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Methods for structural studies of an antibody, screening metabolites in rat urine and analysis of spent cell cultivation media using LC/ESI-MS and chemometricsZamani, Leila January 2009 (has links)
This thesis describes bioanalytical methods for generating fingerprints of biological systems for extracting relevant information with (protein) drugs in focus. Similarities and differences between samples can reveal the hidden relevant information, which can be used to optimize the production and facilitate the quality control of such protein drugs during their development and manufacture. Metabolic fingerprinting and multivariate data analysis (MVDA) can also facilitate early diagnosis of diseases and the effects and toxicity of drugs. Currently, several protein drugs are available on the global market. Nevertheless, despite, the success of such biotherapeutics significant challenges remain to be overcome in maintaining their stability and efficacity throughout their production cycle and long-term storage. The native structure and functional activity of therapeutic proteins is affected by many variables from production to delivery, incl. variables assoc. with conditions in bioreactors, purification, storage and delivery. Thus, part of the work underlying this thesis focused on structural analysis of a protein drug using chemical labeling, peptide mapping, and evaluation of the charge state distributions of the whole protein generated by ESI. The other part focuses on non-targeted metabolomics with a view to optimizing the cell cultivation process and assessment of the drug’s toxicity. A combination of appropriate analytical methods and MVDA is needed to find markers that can facilitate optimization of the cultivation system and expression of the target proteins in early stages of process development. Rapid methods for characterizing the protein drugs in different stages of the process are also required for quality control. In order to obtain high quality fingerprints analytical separation techniques with high resolution (such as HPLC or UHPLC) and sensitive analytical detection techniques (such as ESI, quadrupole or TOF MS) have been used, singly or in combination. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript.
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Establishment and Characterization of Immortalized Non-Transplantable Mouse Mammary Cell Lines Cloned from a MMTV-induced Tumor Cell Line Cultured for A Long DurationHOSHINO, MUNEMITSU, MATSUYAMA, MUTSUSHI, TAGUCHI, OSAMU, KUSAKABE, MORIAKI, WAJJWALKU, WORAWIDH, LU, JIN, YOKOI, TOYOHARU, IMAI, MASAO, MIYAISHI, OSAMU, SAGA, SHINSUKE, TAKENAKA, TOKUYA 03 1900 (has links)
名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成2年11月22日 竹中徳哉氏の博士論文として提出された
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A comparative membrane surface analysis between two human hepatocarcinoma cell lines ( SK-HEP-1 and Hep G2 cells ) using Atomic Force MicroscopeLi, I-Ting 03 September 2010 (has links)
Atomic force microscopy (AFM) can be used to acquire high-resolution topographical images of surfaces, but has the additional capability of detecting the local nanometer scale mechanical properties. For these reasons, it becomes a standard research tool in the surface science recently. In this paper, we used AFM to measure the several properties of two different human hepatocellular
carcinoma cell lines, Hep G2 ( known as well differentiated and more highly carcinomatous hepatoma cell lines ) and SK-HEP-1 ( known as poorly differentiated and more lightly carcinomatous hepatoma cell lines ) cells fixed on the glass substrate, which including the surface morphology and the relationship between the cantilever deflections and loading forces ( force curve ). Considered the heterogeneous characteristics of the cell surface, the
preferred experimental method is to make pixel-by-pixel force curves in a designated area ( force map ) , both adhesion forces and elasticity associated with different locations on the cell surfaces will be obtained. Finally, we use Hertzian model to calculate Young's modulus of Hep G2 and SK-HEP-1 respectively. Based on these results, we can understand the surface properties of two human hepatocarcinoma cell lines with different differentiated stage. The results showed the difference of the morphology, height, cell migration, degree of cell aggregation, roughness, elasticity, adhesive force of two cells. SK-HEP-1 cell has the wide distance of the folds, better cell migration, homogeneous properties of elasticity. It can be assumed that the SK-HEP-1 cells have a dense network structure of actin filaments under the cell membrane like branches (branched networks); Hep G2 cell has the narrow distance of the folds, poor cell migration, heterogeneous properties of elasticity. It can be assumed that the Hep G2 cells have the individual actin
filaments and cross-linked network structure of actin filaments under the cell membrane. The above results can be speculated that the elastic properties of the membrane surface will be influenced of actin filaments.
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Cytogenetic and molecular alterations in immortalization of normal esophageal epithelial cellsZhang, Hao, 張浩 January 2005 (has links)
published_or_final_version / abstract / Medicine / Master / Master of Philosophy
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Characterization of Effects of Muc1 Expression on Epidermal Growth Factor Receptor Signaling in Breast CancerPochampalli, Mamata Rani January 2006 (has links)
EGF receptors are key regulators of cell survival and growth in normal and transformed tissues. Ligand binding results in formation of homo/hetero dimers of these receptors, followed by activation of the kinase activity and subsequent tyrosine phosphorylation of many downstream molecules. The activation of these receptors is not only mediated by the binding of their cognate ligands, but by transactivaton by other molecules as well. Recent studies have identified an oncogenic glycoprotein MUC1 as a binding partner for EGFR and that MUC1 expression can potentiate EGFR-dependent signal transduction. After receptor activation, EGFR is typically downregulated via an endocytic pathway that results in receptor degradation or recycling. We report here that MUC1 expression inhibits the degradation of ligand-activated erbB1. In addition, MUC1 expression results in prolonged activation of Akt, but not ERK1,2 MAPKinase. The MUC1-mediated protection against degradation occurs with a decrease in EGF-stimulated ubiquitination of erbB1, and an increase in erbB1 recycling. We then utilized the WAP-TGFα transgenic mouse model of breast cancer and determined that a loss of Muc1 expression dramatically alters mammary tumor progression. While 100% of WAP-TGFα/Muc1^(+/+) mice form mammary gland tumors, only 37% of WAP-TGFα/Muc1^(-/-) form tumors. Furthermore, expression of cyclin D1 expression is significantly suppressed in tumors derived from WAPTGFα/Muc1^(-/-) animals, and loss of Muc1 expression resulted in a significant inhibition in the formation of hyperplastic lesions in the mammary gland. We also observed metastatic pulmonary adenocarcinoma (1/29) and perivascular lymphoma of unknown origin (28/29) in the WAP-TGFα transgenic mice but not in the WAP TGFα/Muc1^(-/-) animals. To determine the effects of Muc1 expression on metastasis in a model lacking perivascular lymphoma, we crossed MMTV-Wnt-1 and MMTV-MUC1 transgenic mice and evaluated interactions between Muc1 and EGFR. Although the MMTV-Wnt-1 mice are non-metastatic, a majority (6/10) of the bitransgenic MMTVWnt- 1/MMTV-MUC1 formed pulmonary metastases. Furthermore, overexpression of MUC1 increases the breast cancer cell invasion in vitro. The MUC1 induced increase in invasion is found to be EGF and EGFR-kinase dependent. Collectively, these data indicate that MUC1 expression contributes to many of the hallmarks of cancer and in addition, is an important modulator of EGFR-associated mammary tumor progression.
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Expression of anti-HIV peptides in tobacco cell culture systemsMoodley, Nadine January 2009 (has links)
Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, South Africa,2009. / Nearly half of all individuals living with HIV worldwide at present are woman and
the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs).
Microbicides are ARV’s which directly target viral entry and avert infection at mucosal
surfaces. However, most promising ARV entry inhibitors are biologicals which are costly
to manufacture and deliver to resource-poor areas. Microbicides formulated as simple
gels, which are currently not commonly used in ARV therapy, show immense potential
for use in prevention and treatment of multidrug-resistant viral infections in developing
countries.
Among the most potent HIV entry inhibitory molecules are lectins, which target the
high mannose N-linked glycans which are displayed on the surface of HIV envelope
glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent
anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on
high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per
homodimer, which likely accounts for its unparalleled potency. The antiviral potency of
GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability
make it an ideal active ingredient of a topical HIV microbicide.
v
Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous
extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of
SVN have been reported to inactivate laboratory strains and primary isolates of HIV-
1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1
envelope glycoproteins gp120, gp160 and gp41.
Current recombinant production methods for GRFT and SVN molecules are
unfortunately hampered by inadequate production capacities. This project therefore
aimed to determine if these molecules can be produced in plant cell culture systems.
The transgenic tobacco cell culture system was evaluated to determine if it can be an
alternative, cost effective production system for these molecules.
Results of the study show that the microbicide genes can be cloned into plant
transformation vectors, used to successfully transform SR1 tobacco cell lines and
adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per
gram of SR1 tobacco callus fresh weight.
The promising results attained in this study form the basis for further work in
optimising plant cell based production systems for producing valuable anti-HIV
microbicides, a possible means to curbing the elevated HIV infection rates worldwide.
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Investigations of telomere maintenance in DNA damage response defective cells and telomerase in brain tumoursCabuy, Erik January 2005 (has links)
Telomeres are nucleoprotein complexes located at the end of chromosomes. They have an essential role in protecting chromosome ends. Telomerase or ALT (alternative lengthening of telomeres) mechanisms maintain telomeres by compensating natural telomeric loss. We have set up a flow-FISH method and using mouse lymphoma cell lines we identified unexpectedly the presence of subpopulations of cells with different telomere lengths. Subpopulations of cells with different telomere lengths were also observed in a human ALT and non-ALT cell line. Differences in telomere length between subpopulations of cells were significant and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). By applying flow-FISH we could successfully measure telomere lengths during replicative senescence in human primary fibroblasts with different genetic defects that confer sensitivity to ionising radiation (IR). The results from this study, based on flow-FISH and Southern hybridisation measurements, revealed an accelerated rate of telomere shortening in radiosensitive fibroblasts. We also observed accelerated telomere shortening in murine BRCA1 deficient cells, another defect conferring radiosensitivity, in comparison with a BRCA1 proficient cell line. We transiently depleted BRCA1 by siRNAs in two human mammary epithelial cell lines but could not find changes in telomere length in comparison with control cells. Cytological evidence of telomere dysfunction was observed in all radiosensitive cell lines. These results suggest that mechanisms that confer sensitivity to IR may be linked with mechanisms that cause telomere dysfunction. Furthermore, we have been able to show that human ALT positive cell lines show dysfunctional telomeres as detected by either the presence of DSBs at their telomeres or cytogenetic analysis and usually cells with dysfunctional telomeres are sensitive to IR. Finally, we assessed hTERT mRNA splicing variants and telomerase activity in brain tumours, which exhibit considerable chromosome instability suggesting that DNA repair mechanisms may be impaired. We demonstrated that high levels of hTERT mRNAs and telomerase activity correlate with proliferation rate. The presence of hTERT splice variants did not strictly correlate with absence of telomerase activity but hTERT spliced transcripts were observed in some telomerase negative brain tumours suggesting that hTERT splicing may contribute to activation of ALT mechanisms.
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Effects of Insulin Resistance on Leptin Modulation of Hypothalamic NeuronsNazarians-Armavil, Anaies 10 July 2013 (has links)
Central resistance to the actions of insulin and leptin is strongly associated with obesity and type 2 diabetes mellitus (T2DM). These anorexigenic hormones modulate one another’s actions at the neuronal level. To investigate the cellular events underlying the effect of insulin resistance on leptin modulation of hypothalamic neurons, a neuronal cell model was established. The rHypoE-19 cell line expresses the insulin and leptin receptors alongside a complement of signaling molecules rendering it an appropriate model to study the molecular events underlying leptin and insulin crosstalk. Hyperinsulinemia was used to induce insulin resistance and leptin
regulation of the rHypoE-19 neurons was analyzed prior to and following the induction of insulin resistance. It was found that the attenuation of insulin signal transduction affects leptin signaling and transcriptional modulation of the rHypoE-19 neurons. These studies will ultimately lend itself to an improved understanding of the complex cellular events that accompany neuronal hormone resistance.
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Effects of Insulin Resistance on Leptin Modulation of Hypothalamic NeuronsNazarians-Armavil, Anaies 10 July 2013 (has links)
Central resistance to the actions of insulin and leptin is strongly associated with obesity and type 2 diabetes mellitus (T2DM). These anorexigenic hormones modulate one another’s actions at the neuronal level. To investigate the cellular events underlying the effect of insulin resistance on leptin modulation of hypothalamic neurons, a neuronal cell model was established. The rHypoE-19 cell line expresses the insulin and leptin receptors alongside a complement of signaling molecules rendering it an appropriate model to study the molecular events underlying leptin and insulin crosstalk. Hyperinsulinemia was used to induce insulin resistance and leptin
regulation of the rHypoE-19 neurons was analyzed prior to and following the induction of insulin resistance. It was found that the attenuation of insulin signal transduction affects leptin signaling and transcriptional modulation of the rHypoE-19 neurons. These studies will ultimately lend itself to an improved understanding of the complex cellular events that accompany neuronal hormone resistance.
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Development of models for the study of anesthetic preconditioning using rat pheochromocytoma and mouse neuroblastomaKam, Sarah Anne. January 2009 (has links)
Honors Project--Smith College, Northampton, Mass., 2009. / Includes bibliographical references (p. 54-57).
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