251 |
Role of T-Box 3 in Cardiomyocyte ApoptosisXia, Ying 21 August 2023 (has links)
No description available.
|
252 |
Development and Characterization of Caspase Activatable GFP and a Family of Fluorescent ReportersNicholls, Samantha Elizabeth Bernard 01 February 2013 (has links)
The cellular process of programmed cell death, or apoptosis, is critical in homeostasis and development. In addition it's misfunction is implicated in an array of disease states from cancer to neurodegeration, making it an attractive pathway for drug targeting. A family of proteases, known as caspases, plays a central role in the apoptotic cascade resulting in the ultimate destruction of the cell. We report a genetically encoded dark-to-bright reporter of caspase activity used in E.coli, mammalian cells, and whole organisms which can be used to monitor apoptosis. This reporter, caspase activatable green fluorescent protein (CA-GFP) consists of GFP fused through a flexible linker containing the caspase-3 and -7 recognition sequence, DEVD, to a hydrophobic peptide derived from the influenza A viral M2 protein. This fusion reporter shows a significant fluorescent response in the presence of active caspase. CA-GFP is unique in its ability to hold GFP in a dark state prior to cleavage by active protease. We investigate the mechanism of quenching, examining the structural characteristics which lead to the inability of the GFP chromophore to mature in the presence of the peptide. In better understanding the mechanism of quenching we can engineer CA-GFP to ultimately be used in transgenic animal models. This requires the development of a palette of protease-activatable fluorescent proteins (PrA-FP) which would enable the monitoring of multiple proteolytic events within a cell or organism in real time. Our development of this palette of reporters, varying in their fluorescence and proteolytic response shows that CA-GFP has the potential to be a powerful tool for the study of the role of apoptosis during development in whole organism models and could be an important tool in understanding the role of individual proteases within the complex biochemical environment in the cell.
|
253 |
The Fate of Midline Lineages in the Embryos Deficient for Apoptosis in Drosophila melanogaster / Midline Lineages in Embryos Deficient for Apoptosis in DrosophilaDong, Rong 01 1900 (has links)
One function of programmed cell death in the developing CNS is the removal of excess cells that provide transient function. Previous studies in Drosophila observed an overpopulation of midline glia cells in embryos deficient for apoptosis. Where do these extra glia cells come from? Using different enhancer traps and antibodies as cell identity markers, the cell number of different CNS midline lineages was assessed in both wild type and Df(3L)H99 embryos which are deficient for apoptosis. The results show that at stage 16 there are approximately 3 cells labeled by midline glia specific markers (AA142 enhancer trap & P[slit1.0/lacZ] reporter construct) in wild type while there are up to 12 cells in embryos deficient for apoptosis. Comparing the number of midline neurons of mutants with that of wild type embryos, there are no detectable changes labeled by the enhancer trap P223, antibody 22C10, or an antibody to Engrailed. Exceptionally, there is one more neuron labeled by enhancer trap XS 5 in Df(3L)H99 embryos. Therefore, apoptosis is restricted to the midline glia lineage. Using P[slit1.0/lacZ] as MG marker, I observed that the extra midline glia in Df(3L)H99 initially appear at late stage 12 or early stage 13. The expression of reaper mRNA precedes programmed cell death. In wild type embryos, the initial expression of reaper mRNA of midline cells is at late stage 11 as revealed by in situ hybridization. These indicate that the first programmed cell death in the midline occurs approximately at stage 12. The supernumerary cells labeled by midline glia specific markers in Df(3L)H99 embryos share featur,es ofthe midline glia. These extra midline glia may be divided into two groups according to their differentiation. The cells of the first group strongly express the AA142 enhancer trap and ensheath the commissures. These cells are functional midline glia corresponding to the surviving midline glia in wild type embryos. The cells of the second group weakly express the AA142 and associate with but do not ensheath the commissures. These are likely the cells which normally undergo apoptosis in wild type. The results of this study indicate that the supernumerary midline glia come from neither midline glia proliferation nor other lineages. They may come from a midline glia progenitor pool in which midline glia marker expression begins at different stages. In wild type embryos, these potential midline glia die by apoptosis before activating midline glia specific genes. In Df(3L)H99 embryos, these midline glia survive and express midline glia markers.
All the midline glia die in embryos mutant for spitz group genes. In embryos double mutant for spitz group genes and Df(3L)H99, supernumerary midline glia cells survive. These cells cannot totally rescue the axon tract phenotype of spitz group gene mutants indicating that spitz group genes are necessary for producing 'mature' midline glia. In Df(3L)H99 embryos, approximately 12 midline cells labeled with the midline glia specific marker P[slit1.0/lacZ]. However, there is not a significant increase in the number of midline glia expressingpnt or argos compared with wild type. Therefore, the survival of supernumerary midline glia in embryos deficient for apoptosis does not require DER signaling. However, the DER pathway seems to specify which and how many midline glia progenitors avoid apoptosis. / Thesis / Master of Science (MSc)
|
254 |
Rol de la hormona paratiroidea en la apoptosis de células intestinalesCalvo, Natalia Graciela 17 December 2009 (has links)
La hormona paratiroidea (PTH) es un importante mediador de la
remodelación ósea y actúa como regulador esencial de la homeostasis del calcio. Dependiendo del tipo y del contexto celular, PTH puede también inhibir o promover la apoptosis. La apoptosis, una forma de muerte celular programada, es un proceso fundamental para el crecimiento y desarrollo
normal, la respuesta inmune, la remodelación de los tejidos, y la homeostasis del epitelio intestinal.
En este trabajo de tesis se demostró, por primera vez, la localización y expresión del receptor de PTH tipo 1 (RPTH1) en las células epiteliales Caco-2, derivadas de adenocarcinoma de colon humano. El receptor, se localiza en la membrana plasmática, el citoplasma y el núcleo de estas células intestinales. Se comprobó que en las células Caco-2 el tratamiento con PTH disminuye el número de células viables e induce cambios morfológicos consistentes con la apoptosis: alteración de los filamentos de actina y consecuentemente de la forma celular, pérdida de las uniones intercelulares, externalización de la fosfatidilserina de membrana, distribución perinuclear de las mitocondrias, condensación nuclear y fragmentación del ADN.
Además, el tratamiento con PTH en estas células resultó en la
desfosforilación de la proteína pro-apoptótica Bad, su disociación de la proteína 14-3-3 y su translocación a las mitocondrias con la consecuente liberación de los factores pro-apoptóticos citocromo c y Smac/Diablo desde las mitocondrias al citosol resultando finalmente en la activación de la enzima pro-apoptótica caspasa-3 y el clivaje de su sustrato PARP. En estas células, la hormona provocó la desfosforilación de Akt (quinasa
que controla el balance entre la supervivencia celular y la apoptosis e induce la
fosforilación de Bad) mediante la serina/treonina fosfatasa PP2A. PTH además
indujo la asociación de Akt con la subunidad catalítica de PP2A (PP2Ac),
promovió la translocación de PP2Ac del citosol a las mitocondrias e incrementó
la actividad de esta fosfatasa vía el AMPC. PP2A también participó en la
regulación de la viabilidad de las células Caco-2 dependiente de PTH y en el
clivaje del sustrato de caspasa-3, PARP.
PTH no modificó los niveles de expresión proteica de p53, sugiriendo que,
en estas células, la hormona es un estímulo apoptótico que induce la muerte
celular por un mecanismo independiente de la expresión de p53.
PTH aumentó los niveles de la proteína 14-3-3 en el citosol y no se detectó
la asociación entre 14-3-3 y RPTH1 en condiciones basales ni luego del
tratamiento hormonal, sugiriendo que 14-3-3 no regularía la localización
subcelular del RPTH1 en las células Caco-2.
Los resultados de este trabajo de tesis demuestran que PTH desencadena
efectos pro-apoptóticos en las células Caco-2, y aportan información sobre los
mecanismos moleculares de señalización que son estimulados por la hormona
en estas células intestinales. / Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. Depending on the cell type involved, PTH also inhibits or promotes the apoptosis. Apoptosis, a form of programmed cell death, is a fundamental process to
normal growth and development, immune response, tissue remodeling, and homeostasis of the intestinal epithelium.
In this thesis work, we demonstrated, for the first time, the localization and expression of the PTH receptor type 1 (PTHR1) in the epithelial Caco-2 cells, a cell line derived from human colorectal adenocarcinoma. The receptor is
present in plasma membrane, cytoplasm and nucleus of these intestinal cells. In Caco-2 cells, PTH treatment diminished the number of viable cells. Moreover, the hormone induced disruption of actin filaments with changes to cellular shape, alteration of cell-to-cell junctions, externalization of membrane
phosphatidylserine, mitochondrial cellular distribution to the perinuclear region, chromatin condensation and DNA fragmentation of the nucleus, which are morphological features consistent with apoptosis.
In addition, the treatment with PTH in these cells resulted in Bad dephosphorylation, its dissociation of 14-3-3 protein and its translocation to the mitochondria with the subsequent release of cytochrome c and Smac/Diablo to the cytosol which resulted in activation of downstream caspase-3 and
degradation of its substrate PARP.
The hormone also decreased the phosphorylation of Akt (a kinase which controls the balance between cell survival and apoptosis and induces the phosphorylation of Bad) via the serine/threonine phosphatase PP2A. PTH also induced an association of Akt with the catalytic subunit of PP2A (PP2Ac),
promoted the translocation of PP2Ac from the cytosol to the mitochondria and increased its phosphatase activity via the AMPc pathway. Furthermore, PP2A plays a role in hormone-dependent Caco-2 cells viability and in the cleavage of
caspase-3 substrate PARP.
PTH did not modify the protein levels of p53, suggesting that the hormone is an apoptotic stimulus that induces cell death by a p53-expression independent mechanism in these cells.
PTH increased the amount 14-3-3 protein in the cytosol. However, the association of 14-3-3 with PTHR1 was not detectable under basal conditions or after PTH treatment, suggesting that 14-3-3 does not regulate the subcellular
localization of PTHR1 in Caco-2 cells. The results of this tesis provide, for the first time, evidence of pro-apoptotic
effects of PTH in Caco-2 cells and information about the molecular mechanisms of PTH-mediated signaling pathway in these intestinal cells.
|
255 |
The Role of CD44 in Concanavalin A-Induced HepatitisChen, Dawei 08 May 2000 (has links)
Administration of Concanavalin-A (Con A) induces severe injury to the hepatocytes in mice and is considered to be a model for human hepatitis. In the current study, we investigated the role of CD44 in Con A induced hepatitis. Although immune cells have been identified as the causative agent of Con A-induced hepatitis, the exact mechanism of pathogenesis remains unclear. When Con A was injected into CD44 wild type (WT) mice, it induced hepatitis as evident from increased plasma aspartate aminotransferase (AST) levels accompanied by active infiltration of mononuclear cells in the liver and significant induction of apoptosis. Interestingly, Con A injected C57BL/6 CD44-knockout (KO) mice exhibited increased hepatitis with higher levels of apoptosis in the liver and increased plasma AST levels when compared to the CD44 WT mice. Also, transfer of T cells from Con A injected CD44-KO mice into CD44 WT mice induced higher levels of hepatitis when compared to transfer of similar cells from CD44 WT mice into CD44 WT mice. The increased hepatitis seen in CD44-KO mice was partially due to increased production of cytokines such as TNF-a, IL-2 and IFN-g, but not Fas or FasL. Also, it was not caused by altered presence of T cell subsets. The increased susceptibility of CD44 KO mice to hepatitis correlated with increased resistance of T cells from CD44 KO mice to undergo apoptosis when compared to the CD44 WT mice. Together, these data demonstrate that activated T cells use CD44 to undergo apoptosis, and dysregulation in this pathway could lead to increased pathogenesis in a number of diseases, including hepatitis. / Master of Science
|
256 |
Apoptosis in the equine small intestine following experimental ischemia-reperfusion injuryNagy, Amy Dae 05 November 2008 (has links)
This study was aimed at characterizing the apoptotic response equine small intestine subjected to experimental ischemia-reperfusion injury and determining if use of an angiotensin converting enzyme inhibitor (enalaprilat) would ameliorate the apoptotic response. It was designed to determine if mucosal epithelial cells undergo apoptosis during the ischemia phase and also examined if apoptosis is significantly exacerbated by reperfusion. It also investigated whether administration of enalaprilat decreased reperfusion injury secondary to reduced enterocyte apoptosis. Injury was induced using a low flow model of I-R. During celiotomy a single loop of jejunum was isolated and arterial flow decreased to 20% of baseline for one hour and complete occlusion for a second hour. Reperfusion was monitored for 3 hours. A control group (n=6) were not treated while the treatment group (n=6) received 0.5 mg/kg enalaprilat in 0.9% NaCl immediately following ischemia. Jejunal samples were taken prior to the induction of ischemia, immediately post-ischemia and at 1, 2 and 3 hours of reperfusion. Samples were evaluated for gross tissue pathology with standard hematoxylin and eosin staining, the presence of apoptotic cells via TUNEL staining, and gene expression of three apoptosis related genes (bax, bcl-2, p53) using qPCR. Serum enalaprilat and ACE concentrations were determined from blood samples drawn concurrent with jejunal sampling using HPLC/MS and standard HPLC. Plasma enalaprilat concentrations were comparable to previous reports in awake horses. Enalaprilat appeared to have no effect on serum ACE concentrations, however a significant spike in ACE concentration occurred in the treatment group at 1 hour of reperfusion (P=0.0001). Grade of mucosal damage was not significantly different between control and treatment groups at any time point. Subjectively apoptotic index appeared to be higher in the treatment group at end ischemia and during reperfusion. There were no changes in expression of p53 or bcl-2 in either group. Bax expression was significantly decreased (P= 0.02) in the control group at 2 hours of reperfusion. Based on our data administration of an ACE inhibitor during anesthesia in horses with an ischemic segment of intestine confers no protective benefit and may be associated with increased intestinal injury and apoptosis. Lack of expression of p53, bax and bcl-2 suggests another apoptotic mechanism in equine ischemic intestine. / Master of Science
|
257 |
Reversal of apoptosis: a potential link to carcinogenesis and cancer recurrence. / CUHK electronic theses & dissertations collectionJanuary 2011 (has links)
Tang, Ho Lam. / "December 2010." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 119-132). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
|
258 |
The role of growth hormone secretagogue receptor (GHSR) in apoptosis.January 2005 (has links)
Lau Pui Ngan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 171-181). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / Abbreviations --- p.viii / Publications Based on work in this thesis --- p.xii / Chapter Chapter 1 --- Introduction and project overview --- p.1 / Chapter 1.1 --- Ghrelin structure and its synthesis --- p.3 / Chapter 1.2 --- Types of growth hormone secretagogues (GHSs) --- p.6 / Chapter 1.3 --- Characterization of GHS-R --- p.7 / Chapter 1.3.1 --- Cloning of GHS-Rla and GHS-Rlb --- p.7 / Chapter 1.3.1.1 --- GHS-R subtypes --- p.7 / Chapter 1.3.1.2 --- Properties of GHS-R subtypes --- p.7 / Chapter 1.3.1.3 --- Evidence of non-GHS-Rla stimulated by ghrelin and GHSs --- p.9 / Chapter 1.3.1.4 --- Distribution of GHS-R --- p.10 / Chapter 1.3.2 --- Signal transduction pathways of GHS-R --- p.11 / Chapter 1.3.3 --- Comparison between human and seabream GHS-R --- p.12 / Chapter 1.4 --- Is adenosine a partial agonist at GHS-Rla? --- p.15 / Chapter 1.5 --- Physiological effects of ghrelin --- p.17 / Chapter 1.6 --- Apoptosis --- p.19 / Chapter 1.6.1 --- Introduction --- p.19 / Chapter 1.6.2 --- Apoptosis versus necrosis --- p.19 / Chapter 1.6.3 --- Mechanisms of apoptosis --- p.20 / Chapter 1.6.4 --- Methods to study apoptosis --- p.23 / Chapter 1.6.5 --- Different types of apoptotic inducers --- p.24 / Chapter 1.7 --- Apoptotic and anti-apoptotic pathways regulated by GPCRs --- p.27 / Chapter 1.7.1 --- Bcl-2 family pathway --- p.27 / Chapter 1.7.2 --- Caspase pathway --- p.27 / Chapter 1.7.3 --- ERK pathway --- p.28 / Chapter 1.7.4 --- PI3K/Akt pathway --- p.29 / Chapter Chapter 2 --- Materials and solutions --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.2 --- "Culture medium, buffer and solutions" --- p.37 / Chapter 2.2.1 --- Culture medium --- p.37 / Chapter 2.2.2 --- Buffers --- p.37 / Chapter 2.2.3 --- Solutions --- p.38 / Chapter Chapter 3 --- Methods --- p.41 / Chapter 3.1 --- Maintenance of cell lines --- p.41 / Chapter 3.1.1 --- Human Embryonic kidney (HEK293) cells --- p.41 / Chapter 3.1.2 --- HEK293 cells stably expressing black seabream growth hormone secretagogues receptors (HEK-sbGHS-Rla and HEK-sbGHS-Rlb) --- p.41 / Chapter 3.2 --- Preparation of plasmid DNA --- p.42 / Chapter 3.2.1 --- Preparation of competent E. coli --- p.42 / Chapter 3.2.2 --- Transformation of DNA into competent cells --- p.42 / Chapter 3.2.3 --- Small-scale and large-scale plasmid DNA preparation --- p.43 / Chapter 3.2.4 --- Confirmation of the purity and the identity of the plasmid DNA --- p.43 / Chapter 3.3 --- Transient transfection of mammalian cells --- p.45 / Chapter 3.4 --- Development of stable cell lines --- p.46 / Chapter 3.4.1 --- Determination of the optimum concentration of each antibiotic used in selection of clones --- p.46 / Chapter 3.4.2 --- Development of monoclonal stable cell line --- p.46 / Chapter 3.4.3 --- Confirmation the expression of 2myc-hGHS-Rla and myc-hGHS-Rlb --- p.48 / Chapter 3.5 --- Measurement of phospbolipase C activity --- p.49 / Chapter 3.5.1 --- Introduction --- p.49 / Chapter 3.5.2 --- Preparation of columns --- p.49 / Chapter 3.5.3 --- [3 H]-inositol phosphate assay --- p.49 / Chapter 3.5.4 --- Measurement of [3H]-inositol phosphates production --- p.50 / Chapter 3.5.5 --- Data analysis --- p.50 / Chapter 3.6 --- Determination of transient transfection efficiency --- p.51 / Chapter 3.7 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.52 / Chapter 3.7.1 --- RNA extraction and first strand cDNA production --- p.52 / Chapter 3.7.2 --- PCR and visualization of amplicons --- p.52 / Chapter 3.7.3 --- Real-time PCR --- p.59 / Chapter 3.7.3.1 --- Construction of standard curve --- p.60 / Chapter 3.7.3.2 --- Data analysis --- p.60 / Chapter 3.8 --- Measurement of caspase-3 activity --- p.65 / Chapter 3.8.1 --- Determination of caspase-3 activity using colorimetric assay --- p.65 / Chapter 3.8.1.1 --- Introduction --- p.65 / Chapter 3.8.1.2 --- Induction of apoptosis --- p.65 / Chapter 3.8.1.3 --- Preparation of cell lysates --- p.65 / Chapter 3.8.1.4 --- Quantification of caspase-3 activity by measuring pNA absorbance --- p.66 / Chapter 3.8.1.5 --- Data analysis --- p.67 / Chapter 3.8.2 --- Determination of caspase-3 activity using bioluminescence resonance energy transfer (BRET2) assay --- p.67 / Chapter 3.8.2.1 --- Introduction --- p.67 / Chapter 3.8.2.2 --- Quantification of caspase-3 activity using BRET2 assay --- p.68 / Chapter 3.8.2.3 --- Data analysis --- p.69 / Chapter 3.8.3 --- Determination of caspase-3 activity using fluorescence resonance energy transfer (FERT) assay --- p.70 / Chapter 3.8.3.1 --- Introduction --- p.70 / Chapter 3.8.3.2 --- Quantification of caspase-3 activity using FRET assay --- p.70 / Chapter 3.8.3.3 --- Data analysis --- p.71 / Chapter Chapter 4 --- Results --- p.72 / Chapter 4.1 --- Characterization of GHS-R --- p.72 / Chapter 4.1.1 --- Properties of GHS-Rla --- p.72 / Chapter 4.1.1.1 --- Constitutively active receptor --- p.72 / Chapter 4.1.1.2 --- Characterization of epitope-tagged hGHS-Rla --- p.73 / Chapter 4.1.2 --- Properties of GHS-Rlb --- p.75 / Chapter 4.1.3 --- Conclusions --- p.75 / Chapter 4.2 --- Effect of co-transfection of HEK293 cells --- p.85 / Chapter 4.2.1 --- Effect of balancing DNA concentrations transfected into HEK293 cells --- p.85 / Chapter 4.2.2 --- Effect of balancing DNA concentration using another Gq-coupled receptor --- p.87 / Chapter 4.2.3 --- Effect of Gi- and Gs-coupled receptor on GHS-Rla signaling --- p.88 / Chapter 4.2.4 --- Potentiating effect of co-transfection appeared using different transfection reagents --- p.88 / Chapter 4.2.5 --- Co-transfection improves transfection efficiency --- p.89 / Chapter 4.2.6 --- Discussions --- p.91 / Chapter 4.3 --- Development of cell lines stably expressing hGHS-Rla or hGHS-Rlb --- p.102 / Chapter 4.3.1 --- Advantages of using a monoclonal cell line --- p.102 / Chapter 4.3.2 --- Sensitivity of HEK293 cells to antibiotics --- p.102 / Chapter 4.3.3 --- Production of polyclonal stable cell line --- p.103 / Chapter 4.3.4 --- Monoclonal stable cell line selection --- p.104 / Chapter 4.3.5 --- Discussions --- p.105 / Chapter 4.4 --- Effect of adenosine on GHS-Rla signaling --- p.111 / Chapter 4.4.1 --- Adenosine acts as partial agonist --- p.111 / Chapter 4.4.2 --- Effect of substance P analog on adenosine-mediated GHS-Rla signaling --- p.112 / Chapter 4.4.3 --- Effect of adenosine deaminase (ADA) on adenosine- and ghrelin-stimulated GHS-Rla signaling --- p.113 / Chapter 4.4.4 --- Specificity of ADA --- p.115 / Chapter 4.4.5 --- Conclusions --- p.116 / Chapter 4.5 --- Role of GHS-R in apoptosis --- p.124 / Chapter 4.5.1 --- Different methods to measure caspase-3 activity --- p.124 / Chapter 4.5.1.1 --- Colorimetric assay --- p.124 / Chapter 4.5.1.1.1 --- Time course for staurosporine and etoposide in HEK293 cells --- p.125 / Chapter 4.5.1.1.2 --- Effect of 2myc-hGHS-Rla on staurosporine- and etoposide-induced caspase-3 activity --- p.127 / Chapter 4.5.1.1.3 --- Time course for staurosporine and etoposide in sbGHS-R monoclonal stable cell line --- p.128 / Chapter 4.5.1.1.4 --- Effect of sbGHS-Rs on staurosporine- and etoposide- induced caspase-3 activityin HEK 293 cells --- p.129 / Chapter 4.5.1.1.5 --- Effect of sbGHS-Rs on staurosporine- induced caspase-3 activity in sbGHS-R monoclonal stable cell line --- p.130 / Chapter 4.5.1.1.6 --- Differences between epitope-tagged and non-tagged sbGHS-Rs in staurosporine- induced caspase-3 activity --- p.131 / Chapter 4.5.1.1.7 --- The role of epitope-tagged sbGHS-Rlbin staurosporine-induced caspase-3 activity --- p.132 / Chapter 4.5.1.1.8 --- Effect of staurosporine and etoposide on GHS-Rla signaling --- p.133 / Chapter 4.5.1.2 --- BRET2 assay --- p.135 / Chapter 4.5.1.3 --- FRET assay --- p.136 / Chapter 4.5.1.4 --- Conclusions --- p.136 / Chapter 4.6 --- Determination of GHS-R amount in terms of mRNA --- p.155 / Chapter 4.6.1 --- Determination of GHS-R amount in stable cell lines --- p.155 / Chapter 4.6.2 --- Transfected DNA amount match with stable cell lines --- p.155 / Chapter Chapter 5 --- "Discussion, Conclusions and Future Plan" --- p.159 / Chapter 5.1 --- General Discussion and Conclusions --- p.159 / Chapter 5.2 --- Future Plan and Experimental Design --- p.168 / References --- p.171
|
259 |
Induction of suicide in an autoantibody-producing hybridoma. / CUHK electronic theses & dissertations collectionJanuary 2005 (has links)
Autoantibodies are antibodies made abnormally to self-antigens that are expressed on the surface, in the cytoplasm, or in the nucleus of the cell. These antibodies are produced in certain people whose immune system has failed to work properly. It is a puzzle, however, how the B cells that produce autoantibodies which target the vital intracellular autoantigens and with potential deleterious effects to the cell, are able to exist in nature and not succumb to suicide. We have found a suitable model to answer this question. This is a newly-constructed mouse hybridoma (#476), which produces an IgG1 monoclonal antibody (mAb 476) to an epitope in the catalytic subunit of telomerase (TERT). This mAb stains TERT very well in both mouse and human cells. The unusual thing about hybridoma 476 is that, although it can grow and produce the antibody normally in culture, however, it fails repeatedly to grow in the peritoneum of normal BALB/c mice primed with an adjuvant, unlike other hybridomas of similar backgrounds that we have used. Control cell lines used in the study include a hybridoma (Mab2) which makes an IgG1 antibody to phosphorylcholine, and the myeloma fusion partner (Sp2/0) used to construct hybridoma 476. / When the antibody specificity was abrogated by genetic manipulation or by natural selection, the 476 cells were able to grow and produce ascites in vivo normally. This shows that the problem was specific, due to binding of the mAb with its antigen, TERT. Telomerase is a housekeeping enzyme in cells essential for maintaining the stability of the telomere, the DNA repeats found at the ends of chromosomes, failing which the cell undergoes senescence and eventually dies. It appeared that the peritoneum, unlike the culture medium, contained factors that presumably up-regulated the antibody production in the cells, which consequently increased the complex formation between TERT and mAb 476 in these cells, and led to cell apoptosis. Indeed, examination of the culture supernatant ("soup") of the peritoneal cells harvested from the adjuvant-primed mice, revealed no cytotoxic cytokines or chemokines present, but an abundant amount of IL-6. In parental 476 cells in culture, both soup and purified recombinant IL-6 were found to up-regulate the production of the intracellular antibodies, but not the secreted antibodies. The effect of these reagents on the Mab2 cells was different; both the intracellular and secreted antibodies were increased. (Abstract shortened by UMI.) / Niu Haitao. / "April 2005." / Adviser: Pak-Leong Lim. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0169. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 199-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
|
260 |
Use of fluorescent imaging to monitor drug responses in mouse models of tumourigenesisBalderstone, Lucy Anne January 2014 (has links)
As our understanding of the complexities of cancer biology has increased, the ability to exploit unique features of tumour cells with molecularly targeted therapies has become a reality. However, despite unprecedented volumes of new molecules in clinical trials, the number of highly effective drugs approved by the regulatory authorities remains disappointingly low. Moreover, oncology drug development is plagued by high levels of attrition in late phase clinical development. Failure due to poor efficacy and toxicity issues are not believed to be a result of the development of molecules with inadequate pharmaceutical properties, but rather due to a lack of understanding of their full mechanism of action. All of this points to imprecise analysis of the drugs during the preclinical phase, highlighting the need for better preclinical drug development tools. Animal models provide a key preclinical tool, and as a therapeutic area, oncology is characterised by models which are not predictive of the true human pathology. Overexpression of the human epidermal growth factor receptor two (HER2) oncogene, and inactivation of the phosphatase and tensin (PTEN) tumour suppressor, are two important events in human breast cancer. A novel conditional mouse model driven by overexpression of HER2 coupled with / without the loss of PTEN has been characterised to interrogate the importance of these two cellular perturbations. Multifocal tumours arose in mice from both lines, while luminal tumour characteristics were shown to be reduced and basal characteristics increased with a reduction in PTEN expression. Disruption of PTEN rapidly accelerated tumour onset (from 138 to 82 days) and tumour growth (with the time from tumour onset to maximum tumour size reduced from 38 to 21 days), significantly reducing overall survival (from 165 to 102 days). The ability of tumour cells to colonize the lungs was not significantly affected by the loss of PTEN. Tumours arising in both mice genotypes were utilized to generate cell lines. These failed to provide an in vitro representation of the tumours, and found little utility in drug efficacy studies with HER family targeted agents, a situation which could be improved by the use of different culture methods. Since suppression of apoptosis is a hallmark of human cancer, and a desired endpoint of many anticancer therapies is the induction of cell death, the generation of cell lines inherently capable of sensing caspase-mediated apoptotic cell death would be a valuable drug development tool. Given that fluorescence imaging is also emerging as a potentially powerful modality for preclinical drug development, a novel fluorescent in house apoptosis reporter construct was generated (pCasFSwitch). Initial validation of pCasFSwitch by transient transfection into murine mammary carcinoma cells proved difficult due to transfection associated toxicity, yet proof-of-principle was indicated. Transfer of pCasFSwitch into a retroviral backbone vector enabled the generation of stably transfected squamous carcinoma cells more suitable for further analysis. Incubation of lysates from these cells with recombinant enzymes revealed the construct could be cleaved by caspase-3, but not by other members of the cysteine protease family. Furthermore, assessment of apoptosis levels in the cells upon staurosporine treatment proved the utility of the construct to quantify cell death, and was validated against data generated with a commercial competitor, NucView. Further comparison of the specificity of the imaging agents using caspase inhibitors was limited by the functionality of currently available inhibitors, but did reveal that in common with NucView, construct quantified levels of apoptosis were affected by inhibition. This thesis details the development of two preclinical drug development tools. A novel mouse model enables biological interrogation of two key events in human breast carcinogenesis. Since PTEN loss is associated with resistance to HER2 targeted therapies, it is ideally suited for efficacy testing to overcome such resistance. The in house fluorescent apoptosis imaging agent allows a temporal read-out of drug effects in live single cells. While the use of intravital imaging of stable cell lines implanted under imaging windows would allow in vivo validation of in vitro data. Taken together, such facilitation of thorough evaluation of therapies at the preclinical stage, will reduce the adverse effects felt by the pharmaceutical industry of failure late in the drug development pipeline.
|
Page generated in 0.0344 seconds