A proteomics approach was used to identify proteins potentially implicated in the cellular response concurrent with elevated production levels of human follicle stimulating hormone in a recombinant Chinese hamster ovary cell line (Darren cells), using zinc and sodium butyrate in the production media to increase expression. To this end, 2-dimensional gel electrophoresis (2-DGE) was utilized. Firstly, several aspects of 2-DGE were developed for this investigation. Gel drying conditions were optimized, and a glycine-free blotting method is described which achieved greater efficiency in rapid transfer of proteins than those previously described. Next, hFSH expression was characterized in Darren cells. An ELISA developed for this investigation examined intracellular (expression) and extracellular (secretion) of hFSH during increased expression. These results show a disproportionate increase in intracellular hFSH (188%) expression above extracellular hFSH (41%).

No description available.

A proteomics approach was used to identify proteins potentially implicated in the cellular response concurrent with elevated production levels of human follicle stimulating hormone in a recombinant Chinese hamster ovary cell line (Darren cells), using zinc and sodium butyrate in the production media to increase expression. To this end, 2-dimensional gel electrophoresis (2-DGE) was utilized. Firstly, several aspects of 2-DGE were developed for this investigation. Gel drying conditions were optimized, and a glycine-free blotting method is described which achieved greater efficiency in rapid transfer of proteins than those previously described. Next, hFSH expression was characterized in Darren cells. An ELISA developed for this investigation examined intracellular (expression) and extracellular (secretion) of hFSH during increased expression. These results show a disproportionate increase in intracellular hFSH (188%) expression above extracellular hFSH (41%).

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 388-416).

Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

<p>A saugeye is the progeny of a female walleye (<i>Sander vitreus)</i> and male sauger (<i>Sander canadensis)</i>. In the United States, hybrid saugeyes are considered important for recreational fisheries and as a potential food source. Saugeyes grow exceptionally faster than their non-hybrid parents and are more tolerant of a broader range of water conditions. They are also of interest to anglers due to their increased growth rate and ease to catch. Rather unexpectedly, biologists have recently observed fish that they believe to be saugeye in the Fort Wayne Rivers even though only walleye have been stocked in the area. The fish in Hurshtown Reservoir are believed to be walleye and the identification of those in the Three Rivers is unknown. A potential source for saugeye in the Fort Wayne Rivers is St. Marys State Fish Hatchery in Ohio. This research aims to determine if the fish found in the Fort Wayne Rivers are walleye or saugeye using microsatellite analysis. Microsatellites at seven loci were genotyped for 20 reference walleye, sauger, and saugeye as well as 21 unknown fish caught near Fort Wayne. Of the fish caught near Fort Wayne, three are from Hurshtown Reservoir and 18 are from the Three Rivers. Assignment tests of genotypes were completed using model and non-model based cluster analysis. Genotypic variation clearly resolved the two parent species from their hybrid offspring. Sixteen of eighteen <i>Sander</i> (unknown species) caught in Fort Wayne Rivers between 2018 and 2019 were determined to be first generation saugeye. The other two were walleye found in the Maumee River downstream of Hosey Dam. The three <i>Sander</i> caught in Hurshtown Reservoir were verified to be walleye. Sauger have never been stocked in the Fort Wayne Rivers and connecting waterways. Therefore, it is not likely that the saugeye found in the analysis are from natural reproduction. It is speculated that saugeye are swimming to Fort Wayne from hatcheries within the Maumee watershed. There are many potential sources for walleye in the Fort Wayne Rivers. </p>

<p>The production of developmentally competent in vitro derived embryos is necessary to decreasing both economic and emotional losses. Epigenetic abnormalities/insults have been shown to occur at a higher incidence in in vitro embryos. An increased prevalence of epigenetic derived disorders such as Parkinson’s disease, Prader-Willi syndrome, and α-thalassemia as well as elevated preimplantation embryo arrest and reduced developmental rates are theorized to be caused by errors in the mediation of chromatin remodeling. Chromatin remodeling refers to the restructuring of packaged DNA so that transcription factors are either given more or less access to specific sequences. This can be done by covalent modification through histone methylation, acetylation, and phosphorylation as well as noncovalent modifications which employ ATP dependent chromatin remodeling complexes. The purpose of this thesis was to characterize two structurally integral core subunits, BAF155 and BAF170, of the SWI/SNF chromatin remodeling complex in porcine oocytes and preimplantation embryos. </p> <p>The first study concentrated on the transcript abundance of BAF155 and BAF170 in porcine oocytes and embryos. First, BAF155 and BAF170 transcript sequences were identified in porcine muscle and heart tissues. Those sequences were used to create quantitative polymerase chain reaction (qPCR) primers. mRNA from pools of GV oocytes (100-800) was converted to cDNA for transcript abundance measurements. However, transcript abundance remained too low for either BAF155 or BAF170 to be accurately quantified. </p> <p>The second study focused on developmental competency of embryos post interfering RNA (RNAi) knockdown of BAF155, BAF170, or both BAF155/BAF170 combined. After 7 days of culture, an analysis of variance (ANOVA) was performed to determine differences in mean nuclei numbers and morphological blastocyst percentages across the three groups. No significant difference was seen between means of treatment groups vs. both control groups. Significant differences were seen between siRNA and Non-Injected groups as well as Non-Injected and Scramble RNA groups. However this indicates that loss of BAF155, BAF170, or a combination of the two transcripts is not the driving force of the significant differences, rather the microinjection itself caused the differences.</p> <p>The third study examined the process by which BAF155 and BAF170 proteins are imported from the cytoplasm into the nucleus. It was hypothesized that karyopherin α 7 (KPNA7), a nuclear importer known to be prevalent in the porcine oocyte and early embryo, is the main importer of both subunits. A dominant-negative KPNA7 construct missing the importin beta binding (IBB) domain was microinjected into parthenogenetically activated embryos to outcompete competent wild-type KPNA7. No change in protein localization was seen at the 4-cell stage of development (48 hours post-injection) for either BAF155 or BAF170. To reinforce these results, an RNAi targeting KPNA7 was also microinjected into parthenogenetically activated embryos. Again, no change was shown in protein localization at the 4-cell stage (48 hours post-injection), indicating that KPNA7 was not the main nuclear importer of either BAF155 or BAF170.</p> <p>Further study is necessary to determine transcript abundance and the mechanism of nuclear import of both BAF155 and BAF170.</p><div><br></div>

The Insect Cell - Baculovirus Expression Vector System (IC-BEVS) is widely used for the production of complex recombinant (glyco)proteins. The simplicity of insect cell cultivation in suspension serum-free media and the easy construction of recombinant baculovirus vectors have made the BEVS quite an effective expression system. On the other hand, the BEVS is a transient lytic system that may present some drawbacks in purification and potential degradation of the products. Among the various insect cell lines, the High-Five cell line has a great potential for the production of recombinant proteins using the BEVS in stirred bioreactors, reaching high cell densities and high protein production levels. Moreover, these cells can tolerate environmental stresses and can be cultivated on a large scale (Chapter 1). Unfortunately, up to now, there have been limited data available regarding suitable culture conditions and the metabolism of High-Five cells, a key requirement for the rational development of new processes. The overall goal of the present work was the study of these High-Five cells, in order to develop sophisticated new processes as alternatives to batch cultivation. The original contributions have been developed along two axes. The first axis concerns the study of the physiology and metabolism of High-Five cells. At first, we undertook a study aiming to prevent cell ring formation on suspension culture recipient walls (Chapter 2). Next, we analyzed environmental factors affecting insect cell growth and death, by comparing and developing methods able to distinguish between apoptosis and necrosis of cells (Chapter 3). The comprehensive study of the extended metabolism of High-Five cells was done using a metabolic flux network that takes account of the catabolism but also the anabolism of uninfected and baculovirus-infected cells (Chapter 4). The second axis was the application of the previously gained knowledge on High-Five cells to develop high-density systems specifically adapted to them: a fed-batch feeding strategy consisting of different pulses developed to increase the productivity of cells during infection (Chapter 5) and a fixed-bed reactor system (Chapter 6), as an alternative to classic perfusion, adapted to High-Five cells for recombinant protein production. In sum, new physiological and metabolic knowledge has been translated into new process options for High-Five cells.

In this study, we introduce novel, truly non-ionic hydrogen-bonded layer-by-layer (LbL) coatings for cell surface engineering capable of long-term support of cell function. Utilizing the LbL technique imparts the ability to tailor membrane permeability, which is of particular importance for encapsulation of living cells as cell viability critically depends on the diffusion of nutrients through the artificial polymer membrane. Ultrathin, permeable polymer membranes are constructed on living cells without a cationic pre-layer, which is usually employed to increase the stability of LbL coatings. In the absence of the cytotoxic PEI pre-layer, viability of encapsulated cells drastically increases to 94%, as compared to 20-50% in electrostatically-bonded shells. Engineering surfaces of living cells with natural or synthetic compounds can mediate intercellular communication, render the cells less sensitive to environmental changes, and provide a protective barrier from hostile agents. Surface engineered cells show great potential for biomedical applications, including biomimetics, biosensing, enhancing biocompatibility of implantable materials, and may represent an important step toward construction of an artificial cell.

<p>During gestation, the maternal liver undergoes various adaptive changes to cope with the increasing physiological and metabolic demands from both maternal and fetal compartments. Among these changes are robust growth and changes in transcriptome profile. However, how these events happen, and other aspects of this physiological phenomenon remains unexplored. Therefore, we aimed at further understanding how maternal liver responds to pregnancy. We used BrdU labeling combined with a virus-based tracing approach to quantify the percentage of maternal hepatocytes undergoing DNA synthesis and division over the course of gestation in mice. </p> <p>We found that ~50% maternal hepatocytes entered S-phase but, unexpectedly, did not undergo cytokinesis. This strongly suggests that maternal hepatocytes in fact undergo endoreplication instead of hyperplasia, as believed previously. Pericentral Axin2⁺ hepatocytes were reported to behave as liver stem cells responsible for liver homeostasis and turnover. We generated an <i>in vivo</i> fate-tracing mouse model to monitor the behavior of these cells in the maternal liver. Our results showed that they did not proliferate during pregnancy, homeostasis, and following partial hepatectomy. Curiously, we uncovered that, hepatocytes exhibit developmental phenotypes at mRNA level pre-pregnancy and at both mRNA and protein level during pregnancy. In the non-pregnant state, hepatocytes reserved mRNA expression of liver progenitor marker genes <i>Cd133</i> and <i>Afp</i>, which are localized in the nuclei, without protein translation. During gestation, maternal hepatocytes displayed cytoplasmic translocation of <i>Cd133</i> and <i>Afp</i> transcripts, concomitant with corresponding protein expression. </p> <p>Overall, all maternal hepatocytes became CD133⁺, and a subset of them express AFP. Additionally, in non-pregnant livers, mRNA of <i>Epcam</i>, another liver progenitor marker, was expressed within majority of hepatocytes, whereas its protein was solely translated in the pericentral region. In contrast, by end-gestation, EPCAM protein expression switched to the periportal region. These observations indicate that maternal hepatocytes exhibit heterogeneous developmental phenotypes, partially resembling fetal hepatocytes. It is intriguing why mature hepatocytes dedifferentiate into a progenitor state in response to pregnancy. AFP is considered to be produced primarily from fetal liver and thus is used to evaluate fetal development health. </p> A potential clinical relevance of our data is that we identified maternal liver as a new source of AFP. The hippo signaling pathway has been shown to potently control liver growth and hepatocyte heterogenicity. Surprisingly, we found that pregnancy neither altered the expression nor activities of the components of this pathway and its effector YAP1/TAZ. This finding indicates that pregnancy-induced maternal liver growth is not driven by hippo-YAP1 pathway. However, we demonstrate that the presence of YAP1 is essential for CD133 protein expression in maternal hepatocytes. Collectively, we revealed that, as pregnancy advances, maternal hepatocytes likely undergo endoreplication and display developmental phenotypes. Mechanistically, YAP1 dictates the expression of CD133, contributing to the pregnancy-dependent phenotypic changes of maternal hepatocytes.