Calcium ([Ca2+]i) oscillations, a hallmark of mammalian fertilization, are essential to induce egg activation and embryonic development. As the zygotes transition through the cell cycle, these [Ca 2+]i oscillations progressively diminish until they cease in interphase zygotes. While the mechanism(s) underlying the regulation of [Ca2+]i oscillations may be multi-layered, inositol 1,4,5-trisphosphate receptors (IP3Rs) emerge as a focal point in this regulation. IP3Rs, the calcium channels expressed in all cell types and abundantly expressed in mammalian eggs, contain consensus sequences for phosphorylation by various kinases and interact with members of the cytoskeleton, serving as an integrator of regulatory signals. This dissertation highlights the impact of biochemical and cellular changes in IP3R-1 on fertilization-induced [Ca2+]i oscillations. Together with the changes in the cellular distribution of the sperm factor, these cell cycle-dependent modifications in IP3R-1 may underlie the regulation of [Ca 2+]i oscillations in fertilized mammalian eggs.

The nuclear receptor superfamily is a large group of related receptors that bind steroid hormones, signaling molecules, or xenobiotic chemicals and are expressed across many mammalian tissues. The impact of nuclear receptor activation using two different mouse model systems is explored in this thesis: (1) in utero exposure of the environmental xenoestrogen and proposed endocrine disruptor bisphenol A (BPA) and (2) short adult exposures to the mouse constitutive androstane receptor (CAR) specific agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). First, experiments involving the impact of in utero BPA exposure on the male mouse reproductive tract are described. Minimal changes to long-term mouse testis morphology and function were observed as mice treated with BPA in utero did not show significant changes in spermatozoa production or testis histopathology. Microarray analysis showed few persistently dysregulated genes, none of which were validated using qPCR due to high variability among biological replicates. Next, nuclear RNA-seq was used to characterize global changes in the mouse liver transcriptome following exposure to TCPOBOP, including changes in novel long non-coding RNAs that may contribute to xenobiotic-induced pathophysiology. Dysregulated protein coding genes were associated with a striking male-biased pro-tumor response, including activation of pro-tumor upstream regulators such as cyclin D1 and inhibition of tumor suppressors such as p21 and p53, consistent with the reported male-biased susceptibility to CAR-dependent mouse liver tumorigenesis. Novel long non-coding RNAs were identified in livers of mice exposed to TCPOBOP, including lncRNAs proximal to the CAR target genes like Cyp2b10. Then, DNase-seq was used to identify DHS in male and female mouse liver that open or close following TCPOBOP treatment proximal to CAR responsive coding and non-coding genes. Finally, a series of ChIP-seq experiments targeting the activating histone modifications H3K4me1, H3K4me3 and H3K27ac, and the repressive chromatin modification H3K27me3 were performed in male mice to characterize the corresponding changes in local chromatin environment around DHS and responsive genes. Using a combination of DNase-seq and ChIP-seq, several classes of DNA regulatory elements have been identified, including active enhancers and promoter regions that may play a function role in regulating nearby CAR-responsive protein-coding and lncRNA genes. / 2020-10-08T00:00:00Z

There are various novel approaches to cancer immunotherapy. These potential treatments for malignant cancers include the adoptive transfer of natural killer cells, T cell receptor T cells, chimeric antigen receptor T cells, and many more. It is important to note that conducting these revolutionizing treatments became possible due to the humanized mouse models, which provide the experimental base models for investigating anti-tumor research. To further enhance the measure of treatment for malignant cancers, it is vital to understand how the humanized mouse models work and represent the human immune microenvironment. Here, I review the history of the humanized mouse models, their variations and advantages and limitations that follow with the different types of models, their roles in evaluating the potential cancer immunotherapy treatments, and their anticipated roles in the future to further assist in developing a more personalized antitumor treatment.

As the principal cellular agents initiating thrombus (clot) formation, the primary function of platelets is maintenance of blood hemostasis. These anucleate cell fragments lack nuclear DNA and so provide a unique model for investigating post-transcriptional regulation of gene expression via RNA interference (RNAi). Although platelets are not capable of transcribing nascent mRNA, they are able to maintain protein homeostasis throughout their ~10-day lifespan in circulation; how platelets are able to do so, considering they contain a functional proteasome, remain unclear. We hypothesized that platelet protein translation and homeostasis is driven by ongoing plasma-derived growth factor signaling balanced with degradation, which is regulated by Argonaute 2 (Ago2)-mediated RNAi. In nucleated cells, RNAi occurs via RNA-induced silencing complexes (RISC), in which a guide RNA such as a microRNA (miRNA) docked in an effector protein, typically Ago2, recruits a target mRNA via guide:target sequence complementarity and results in suppression of mRNA translation via multiple mechanisms. Though RNAi has not yet been fully explored in the context of anucleate cells, platelets contain the machinery and proteins necessary for RNAi as well as being highly enriched in a diverse array of miRNAs. Additionally, variations in platelet miRNAs in humans are associated with variable platelet reactivity and risk of thrombosis.   We have found platelets to be capable of taking up exogenous double-stranded miRNA and have published on their subsequent ability to utilize the miRNA to modulate signal-activated translation and platelet function (Aim 1). In order to investigate the role of constitutive translation in circulating platelets (Aim 2) and the effect of Ago2 in regulation of platelet reactivity (Aim 3) we generated a novel platelet-specific Ago2 deleted (Ago2fl/fl/Pf4-Cre) mouse line and compared the proteome of knockout (KO) mice to wild-type (WT), by sex. Based on Ago2 roles in RNAi and miRNA enrichment in platelets, we predicted large-scale increases in platelet protein expression with megakaryocytic Ago2 deletion. However, only a small subset of genes was significantly upregulated. Among these was Ago1, which was significantly increased in both male and female Ago2 KO platelets. These results strongly suggest that Ago-mediated RNAi is essential for platelet development. Interestingly, deletion of platelet Ago2 resulted in decreased protein expression of a different subset of genes in platelet from female mice only; this sex-specific effect may be due to hormonal differences, namely estrogen, as many of these regulated proteins are encoded by estrogen-responsive genes. The gene set downregulated in Ago2 KO female platelets vs WT, but not males, corresponded to genes known to be coordinately regulated at the transcriptional level by ERβ in conjunction with Ago2 in breast cancer cells. These results point to a novel role for Ago2 in modulating the platelet transcriptome in megakaryocytes, affecting the mRNAome in the platelets they generate, in a hormone-dependent manner.   Platelets from both male and female KO mice had significantly increased mean platelet volumes (MPV), not explained by changes in platelet production, lifespan, or protein content; however, KO megakaryocytes (MKs) had significantly increased size & ploidy, accounting for increased MPV, although femurs in the KO mice had fewer MKs. Furthermore, KO mice exhibited faster clot retraction and increased surface expression of integrin αIIbβ3. Platelets from male KO mice showed increased αIIbβ3 integrin activity and P-selectin expression following thrombin and U46619 stimulation, a thromboxane analogue, but these effects were not seen with collagen or ADP, pointing to agonist pathway-specific effects. Bioinformatics analysis revealed sex-specific changes in the platelet proteome of KOs to account for this change in reactivity; a cluster of mitochondrial enzymes modulated in male KOs only, involved in ROS metabolism and respiration, may be responsible for the observed difference in the thromboxane response. Taken together, our findings suggest Ago2-mediated RNAi to be an important regulator of megakaryopoeisis and platelet reactivity. / Biomedical Sciences

The work undertaken in this thesis was directed towards understanding the organization of lipids in cell membranes and the lipid specificity of perforin. Three aspects of the organization of membrane lipids were investigated, (1) the asymmetrical distribution of lipids between the outer and inner leaflets of the membrane, (2) the bobbing motion of lipids perpendicular to the plane of the membrane, and (3) the spacing of lipids in the external leaflet of the membrane. The binding of perforin to membranes was studied by (1) comparing the ability of liposomes constructed from defined lipids to bind perforin, and (2) the ability of perforin to bind to and lyse lipid symmetric and lipid asymmetric erythrocyte membranes. The asymmetrical distribution of lipids in the plasma membrane of erythrocytes was investigated by examining two mechanisms for maintenance of lipid asymmetry: binding of internal lipids to cytoskeletal proteins, and pumping of internal lipids from the outside to the inside of the cell. Analysis of the kinetics of lipid internalization suggested the presence of an ATP-dependent lipid flip/flop catalyst. The thermal energy of lipids results in oscillatory motion perpendicular to the plane of the membrane, and in movement in the plane of the membrane. The vertical distribution of lipids arising from this oscillatory motion was calculated. The motion of lipids in the plane of the membrane results in an average spacing between the lipid molecules. Previous reports, had demonstrated that the fluorescent dye merocyanine 540 bound preferentially to widely spaced phosphatidylcholine lipids. MC540 was shown to have little headgroup, sidechain or cholesterol specificity per se, but bound preferentially to membranes whose lipids are widely spaced, suggesting its use as a probe of lipid spacing. Comparison of perforin binding to liposomes constructed from defined lipids revealed that perforin bound preferentially to liposomes whose lipids were widely spaced. Using erythrocytes as a model, it was observed that increasing the spacing of lipids in the outer leaflet of the plasma membrane resulted in increased perforin binding and susceptibility to perforin-mediated lysis, confirming the conclusions derived from the liposome studies and further suggesting that increased lipid spacing increases overall susceptibility to the more complex processes involved in cellular lysis by perforin. The external phospholipids of the plasma membrane of the CTL were shown to be more tightly packed than the lipids of the more-susceptible target cell, suggesting that the resistance of CTL to perforin is at least in part due to the presence of closely spaced lipids in its membrane.

This study evaluated procedures to improve nuclear transplantation efficiency in rabbit embryos. Recently ovulated oocytes were enucleated and fused at a higher rate than aged oocytes and were efficiently activated by multiple electrical pulses. Repeated stimulations also improved development of reconstituted embryos in vitro. Also, manipulating oocytes in bicarbonate-buffered medium, and cytochalasin B in the post-fusion medium enhanced development in vitro and to term. The profile of nuclear remodelling in reconstituted embryos was characterized and the relationship between chromatin behavior after transfer and embryo development determined. Upon blastomere fusion to non-activated cytoplasm, nuclear remodelling was characterized by premature chromosome condensation (PCC) and nuclear swelling. Fusion to activated cytoplasm prevented PCC and extensive nuclear swelling, but allowed development to blastocysts. The results indicate that remodelling of the donor nucleus was not essential for development to blastocysts; however, it was beneficial. The influence of cell cycle stage of the donor nucleus on development of reconstituted embryos was determined. Use of early cell cycle nuclei resulted in high rate of development to blastocysts, whereas mid and late cell cycle donors led to reduced development. Synchronizing blastomeres in G1 entailed incubation in colcemid to arrest embryos in M phase and in aphidicolin to synchronize embryos at the G1/S boundary. Development to blastocysts was enhanced with G1 nuclei, as opposed to late S. Therefore, extent of development was reduced as donor nuclei progressed in the cell cycle. The effect of donor cell cycle stage on chromatin and spindle morphology in manipulated embryos was determined. After blastomere fusion, an attempt to form a spindle and a metaphase plate occurred in G1, early S and, to some extent, late S transplants. These structures displayed minor and gross abnormalities in early and late S transplants, respectively. G1 and early S transplants contained normal chromosomes, whereas defects occurred in late S transplants. Therefore, PCC in G1 and early S had a minor influence on chromosome constitution of manipulated embryos. PCC in late S, however, affected chromosome conformation and may account for impaired embryo development.

The growth control and response to extracellular adenosine stimulation in pig aortic smooth muscle cells cultured in different conditions were tested. Cells that were cultured in medium containing 10% fetal bovine serum were proliferative. Cells were quiescent when cultured in a chemically defined medium, which contains 10$\sp{-6}$ M insulin, 5 $\mu$g/ml transferrin, and 0.2 mM ascorbate. The response to adenosine stimulation in the quiescent cells was lower than that observed in cells cultured in serum-containing medium. Both the cell growth index and the response to adenosine of cells cultured in defined medium were reversible after replacing the medium with 10% fetal bovine serum-containing medium. These results suggested that the cells in defined medium were healthy and were capable of modulating cellular metabolism depending on culture conditions. The role of phosphodiesterase (PDE) in modulating cellular cAMP levels was also studied in pig aortic smooth muscle cells cultured in serum-containing medium. Three different PDE isozymes were separated by Mono Q HPLC chromatography and identified by selective PDE inhibitors. They are calmodulin-dependent, cGMP-inhibited, and rolipram-sensitive PDEs. Each of the isozymes was differentially distributed in subcellular fractions. These three PDE isozymes present in smooth muscle cells play an important role in modulating cellular cAMP levels after adenosine stimulation. The differential importance of each isozyme may depend on cellular cAMP levels.

HeLa cells, a transformed human epithelial cell line, attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen receptor upregulation, clustering, and binding to the cytoskeleton. The purpose of this research is to examine whether collagen receptor interaction with gelatin induces formation of a second messenger(s) which turns on cell functions leading to spreading. Levels of arachidonic acid (AA) are increased upon attachment and prior to spreading of HeLa cells on a gelatin substratum. Inhibition of phospholipase A2 (PLA2) blocks AA release and cell spreading. Among the inhibitors of AA metabolic pathways, only inhibitors of lipoxygenase (LOX) block cell spreading indicating that a LOX metabolite(s) of AA is a second messenger(s) which initiates HeLa cell spreading. AA appears to be released from phosphatidylcholine (PC) since levels of PC which contain AA decrease during cell spreading. Also, lysophosphatidylcholine is the only lysophospholipid which is detected during cell spreading. AA is released upon clustering of collagen receptors. Receptor occupancy is not sufficient to release AA. The formation of a LOX metabolite(s) appears to induce production of diacylglycerol (DG) which is correlated with activation of protein kinase C (PKC). PKC is activated upon attachment and prior to spreading. Inhibition of PKC blocks cell spreading, and the inhibition is not reversed by addition of AA. However, inhibition of cell spreading by inhibitors of PLA2 or LOX is overcome by PKC activation. This indicates that PLA2 activation occurs prior to PKC activation, and that direct activation of PKC bypasses the requirement for AA release and LOX metabolite formation. Activation of PKC with phorbol ester also enhances cell spreading. The ability of PKC to induce cell spreading appears to be mediated by the modulation of F-actin formation. Cell spreading is accompanied by an increase in F-actin content. Inhibition of PKC blocks the relative increase in F-actin content. By contrast, treating cells with PKC activator prior to spreading assays not only enhances cell spreading but also increases the relative F-actin content. Cell spreading is an amplification process centered on the production of AA. AA production is amplified by hydrolysis of DG and activation of PKC.

Microtubules are dynamic polymers which play important roles in mitosis, cell locomotion, and the determination and maintenance of cell polarity. To understand the role of microtubule assembly and disassembly in these processes I have localized sites of microtubule growth by injecting labeled tubulin subunits into cells during spindle elongation (anaphase B) and chromosome separation (anaphase A). The behavior of microtubules in interphase cells was also examined by injection of fluorescent tubulin subunits, and observation of the resulting fluorescent microtubules using low light level fluorescence microscopy. These experiments demonstrate that rapid assembly of interzonal microtubules occurs concomitantly with the maximal rate of spindle elongation. Furthermore, the rate of interzonal microtubule elongation is greater than the rate of spindle pole separation, and the pattern of microtubule assembly is most consistent with the dynamic instability model of microtubule behavior. These observations demonstrate that interzonal microtubule assembly is not directly coupled to spindle elongation in these cells and place constraints on existing models of spindle pole separation. Electron microscopic and confocal fluorescence observations of cells injected during anaphase A reveal that injection of biotin-tubulin can induce the assembly of kinetochore microtubules at their plus-ends--normally the site of microtubule disassembly at this stage--in a concentration dependent manner. The elongation of kinetochore microtubules is accompanied by the reversal of chromosome-to-pole motion. Thus, anaphase onset does not prevent addition of tubulin subunits at the plus-end of kinetochore microtubules, and some aspects of mitosis can be regulated by the concentration of free tubulin in vivo. Finally, fluorescent microtubules in living CHO fibroblasts and PtK$\sb1$ epithelial cells have been examined using a 2 second observation interval. These observations confirm results of earlier studies which demonstrate that microtubules turn over more rapidly in fibroblasts than in epithelial cells. Surprisingly, quantitative analysis of individual microtubule behavior reveals that microtubules in epithelial cells undergo depolymerization more frequently than microtubules in fibroblasts. However, microtubules undergoing disassembly are rapidly rescued in epithelial cells but not fibroblasts. These results demonstrate that microtubule behavior is regulated in a cell type specific manner and suggest that epithelial cells contain regulatory factors not found in fibroblasts.

Macrophages are a heterogeneous population of immune cells that play a critical role in our innate and adaptive immune systems. The classical understanding of macrophages was centered around the idea that all macrophages are derived from circulating monocytes produced in the bone marrow. In the last decade, our understanding has changed, and studies have indicated that tissue-resident macrophages (TRMs) are of embryonic origin, not bone marrow-derived. The characteristics of TRMs are well-defined in mice, however not all mouse-related information applies to humans. We aim to clearly define the characteristics of human tissue-resident macrophages. This will be accomplished through morphological observations, assessing the immunophenotypic profile, gene expression, macrophage polarization state, and phagocytic capabilities. Our findings indicate that fetal-derived macrophages display a TRM phenotype, further expanding our understanding of human macrophages. / 2026-02-14T00:00:00Z