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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

Proteomic Analysis of Chinese Hamster Ovary Cells Producing Glycosylated Monoclonal Antibodies

Ho, Raymond January 2013 (has links)
Therapeutic monoclonal antibodies (MAb) are produced as secreted complex glycoproteins from mammalian cell systems and represent one of the most important classes of therapeutic medicines for the treatment of a variety of human diseases. Their benefit in health care and high economic impact provide the driving force for the development of improved production levels with the focus of optimizing clinical efficacy. One important issue is the optimization of monoclonal antibody production. A frequent approach used to address this challenge is the engineering of mammalian cell lines to increase antibody production levels through genetic manipulation. Valuable information can then be obtained by monitoring the effects of genetic changes on the biochemistry of the cell associated with MAb production. Global protein expression profiling of mammalian cells used for the production of biopharmaceuticals may reveal key biochemical characteristics associated with MAb-producing cell lines. A better understanding of these characteristics can in turn lead to more rational strategies for cell line and process development. The proposed research relates to a larger NSERC Strategic Network (MAbNet) Grant to develop and establish a novel platform for the large-scale manufacture of specific glycoforms of therapeutic monoclonal antibodies. The efficacy of these recombinant MAbs will be enhanced by the control of their glycosylation profiles. The work presented in this thesis will assist MAbNet in meeting their objectives. Specifically, we use 2D-Differential In-Gel Electrophoresis (2D-DIGE) to quantify protein expression differences between EG2-hFc1-producing Chinese Hamster Ovary cells (CHO-1A7) with its parental cell line (CHO-BRI). Here, we identified 34 unique differentially expressed proteins associated with EG2-hFc1 production that relate to various biological processes including protein processing, carbohydrate metabolism, amino acid metabolism, energy metabolism, apoptosis, and cell proliferation pathways. The majority of identified significant protein expression changes and their associated metabolic processes seem to prioritize energy production in CHO-1A7 cells. Due to the metabolic load of recombinant antibody production, the CHO-1A7 cell line attempts to meet the energy requirements needed for recombinant protein biosynthesis while maintaining cell viability and efficient protein folding mechanisms. A 2-D proteome reference map was also constructed for the CHO-BRI host cell line containing 131 identified protein spots. The map provides information that will further expand our understanding of this particular cell line. It will be a useful tool for studies investigating physiological responses and protein expression patterns of CHO-BRI to genetic and environmental perturbations. The set of identified differentially expressed proteins provides data on the downstream changes in protein expression due to genetic manipulation, and furthermore can provide targets for cell-line specific optimization of antibody production. The work described in this thesis furthers our understanding of antibody production in a specific CHO cell line.

Transient production of biopharmaceutical proteins

Wei, Tzu-Hsiang, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2009 (has links)
The creation of stable mammalian cell lines for biopharmaceutical production often require several months, and is unfavourable for the rapid production of multiple drug candidates for screening in the early stages of development. Biopharmaceutical production by transient transfection provides a possible alternative of quickly producing these early stage drug candidates. The Epi-CHO transient expression system, which consists of a Chinese hamster ovary (CHO) cell line (CHO-T) expressing the murine polyomavirus Large T-Antigen (LT), emonstrated enhanced transient recombinant protein production. The aim of this study was to prolong transient recombinant protein prod.Jction of the Epi-CHO expression system by creating a CHO cell line expressing both LT and EBNA1 (ECHO-T). The pEBNA1-LT expression vector encoding LT and EBNA1 was constructed and transfected into CHO-K1. A total of 20 clones were isolated from the antibioticresistant pool and screened for the expression of functional LT and EBNA1. PCR analysis showed 16 of the 20 clones was positive for EBNA1 and LT DNA. Of the 16 clones, six were positive for EBNA1 and LT expression by RT-PCR. Detection of LT and EBNA1 by immunofluorescence showed positive staining for the P7-G3 clone. Western blotting suggested the P7-G3 clone was: positive for EBNA1, and clones P3-C7 and P7-E2 were positive for LT. A plasmid replication assay confirmed the expression of functional LT in all six clones. Plasmid maintenance assay confirmed clone P7-G3 as the ECHO-T clones to express functional EBNA1. The P7-G3 clone demonstrated prolonged and sustained transient recombinant protein expression when compared to CHO-T. The P7-G3 clone achieved sustained transient protein expression for 32 days in the absence of selection, the longest currently reported for CHO cells.

Polycystic ovary syndrome : a study of adipocyte lipolysis in relation to endocrine and metabolic status /

Ek, Ingvar, January 2003 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Human ovulation : studies on collagens, gelatinases and tissue inhibitors of metalloproteinases /

Lind, Anna Karin, January 2006 (has links)
Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2006. / Härtill 4 uppsatser.

Effecten van een oraal contraceptivum bij de rhesusaap een morfologisch en biochemisch onderzoek /

Vooijs, Gijsbert Peter. January 1971 (has links)
Thesis--University of Nijmegen. / Summary in English and Dutch. Includes bibliographical references (p. 101-108).

Effects of the common pesticide methoxychlor on ovarian steroidogenesis

Akgul, Yucel. January 2007 (has links)
Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains xi, 137 p. : ill. Includes abstract. Includes bibliographical references.

Investigating follicle development using in vitro technologies

Lo, Belinda January 2017 (has links)
Patients with dysfunctional ovaries, such as those with premature ovarian insufficiency and granulosa cell tumours, do not have normal follicle development and may not respond to traditional assisted reproductive techniques. Using the reaggregated ovary (RO) technique, these patients' oocytes may be reaggregated with functional supporting cells and cultured in vitro to develop fertilisable eggs. However, current research using ROs have only used murine ovaries as a somatic cell source. In this thesis, with the aim of moving towards a clinical treatment, we assessed follicle development in ROs in vitro and progressed to using the technique with human tissues. To assess whether an older murine somatic cell source resulted in advanced follicle development, and how follicle development differed between transplanted and cultured ROs, ROs were generated using postnatal day 2 (P2) and P6 mouse ovaries. To investigate theca cell development in follicles from cultured tissue, mouse ovaries were cultured with mouse serum or encapsulated in hyaluronan hydrogels. Prior to generating and culturing chimeric human-mouse ROs (HuMoROs), competent handling and digestion of bovine cortical tissue was required. Broadly, ROs generated from both P2 and P6 exhibited similar follicle development in vitro after 14 d of culture, and follicles from cultured ROs were more developed than those from transplanted ROs. Theca cell development observed in follicles from cultured ovaries was still poorer than those from in vivo ovaries, even when ovaries were cultured in mouse serum or encapsulated in a hyaluronan hydrogel. Finally, some follicles containing potential human oocytes developed within the generated HuMoROs after 7 d of culture. These results have highlighted the potential of the RO technique as a method to generate fertilisable eggs and identified further aspects which need to be targeted in order to improve the success of the technique.

Fetal programming of adult disease : causes and consequences of metabolic dysregulation in an ovine model of PCOS

Siemienowicz, Katarzyna Joanna January 2018 (has links)
Polycystic ovary syndrome (PCOS) is a common and complex endocrine condition with reproductive and metabolic complications, affecting up to 10% of reproductive-age women. Hyperandrogenemia, ovulatory dysfunction, and luteinising hormone hypersecretion are characteristic traits of PCOS however, it seems that the most concerning long-term key issues are metabolic problems associated with the syndrome, such as hyperinsulinemia, insulin resistance, obesity, dyslipidaemia and non-alcoholic liver disease. Despite the numerous studies on PCOS, its origin and pathophysiology are still not fully understood. However, there is increasing evidence that the adult PCOS phenotype is programmed in fetal life by androgen excess. Exposure to increased levels of testosterone in utero in rodents, sheep and monkeys result in adult reproductive and metabolic pathologies that parallel those seen in PCOS women. Since hyperandrogenemia is a hallmark of PCOS and daughters of PCOS mothers have elevated levels of androgens at birth, it is likely that prenatal androgenisation during early life predispose to the future development of PCOS. Animal models of PCOS provide an opportunity to examine the developmental aetiology and molecular mechanisms underlying the pathogenesis of this condition. Over last 10 years our lab has successfully utilised a well-established ovine model of PCOS, where pregnant ewes were treated with testosterone propionate (TP) through mid-gestation. From this model, we had a large sample bank of fixed and frozen tissues from the fetal, lamb and adolescent prenatally androgenised animals that allowed to carry a broad range of experiments. In addition, a new cohort of prenatally androgenised adult sheep enabled additional in vivo analysis. Past research documented that prenatal androgenisation result in hyperinsulinemia with altered pancreas structure and function, and early fatty liver without difference in body weight in adolescent sheep. This thesis examines the effects and consequences of increased in utero androgen exposure on metabolic dysregulation in adolescent and adult female sheep. During puberty, but not fetal or early life, there was decreased adipogenesis in subcutaneous adipose tissue (SAT), but not visceral adipose tissue (VAT), accompanied by decreased circulating concentrations of fibroblast growth factor 21 (FGF21), leptin and adiponectin, and increased concentrations of fasting free fatty acids (FFA) in prenatally androgenised sheep. This was countered by upregulated expression of FFA transporters in liver. As adults, TP-exposed animals had increased body weight, elevated fasting insulin and FFA concentrations but normal FGF21, leptin and adiponectin levels. Histological analysis revealed that adult TP-exposed animals had SAT hypertrophy, which was associated with increased expression of inflammatory markers and correlated with increased fasting FFA. Therefore, it is likely that impaired preadipocyte differentiation in SAT during adolescence resulted in hypertrophy and inflammation of adult SAT. This consequently lowered capacity of SAT to safely store fat and potentially explains metabolic perturbations observed in PCOS-like female sheep. To further investigate potential causes of obesity in adult PCOS-like sheep postprandial thermogenesis (PPT), an important constituent of energy expenditure, was measured through implantation of datalogger thermometers into interscapular adipose tissue. Adult prenatally androgenised sheep had decreased amplitude of PPT, without difference in basal body temperature, despite receiving the same caloric intake, and independent of obesity. These findings indicate that adult PCOS-like sheep have reduced capacity for energy expenditure, which is mirrored in women with PCOS. This reduced capacity for postprandial thermogenesis was correlated with hyperinsulinemia decreased noradrenaline levels and reduced thermogenic potential of brown and/or beige adipose tissue. This suggests that women with PCOS might be prenatally programmed to become obese. In summary, findings documented in this thesis provide better understanding into the pathophysiology of PCOS from puberty to adulthood and give opportunities for early clinical intervention to ameliorate the metabolic phenotype of PCOS.


Doan, Huyen Van 01 May 2013 (has links)
Blastocyst implantation is the process in which a competent blastocyst acquires the ability to tether into the mother endometrium. At the same time, the endometrial tissue undergoes the process of decidualization to support the anchoring of the blastocyst and provides the blastocyst with nutrition until the fully functional placenta is formed. Although the process of implantation and decidualization are under control of progesterone and estrogen, the precise mechanisms involved in this regulation are not fully understood. Here, we report the expression and function of a transcription factor, HAND2, in sensitizing mouse uterus for implantation and decidualization. In mouse, HAND2 expression was localized mainly in the endometrial stromal cells even before the blastocyst implantation. The expression of HAND2 increased after blastocyst implantation and correlated with the increase in decidual compartment. The expression of HAND2 depended on progesterone but not estrogen. Further investigation using conditional knockout mouse revealed that HAND2 was important for both implantation and decidualization. Hand2d/d mice were infertile and had defects in decidualization. It seemed that HAND2 was an important factor that mediates the anti-estrogenic effect of progesterone on luminal epithelial proliferation. The abnormal in expression of Mucin 1, Calcitonin and E-Cadherin in Hand KO uterus may be responsible for defects in the uterine receptivity. The expression of HAND2 was also critical in decidualization in vitro. Silencing and over-expression HAND2 disclosed the roles of HAND2 in regulating the expression of FOXO1A, IGFBP1, BMP2 as well as WNT4. It seemed that HAND2 promoter worked in tissue specific manner and although both HOXA10 and cAMP binding sites were found in proposed HAND2 promoters, its activity was stimulated by cAMP and steroid hormones rather than the expression of HOXA10.

Immunohistochemical Localization of Prolactin Receptors Within the Equine Ovary

Oberhaus, Erin Lea 01 August 2012 (has links)
Prolactin receptors (PRLr) were detected in anestrous (n=3), winter cycling (n=2), follicular (n=3) and luteal phase (n=3) equine ovaries by IHC. Follicle stages evaluated were primordial, preantral and antral. Receptors were detected in all follicle stages and in CL. PRLr staining was not different (P > 0.05) between primordial and preantral, but was greater (P < 0.001) in antral follicles. Primordial follicles stained weakest in anestrous and follicular phase ovaries, followed by luteal phase ovaries and was most intense in winter cycling. Staining in preantral follicles was weakest in anestrus, followed by follicular phase and highest in winter cycling and luteal phase. Staining was most intense in antral follicles with no difference (P > 0.05) between any of the reproductive states. Oocytes and ovulation fossa also possessed PRLr. In conclusion, concentrations of PRLr are highest in large, antral follicles suggesting a mechanistic role for PRL around the time of ovulation.

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