Thesis advisor: David R. Burgess / Centration of the nucleus after fertilization is an essential step for setting-up cell division and proper embryonic development in many proliferating cells such as the sea urchin. The sperm aster must capture the female pronucleus for fusion as well as the nucleus becoming positioned at the center of the cell. Microtubules (MTs) are known to play a role in this centration but the exact mechanism remains unknown. This begins to investigate current models of nuclear centration and the role of various interactions. Three phases of migration were observed as the male aster migrated with support in independent movements of the male and female pronuclei. Dimpling affects present that altered the morphology of the cell were observed when engagement occurred between the male and female pronuclei. It was discovered that this dimpling effect was a result of an interaction between MTs and the cortex, as confirmed by visualization of sheared cells in which only the cortex remained. Stemming from previous and current research in the lab, the role of post-translational modifications (PMTs) in nuclear centration was investigated for the different forces exerted due to various factors. Tyrosinated and detyrosinated populations were observed with and without the presence of parthenolide (PTL), an agent that inhibits detyrosination. PTL was observed to not only prevent the proper migration, but also that it expanded tyrosination of tubulin – which would further disrupt the force vectors created through the PMTs promotion of dyneins and kinesins. The results have lead to a new hypothesis to be furthered in order to gain an in-depth understanding in the mechanism(s) for pronuclear migration. / Thesis (BS) — Boston College, 2018. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Made available in DSpace on 2014-06-11T19:35:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-12-19Bitstream added on 2014-06-13T19:24:36Z : No. of bitstreams: 1 niciura_scm_dr_jabo.pdf: 1029270 bytes, checksum: 8a93cc4207fdb3426be68df4fa064ef9 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A maturação oocitária é marcada pela retomada da primeira divisão da meiose, com progressão do estádio de Vesícula Germinativa (GV) da Prófase I até a Metáfase II (MII), e inclui todos os eventos necessários para que o oócito expresse seu potencial máximo de desenvolvimento após a fecundação. Para avaliarmos a eficiência da maturação in vitro (MIV), utilizamos oócitos classificados em viáveis (graus I, II e III) e inviáveis (atrésico e desnudo), e acompanhamos a progressão nuclear e a distribuição dos grânulos corticais (GC) como indício de maturação citoplasmática, após MIV em TCM 199 com soro fetal bovino, hormônios, antibiótico e piruvato, por 24h em 5% de CO2 em ar. Maturação nuclear (78,4-87,8%) e citoplasmática (GC periféricos; 67,2-79,3%) foram semelhantes entre as diferentes classes de oócitos e apresentaramse como eventos independentes. Para o acompanhamento dos eventos desencadeados pelo espermatozóide, avaliamos a dinâmica nuclear e de microtúbulos, em intervalos de 2h, após fecundação in vitro (FIV), em meio TALP com heparina, PHE e sêmen preparado em gradiente de Percoll. Observamos que o estádio de MII foi predominante de 2 a 8h; MII e Anáfase/Telófase (A/T) predominaram às 10h; MII, A/T e estádio pronuclear (PN) de 14 a 16h; e PN a partir de 18h. A penetração do espermatozóide ocorreu após 4h da inseminação dos oócitos; a diferenciação dos PN 14 masculino e feminino pelo tamanho foi possível de 14 a 18h e a singamia ocorreu a partir de 24h. O período de 10h pode ser suficiente para que a FIV seja efetiva em oócitos bovinos, nas condições aqui descritas. / We aimed to evaluate events involved in in vitro maturation, fertilization and development, and parthenogenetic activation of bovine oocytes assessed by nuclear-cytoplasmic interaction and gene expression. Oocyte morphological selection did not affect nuclear maturation (78.4-87.8%) and cytoplasmic cortical granule distribution (67.2-79.3%). Following nuclear and microtubular dynamics after fertilization (IVF), we observed sperm penetration 4h after insemination; male and female pronuclei differentiation by size from 14 to 18h; syngamy after 24h; and sufficient co-incubation of spermatozoa and oocytes for 10h. Pronuclear transfer to study the interaction between nucleus (N) and cytoplasm (C) in parthenogenetic embryos produced by ionomycin followed by strontium (S) or 6-DMAP (D) was assessed by cleavage, eight-cell, and blastocyst development rates: CSND (76.5, 36.4, and 6.8%) and CDNS (69.5, 25.0, and 4.9%). S cytoplasm promoted dominant effect on D nucleus. Higher rates of developmental arrest up to the eight-cell stage were observed by the combination of cytoplasm and nucleus produced by the two different activation treatments. We recovered parthenogenetic D fetuses on Day 35, which were small but normal in formation and in appearance of chorio-alantoic membranes. Genomic imprinting of IGF2 was observed, but XIST was maternally expressed in extra-embryonic tissues. In vitro culture promoted higher expression of IGF2 and H19 genes and also increased IGF2/IGF2r ratio in IVF embryos compared to in vivo produced ones.

No description available.

Tesis por compendio / [EN] In vitro production (IVP) of embryos has multiple applications, including the development of transgenic / genetically modified animals that are of broad utility in the biomedical field and could reach to be so in agriculture. In this sense, the intracytoplasmic sperm injection (ICSI) with transfected sperm, such as somatic cell nuclear transfer (SCNT) with transfected cells, have proved to be valid strategies to generate animals with stably integrated exogenous DNA, capable of transmitting to their offspring. However, to make it feasible the availability of a large number of IVP embryos it's necessary. Despite the development of IVP systems of pig embryos for these strategies in the last decades, and a high variety of protocols, there are still some critical issues that limit embryonic development and therefore its application on a large scale. The main objective of this thesis has then pursued, optimizing the ICSI and SCNT protocols in order to improve the viability of IVP porcine embryos. The initial approach was to address the study both in vitro maturation (IVM) and oocyte activation within the SCNT frame. To this end, the most efficient electrical activation protocol under our working conditions was selected from among three proposals, and we then studied the effect of serum supplement on IVM medium, noticing that it doesn't increase the development of partenogenetic embryos produced by electrical activation nor of those produced by SCNT. Moreover, and in order to optimize the ICSI technique, different aspects such as Ca2+ concentration in the microinjection medium, sperm pretreatment with Triton X-100 (TX) and the addition of cysteine or caffeine supplements to the in vitro culture (IVC) medium post-ICSI, were studied in order to promote sperm nucleus decondensation and oocyte activation. Noticing that: i) the concentration of Ca2+ did not influence the cleavage rate, or the development of partenogenic embryos from microinjected oocytes without sperm (sham injection); ii) caffeine did not increase either the pronuclear formation neither the subsequent embryo development, in combination with TX sperm pre-treatment it reduced oocyte activation and embryo development rates; iii) the pronuclear formation was not increased by the sperm pre-treatment with TX nor by the cysteine treatment, regardless of the time of the study assessment. / [ES] La producción in vitro (PIV) de embriones goza de múltiples aplicaciones, entre las que cabe destacar la obtención de animales transgénicos/modificados genéticamente que resultan de amplia utilidad en el ámbito biomédico y podrían llegar a resultarlo en el agropecuario. En este sentido, tanto la inyección intracitoplasmática de espermatozoides (ICSI) con espermatozoides transfectados, como la transferencia nuclear de células somáticas (SCNT) con células transfectadas, han demostrado ser estrategias válidas para generar animales con ADN exógeno integrado de manera estable, capaces de transmitirlo a su descendencia. Sin embargo, para lograrlo es necesario disponer de un gran número de embriones PIV. A pesar del desarrollo durante las últimas décadas de sistemas de PIV de embriones de porcino para estas estrategias, y la disponibilidad de gran variedad de protocolos, persisten aún ciertos puntos críticos que limitan el desarrollo embrionario y por lo tanto su aplicación a gran escala. El objetivo principal de esta tesis ha perseguido por lo tanto, la optimización de los protocolos de ICSI y SCNT con el fin de mejorar la viabilidad de los embriones PIV de porcino. El planteamiento inicial consistió en abordar el estudio tanto de la maduración in vitro (MIV) como de la activación oocitaria en el marco de la SCNT. Para ello se seleccionó de entre tres propuestas de activación artificial, el protocolo de activación eléctrica (que resultó ser el más eficiente bajo nuestras condiciones de trabajo), y se estudió a continuación el efecto del suplemento de suero en el medio de MIV, constatando que no se incrementaba el desarrollo de embriones partenogenotas producidos mediante activación eléctrica ni de aquellos producidos mediante SCNT. Por otra parte, y con el fin de optimizar la técnica de ICSI se abordaron diferentes aspectos como la concentración de Ca2+ en el medio de microinyección, el pre-tratamiento espermático con Triton X-100 (TX), así como la adición de suplementos de cafeína o cisteína al medio de cultivo in vitro (CIV) pos-ICSI, con el fin de favorecer la descondensación del núcleo espermático y la activación oocitaria. Observando que: i) la concentración de Ca2+ no influyó en la tasa de división, ni en la de desarrollo de embriones partenogenotas procedentes de oocitos microinyectados sin espermatozoide (sham injection); ii) la cafeína no incrementó la formación pronuclear ni en el desarrollo embrionario posterior, y en combinación con el pre-tratamiento espermático con TX redujo la activación oocitaria y el desarrollo embrionario; iii) la formación pronuclear no se vio incrementada por el pre-tratamiento espermático con TX ni por el tratamiento con cisteína, independientemente del momento de la evaluación estudiado. / [CA] La producció in vitro (PIV) d'embrions gaudeix de múltiples aplicacions, entre les quals cap assenyalar l'obtenció d'animals transgènics/modificats genèticament que resulten d'àmplia utilitat en el àmbit biomèdic y podrien arribar a ser-ho en el àmbit agropecuari. En aquest sentit, tant la injecció intracitoplasmàtica d'espermatozoïds (ICSI) amb espermatozoïds transfectats, com la transferència nuclear de cèl.lules somàtiques (SCNT) amb cèl.lules transfectades, han demostrat ser estratègies vàlides per a l'obtenció d'animals amb ADN exogen integrat de manera estable, capaços de transmetrela a la seva descendència. No obstant això, per aconseguir-ho és necessari disposar d'un gran nombre d'embrions PIV. Malgrat el desenvolupament durant les últimes dècades de sistemes de PIV d'embrions de porcí per a aquestes estratègies, i la disponibilitat de gran varietat de protocols, persisteixen encara certs punts crítics que limiten el desenvolupament embrionari i per tant la seva aplicació a gran escala. L'objectiu principal d'aquesta tesi ha perseguit per tant, l'optimització dels protocols d' ICSI i SCNT per tal de millorar la viabilitat dels embrions PIV de porcí. El plantejament inicial va consistir en abordar l'estudi tant de la maduració in vitro (MIV) com de l'activació oocitària en el marc de la SCNT. Per a això es va seleccionar entre tres propostes d'activació artificial, el protocol d'activació elèctrica (que va resultar ser el més eficient sota les nostres condicions de treball), i es va estudiar a continuació l'efecte del suplement de sèrum en el medi de MIV, constatant que no va incrementar el desenvolupament d'embrions partenogenotes produïts mitjançant activació elèctrica ni d'aquells produïts mitjançant SCNT. D'altra banda, i per tal d'optimitzar la tècnica de l'ICSI es van abordar diferents aspectes com la concentració de Ca2 + en el medi de microinjecció, el pretractament espermàtic amb Triton X-100 (TX), així com l'addició de suplements de cafeïna o cisteïna al mitjà de cultiu in vitro (CIV) pos-ICSI, per tal d'afavorir la descondensació del nucli espermàtic i l'activació oocitària. Observant que: i) la concentració de Ca2+ no va influir en la taxa de divisió, ni en la de desenvolupament d'embrions partenogenotes procedents d'oòcits microinjectats sense espermatozoïd (sham injection); ii) la cafeïna no va incrementar la formació pronuclear ni en el desenvolupament embrionari posterior, i en combinació amb el pretractament espermàtic amb TX va reduir l'activació oocitària i el desenvolupament embrionari; iii) la formació pronuclear no es va veure incrementada pel pretractament espermàtic amb TX ni pel tractament amb cisteïna, independentment del moment de l'avaluació estudiat. / García Mengual, ME. (2016). OPTIMIZACIÓN DE LAS ESTRATEGIAS REPRODUCTIVAS DE MICROMANIPULACIÓN PARA LA PRODUCCIÓN IN VITRO DE EMBRIONES PORCINOS [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61448 / Compendio

Orientador: Joaquim Mansano Garcia / Banca: Flávio Vieira Meirelles / Banca: Claudia Lima Verde Leal / Banca: Vera Fernanda Martins Hossepian de Lima / Banca: Gisele Zoccal Mingoti / Resumo: A maturação oocitária é marcada pela retomada da primeira divisão da meiose, com progressão do estádio de Vesícula Germinativa (GV) da Prófase I até a Metáfase II (MII), e inclui todos os eventos necessários para que o oócito expresse seu potencial máximo de desenvolvimento após a fecundação. Para avaliarmos a eficiência da maturação in vitro (MIV), utilizamos oócitos classificados em viáveis (graus I, II e III) e inviáveis (atrésico e desnudo), e acompanhamos a progressão nuclear e a distribuição dos grânulos corticais (GC) como indício de maturação citoplasmática, após MIV em TCM 199 com soro fetal bovino, hormônios, antibiótico e piruvato, por 24h em 5% de CO2 em ar. Maturação nuclear (78,4-87,8%) e citoplasmática (GC periféricos; 67,2-79,3%) foram semelhantes entre as diferentes classes de oócitos e apresentaramse como eventos independentes. Para o acompanhamento dos eventos desencadeados pelo espermatozóide, avaliamos a dinâmica nuclear e de microtúbulos, em intervalos de 2h, após fecundação in vitro (FIV), em meio TALP com heparina, PHE e sêmen preparado em gradiente de Percoll. Observamos que o estádio de MII foi predominante de 2 a 8h; MII e Anáfase/Telófase (A/T) predominaram às 10h; MII, A/T e estádio pronuclear (PN) de 14 a 16h; e PN a partir de 18h. A penetração do espermatozóide ocorreu após 4h da inseminação dos oócitos; a diferenciação dos PN 14 masculino e feminino pelo tamanho foi possível de 14 a 18h e a singamia ocorreu a partir de 24h. O período de 10h pode ser suficiente para que a FIV seja efetiva em oócitos bovinos, nas condições aqui descritas. / Abstract: We aimed to evaluate events involved in in vitro maturation, fertilization and development, and parthenogenetic activation of bovine oocytes assessed by nuclear-cytoplasmic interaction and gene expression. Oocyte morphological selection did not affect nuclear maturation (78.4-87.8%) and cytoplasmic cortical granule distribution (67.2-79.3%). Following nuclear and microtubular dynamics after fertilization (IVF), we observed sperm penetration 4h after insemination; male and female pronuclei differentiation by size from 14 to 18h; syngamy after 24h; and sufficient co-incubation of spermatozoa and oocytes for 10h. Pronuclear transfer to study the interaction between nucleus (N) and cytoplasm (C) in parthenogenetic embryos produced by ionomycin followed by strontium (S) or 6-DMAP (D) was assessed by cleavage, eight-cell, and blastocyst development rates: CSND (76.5, 36.4, and 6.8%) and CDNS (69.5, 25.0, and 4.9%). S cytoplasm promoted dominant effect on D nucleus. Higher rates of developmental arrest up to the eight-cell stage were observed by the combination of cytoplasm and nucleus produced by the two different activation treatments. We recovered parthenogenetic D fetuses on Day 35, which were small but normal in formation and in appearance of chorio-alantoic membranes. Genomic imprinting of IGF2 was observed, but XIST was maternally expressed in extra-embryonic tissues. In vitro culture promoted higher expression of IGF2 and H19 genes and also increased IGF2/IGF2r ratio in IVF embryos compared to in vivo produced ones. / Doutor

Research report submitted in partial fulfilment of the requirements Of the Master of Arts Degree In Sociology At the University of Witwatersrand Department of sociology / The main purpose of this research is to provide an analysis of opposition to nuclear energy in South Africa, focusing on organisations and activists opposed to the current nuclear build programme. The organisations opposed to nuclear include Earthlife Africa, Greenpeace, Coalition Against nuclear Energy, national Union of Mineworkers, the Economic Freedom fighters and Democratic Alliance, COSATU and other organisations. The study utilises a qualitative approach, mainly document analysis complimented by indepth expert interviews. The South African nuclear energy programme is a polarised issue triggering intense and often emotive debate on both sides, anti-nuclear activists and pronuclear proponents. The research pays specific attention to anti-nuclear activists and organisations opposed to South Africa's nuclear energy programme. In order to understand the anti-nuclear position, I have also incorporated pro-nuclear arguments in the study to have a better grasp of the concerns raised by organisations opposing nuclear energy. There are three main points of conflict: economic, environmental, and political. I show how opponents have framed resistance around cost and safety concerns, environmental impacts associated with nuclear and the nexus of elite political capture of state resources. / Andrew Chakane 2021