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In pigs and other mammals, embryonic losses often occur during implantation when the conceptus (embryo plus its extra-embryonic membranes) attaches to the maternal uterine epithelium. Mechanisms controlling this process are not completely understood. Integrins and growth factors are among many molecules likely involved in controlling implantation. Numerous integrins (ITG), including subunits ITGAV (alpha v), ITGB1 (beta 1), ITGB3 (beta 3), and ITGB5 (beta 5), and transforming growth factor betas (TGFBs), in both latent and active forms, are present at the porcine conceptus-maternal interface. TGFBs are released as latent precursors which cannot interact with TGFBRs prior to their activation. Latency associated peptide (LAP), part of the TGFB latent complex, contains an amino acid sequence Arg-Gly-Asp (RGD) that is found in other extracellular matrix molecules and may interact with and signal through integrins. We hypothesize that LAP will bind to and activate ITGAV-containing heterodimers at the conceptus-maternal interface and that these interactions are a functional component of implantation. We also hypothesize that TGFB acting via TGFBRs has critical roles during peri-implantation, and such roles may include promoting conceptus development, survival, and adhesion. Immunofluorescence was used to colocalize TGFB, LAP, and integrins in porcine peri-implantation uterus and conceptus; immunohistochemistry of phosphorylated SMAD2/3 provided evidence of TGFB activity. Affinity chromatography identified cell surface integrins on porcine trophectoderm that are capable of binding LAP. In vivo, intrauterine infusions of LAP with its native RGD site (LAP-RGD) resulted in inhibition of conceptus elongation; LAP-RGE infusions yielded normal-appearing filamentous conceptuses at d13 of pregnancy. At d24, allantois length and fetal weights were greater in gilts which received LAP-RGE infusions compared to controls which received vehicle only. Results provide evidence for 1) active and latent TGFB in porcine conceptus and uterus; 2) receptor-ligand interactions of integrins and LAP; 3) integrin aggregation and potential focal adhesion formation at the conceptus-maternal interface; and 4) TGFB- and/or integrin-associated mechanisms which regulate conceptus elongation and placental and fetal size. Collectively, results suggest that TGFB and integrins are extensively involved in communication at the porcine conceptus-maternal interface, particularly regulating conceptus development, adhesion, and placental and fetal development.

Unique among the intracellular lipid binding proteins, acyl CoA binding protein (ACBP) exclusively binds long chain fatty acyl CoAs (LCFA-CoAs). To test if ACBP is an essential protein in mammals, the ACBP gene was ablated by homologous recombination in mice. While ACBP heterozygotes appeared phenotypically normal, intercrossing of the heterozygotes did not result in any live homozygous deficient (null) ACBP^(-/-) pups. Heterozygous and wild type embryos were detected at all postimplantation stages, but no homozygous ACBP null embryos were obtained– suggesting that an embryonic lethality occurred at a preimplantation stage of development, or that embryos never formed. While ACBP null embryos were not detected at any blastocyst stage, ACBP null embryos were detected at the morula (8- cell), cleavage (2-cell), and zygote (1-cell) preimplantation stages. Two other LCFACoA binding proteins, sterol carrier protein-2 (SCP-2) and sterol carrier protein-x (SCPx) were significantly upregulated at these stages. These findings demonstrate for the first time that ACBP is an essential protein required for embryonic development and its loss of function may be initially compensated by concomitant upregulation of two other LCFA-CoA binding proteins only at the earliest preimplantation stages. The fact that ACBP is the first known intracellular lipid binding protein whose deletion results in embryonic lethality suggests its vital importance in mammals.

Thèse d'exercice : Médecine. Ophtalmologie : Paris 12 : 2006. / Titre provenant de l'écran-titre. Bibliogr. f. 67-75.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 104-125).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

Zinc Oxide (ZnO)is a wide band gap semiconductor which has attracted great attention because of its wide applicability. In order to obtain semiconductor devices with stable and reproducible properties further study of deep level defects is essential. DLTS (Deep level Transient Spectroscopy) is a direct and straightforward techniqueto determine the energy level of the deep level defects. Other information such as activation energy and capture cross section of the defect can also be obtained through this method. In our study ZnO single crystal samples were implanted by oxygen with the energy of 150keV. After the pretreatment of hydrogen peroxide, Schottky contacts were fabricated with Au film deposited by thermal evaporation. Deep level defects were studied by deep level transient spectroscopy (DLTS). Single peak spectra were observed in the as-implanted sample and samples anneal at 350oC, 650oC and 750oC with the corresponding activation energy decreasing with the annealing temperature from ~0.29eV as found in theas-implanted sample. Three peaks were identified in the DLTS spectra of the 900oC sample, with the activation energies of 0.11eV, 0.16eV and 0.37eV respectively.After analysis in detail we found some peaks in the DLTS spectra were the combination of two other peaks, dominated in different temperature range. The thermal evolutions of the deep levels up to the temperature of 1200oC were also investigated. / published_or_final_version / Physics / Master / Master of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

MicroRNAs are small regulatory RNAs that bind to the seeding regions within the 3’-untranslated region (3’-UTR) of their target transcripts to modulate transcript stability and/or inhibit protein translation. MicroRNA Let-7a belonging to the Lethal-7 (Let-7) family is down-regulated at the blastocyst stage, suggesting its suppression is crucial for embryo implantation. Yet, the underlying mechanism on how Let-7a modulates blastocyst implantation remains largely unknown. In silico analysis identified attachment related integrin beta-3, outgrowth related vav3, and the microRNA processing dicer, as Let-7a targets. Therefore, it is hypothesized that down-regulation of Let-7a promotes embryo implantation by stimulating these target proteins. Let-7a is down-regulated during blastulation and at 3-hour post-estradiol activation of the dormant blastocysts. Force-expression of Let-7a in mouse blastocysts suppressed blastocyst attachment, outgrowth on fibronectin-coated plates and compromised pregnancy in vivo. Dual luciferase assay using the 3’-UTR reporter constructs of the integrin beta-3, vav3, and dicer confirmed the interaction between Let-7a and the three targets. Force-expressing or inhibiting Let-7a expression in mouse blastocysts by electroporating the Let-7a precursor or inhibitor respectively illustrated post-transcriptional regulation of Let-7a on integrin beta-3 and vav3, and transcriptional regulation on dicer. Dormant blastocysts retrieved from the delayed implanting mice expressed high Let-7a levels, which was suppressed in the first 12-hours of estradiol activation. Concomitantly, dormant blastocysts expressed low levels of integrin beta-3, vav3, and dicer, yet, their protein expression was up-regulated from 3 hours-post estradiol activation. Furthermore, addition of integrin beta-3 antibody suppressed attachment of trophoblast spheroids (blastocyst surrogate) onto endometrial epithelial cells in a co-culture model and the outgrowth of the spheroids on fibronectin-coated plates. Knockdown of Vav3 with siRNA decreased blastulation, hatching, and outgrowth rates of the embryos in vitro. Although the loss of vav3 activities did not affect embryo implantation, it suppressed trophoblast migration on fibronectin-coated plates and invasion into collagen matrix. In contrast, force-expression of vav3 enhanced blastocyst outgrowth, and promoted cell proliferation. Blocking integrin beta-3 activities in the vav3 knock-down embryos further suppressed blastocyst outgrowth, suggesting the intertwining effect of the integrins and vav3. Dicer knock-down in mouse blastocysts decreased mature Let-7a expression and suppressed blastulation and hatching in vitro and implantation in vivo. Dicer knock-down in estradiol activated mouse blastocysts decreased the epidermal growth factor receptor expression and lowered the affinity of the embryos to EGF, and suppressed the implantation potential to a level similar to that of dormant blastocysts. Taken together, the suppression of Let-7a by estradiol up-regulates integrin beta-3, vav3, and dicer. The increased Itgb3 expression promotes blastocyst attachment and intertwined with the up-regulated vav3 expression to enhance blastocyst outgrowth. The increased vav3 expression further stimulates cell proliferation and confers blastocyst invasion into the collagen matrix. Dicer, by altering microRNA processing, facilitates blastulation and thereby embryo implantation. Thus, the loss of Let-7a biological activities during blastulation is crucial to enhance blastulation and stimulate trophoblast activities for successful embryo implantation. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Traditional Chinese medicine (TCM) plays an important role in the Chinese healthcare system for over five thousand years. It includes the use of herbal medicine, acupuncture, Tui Na (推拿), and diet therapy. TCM helps to maintain a balance of Yin-Yang (阴阳), Five Phases (五行), Meridians (经络) and Qi (气) inside the body. In practise, pregnant women take tocolytic drugs to tonify the blood and qi to provide a continuous supply of nutrients for baby. Traditional Chinese herbal medicines usually prescribed as a complex formula to produce synergistic or agonistic effect to maintain a well balance of the above components in human bodies. Moreover, TCM usually cannot produce immediate effect on patients, therefore, the efficacy of individual component remains largely unknown. This study aims to investigate whether Chinese tocolytic drug components could modulate fertility by affecting the in vitro spheroid (blastocyte surrogate) attachment process by using trophoblastic (JEG-3) and endometrial epithelial (Ishikawa) cells to mimic the embryo-endometrial implantation process. Nine Chinese herbal medicinal compounds (Atractylenolide I(白术内酯), Atractylenolide II(白术内酯II), Atractylenolide III(白术内酯III), Paeoniflorin(芍药苷), Albiflorin(芍药内酯苷), Nuzhenide(女贞子甙), Ecliptasaponin A(旱莲甙A), Wedelolactone(蟛蜞菊内酯) and Columbianadin(二氢欧山芹醇当归酸酯)) which are commonly found in traditional Chinese tocolytic drug formula were selected to study (1) the toxicity of the drugs on trophoblastic (JEG-3) and endometrial epithelial (Ishikawa) cells growth, (2) the effect of three tocolytic drugs (Atractylenolide I, Atractylenolide II and Atractylenolide III) on spheroid attachment, and (3) their effect of the expression of Wingless (Wnt) signaling molecules (Active-β-Catenin, Axin-2, β-catenin, E-cadherin, GSK-3β, and Mucin-1). It was found that the nine compounds, Atractylenolide I, Atractylenolide II, Atractylenolide III, Paeoniflorin, Albiflorin, Nuzhenide, Ecliptasaponin A, Wedelolactone and Columbianadin did not affect cell viability at 25μM, 25μM, 5μM, 0.2μM, 125μM, 125μM, 125μM, 5μM and 25μM, respectively, by cell proliferation assay. However, at these concentrations, the spheroid attachment was not significantly increased by Atractylenolide I, Atractylenolide II and Atractylenolide III. Interestingly, the protein expression of GSK-3β and Active-β-catenin were up-regulated by the three compounds in both cells and JEG-3 cells respectively. The expressions of Axin-2 and E-cadherin were up-regulated by Atractylenolide III in Ishikawa cells and Atractylenolide II in JEG-3 cells. Atractylenolide I and Atractylenolide III increase the Ishikawa cells expression of Active-β-catenin and β-catenin respectively and together suppress the JEG-3 cells Mucin-1 and β-catenin expression. In conclusion, the nine tocolytic compounds have different effect on cell proliferation. Atractylenolide I, Atractylenolide II and Atractylenolide III did not enhance the attachment rate of JEG-3 spheroid onto Ishikawa monolayer. However, they affected Wnt-signaling molecules expression, suggesting that they may modulate endometrial receptivity. Further experiments are needed to study their combined effect on co-culture and expression of Wnt-signaling molecules. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Medical Sciences