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 dir="ltr">Delta-like 1 (DLL1) is a protein on the surface of the cell that serves as a ligand for NOTCH receptors. Like NOTCH receptors, NOTCH ligands span the membrane of the cell and contain extracellular, transmembrane, and intracellular domains. NOTCH activation occurs through contact-dependent interactions between the NOTCH receptor on one cell and ligand on an adjacent cell. Previous studies have demonstrated that DLL1 predominantly functions cell non-autonomously to trigger NOTCH signaling in a neighbor cell but a cell-autonomous function of DLL1 has also been proposed in recent years. However, there is no direct evidence to support a cell-autonomous function of DLL1 in vivo. The overall goal of this thesis was to elucidate the cell-autonomous function of DLL1 by testing the hypothesis that the intracellular domain of DLL1 (DLL1ICD) can be cleaved and function in the DLL1-expressing cell in addition to triggering NOTCH signaling in a neighbor cell. The research strategy to test the hypothesis is by overexpression of full-length (FL) and cleaved Dll1 (Dll1ICD) in the murine myoblast cell line (C2C12). These plasmids utilize a tet-on system for inducible overexpression. Transfected myoblasts were used to analyze how overexpression of Dll1-FL and Dll1ICD affects proliferation, differentiation, fusion, and gene expression. The results show that Dll1-FL can be cleaved to generate an ICD. DLL1ICD overexpression promotes fusion without affecting proliferation. Further investigation reveals that overexpression of Dll1ICD affects the expression of NOTCH and myogenic-specific genes during differentiation, confirming the cell-autonomous role of DLL1ICD in myoblasts. These results together show that DLL1 can function cell autonomously through its intracellular domain to regulate myogenesis.</p>

<p dir="ltr">Adipose tissue consists of adipocytes that store energy within lipid droplets and are a central component of lipid metabolism. Mammals contain white, brown and beige adipocytes, which differ in their metabolic roles. White adipocytes store energy, in the form of triglycerides, within lipid droplets and predominantly take on an energy storage role. Brown and beige adipocytes promote energy expenditure and the dissipation of energy as heat through non-shivering thermogenesis. Since energy expenditure combats excess caloric intake and overeating, non-shivering thermogenesis has become heavily researched for its potential therapeutic use in combatting the continued increase in obesity and metabolic disorders worldwide.</p><p dir="ltr">In addition to ATP synthesis, mitochondria are required for a multitude of metabolic processes that maintain cellular homeostasis, including non-shivering thermogenesis. Brown and beige adipocyte mitochondria are specialized to perform non-shivering thermogenesis in response to an environmental stressor like cold exposure. Uncoupling protein 1 (UCP1) is uniquely characteristic of brown and beige adipocyte mitochondria, because it allows oxidative phosphorylation to be uncoupled from ATP synthesis. In order to enhance non-shivering thermogenesis, ongoing molecular characterization of brown adipose tissue (BAT) is being conducted to identify proteins that regulate mitochondrial function and UCP1 activity. In this study, I explored the function of LETM1-domain containing 1 (LETMD1), a novel mitochondrial inner membrane protein with unknown function in BAT. We generated a global (<i>Letmd1</i>^<em>KO</em>) and UCP1+ cell-specific <i>(Letmd1</i>^<em>UKO</em><i>) knockout</i><i> </i>mouse model to study the whole-body and cell-autonomous role of LETMD1 in BAT, respectively. Loss-of-function studies resulted in striking, BAT-specific phenotypic differences, including whitened BAT under thermoneutral, room temperature and cold exposure. Both knockout models were cold intolerant without access to food, and became hypothermic within a few hours of fasted cold exposure. Loss of normal mitochondria structure and cristae arrangement were also evident in knockout BAT, resulting in a decreased number of mitochondria and decreased number of cristae per mitochondrion. Mitochondrial DNA copy number was also significantly decreased in both knockout models. Abnormal mitochondria morphology was supported by increased reactive oxygen species (ROS) accumulation in both knockout models and the visualization of protein aggregates and mitophagy-like morphologies in <i>Letmd1</i>^<em>UKO</em><i> </i><i>mice specifically</i>. TurboID proximity labeling of brown adipocytes revealed enrichment of several respiratory chain complex proteins, mitochondrial ribosome proteins and mitochondrial protein import machinery. Moreover, the aggregation of misfolded nuclear-encoded mitochondrial proteins, including several respiratory chain and mitochondrial ribosome proteins, suggested that LETMD1 facilitates mitochondrial protein import and mitochondrial ribosome assembly, thereby compromising respiratory chain assembly and function during non-shivering thermogenesis. Overall, this study identifies LETMD1 as a novel regulator of brown adipocyte mitochondrial structure and thermogenic function and highlights the requirement of LETMD1 for mitochondrial biogenesis.</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.