re-annotiert. Dabei wurden 0.53% der annotierten Proteine der GO-Unterkategorie “Prozesse des Immunsystems” zugeordnet. Basierend auf Homologieanalysen wurden Gene identifiziert, die für 510 Immunproteine kodieren. Die Genprodukte spielen eine Rolle bei der Erkennung von Mikroben und in den Signalwegen des Immunsystems, sind jedoch auch an Prozessen der zellulären Immunantwort, wie beispielsweise Phagozytose und Melanisierung, beteiligt. Dabei sind Komponenten der Hauptsignalwege hoch konserviert. Obwohl die Anzahl der identifizierten Proteine, die Fremdorganismen erkennen (PRRs), und die Anzahl an antimikrobiellen Peptiden (AMPs) vergleichsweise gering ist, verfügt C. floridanus insgesamt über ein umfangreiches Immunrepertoire. Neben drei Genen, die für Thioester-enthaltende Proteine (TEPs) kodieren und wie in anderen Insekten möglicherweise eine Rolle als Opsonine bei der Phagozytose spielen, wurden sechs AMP-Gene identifiziert. Diese kodieren für Defesin-1 und Defensin-2, Hymenoptaecin, zwei Tachystatin-ähnliche und ein Crustin-ähnliches Peptid. Die geringe Anzahl an bekannten AMPs im Vergleich zur Honigbiene Apis mellifera (13 AMPs) und Wespe Nasonia vitripennis (46 AMPs) könnte ein möglicherweise geringeres Potential des Immunsystems anzeigen. Allerdings könnten zusätzliche Maßnahmen, die unter dem Begriff „Soziale Immunität“ zusammengefasst werden, das Immunrepertoire von C. floridanus ergänzen, wie es schon für andere Insekten diskutiert wurde. Zudem könnten durch proteolytische Prozessierung des Hymenoptaecin Multipeptid Präkursormoleküls sieben mögliche antimikrobielle Peptide freigesetzt werden. Für die vorliegende Arbeit wurden zwei verschiedene dieser Hymenoptaecin Peptide heterolog exprimiert und aufgereinigt. Die vorläufige funktionelle Charakterisierung der Peptide zeigt, dass diese Peptide möglicherweise bakteriostatische Wirkung mit einem unterschiedlichen Wirkspektrum gegen Escherichia coli D31 und Staphylococcus aureus entfalten. Dies erlaubt die Annahme, dass die Expression des Hymenoptaecins zu einer funktionellen Amplifikation der Immunantwort führt und das Immunrepertoire der Ameisen erweitert. Nach Injektion von bakteriellem Material in die Ameisen wurde die Expression von 257 Genen reguliert. Viele dieser Gene kodieren für Proteine zur Erkennung von Pathogenen oder kodieren für Effektoren des Immunsystems. Komponenten der Signalwege zeigten dagegen kaum Veränderungen in ihrer Expression auf. Außerdem zeigten Gene, die für Speicherproteine oder Proteine des Metabolismus kodieren, generell eine geringere Expression nach Stimulierung des Immunsystems auf. Dies lässt einen Ausgleich zwischen zwei energieintensiven Prozessen vermuten, um eine effektive Immunantwort zu ermöglichen. Darüber hinaus zeigt die Validierung der Expressionsdaten mittels qRT-PCR eine Abhängigkeit der Expression mehrerer Gene vom Entwicklungsstadium der Ameisen auf. Generell wurden die gleichen Tendenzen in der Regulation der Expression dieser Gene nach Immunstimulierung beobachtet. Allerdings wurde die Expression mehrerer immunrelevanter Gene in Larven weit stärker induziert als in Adulten. Wie es auch schon für andere Insekten gezeigt wurde, scheinen C. floridanus Larven und Arbeiterinnen auf individuelle Kombinationen externer und interner Immunfaktoren zurückzugreifen. Die vorher beschriebenen Transkriptomdaten wurden durch die Charakterisierung des Hämolymph-Proteoms von C. floridanus nach Immunstimulation ergänzt, wodurch die Immunrelevanz vieler Faktoren auch auf Proteinebene bestätigt werden konnte. Beispielsweise wurden zahlreiche PRRs und extrazellulär aktive Proteasen des Toll-Signalwegs, aber auch Immuneffektoren wie AMPs, Lysozyme und TEPs in der Hämolymphe identifiziert. Zusätzlich führte die Immunstimulation in Larven und Adulten zur Anreicherung nicht-kanonischer Proteine mit möglicher Immunfunktion, beispielsweise Vitellogenine, NPC2-ähnliche Proteine und Hemocytin. Aus einer früheren Arbeit ist bekannt, dass septische Verwundungen zusätzlich die transkriptionelle Aktivierung von Genen der Stressantwort hervorrufen können. So wurden auch in der Hämolymphe Proteine entdeckt, die eine Rolle bei der Stabilisierung von Proteinen, und dem Schutz gegen oxidativen Stress und Insektizide spielen. Zur Identifizierung weiterer möglicher Peptideffektoren wurden Hämolymphpeptid-Proben von immunstimulierten Larven und Adulten analysiert. Der Fokus der Analyse lag dabei auf der Identifizierung von Peptiden, die auf dem sekretorischen Weg gebildet werden, wie es zuvor für Neuropeptide von C. floridanus beschrieben worden war. 567 differentiell regulierte Peptide, die von 39 Proteinen abstammen, wurden in Larvenhämolymphe identifiziert, wohingegen in der Hämolymphe von Adulttieren 342 derartige Peptide, die 13 Proteinen zugeordnet werden können, gefunden wurden. Die meisten dieser Peptide können Hymenoptaecin oder bisher noch nicht charakterisierte Proteinen zugeordnet werden. Jedoch wurde ein Peptid in larvaler Hämolymphe identifiziert, dessen Aminosäuresequenz vollständig mit der Sequenz eines vorhergesagten, von Vitellogenin stammenden Peptids übereinstimmt. Weil dieses Peptid keine Ähnlichkeiten zu anderen bereits charakterisierten antimikrobiellen Peptiden aufweist, stellt es einen geeigneten Kandidaten für weitere funktionelle Analysen dar. Die bakterielle Infektion von Oozyten ermöglicht die transovariale Übertragung von B. floridanus und ermöglicht damit die Etablierung einer stabilen Infektion in der nächsten Wirtsgeneration. Die vorliegende Arbeit beinhaltet die erste umfassende und detaillierte Beschreibung der Lokalisation bakterieller Endosymbionten in Ovarien von C. floridanus. Im apikalen Germarium, in welchem sich die Keimbahn-Stammzellen befinden, liegt noch keine bakterielle Infektion des Gewebes vor. In späteren Segmenten des Germariums jedoch können Blochmannien das erste Mal in kleinen somatischen Zellen der äußeren Schicht jeder Ovariole detektiert werden. Mit beginnender Zystozytendifferenzierung werden die Endosymbionten von Follikelzellen ausschließlich in die heranwachsenden Oozyten transportiert, wobei sehr wahrscheinlich Exozytose-Endozytose-Prozesse involviert sind. Nährzellen zeigen zu keinem Zeitpunkt während der Oogenese eine bakterielle Infektion auf. Da in einer früheren Studie vorgeschlagen wurde, dass eine signifikant reduzierte Anregung der Immunantwort im Mitteldarmgewebe zur Toleranz der Endosymbionten beitragen könnte, wurde auch die Expression ausgewählter Immungene in den Ovarien durch qRT-PCR untersucht. Die relativ geringe Expression von Genen des Toll- und des IMD-Signalwegs und die zusätzlich vergleichsweise starke Genexpression negativer Regulatoren des Immunsystems, wie PGRP-LB, PGRP-SC2 und tollip, sind Indikatoren einer reduzierten Immunantwort in den Ovarien von C. floridanus. Wie schon für den Mitteldarm der Tiere vorgeschlagen, könnte dies möglicherweise sowohl zur Toleranz von Blochmannia als auch zur vertikalen Übertragung der Endosymbionten beitragen. Die vorliegende Doktorarbeit erweitert das Wissen über das Immunrepertoire von C. floridanus und es konnten vielversprechende Kandidaten für weitere funktionelle Analysen von möglichen Immunfaktoren identifiziert werden. Darüber hinaus konnten weitere Hinweise auf die Bedeutung von Immunfaktoren der Ameisen bei der Toleranz gegenüber den symbiontischen Bakterien gefunden und auf die Ovarien der Tiere ausgeweitet werden.

Made available in DSpace on 2014-06-11T19:25:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-03Bitstream added on 2014-06-13T20:32:58Z : No. of bitstreams: 1 lima_rs_me_botib.pdf: 532125 bytes, checksum: 43ee357752dc07c85fbf032817d7466d (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O estresse térmico materno compromete a fertilidade de vacas leiteiras. Os oócitos nas fases de vesícula germinativa (VG) e maturação são susceptíveis aos efeitos deletérios causados pelo estresse térmico, entretanto os mecanismos celulares desencadeados pela temperatura elevada são pouco conhecidos. Os danos celulares induzidos pelo estresse térmico podem ser manipulados por amplo espectro de fatores biológicos, incluindo o fator de crescimento semelhante à insulina- I (IGF-I). Dessa forma, os objetivos gerais desta proposta foram caracterizar as alterações celulares e de desenvolvimento induzidas pela temperatura elevada em oócitos na fase de VG e avaliar o papel termoprotetor do IGF-I neste contexto. Para tanto, os experimentos 1 e 2 visaram estabelecer o modelo de bloqueio meiótico in vitro. No primeiro experimento foram avaliadas concentrações crescentes (50; 75; 100; 150 e 200 μM) do bloqueador meiótico roscovitina. A porcentagem de oócitos em VG (taxa de bloqueio meiótico) foi baixa em todas as doses de roscovitina testadas. No segundo experimento foram avaliadas diferentes concentrações de butirolactona (0; 12,5; 25; 50 e 100 μM) em meio de inibição meiótica contendo 0 ou 3 mg/ml de albumina sérica bovina (BSA) na porcentagem de oócitos em VG e na porcentagem de oócitos em metáfase II (taxa de reversão do bloqueio meiótico após a maturação in vitro). A eficiência do bloqueio meiótico foi alta para todas as doses de butirolactona avaliadas, exceto 12,5 μM de butirolactona com BSA. De maneira similar, a porcentagem de oócitos em MII foi alta para todos os tratamentos, exceto para dose de 100 μM de butirolactona sem BSA, indicando possível efeito tóxico desta concentração. Com base nestes resultados o modelo de inibição meiótica utilizado para todos os demais experimentos fez uso... / Maternal heat stress compromises fertility of lactating dairy cows. Germinal vesicle stage (GV) and mature oocytes are susceptible to deleterious effects of maternal heat stress. However, the cellular mechanisms triggered by elevated temperature are not well known. The cellular damage induced by heat stress can be manipulated by a wide range of biological factors, such as insulin-like growth factor-I (IGF-I). Therefore, overall objectives of this proposal were to characterize cellular and developmental changes induced by elevated temperature on GV oocytes and evaluate the role of IGF-I in this context. Experiments 1 and 2 aimed to establish an in vitro meiotic arrest model. The first study evaluated the effect of increasing concentrations (50, 75, 100, 150 and 200 μM) of the meiotic inhibitor roscovitine. The percentage of GV oocytes (rate of meiotic arrest) was low at all roscovitine doses tested. In the second experiment different butyrolactoneconcentrations (0, 12.5, 25, 50 and 100 uM) were evaluated in meiotic inhibition medium containing 0 or 3 mg/mL bovine serum albumin (BSA) in percentage of GV oocytes and percentage of metaphase II oocytes (rate of meiotic arrest reversibly after in vitro maturation). Meiotic arrest efficiency was high for all butyrolactone doses tested, except 12.5 μM butyrolactone with BSA. Similarly, the percentage of MII oocytes was high for all doses, except for 100 μM butyrolactone without BSA, indicating a possible toxic effect of this concentration. Based on these results the meiotic arrest model used for all subsequent experiments used 12.5 μM butyrolactone in the absence of BSA. The third experiment evaluated the effect of various IGF-I concentrations (0, 12.5, 25, 50 and 100 ng/ml) on heat-induced DNA fragmentation on GV oocyte. In the absence of IGF-I, heat shock of 41°C for 14 hours increased... (Complete abstract click electronic access below)

Made available in DSpace on 2014-11-10T11:09:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-21Bitstream added on 2014-11-10T11:58:44Z : No. of bitstreams: 1 000794186.pdf: 3889062 bytes, checksum: 2cc9d4ef4d96b18c583d8f9cc3727adf (MD5) / Laetacara araguaiae é uma espécie de ciclídeo sul-americano descrito na bacia do rio Araguaia, São Miguel do Araguaia, Goiás, Brasil por Ottoni e Costa (2009). A descrição das fases reprodutivas vem sendo realizada há algum tempo com a análise do ciclo reprodutivo anual, que se baseia nas fases de maturação gonadal de espécies analisadas. O objetivo do trabalho foi descrever morfologicamente o ciclo reprodutivo de fêmeas de L. araguaiae. Foram coletadas 80 fêmeas de L. araguaiae, no período abril/2012 a março/2013, no rio Dois Córregos, Selvíria, Mato Grosso do Sul, Brasil. Os ovários foram processados, utilizando-se técnicas para microscopia de luz e microscopia eletrônica de transmissão. Ainda foram identificadas as fases do ciclo reprodutivo que se aplicam as fêmeas, índice gonadossomático (IGS) e tamanho da primeira maturação ovariana. A oogênese apresentou epitélio germinativo com três tipos de oogônias e oócitos em fase de prófase da meiose para a formação dos folículos ovarianos. Crescimento primário com a presença de oócitos pré-vitelogênicos e oócitos alvéolos corticais e crescimento secundário ou vitelogênese, caracterizado pela deposição inicial de grânulos de vitelo. O evento, que caracteriza o estágio de maturação é a migração do núcleo para a periferia do oócito em direção à micrópila. O complexo folicular sofreu modificações morfológicas ao longo dos estágios dos oócitos. Alem disso, para Laetacara araguaiae foram definidas cinco fases reprodutivas ao longo do ano: imaturo, desenvolvimento, apto a desova, regressão e regeneração. O comprimento médio da primeira maturação ovariana foi estimado em 2,0 cm de comprimento padrão. Os indivíduos maduros (aptos à desova) foram capturados a partir de setembro que coincidem com as estações de primavera e verão. O IGS apresentou valores médios elevados na fase de apto a desova (4,528) e os menores valores médios ... / Laetacara araguaiae is a species of south American ciclideo described in the Araguaia river basin, São Miguel do Araguaia, Goias, Brazil by Ottoni and Costa (2009). The description of reproductive phases is being performed some time ago with the analysis of the annual reproductive cycle, that is based on stages of gonadal maturation of analyzed species. The objective of this work was to describe morphologically the reproductive cycle of female L. araguaiae. Were collected from 80 females of L. araguaiae, in the period April/2012 to March/2013, in Two Streams, river Selviria, Mato Grosso do Sul, Brazil. The ovaries were processed, using techniques for light microscopy and transmission electron microscopy. Even were identified phases of the reproductive cycle that apply to females, gonadossomatico index (GSI) and size of first ovarian maturation. The oogenesis presented germinative epithelium with three types of oogonias and oocytes in phase of profase of meiosis for the formation of the ovarian follicles. Primary Growth with the presence of oocytes pre-vitelogenicos and oocytes cortical alveoli and secondary growth or vitelogenese, characterized by initial deposition of granules of yolk. The event which characterizes the maturation stage is the migration from the nucleus to the periphery of the oocyte toward through. The follicular suffered complex morphological changes along the stages of oocytes. In addition, for Laetacara araguaiae were defined five reproductive phases throughout the year: immature, development, able to spawning, regression and regeneration. The average length of first ovarian maturation was estimated at 2.0 cm standard length. The mature individuals (able to spawn) were captured from September to coincide with the seasons of spring and summer. The IGS showed high average values in phase of able to spawn (4,528) and the lowest average values in immature (0.502). The seasons of the year in which the individual is able ...

During animal development precise coordination and regulation of cell proliferation, differentiation and cell death is required for proper tissue organization. This is achieved through specific cell communication by intercellular signals. Cell death, for example, is a mechanism utilized by multicellular organisms for several developmental processes such as elimination of damaged cells or morphological shaping. Apoptosis can be induced by intrinsic signals generated within the cells or from extrinsic signals received from the surrounding environment. This work centered on the analysis of the mechanisms and signals that trigger apoptosis during Drosophila oogenesis. Drosophila ovaries are composed of approximately 16-20 ovarioles, each of which contains a series of egg chambers that are proceeding through the 14 stages of oogenesis moving from the germarium toward the oviduct. For the mature egg to be formed cell death has to occur at specific stages both in the germline (nurse cells) and in the somatic cells (follicle cells). However, whether this apoptosis is caused by intrinsic or extrinsic signals is not known. In addition to this developmentally controlled cell death, apoptosis can also be induced by environmental cues. Under starvation, for example, there is an increase of apoptosis at particular stages: in the germarium and at mid-oogenesis. Mid-oogenesis (stage8/9) is when vitellogenesis starts. At these stages the state of the egg chambers are checked in order to eliminate, through apoptosis of the germline, defective egg chambers. In starved flies, through activation of this “check-point”, oogenesis is blocked before vitellogeneis starts. It is not clear what are the signals that prevent apoptosis at mid-oogenesis in well-fed flies. In this study I have analyzed the function of a newly identified signaling molecule CG7447 during Drosophila oogenesis. My results indicate that CG7447 is required to prevent apoptosis at mid-oogenesis in well-fed flies. CG7447 RNA is only detectable in cells of the germarium, but not at later stages of oogenesis. When an HA tagged version of CG7447 (CG7447-HA) was expressed in the follicle cells of the germarium, this protein was found to be enriched in the oocyte during stages 2-8. These data suggest that CG7447 might be a secreted protein produced in the germarium, which is then secreted into the oocyte. To test the function of CG7447 during oogenesis, I generated a mutant allele. The mutation in CG7447 reduced female fertility. Mutant ovaries showed a block of egg chamber development at mid-oogenesis and this block correlated with apoptosis of the follicle cells. These mutant phenotypes could be reversed by expression of CG7447-HA, showing that these defects are due to the mutation in CG7447. Surprisingly, expression of CG7447-HA in the follicle cells only from stage 9 onward could restore fertility and normal oogenesis in a CG7447 mutant, indicating that CG7447 is required for follicle cell survival at later stages. Proper nutritional conditions are required to prevent apoptosis in the germline. Our data suggest that CG7447 is instead required to prevent apoptosis in the follicle cells. Thus, our analysis appears to have identified a novel signaling pathway that prevents survival of follicle cells in well-fed flies. Finally, our bioinformatic analysis showed that CG7447 is homologous to vertebrate EGF-like domain 7 (Egfl7) and EGF-like domain 8 (Egfl8) proteins. Importantly, expression of mouse Egfl7 or Egfl8 were able to confer normal oogenesis and fertility to CG7447 mutant flies. We therefore conclude that CG7447 is an evolutionary conserved protein and that CG7447 and Egfl7/8 share a common molecular function. CG7447 is a newly identified signaling molecule required during Drosophila oogenesis to promote the survival of follicle cells and to allow entry into vitellogenesis. Identification of the signaling cascade triggered by CG7447 will be important to more precisely understand its function during oogenesis. It may also help to reveal the molecular role of Egfl7/8 during vertebrate development.

Made available in DSpace on 2014-06-11T19:25:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-02Bitstream added on 2014-06-13T19:12:07Z : No. of bitstreams: 1 pinto_rfp_me_botib.pdf: 235047 bytes, checksum: f66f9dd3e29764cee35ce259fd850b2c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O fator de crescimento fibroblástico (FGF10) atua de forma parácrina no complexo cumulus-oócito, aumentando a expressão de genes relacionados à expansão das células do cumulus e à competência oócitária. Objetivou-se com o presente estudo testar se a adição do FGF10 ao meio de maturação melhora a maturação, diminui as taxas de oócitos apoptóticos e influencia na produção de embriões in vitro e na expressão de genes relacionados à competência e implantação embrionárias (COX2, CDX2 e PLAC8). Em todos os experimentos, oócitos foram obtidos de abatedouro, coletados de vacas adultas e maturados por 22 h em meio TCM 199 suplementado com: 2.5 ng/mL FGF10, 10 ng/mL FGF10, 50 ng/mL FGF10 ou na ausência de FGF10 (grupo controle). No Experimento 1, depois da maturação, os oócitos foram fixados e corados com TUNEL para determinar a porcentagem de oócitos apoptóticos e posteriormente com Hoechst-33342 para avaliação dos diferentes estágios da meiose (metáfase 1, metáfase 2 ou metáfase 2 com extrusão do primeiro corpúsculo polar). No Experimento 2, os oócitos foram fertilizados e cultivados até o estágio de blastocisto inicial. No experimento 3 avaliou-se o efeito da suplementação do FGF10 ao meio de maturação in vitro sobre a expressão dos genes COX2, CDX2 e PLAC8 em embriões bovinos. No experimento 1, os resultados demonstraram que a dose de 2,5 ng/mL FGF10 aumentou a porcentagem de oócitos com extrusão do primeiro corpúsculo polar (36%) quando comparado as doses de 10 ng/mL FGF10 (13%), 50 ng/mL FGF10 (12%) e grupo controle (19%; p≤0.05). Com relação ao número de oócitos apoptóticos, as maiores doses de FGF10 apresentaram menor quantidade de oócitos TUNEL-positivos (10 ng/mL and 50 ng/mL FGF10, 5% e 6%, respectivamente) quando comparados a menor... / Not available

Made available in DSpace on 2014-06-11T19:35:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-12Bitstream added on 2014-06-13T19:05:26Z : No. of bitstreams: 1 blanco_idp_dr_botfmvz.pdf: 464375 bytes, checksum: 98e141ce5fa2b921f8f4ddf393292d45 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As alterações observadas nos folículos pré-ovulatórios dos mamíferos a partir da onda pré-ovulatória de LH são mediadas por fatores de crescimento como AR e ER, que induzem a expansão das células do cumulus e maturação oocitária. O LH também estimula a deposição de ácido hialurônico mediada pela enzima HAS-2. Em eqüinos, já foi caracterizada a expressão de HAS-2, AR e ER em resposta à administração de hCG em folículos pré-ovulatórios, mas sua ação em folículos imaturos não está esclarecida, embora tenha sido demonstrada a expansão precoce do cumulus após sua administração em folículos menores de 30mm. O objetivo deste estudo foi caracterizar o padrão de expressão de ER, AR e HAS-2 em folículos de eqüinos em diferentes estágios de maturação, submetidos ou não ao tratamento com EPE e hCG. Para isso, recuperamos células foliculares por OPU de folículos de diversos tamanhos, em 3 ou 24 horas após estímulo gonadotrófico exógeno, e avaliamos a expressão gênica de ER, AR e HAS-2 por PCR em tempo real. O tratamento com hCG induziu a expressão de ER em folículos imaturos e pré-ovulatórios, demonstrando sua capacidade de indução da maturação folicular precoce, e houve tendência do mesmo efeito ser obtido com a expressão de HAS-2, embora não tenha sido estatisticamente comprovado. O EPE apresentou efeito menor que a hCG na indução da expressão de ER, o que pode ser justificado pelo mecanismo de down regulation que o LH exerce sobre a ativação dos fatores de crescimento ligados ao EGF, como a ER e a AR / The modifications observed on mammalia n follicles after the pré-ovulatory surge of LH are mediated by growth factors like epiregulin (ER), which is responsible for the cumulus cells expansion and interfere with the oocyte maturation . The LH also stimulates the deposition of hyaluronic acid mediated by the hialuronan synthase -2 (HAS-2). In horses , the expression of HAS-2 and ER was already characterized in pre-ovulatory follicles , after the administration of hCG. In the present experiment we were able to demonstrate that the hCG is also able to induce the expression of ER in equine follicles smaller than 30mm in diameter , confirming an ability of immature follicles in responding to hormonal stimuli. Although no statistic differences were observed among groups a tendency to the same effect was also observed for the HAS-2. Similarly, the use of EPE stimulated the expression of ER, but when EPE and hCG were combined a decrease in expression was observed. This observation indicates a mechanism of down regulation exerted by the LH on the ER and HAS expression