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

Leucine Zipper-like Transcription Regulator 1 (LZTR1) is responsible for encoding a member of the BTB-Kelch superfamily, which interacts with the Cullin3 (CUL3)-based E3 ubiquitin ligase complex. Researchers have discovered mutations in LZTR1 in glioblastoma, schwannomatosis, and Noonan syndrome. However, the exact function of LZTR1 in cancer development or human growth remains unclear. A previous study in the Neel lab showed there was a cell-context dependent regulation of pan RAS (K/N/H RAS) levels by LZTR1. This thesis's main objective was to understand the underlying mechanisms for the above observation. The LZTR1 gene has gained importance in human well-being due to its essential role in cellular activities and its link to various diseases. Investigating the range of functions of LZTR1 and the underlying mechanisms will offer new perspectives on disease prevention and therapeutic approaches. These results provide hints for deciphering the mechanisms of RAS degradation and controlling the RAS/MAPK signaling pathway.

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