Proefschrift--Utrecht. / Summary in Dutch. "Stellingen": [2] L. laid in. Bibliography: p. [325]-340.

Proefschrift--Utrecht. / Summary in Dutch. "Stellingen": [2] L. laid in. Bibliography: p. [325]-340.

After the discovery that Zika virus (ZIKV) infection in pregnant women may result in severe adverse outcomes such as fetal microcephaly, ZIKV must be added to an ever-expanding list of teratogenic viruses. As only a small minority of newborns will display congenital abnormalities after maternal primary infection, innate immune mechanisms must exist in the placenta to prevent viral transmission to the fetus. Understanding the innate antiviral defenses of the placenta is critical to improving diagnosis, treatment, and prevention of adverse pregnancy outcomes stemming from viral infections. We hypothesized that RLRs are expressed in either one or both of the outer cell layers of the chorionic villi, either the syncytiotrophoblast (STB, outermost layer) or in the villous cytotrophoblast (CTB, inner layer), and that expression of these receptors will increase with advancing gestational age.  In order to determine the expression of RLRs in placental tissue (6-32 weeks and full-term), we used immunohistochemistry to stain for the RIG-I-like receptors (RLRs) RIG-I, DHX58/LGP2, and MDA5, as well as the endosomal Toll-like receptor TLR7, that serve as antiviral innate immune receptors involved in detecting microbial ligands and cytoplasmic viral nucleic acids. Hofbauer (HB) cells stained positive for all receptors and served as a positive internal control. TLR7 was not present in either the STB or CTB throughout gestation. MDA5 was localized to the STB cytoplasm up to 13 weeks. After 13 weeks, MDA5 was localized to the CTB cytoplasm. DHX58/LGP2 was localized to the STB cytoplasm at 6 weeks of gestation, the STB apical and basal membranes in addition to cytoplasm at 7 weeks of gestation, and also CTB cytoplasm after 7 weeks of gestation. Lastly, RIG-I was localized to the CTB cytoplasm throughout gestation. The differential expression of these RLRs suggest an innate immune defense system unique to the placenta that is responsible for protecting the conceptus from viral attack.  These findings will complement ongoing work in characterizing replication of teratogenic viruses in the placenta.

Induced pluripotent stem (iPS) cells that possess embryonic stem (ES) cell-like properties are generated through the use of the Yamanaka transcription factors, OCT4, SOX2, KLF4, and MYC (OSKM). Advanced transgene delivery methods utilizing non-integrating viruses for transduction of target cells has provided new opportunities for regenerative medicine in humans and other species. We sought to use this technology to generate equine iPS cells to address challenges in equine regenerative medicine. Umbilical cord matrix mesenchymal stromal cells (MSC) were transduced with the non-integrating Sendai virus encoding for the OSKM transcription factors. The cells initially were cultured on mouse embryonic feeder cells supplemented with LIF (10 ng/mL) and FGF2 (4 ng/mL). Transduction generated 21 initial colonies. Of these, four survived beyond 20 passages. The transduced equine cells morphologically resembled ES cells and expressed cell surface antigens indicative of ES cells. Molecular evaluation revealed the cells maintained expression of endogenous OSKM while the exogenous OSK transgenes were extinguished, but MYC was maintained. The transduced equine cells did not express the ES marker NANOG, but did express the trophectoderm markers CDX2 and TFAP2A. Both OCT4 and CDX2 were colocalized to the nucleus. The transduced equine cells were termed equine induced trophoblast (iTr) cells. Culture of the iTr cell in suspension resulted in formation of blastocyst-like spheres rather than solid cell aggregates indicative of ES and iPS cells. The iTr cells were transitioned to a feeder free monolayer culture. Transformation of the iTr cells to the spherical arrangement stimulated expression of genes that mark differentiation of trophoblast cells and up-regulated 250 transcripts over the monolayer arrangement. The iTr monolayer arrangement up-regulated 50 transcripts compared to the spherical arrangement. The iTr spheres respond to BMP4, EGF, and FGF2 by phosphorylating signal transduction proteins. Addition of BMP4, EGF, or FGF2 in combined pairs was able to alter TFAP2A, NEU1, and SLC35A1 expression. The generation of iTr cells by transduction of the Yamanaka reprogramming factors is not unique to equine cells. However, this report marks the generation of the first equine trophoblast cell line capable of recapitulating early equine trophoblast development. The new iTr line could prove valuable in gaining greater understanding of equine trophectoderm development. / Ph. D.

No description available.

Thesis (Ph.D.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR46006

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The fetal component of the human placenta is comprised of cells termed trophoblasts and the proper differentiation of these cells is pivotal for maintaining maternal and fetal health throughout pregnancy. The placental syncytiotrophoblast layer is a key secretory portion of the placenta and is produced through fusion of underlying progenitor cytotrophoblast cells with the multinucleated syncytiotrophoblast layer. The MacPhee laboratory has previously established that fusion or syncytialization of cytotrophoblasts is aided by expression of integrin-linked kinase (ILK), a cytoplasmic adapter protein. Nuclear enzyme Poly ADP-ribose Polymerase-1 (PARP-1) and the transcriptional repressor Snail-1 work with ILK to downregulate epithelial cell-cell adhesion. This process would also be necessary for proper trophoblast syncytialization. Thus, it was hypothesized that PARP-1 would be an important mediator of syncytiotrophoblast development. To test this hypothesis, immunofluorescence analysis of PARP-1 and Snail expression in human chorionic villi from first and second trimester as well as from term pregnancy was conducted. Furthermore, co-localization between PARP-1 and Snail-1 were evaluated. Lastly, human PARP-1 was overexpressed in the BeWo trophoblast derived cell line, under fusion promoting conditions, to directly test the ability of PARP-1 to regulate trophoblast fusion. Throughout the first and second trimester, PARP-1 and Snail were highly expressed and co-localized in villous cytotrophoblast nuclei. In contrast, PARP-1 was rarely detectable in syncytiotrophoblast nuclei, while Snail was highly expressed. Upon transient overexpression of PARP-1 in BeWo cells, fusion was robustly promoted. Furthermore, the mean number of nuclei per syncytium was markedly higher in PARP-overexpressing cells compared to control cells. However, PARP-1 overexpression did not regulate trophoblast hormonal differentiation. In conclusion, PARP-1 does appear to be a key enzyme in the process of trophoblast syncytialization. Lastly, a comparative analysis of PARP-1 was conducted in the mouse placenta. It was found that PARP-1 was highly detectable in nuclei of mononuclear trophoblast as well as the nuclei in the syncytiotrophoblast bilayer of the mouse labyrinth. Additionally, PARP-1 was localized to the nuclei of other trophoblast populations, including spongiotrophoblasts, trophoblast giant cell, and glycogen trophoblast giant cells, which are involved in the invasive pathway of trophoblast differentiation.

Thesis (M. Med. Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 82-86).

Thesis (M.Med.Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 82-86). Also available in print.