The purpose of this thesis is to present the results of a morphological investigation of the megagametophytes of two species of Fritillaria. The author has attempted to present the data in sufficient detail to clearly picture the significant features in the histories of this gametophyte generation without unnecessary repetition. The data are presented as their chronological sequence dictates.

There is but a single species of Iris native to Utah. This is Iris missouriensis Nutt. This plant was chosen for investigation because of its possible significance and because material of this species was readily available. The embryo of Iris missouriensis Nutt. shows some resemblance to the Lily type in its development of an irregular dividing suspensor. It also resembles the Orchid type in the usual development of sixteen cells before the dermatogen is cut off. In general development, however, the writer believes it to have an embryo sequence more similar to the Alisma type than to the dexcriptions given of either of the above.

The purpose of this investigation was to characterize the activities of three enzymes, aromatase, estrone (E1) sulfotransferase and E] sulfatase, in the porcine placenta and endometrium on d 30, 60 and 90 of gestation. These enzymes play key roles in determining in utero concentrations of estrogens. Days 30, 60 and 90 were chosen because previous investigations had determined that these were times of substantial changes in in vitro estrogen production by the porcine placenta and endometrium. / Master of Science

Thirty-three Holstein cows were superovulated with follicle stimulating hormone. Embryos were non-surgically recovered 6 days post-mating. Ninety embryos were morphologically evaluated and placed in culture in Hams F-10 media supplemented with a 10% protein source (steer serum, bovine serum albumin, or uterine flushings). Embryo development was observed every 12 h. Treatment differences were evaluated by assigning a numerical value of Oto 4 to each embryo representing the most advanced stage of development reached in vitro (O=no development, 4= hatched blastocyst). Data were then analyzed by analysis of variance, and Chi-square contingency analysis. Development of embryos was comparable in steer serum and bovine serum albumin but differed significantly from heat-treated or non heat-treated uterine flushings. Since albumin alone was able to support embryo growth it suggests that the albumin component of steer serum may be responsible for the development observed with that treatment. However, the possibility of non-albumin compounds contributing to the success of this protein source cannot be ruled out. Uterine fluids were unable to promote growth of embryos in vitro. Several explanations can be offered. The uterine fluids utilized were collected from ovariectomized hormonally induced cows and may not reflect the naturally occurring proteins of the normal uterus. The proteins may reflect a uterine stage which does not coincide with the age of the embryos. The level at which the uterine proteins were supplemented may have exceeded normal levels in the uterus and may have become inhibitory and lastly, the uterine fluids may have contained an abnormal amount of immunoglobulins which could interfere with normal embryo development. Analysis of the mean time to reach each stage demonstrated the similarity in the time course of development between the embryos supplemented with steer serum or bovine serum albumin in culture and embryos in vivo. This suggests that culture conditions other than uterine flushing supplementation were very favorable to embryo development. / M.S.

Early mouse (n = 501) and bovine (d = 6; n = 172) embryos were obtained to evaluate the effect of a deproteinized hemodialysate (CLBl 107) on embryo development in vitro. Bovine morulae also were cultured to examine the effect of agar embedding and the environment of immature mouse uteri on embryo development. Mouse embryos were cultured for up to 96 h in M2 medium or M2 supplemented with CLBl 107. One-and two-cell embryos did not develop beyond the two-cell stage in vitro. Degeneration of one-cell embryos occurred within 36 h. Two-cell embryos degenerated sooner when cultured in M2 plus I% CLB 1107 (27.4 ± 2.5 h) than in M2 alone (41.7 ± 3.0 h). Mean final development classification of embryos cultured from the morula stage (6) in M2 supplemented with CLBI 107 was higher (9.0 ± 2) than that for morulae (8.2 ± 1) cultured in unsupplemented M2 medium. Development of embryos cultured from the blastocyst (8) or expanded blastocyst (9) stages was not affected by treatment. Agar embedded bovine embryos were cultured in Ham's F-10 and 10% steer serum either 1) immediately after collection or 2) 24 h after storage in immature mouse uteri. Non-embedded embryos were cultured in Ham's F-10 containing 3) 10% steer serum, 4) 1% CLB 1107 or 5) 1% CLB 1107 and 10% steer serum. A greater percentage of the embryos reached the hatched blastocyst stage after culture in treatments 1, 3, 4, and 5 (38.1%, 34.6%, 28.6% and 21.1%) than embryos stored in immature mouse uteri for 24 h prior to in vitro culture (9.5%). Mean final development scores for non-embedded and agar embedded embryos cultured in Ham's F-10 and 10% steer serum were not different (5.46 ± .34 and 4.89 ± .44), but were higher than embryos cultured in CLB 1107 (4.19 ± .39), CLBl 107 and steer serum (4.22 ± .44) or immature mouse uteri (3.39 ± .43). Medium supplemented with CLB 1107 did not support mouse embryo development beyond the 2-cell stage nor did it enhance bovine embryo development. However, it appeared to enhance development of mouse morulae in vitro. Additionally, bovine morulae were not affected by agar embedding in vitro and they were able to develop following short term storage in the immature mouse uterus. / M.S.

糖調節蛋白78 (Glucose-regulated protein 78),也稱之Bip,是70kDa熱休克蛋白家族成员之一。已有的研究表明，Bip 是一個具有多功能的蛋白，參與眾多的生物調控過程，包括蛋白折疊，調節鈣平衡，以及作為內質網緊張（ER stress) 的感應器。有研究表明，Bip可以在細胞膜上定位，作為Nodal信號通路的一個輔助受體發揮作用。大量的研究表明，Bip在疾病和代謝方面也發揮重要作用。它參與胰島素的生物合成，並可以提高長期高血糖下β細胞的功能。同時具有抗細胞凋亡的作用。然而Bip在胚胎髮育中的生物功能卻知之甚少。 / 高等脊椎動物腎臟發育中經歷形成3種腎臟形式：前腎，中腎和後腎。腎單位是這3種形式的基本結構和功能單位。在兩棲類，前腎在胚胎時期發揮作用，在胚胎的兩側各只有一個腎單位。這使得爪蟾成為前腎研究的一個非常好的模型。 / 在此項研究中，我們採用非洲爪蛙作為動物模型來研究Bip在胚胎髮育過程中，尤其是在前腎發育中的生物功能。Bip是一個母性因子，在尾芽期，Bip 表達在粘液腺，前腎，肝以及耳囊。 Bip在前腎清晰明確的表達，表明Bip可能在前腎的發育中發揮作用。我們利用BipMO來進行敲低功能實驗，免疫印記顯示BipMO能阻斷帶Flag標記Bip的翻譯。通過原位雜交技術檢測前腎的不同標記基因的表達發現，敲低Bip抑制前腎的形成，表明Bip的正常表達是前腎發育所必須的。 / 為了研究Bip調節前腎的發育的分子機制， 我們使用Affmetrix基因芯片分析在Bip敲低情況下的不同時期胚胎中基因的表達譜，發現在Bip敲低表達的胚胎中，視黃酸信號通路的一些重要的組分的表達受到抑制。爪蛙胚胎原腸胚的動物帽細胞具有多能性， 使用激活素和視黃酸一同處理動物帽細胞可以誘導其分化成為原腎組織。在此體外分化體系中敲低Bip表達，前腎標記基因表達降低，顯示在這一體外系統中前腎的分化受到抑制。該實驗結果與體內實驗結果一致。在體外培養的HEK293T細胞中敲低Bip，抑制視黃酸處理後視黃酸信號通路螢光素報告的活性。 lhx1是前腎發育早期表達標記之一，對於前腎原基的初始化具有重要的作用，同是它也是視黃酸信號通路的靶基因。共同註射BipMO和lhx1表明，前腎的異常可以明顯降低，顯示lhx1可以部份拯救由於Bip缺失所造成的腎臟發育缺陷。該實驗表明Bip通過調節視黃酸信號通路，來調控lhx1的表達前腎的形成。我們進一不發現，敲低Bip後，前腎異常形成的區域內，細胞凋亡增加，增殖減少。該結果在細胞水平上解釋了Bip敲低表達時前腎形成異常的一個原因。 / 综述所述，Bip正確表達对胚胎前肾的发育極為重要。它胚胎发育过程中通过視黃酸信号通路調控lhx1的表達，從而对前肾的形成发挥重要作用。 / Glucose-regulated protein 78 (Grp78), also known as Bip, belongs to heat shock protein 70kDa family. It has been implicated in various biological processes including protein folding, regulation of calcium homeostasis, and serving as a sensor of ER (Endoplasmic Reticulum) stress. Moreover, it can localize in cell membrane, acting as co-receptor of nodal signaling. It is essential for insulin biosynthesis. In addition, Bip plays important roles in a number of diseases. For example, BIP can improve β-cell function in the prolonged hyperglycemia. Knockdown of BIP in β-cell can induce apoptosis. However, little is known about its function during embryonic development. / In high vertebrate, three sets of nephric forms develop successively during embryonic kidney development. They are pronephros, mesonephros, and metanephros. Nephron is the basic structural and functional unit of all these three forms. In amphibian, the pronephros performs function at the embryonic stages, which has only one nephron on either side of the body. It makes Xenopus a very good model for pronephros study. / In this study, we took advantage of Xenopus leavis as an animal model to investigate Bip function during embryonic development, especially its role in pronephros development. We first examined the expression of Bip in developing embryos. Whole mount in situ hybridization showed that Bip was expressed in the cement gland, pronephros, liver and ear vesicle during tailbud stages. It was expressed in the pronephros strongly and clearly which suggested that Bip might play roles in pronephros development. We performed loss-of-function experiment by using morpholino oligonucleotide (MO) knock down translation of endogenous Bip expression. Depletion of Bip impaired formation of pronephros revealed by reduction expression of different pronephros maker genes. The pluripotent animal caps can differentiate into pronephros tissue when treated with activin and all-trans retinoic acid (atRA) in vitro kidney induction assay. In line with our in vivo observation, knockdown of Bip inhibited pronephros differentiation that can normally achieved by combined effects of activin and atRA in animal cap assay. / In order to investigate the molecular mechanisms as how Bip regulated pronephros development, we performed Affymetrix DNA microarray assay to generate gene expression profile in Bip morphants. We found that some components of RA signaling were inhibited when Bip was knockdown. Moreover, knockdown of Bip caused reduction of RA target genes expression after treatment with RA. Consistent with above observations, luciferase activities of RA signaling reporter was reduced in HEK293T cells when BIP expression was depleted by RNAi. lhx1 is one of RA target genes and has been implicated playing essential roles in pronephros development. The inhibition of pronephros formation induced by Bip depletion can be partially rescued by co-overpression, suggesting 1) lhx1 is downstream of Bip in the regulatory network of pronephros formation; and 2) Bip regulates pronephros formation through RA signaling via lhx1. We also found increased apoptosis and decreased cell proliferation at pronephros-forming region in Bip morphants. That could explain the reason of pronephros malformation when Bip is downregulated. / Taken together, Bip is essential for pronephros development. It functions through RA signaling during the complex developmental processes. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Shi, Weili. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 125-143). / Abstracts also in Chinese.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2002. / Bibliography: leaves 251-271.

The main focus of my PhD studies was aimed at understanding the general mechanism of nuclear translocation and isolating novel components of the nuclear translocation pathway in neurons. Using the Drosophila visual system as an in vivo model to study nuclear motility in developing photoreceptor cells (R-cells), I have identified a novel role for the Ser/Thr kinase Misshapen (Msn) and the endocytic trafficking pathway in regulating the nuclear translocation process. / The development of R-cells in the Drosophila eye disc is an excellent model system for the study of nuclear motility owing to its monolayer organization and the stereotypical translocation of its differentiating R-cell nuclei along the apical-basal plane. Prior to my thesis work, several laboratories had identified dynein and its associating proteins in R-cell nuclear translocation, however nothing was known about the signalling pathway that controlled their function in nuclear migration. Thus, one of my thesis goals was to elucidate the signalling mechanism controlling nuclear translocation in R-cells. / Using a combination of molecular and genetic approaches, I identified Msn as a key component of a novel signalling pathway regulating R-cell nuclear translocation. Loss of msn causes a failure of R-cell nuclei to migrate apically. Msn appears to control R-cell nuclear translocation by regulating the localization of dynein and Bicaudal-D (Bic-D). My results also show that Msn enhances Bic-D phosphorylation in cultured cells, suggesting that Msn regulates R-cell nuclear migration by modulating the phosphorylation state of Bic-D. Consistently, my results show that a Bic-D-phosphorylation-defective mutation disrupted the apical localization of both Bic-D and dynein. I propose a model in which Msn induces the phosphorylation of Bic-D, which in turn modulates the activity and/or subcellular localization of dynein leading to the apical migration of R-cell nuclei. / In addition to studying Msn, I have also searched for additional players in R-cell nuclear migration. From a gain-of-function approach, I found that the misexpression of the GTPase-activating-protein (GAP) RN-Tre caused a severe defect in R-cell nuclear migration. Since mammalian RN-Tre is involved in negatively regulating Rab protein activity, I speculated that the RN-Tre misexpression phenotype reflected a role for Rab-mediated vesicular transport in regulating R-cell nuclear migration. I systematically examined the potential role of Rab family proteins in R-cell nuclear migration and found that interfering with the function of Rab5, Rab11 or Shibire caused a similar nuclear migration phenotype. I propose that an endocytic pathway involving these GTPases is required for the targeting of determinants to specific subcellular locations, which in turn drive the apical migration of R-cell nuclei during development.

Indiana University-Purdue University Indianapolis (IUPUI) / The anatomical sciences are experiencing a notable decrease in the time and resources devoted to embryology in North American medical education. With more changes assured, it is necessary to investigate the current trends in curriculum, pedagogy, and related experiences of embryology teachers and learners. To address these concerns, the researcher developed two online mixed methods surveys: one for current anatomy and embryology faculty and another for first year medical students. The faculty survey was followed by interviews with volunteers from that cohort. The researcher used a grounded theory methodology to analyze the qualitative components of the surveys, and descriptive statistics to analyze the quantitative components of the surveys. Both the faculty and student surveys illuminated the vast differences between the explicit, implicit, and null curricular components found in the numerous medical education programs represented. A combined grounded theory methodology and phenomenological approach was used to analyze the interviews with faculty. This generated a lived experience narrative of the phenomenon of teaching embryological content to medical students in the modern world, which led to a better understanding of the needs and challenges that face this subject matter and those who teach it. In this fluid era of medical education reform and integration, the perceptions and experiences of anatomy and embryology faculty and first year medical students are invaluable to assessing the curriculum and pedagogy of this foundational anatomical science and formulating evidence-based recommendations for the future.

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