Mouse epiblast stem cells (EpiSC) are pluripotent cells derived of the epiblast of post-implantation blastocysts that can self-renew indefinitely in culture, display lineage-restricted differentiation, and appear to closely resemble human embryonic stem cells (ESC). Despite significant advances in the last decade, the precise molecular mechanisms and many master regulator (MR) genes underlying stem cell self-renewal, pluripotency, interactions with surrounding cells, and lineage-specific differentiation still remain elusive. The goal of this thesis is to address these gaps of knowledge using a systematic approach to identify novel MR genes and functionally validate them using genetically modified mouse models.In order to elucidate MR genes that control understudied biological processes, previous work in the Shen lab have computationally reconstructed the regulatory network of EpiSC and interrogated the EpiSC interactome with pluripotency signatures of EpiSC lines. One MR gene of interest from the previous analysis is ZC3H13, which encodes a protein that has been previously shown to be a crucial for N6-methyladenosine modification in RNA (m⁶A). This suggests a novel connection between m⁶A epitranscriptional modifications and primed state pluripotency. In my thesis research, I have shown that Zc3h13 is essential for proper trophoblast lineage differentiation and the importance of m6A modifications in early embryonic development. Using two Zc3h13 knockout mouse lines, I have found that Zc3h13 null embryos are embryonic lethal at the peri-implantation stage due to a failure to implant into the uterus. In vitro outgrowth analysis revealed a lack of trophoblast giant cells in Zc3h13 null outgrowths, and the lack of enlarged nuclei in the Zc3h13 null outgrowth suggests a failure in endoreduplication. Immunofluorescence analysis of Zc3h13 null blastocysts showed that the trophectoderm cells of Zc3h13 null blastocyst expressed trophectoderm specific factors at abnormal levels, indicating a severe dysregulation of the trophectoderm regulatory network. To elucidate the effects of Zc3h13 knockout on pluripotency, I also performed a detailed immunofluorescence analysis of Zc3h13 null inner cell mass (ICM), which expressed pluripotency factors at normal levels. However, Zc3h13 null blastocysts were less efficient at generating ESC lines and the Zc3h13 KO ESC generated were morphologically abnormal. Dot blot and mass spectrometry analysis showed that Zc3h13 KO ESC had a dramatically lower level of m⁶A modification, suggesting a connection between m6A epitranscriptional modification and endoreduplication. Interestingly, chimera and teratoma analysis showed that while Zc3h13 KO ESC can contribute to derivatives of the three primary lineages, Zc3h13 KO ESC has a bias towards neuroectoderm differentiation. In this thesis, I have shown the importance of m6A transcriptional regulation in trophoblast giant cell differentiation. Taken together, my studies can help further the understanding of the biological functions of m⁶A modifications as well as the relationship between transcriptional regulation and cell fate transition. My work highlights another level of gene regulation through epitranscriptional modification and the importance of the epitranscriptomic landscape in cell fate transition and development.

Eye tracking is the process of measuring where people are looking at with an eye tracker device. Eye tracking has been used in many scientific fields, such as education, usability research, sports, psychology, and marketing. Eye tracking data are often obtained from a static eye tracker or are manually extracted from a mobile eye tracker. Visualization usually plays an important role in the analysis of eye tracking data. So far, there existed no software package that contains a whole collection of eye tracking data processing and visualization tools. In this dissertation, we review the eye tracking technology, the eye tracking techniques, the existing software related to eye tracking, and the research on eye tracking for posters and related media. We then discuss the three main goals we have achieved in this dissertation: (i) development of a Matlab toolbox for automatically extracting mobile eye tracking data; (ii) development of the linked microposter plots family as new means for the visualization of eye tracking data; (iii) development of an R package for automatically extracting and visualizing data from mobile and static eye trackers.

The development of a preimplantation embryo is a stepwise process consisting of morphological, biochemical, and genomic changes. Much remains unknown about the attainment of embryo competency to develop and establish pregnancy. To investigate this, we compared methods of selection at the oocyte or embryo level for improved blastocyst production. Brilliant cresyl blue staining was used to sort oocytes by their growth status (not fully grown vs. fully grown) and the timing of the first embryonic cell division to sort embryos. We found that an embryo's cleavage kinetics are more indicative of their competency than the growth status of the oocyte that gave rise to that embryo. We further investigated the cryopreservation survival of embryos with fast or slow cleavage kinetics and found no significant differences in their ability to hatch post-thawing. Next, we used the complete sequence of the cattle Y chromosome to identify oligonucleotides for efficient sexing of samples. These materials may be used to understand sexual dimorphism as a biological factor in future experiments. Finally, we designed a new method to induce targeted DNA sequence deletions and mRNA cleavage in zygotes using CRISPR-Cas. We targeted the gene OCT4, since the literature shows variable knockout outcomes. Our method improved deletion efficiency while accounting for preexisting or maternally inherited mRNA of the target gene. Our findings can be used to better understand early embryo development and biological drivers of quality, which can be leveraged to improve embryo production and transfer outcomes. / Master of Science / The development of an early embryo involves many biological and structural changes. Much remains unknown about the influences on embryo quality and ability to successfully develop. To investigate this, we compared methods for selecting the highest quality cattle eggs or embryos. We found that the observation of an embryo's development speed is better for selecting high quality embryos than egg quality. We further investigated the freezing survival of embryos with fast or slow growth. We found that the freezing survival of fast and slow growing embryos is not different. Next, we used the complete sequence of the cattle Y chromosome to identify PCR primers for determining sample sex. These resources can help us understand how an individual's sex can influence biological differences. Finally, we designed a new method for removing the total function of a gene in embryos. For this, we deleted segments of DNA and cut RNAs. Our findings can be used to better understand early embryo development and biological drivers of quality, which can be leveraged to improve embryo production and transfer outcomes.

<p>Triclosan is an antimicrobial additive found in a number of personal care and household products. Widely detected in humans, the compound has been given increasing attention due to reports of its endocrine-disrupting potential. Recent evidence indicates that triclosan is mildly estrogenic. The carefully timed event of blastocyst implantation in mammals is modulated in part by estrogen and can be disrupted by above optimal elevations in estrogenic stimulation. Here, we examined the influences of triclosan administration in inseminated female mice. Doses of 18 and 27 mg/animal/day on gestation days (GD) 1–3 reduced implantation site numbers as observed on GD 6, relative to vehicle controls and females given lower doses. Single doses of 18 or 27 mg reduced implantations when given on GD 3, whereas only 27 mg did so when given on GD 2. Subsequently, we examined the impacts on early pregnancy of triclosan in combination with the xenoestrogen bisphenol-A, which has been previously found to disrupt implantation, at doses that were individually ineffective. A combination of 4 mg BPA and 9 mg triclosan/animal/day administered on GD 1–3 reduced the number of implantations observed on GD 6 and increased the length of gestation, relative to controls and those animals simply given one or the other compound. All of these effects mimicked stronger effects seen in positive controls given 17β-estradiol. These data are consistent with the notion that triclosan has mild estrogenic properties, and show that it can act together with a known xenoestrogen to disrupt implantation.</p> / Master of Science (MSc)

Factors currently at the forefront of human in vitro fertilization (IVF) that collectively influence treatment success in the form of blastocysts development were investigated during early mammalian embryology with concentration on infertile patients presenting with diminished ovarian reserve or preliminary ovarian failure. A novel experimental technique, Graft Transplant-Embryonic Stem Cells (GT-ESC) was introduced in the mouse model, as the first inclusive approach for embryo selection in IVF treatments resulting in successful graft integration of sibling cells, stage-dependent (day 4) blastocysts. E-Cadherin-catenin bonds play an integral role in trophectoderm cell viability and calcium removal, inducing disruption of cell-to-cell bonds at the blastocyst stage was detrimental to continued blastocyst development. One of the leading methods for embryo selection for uterine transfer in human IVF is application of pre-implantation genetic screening (PGS) methods such as next generation sequencing (NGS). Female patients <35 y do not benefit from this treatment when outcome is measured by presence of fetal heart beats at 10 weeks of gestation. Patients 35-37 y benefit from PGS with no significant difference of outcome based on form of PGS method utilized. Therefore, small nucleotide polymorphism array (snp-array) or targeted-NGS should be selected for this age range to lessen the financial burden of the patient. Embryos from women >40 y have a higher rate of mosaic cell lines which can be detected by NGS. Therefore NGS is most beneficial for women >40 y. Additionally, ovarian stimulation of the patient during human IVF can notably influence outcome. Anti-Müllerian hormone (AMH) is a more conducive indicator of blastocysts development per treatment compared to basal follicle stimulating hormone (FSH). Actionable variables included in a decision tree analysis determined a negative influence (0% success, n=11) of high dose gonadotropin use (>3325 IUs) in good prognosis patients (>12 mature follicles at trigger, AMH >3.15 ng/mL). A positive relationship exists (80% success, n=11) between poor responders (AMH <1.78 ng/mL, <12 mature follicles at trigger) and high dose gonadotropin use (>3025 IUs). Utilizing the decision tree during IVF treatment can be beneficial to treatment success. Moreover, a parallel relationship of the fundamental principles of culture medium pH, pCO2 and pO2 was found with respect to blastocyst development. Human infertility patients' gametes predisposed to primary stressors (i.e., age, genetics and etiology) are negatively impacted (~30% success, n=7) for cleavage stage (day 3) embryo development when primary culture medium has pCO2 <30mmHg given age >31 y and <14 oocytes retrieved. When day 3 embryo development is measured at >65% good quality embryos per treatment (based on SART grading criteria), blastocysts development success is highest when secondary culture medium pO2 is 69-88 mmHg (~90% success, n=12). Thus, IVF treatment outcome can be optimized with utilization of predictive model analyses in the form of decision trees providing greater success for the IVF laboratories, ultimately decreasing the emotional and financial burden to infertility patients.

Neste estudo foi analisada a expressão gênica e protéica, uterina e embrionária da quimiocina Ccl25 e de seu receptor Ccr9 nas fases iniciais da implantação embrionária (dias 3,5, 4,5, 5,5 e 7,5 de gestação). Por meio de reações imunohistoquímicas e de citometria de fluxo, foram identificadas as populações celulares envolvidas nesta expressão. Também, foram realizados ensaios de quimiotaxia com o silenciamento da expressão de Ccl25 (ODNs-Antisense) nas células trofoblásticas, para a avaliação das atividades desta quimocina. Nossos resultados sugerem o estabelecimento de uma comunicação embrião (células trofoblásticas) - endométrio (células do sistema immunológico), via Ccl25/Ccr9. Esses achados são relevantes para a compreensão das interações blastocisto/sistema immunológico materno no estabelecimento dos mecanismos imunoreguladores durante a implantação embrionária. / In this study, we analyzed the gene and protein, uterine and embryonic expression of Ccl25 chemokine and its receptor Ccr9 in early stages of embryo implantation (days 3.5, 4.5, 5.5 and 7.5 of gestation). By using immunohistochemistry and flow cytometric assays, cell populations involved in this expression were identified. In addition, chemotaxis assays were also performed after silencing of Ccl25 (ODNs-antisense) in trophoblast cells. . Our results suggest the establishment of an endometrium (immune cells) - embryo (trophoblast cells) dialogue via Ccl25/CCR9. These findings are relevant for understanding the interactions between blastocyst and maternal immune system in the establishment of immunoregulatory mechanisms during implantation.

O estabelecimento de cultivo de células-tronco embrionárias (ESC) ainda não foi realizado com sucesso. Verificação de marcadores de pluripotência e diferenciação nos três folhetos germinativos são necessárias para validação de uma linhagem celular pluripotente. O objetivo deste estudo foi estabelecer e caracterizar o cultivo de ESC suínas usando Matrigel e comparar a expressão dos marcadores de pluripotência Oct-4, CD9 e &alpha;6-integrina em embriões. Blastocistos in vitro ou in vivo foram submetidos à imunocirurgia para cultura da massa celular interna, fixados para imunocitoquímica ou extração de RNA total para RT-PCR. Nenhuma colônia de ESC foi obtida usando co-cultivo em fibroblastos embrionários murinos (MEF) ou em Matrigel. Expressão de Oct-4, CD9 e &alpha;6-integrina foi detectada por PCR. Os produtos de PCR de CD9 e &alpha;6-integrina tiveram suas sequências nucleotídicas determinadas e comparadas com bases de dados públicas. O produto de CD9 foi idêntico à seqüência do CD9 suíno e o produto de &alpha;66integrina foi similar à humana e eqüina. Reação de Imunocitoquímica revelou a presença de Oct-4 no citoplasma de células da massa celular interna e do trofoblasto. CD9 e &alpha;6-integrina foram observados preferencialmente em células do trofoblasto. Não foi possível comparar a expressão dos marcadores de pluripotência entre ESC e embriões em suínos. Porém, este estudo descreve pela primeira vez a expressão de CD9 e &alpha;6-integrina em blastocistos suínos, os quais podem não estar relacionados com células pluripotentes embrionárias suínas. / Establishment of embryonic stem cell (ESC) culture in pigs has not been achieved. Verification of pluripotency markers and differentiation in the three embryonic layers are necessary for validation of a pluripotent cell line. The objective of this study was to establish and characterize porcine ESC culture using Matrigel and compare the expression of pluripotency markers Oct-4, CD9 and &alpha;6-integrin with embryos. In vitro or in vivo porcine blastocysts were submitted to immunosurgery for culture of inner cell mass, fixation for immunocytochemistry or total RNA extraction for RT-PCR. No ESC colonies were obtained using co-culture on mouse embryonic fibroblasts (MEF) or on Matrigel. Expression of Oct-4, CD9 and &alpha;6-integrin was detected by PCR. CD9 and &alpha;6-integrin PCR products had their nucleotide sequence assessed and compared with public nucleotide database. CD9 product was identical to CD9 porcine sequences and &alpha;6-integrin product was similar to human and equine &alpha;6-integrin. Immunocytochemistry revealed Oct-4 expression in cytoplasm of the inner cell mass and trophoblast cells. CD9 and &alpha;6-integrin were observed preferentially on trophoblast cells. It was not possible to compare expression of pluripotency markers between porcine ESC and embryos. However, this study describes for the first time expression of CD9 and &alpha;6-integrin in porcine blastocysts, which may not be related to pluripotent porcine embryonic cells.

Uma das maiores dificuldades das terapias de reprodução assistida é a seleção de células germinativas de boa qualidade para posterior fertilização e implantação. Atualmente, a seleção de oócitos se dá basicamente por avaliação morfológica, que não reflete satisfatoriamente a competência oocitária. Assim, a busca por bioindicadores da qualidade oocitária é necessária. O cumulus oophorus forma um conjunto de células somáticas que circundam o oócito no folículo antral,mantendo uma relação íntima com a célula germinativa, com comunicação direta via junções tipo GAP. As células do cumulus oophorus são descartadas após técnicas de fertilização in vitro, o que as torna um material de fácil acesso, livre de conflitos éticos. Uma série de metodologias de análise das células do cumulus foram propostas para identificação de anormalidades no oócito. Porém, nenhuma delas é rotineiramente utilizada na clínica. Os processos celulares das células do cumulus refletem as condições do microambiente folicular, e podem, assim, trazer evidências das condições oocitárias. Nosso grupo analisou as células do cumulus primeiramente por meio de abordagens de bioinformática, que revelaram processos celulares relacionados ao desenvolvimento de embriões até o estágio de blastocisto. Com base nestes resultados, análises de parâmetros bioquímicos e expressão gênica das células do cumulus foram realizadas e permitiram a identificação de possíveis biomarcadores da qualidade do oócito que levam em consideração as características clínicas das pacientes. Estas análises indicaram que expressão do gene PTGS2 e a atividade da enzima Glutationa-S-Transferase como indicadores da formação de blastocistos em pacientes com diferentes variáveis clínicas (backgrounds) analisados. Se confirmados, estes parâmetros poderão ser utilizados como biomarcadores no ambiente clínico, elevando as taxas de sucesso em técnicas de reprodução assistida. / One of the great challenges in assisted reproduction techniques is the selection of appropriate germ cells for fertilization and implantation. Nowadays, oocyte selection occurs basically through morphological evaluation, which does not reflect satisfactorily the oocyte competence. Therefore, the search for bioindicators of oocyte quality is necessary. The cumulus oophorus forms a set of somatic cells that surround the oocyte in the antral follicle, participating in the processes of oocyte maturation and folliculogenesis. These cells maintain an intimate relationship with the germ cell, with direct communication via GAP junctions. Cumulus oophorus cells are discarded in in vitro fertilization techniques, which makes them an easy-access material that can be collected in a free-of-ethicalissues way. A series of cumulus cell analysis methodologies were proposed to identify abnormalities in the oocyte. However, none of them is routinely used in clinical environment. The cellular processes of cumulus cells reflect the conditions of the follicular microenvironment, and may thus bring evidences of oocyte conditions. Our group analyzed cumulus cells in search of predictors of oocyte quality primarily through bioinformatics approaches, which revealed cellular processes related to the development of embryos up to the blastocyst stage. Based on these results, analyzes of biochemical parameters and gene expression of cumulus cells were performed and allowed the identification of possible biomarkers of oocyte quality that takes into consideration patients clinical variables. These analysis indicated PTGS2 expression and Glutathione-S-Transferase activity as indicators of blastocyst formation in all patient profiles (backgrounds) analyzed. If confirmed, these parameters may be used as biomarkers in clinical environment, improving assisted reproduction success rates.

Orientador: Marcelo Fabio Gouveia Nogueira / Resumo: Além de permitir a produção em larga escala de embriões, a maturação in vitro (MIV) não utiliza hormônios superestimulatórios, evitando prejuízos econômicos e efeitos iatrogênicos da superestimulação. Entretanto, a MIV ainda está aquém das expectativas devido à incapacidade em simular eventos fisiológicos que ocorrem in vivo. Um artefato da MIV consiste na retomada espontânea da meiose após a remoção do oócito do ambiente folicular. Sem o aparato molecular que retém a meiose, a maturação nuclear ocorre sem o estímulo hormonal fisiológico, impedindo a reorganização adequada das organelas durante a maturação citoplasmática, o que pode comprometer a competência oocitária in vitro. A temática primordial desta tese foi testar um sistema de pré-maturação oocitária (Pré-MIV), no qual dois fármacos (forskolin e 3-isobutil- metilxantina) são utilizados antes da MIV para retardar a meiose. O desempenho da Pré-MIV foi comparado à MIV convencional, em contexto comercial e de pesquisa. Em um primeiro momento, comparamos os sistemas convencional e Pré-MIV de forma holística, ou seja, testamos os sistemas comerciais completos. Aqui, o critério avaliativo foi a análise ultraestrutural de oócitos e blastocistos, e a análise da expressão de 96 genes relacionados à qualidade embrionária. Na segunda abordagem, adaptamos os fármacos da Pré- MIV em nossa MIV rotineira. Por estes fármacos afetarem o metabolismo lipídico, e, frente a importância deste no metabolismo energético celular, a segunda inv... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Assisted reproductive technologies (ARTs) have now been extensively used to promote reproductive efficiency as a fertility treatment not only in human medicine but also animal reproduction. ARTs serve as an important tool to advance the fundamental knowledge of reproductive processes. The quality of female’s eggs defines its ability to undergo maturation, fertilization, and development. This quality is determined by various factors and is crucial for the success of ARTs. Any alternations happening during the egg growth and maturation process can result in the decreased quality, which could have long-lasting effects on development. Improving the developmental efficiency of the egg is quite challenging due to the limited knowledge on the underlying mechanism of how the egg regulates biological processes during the growth and maturation phase. We compared good-quality and poor-quality eggs to detect the key players in determining the egg quality at the molecular level. Our finding also provides information that benefits the understanding of how the nutrients in culture medium facilitate oocyte maturation, which will eventually help optimize the condition for oocyte culture. Based on the results from these comparative studies, we proposed a potential strategy for improving egg quality. The knowledge obtained from our research offers promise for many applications in the treatment of infertility and improvement of ART efficiency.