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The measurement and regulation of cytosolic calcium in plant cellsGilroy, Simon January 1987 (has links)
No description available.
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Cloning of oestrogen regulated mRNA from human breast cancer cellsDaly, R. J. January 1988 (has links)
No description available.
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Dual Regulation of Telomerase Activity By HSF1 And Its Role in Prostate Cancer ProgressionJensen, Keith Douglas Ostergaard 01 January 2006 (has links)
It has been shown that the key components of the hsp90 chaperone complex, including hsp90, p23, hsp70, hsp40, and HOP (p60), associate with telomerase; however, their specific roles in telomerase function and tumor progression have not yet been defined. HSF1, the primary mammalian heat shock protein transcription factor, may affect telomerase activity and transformation by regulating the expression of several hsp90 chaperone complex proteins in response to stress as well as regulating the transcription of hTERT, the protein subunit of telomerase.In our in vitro model of prostate cancer progression, as cells progress from immortal but non-tumorigenic (P69) to tumorigenic (M2182) and eventually metastatic (M12) capabilities, both telomerase activity and global chaperone protein levels increase. Our hypothesis is that HSF1 affects telomerase activity directly at the level of transcription and indirectly at the protein level via its regulation of proteins of the hsp90 chaperone complex. Furthermore, upregulation of HSF1 and/or members of the hsp90 chaperone complex directly contribute to prostate cell transformation and that introduction of chaperone-related genes will convert non-tumorigenic prostate cells to a tumorigenic state.We have shown that ectopic overexpression of HSF1 induces increased expression of endogenous hsp90 in P69 cells. Furthermore, telomerase activity in the overexpressing HSF1 cell lines is increased as well and is the end result of two disparate, yet ultimately cooperating pathways. However, the increased telomerase activity does not correlate with increased tumorigenicity.In conjunction with this study, we have overexpressed hTERT in the P69 cell lines and found that telomerase activity is markedly increased in the absence of chaperone upregulation. We propose that the demand for increased folding and stability of the exogenous hTERT leads to a recruitment of telomerase associated chaperone proteins, which can be measured by increased activity after immunoprecipitations and nuclear translocation of hsp90 chaperone complex proteins.Taken together, these projects indicate a significant role for HSF1 and the hsp90 chaperone complex proteins on telomerase activity, and provide evidence that each may be a viable target for therapeutic intervention.
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A comparative analysis of the regulatory framework of the therapeutic application of stem cell technologiesLaurens, Johannes Bernardus January 2017 (has links)
Stem cell technologies as a branch of regenerative medicine are becoming increasingly popular as the science behind it evolves. Therefore, it is important that the regulatory framework pertaining to stem cell technologies be well defined and appropriate to prevent unethical and unscrupulous behaviour on the part of medical practitioners, which gives rise to stem cell tourism. South African legislation pertaining to stem cell technology is regarded as inadequate and dissonant with the Constitution, exacerbating the problem of stem cell tourism and denying patients access to certain stem cell therapies, which ultimately can be viewed as an infringement of their constitutional rights. The United Kingdom (UK) provides a clear-cut regulatory framework, which is not only centred around consent and patient safety but is also conducive to production of stem cell therapies. For such reasons, this dissertation finds the UK framework to be an appropriate benchmark against which the South African regulatory framework can be evaluated. By means of comparison and elaborating on the biology of stem cells in addition to pertinent ethical principles, legislation and human rights of both South Africa and the UK, an argument will be made out that South African legislation pertaining to stem cell therapy and related matters is wanting. Furthermore, analysis will be made of the definition of biological medicine as put forward by the Medicines and Related Substances Control Act 101 of 1965 to conclude that certain stem cell therapies are best excluded from such a definition as such stringent requirements and protocols encumbers access to stem cell therapies and inflates costs. Lastly, remedial measures are proposed to remedy these injustices by proposing for the institution of a specialist adivisary committee to oversee stem cell and related activities.
Key Words: Regenerative Medicine; Stem Cells; Stem Cell Regulation; National Health Act; Medicines and Related Substances Control Act; Advanced Therapy Medicinal Product; Human Tissue Authority; Human Fertilisation and Embryology Authority; HTA; HFEA; Medicine and Healthcare Products Regulatory Agency; MHRA; European Medicines Agency; Tissue-engineered Products; Doctor-Patient Relationship; Medical Innovation Bill 2014; Experimental Treatments; Innovative Therapy; Hospital Exemption; Informed Consent; Special Exemption; Autologous Stem Cell Therapy; Stem Cell Transplants; Gene Therapy Advisory Committee. / Dissertation (LLM)--University of Pretoria, 2017. / Public Law / LLM / Unrestricted
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The Role of Sphingosine Kinase 2 in Cell Growth and ApoptosisSankala, Heidi M. 01 January 2007 (has links)
Two isoforms of sphingosine kinase (SphK) catalyze the formation of sphingosine-1-phosphate (SIP). Whereas, SphKl stimulates cell growth and survival, it was found that when overexpressed in mouse NIH 3T3 fibroblasts SphK2 enhances caspase-dependent apoptosis in response to serum deprivation, independently of S1P receptors. Sequence analysis revealed that SphK2 contains a 9 amino acid motif similar to that present in BH3-only proteins. Studies showed that the BH3-only domain, catalytic activity, endoplasmic reticulum (ER) stress, and uptake of calcium by the mitochondria may all contribute to the apoptotic effects of overexpressed SphK2 in NIH 3T3 cells. Further studies in human carcinoma cells showed that overexpression of SphK2 increased the expression of the cyclin dependent kinase (cdk) inhibitor p21, but interestingly had no effect on p53 or its phosphorylation. Correspondingly, downregulation of endogenous SphK2 with small interfering RNA (siRNA) targeted to unique mRNA sequences decreased basal and doxorubicin-induced expression of p21 without affecting p53. In addition, downregulation of SphK2 decreased G2/M arrest in response to doxorubicin. Surprisingly however, siSphK2 markedly enhanced apoptosis induced by doxorubicin in MCF7 and HCT-116 cells. This result raises the question of how overexpression of SphK2 decreases cell growth and enhances apoptosis while its downregulation sensitizes cells to apoptosis. A partial answer may come from the possibility that when SphK2 is overexpressed it does not always have the same subcellular distribution as the endogenous protein. It may also be possible that proteolysis of overexpressed SphK2 might induce apoptosis due to liberation of its BH3 peptide domain, which does not occur at the levels at which endogenous SphK2 is expressed. Collectively, these results demonstrate that endogenous SphK2 is important for p53-independent induction of p21 expression by doxorubicin and suggest that SphK2 expression may influence the balance between cytostasis and apoptosis.
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MOLECULAR MECHANISMS THAT GOVERN STEM CELL DIFFERENTIATION AND THEIR IMPLICATIONS IN CANCERLama Abdullah Alabdi (7036082) 02 August 2019 (has links)
<p>Mammalian development is
orchestrated by global transcriptional changes, which drive cellular
differentiation, giving rise to diverse cell types. The mechanisms that mediate
the temporal control of early differentiation can be studied using embryonic
stem cell (ESCs) and embryonal carcinoma cells (ECCs) as model systems. In
these stem cells, differentiation signals induce transcriptional repression of
genes that maintain pluripotency (PpG) and activation of genes required for
lineage specification. Expression of PpGs is controlled by these genes’
proximal and distal regulatory elements, promoters and enhancers, respectively.
Previously published work from our laboratory
showed that during
differentiation of ESCs, the repression of PpGs is accompanied by enhancer
silencing mediated by the Lsd1/Mi2-NuRD-Dnmt3a complex. The enzymes in this
complex catalyze histone H3K27Ac deacetylation and H3K4me1/2 demethylation
followed by a gain of DNA methylation mediated by the DNA methyltransferase,
Dnmt3a. The absence of these chromatin changes at PpG enhancers during ESC
differentiation leads to their incomplete repression. In cancer, abnormal
expression of PpG is commonly observed. Our studies show that in
differentiating F9 embryonal carcinoma cells (F9 ECCs), PpG maintain
substantial expression concomitant with an absence of Lsd1-mediated H3K4me1
demethylation at their respective enhancers. The continued presence of H3K4me1
blocks the downstream activity of Dnmt3a, leading to the absence of DNA
methylation at these sites. The absence of Lsd1 activity at PpG enhancers
establishes a “primed” chromatin state distinguished by the absence of DNA
methylation and the presence of H3K4me1. We further established that the
activity of Lsd1 in these cells was inhibited by Oct3/4, which was partially
repressed post-differentiation. Our data reveal that sustained expression of
the pioneer pluripotency factor Oct3/4 disrupts the enhancer silencing
mechanism. This generates an aberrant “primed” enhancer state, which is susceptible
to activation and supports tumorigenicity. </p>
<p>As differentiation proceeds and
multiple layers of cells are produced in the early embryo, the inner cells are
depleted of O<sub>2</sub>, which triggers endothelial cell differentiation. These
cells form vascular structures that allow transport of O<sub>2</sub> and nutrients to cells. Using
ESC differentiation to endothelial cells as a model system, studies covered in
this thesis work elucidated a mechanism by which the transcription factor
Vascular endothelial zinc finger 1 (Vezf1) regulates endothelial
differentiation and formation of vascular structures. Our data show that
Vezf1-deficient ESCs fail to upregulate the expression of pro-angiogenic genes
in response to endothelial differentiation induction. This defect was shown to
be the result of the elevated expression of the stemness factor Cbp/p300-interacting
transactivator 2 (Cited2)
at the onset of differentiation. The improper expression of Cited2 sequesters
histone acetyltransferase p300 from depositing active histone modifications at
the regulatory elements of angiogenesis-specific genes that, in turn, impedes
their activation. </p>
<p>Besides the discovery of
epigenetic mechanisms that regulate gene expression during differentiation, our
studies also include development of a sensitive method to identify activities
of a specific DNA methyltransferase at genomic regions. In mammals, DNA
methylation occurs at the C5 position of cytosine bases. The addition of this
chemical modification is catalyzed by a family of enzymes called DNA methyltransferases
(Dnmts). Current methodologies, which determine the distribution of Dnmts or
DNA methylation levels in genomes, show the combined activity of multiple Dnmts
at their target sites. To determine the activity of a particular Dnmt in
response to an external stimulus, we developed a method, Transition State
Covalent Crosslinking DNA Immunoprecipitation (TSCC-DIP), which traps
catalytically active Dnmts at their transition state with the DNA substrate.
Our goal is to produce a strategy that would enable the determination of the
direct genomic targets of specific Dnmts, creating a valuable tool for studying
the dynamic changes in DNA methylation in any biological process.</p>
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Stem Cell Regulation Using Nanofibrous Membranes with Defined Structure and Pore SizeBlake, Laurence A 08 1900 (has links)
Electrospun nanofibers have been researched extensively in the culturing of stem cells to understand their behavior since electrospun fibers mimic the native extracellular matrix (ECM) in many types of mammalian tissues. Here, electrospun nanofibers with defined structure (orientation/alignment) and pore size could significantly modulate human mesenchymal stem cell (hMSC) behavior. Controlling the fiber membrane pore size was predominantly influenced by the duration of electrospinning, while the alignment of the fiber membrane was determined by parallel electrode collector design. Electric field simulation data provided information on the electrostatic interactions in this electrospinning apparatus.hMSCs on small-sized pores (~3-10 µm²) tended to promote the cytoplasmic retention of Yes-associated protein (YAP), while larger pores (~30-45 µm²) promoted the nuclear activation of YAP. hMSCs also displayed architecture-mediated behavior, as the cells aligned along with the fiber membranes orientation. Additionally, fiber membranes affected nuclear size and shape, indicating changes in cytoskeletal tension, which coincided with YAP activity. The mechanistic understanding of hMSC behavior on defined nanofiber structures seeks to advance their translation into more clinical settings and increase biomanufacturing efficiencies.
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