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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.
41

The proto-oncoprotein PRAME associates with the Cullin-2E3 ubiquitin ligase complex and is upregulated in response to bacterial pathogen associated molecual patterns

Wadelin, Frances Roswyn January 2012 (has links)
PRAME (preferentially expressed antigen in melanoma) is a cancer-testis antigen that is expressed in normal testis tissue and is detected at high levels in a number of haematological and solid organ malignancies. Little is known about the physiological function of PRAME, although a role in the repression of retinoic acid receptor signalling and modulation of cell proliferation has been proposed. PRAME has been shown to promote leukaemogenesis; therefore it is important to understand its expression is regulated in normal and cancerous cells. The aim of this thesis was to investigate PRAME gene regulation and to discover clues to its function by affinity purification of interacting proteins. Using sequence similarity searches, PRAME is defined here as a leucine-rich repeat protein, and it is shown to undergo both nuclear and cytoplasmic localisation. Transcription of the PRAME gene is shown to be induced by exposure of malignant cell lines to bacterial pathogen-associated molecular patterns (PAMPs) in combination with interferon-y. Evidence is presented that PAMP/interferon-y treatment also upregulates the translation of PRAME, and alters the subcellular localisation of PRAME protein, resulting in its association with the Golgi apparatus. These results suggest that PRAME may have a role in the innate immune response. Using affinity purification and mass spectrometry, novel PRAME- interacting proteins were identified. PRAME is shown to associate with the Cullin-2-E3 ubiquitin ligase complex via interaction with elongin C, as validated by in vitro binding studies, co-immunoprecipitation and immunofluorescence staining. Cytoplasmic PRAME is shown to colocalise with elongins in Golgi-like vesicles. In addition, PRAME is shown to interact with histones. Based on these results, it is proposed that PRAME is a BC-box protein that may function as a substrate targeting component of Cullin-2-E3 ubiquitin ligase complexes, which may aid in trafficking of intracellular proteins to endosomes and Golgi for degradation or modification. The interaction with histones suggests that nuclear PRAME may perform some role in gene regulation, and future studies will explore its potential role in histone ubiquitination.
42

Flavonoid metabolism and bioactivities in a prostate cancer cell model

Xu, Juan January 2012 (has links)
Flavonoids are promising candidate agents for chemoprevention in prostate cancer. However, only a few flavonoids have been investigated and studies on effects of flavonoid metabolites are rarely reported. Glucuronides of eight flavonoids were enzymatically-synthesised and impact on prostate cancer LNCaP cells in comparison with their parent flavonoid was assessed. The molecular mechanism was explored by studying expression of uridine 5'-di phospho-gl ucuronosy I transferase (UOT), catechol-O-methyltransferase (COMT) and estrogen receptor (ER)-β in LNCaP cells. All flavonoids were glucuronidated by LNCaP cells, but to different extents. Catechol flavonoids were methylated prior to glucuronidation, the methyl form predominating. Methylation was inhibited by 3, 5-dinitrocatecol (DNC; a COMT inhibitor), conversely hesperetin, genistein and naringenin glucuronidation was increased by co-incubation of DNC. Apigenin, luteolin, quercetin and isorhamnetin showed cytotoxic effects on LNCaP cells. All metabolites demonstrated reduced cytotoxicity compared to parent compounds, except genistein glucuronide. Co-incubation of catechol-flavonoids with DNC reduced cytotoxicity, and DNC alone dose-dependently stimulated cell proliferation. Ascorbic acid with luteolin or quercetin showed synergistic effects on cytotoxicity at physiologically achievable concentrations. Glucuronidation of testosterone in LNCaP cells was enhanced by flavonoids in a dose-dependent manner. Flavonoid metabolite mixtures had reduced activity however, the purified glucuronides displayed higher activity, showing position of glucuronide conjugation is significant. DNC co-treatment with flavonoids reduced their potential to enhance UGT activity. Luteolin, quercetin, apigenin and genistein significantly increased UGT2B17 expression. UGT2B15 expression was enhanced by eriodictyol, genistein, hesperetin, kaempferol and nanngemn. DNC dose-dependently down-regulated expression of UGT2B 17, while up-regulating UGT2BI5. All flavonoids, except homoeriodictyol, enhanced COMT activity toward luteolin; genistein demonstrated the highest activity. Genistein and hesperetin-treated cells significantly increased COMT expression. COMT was down-regulated by increasing concentration of DNC. Flavonoids up-regulated the expression of ER~ 1 but showed varying effects on ER~2 and ER~5 isoforms. Overall, the data demonstrates an interaction of flavonoids and flavonoid glucuronides with LNCaP cells. Flavonoids differentially regulate the expression of UGT, COMT, and ER~ isoforms; upregulation of UGT2B 17 appears to be correlated to the cytotoxic effects observed. Future studies should continue to determine the bioactive potential for flavonoids and metabolites in chemoprevention of prostate cancer
43

The rapid measurement of LNCAP intracellular PSA as a model for a novel cell based diagnostic exploiting magneto immunoassay

Sharif, Elham Abdullatif Modh January 2009 (has links)
A novel magneto immunoassay was developed to detect intracellular PSA in LNCAP cells. This project was conducted to investigate methods of rapid intracellular protein measurement using a magneto-immuno assay. Cell cultures were established using Jurkat and ECV304 cells and were used as a continuous source of intracellular proteins. As the project progressed LNCAP cells were used as a model for intracellular PSA. To release intracellular protein, experiments were performed to develop a novel method of cell lysis using paramagnetic particles (PMPs). To accomplish this, eight different lysis methods were initially evaluated using a quantitative approach measuring total protein and lactate dehydrogenase LDH activity. A qualitative approach was adapted to study the protein by western blotting and environmental scanning electron microscopy (ESEM). A novel method utilizing a sonicator probe in conjunction with paramagnetic particles to lyse Jurkat cells was investigated; two types of particles were investigated (1 urn and 2.8 urn Dynabeads). This method was compared with other traditional lysing methods such as freeze-thawing, detergent, heat treatment and a sonicator water bath. The results showed that chemical lysis method was the most efficient for releasing protein and yielded the highest LDH activity. But chemical lysis method was not able to release measurable PSA from LNCAP cells. A higher amount of PSA was released when sonicating LNCAP cells with super paramagnetic particles energised with a sonicator probe. Two sizes of paramagnetic particles (2.8 and 1 urn) and particles of the same size with different coating were evaluated. The size of the paramagnetic particles had a significant effect on the sonication process. ESEM studies showed that the cells tend to lose their shape and integrity when sonicated by the sonicator alone. Moreover comparing the micrographs obtained when sonicating the cells with 1 urn particles a membranous structures was observed while a coagualtive structures were observed when sonicating the cells with 2.8 urn particles. The second part of the thesis describes an investigation into a novel magneto immunoassay, using paramagnetic particles as a label for PSA detection, using a prototype devices developed at UWE. The method developed relies on the specificity of antibody-antigen interaction between antigen and antibody on PMPs and the surface of specifically designed reaction vessel containing the PMP to be captured on the surface. The captured PMP was then detected using magnetometer which has a resonant coil situated below the reaction surface. Using this approach a faster and cheaper detection of PSA could be achieved. A disposable reaction chamber, using low sample volume is added advantages in this method. Two detection systems were evaluated, a resonant coil magnetometer (RCM) detection system and a fixed frequency magnetometer (FFM), both developed at UWE. The latter system was used to investigate the effect of testosterone on LNCAP cells and the release of intracellular PSA. The results show that androgens cause a positively enhanced LNCAP cell growth rate, which consecutively leads to an increase in LNCAP intracellular PSA production. Correlation of serum samples using patient's samples from the Bristol Royal Infirmary was performed. The FFM system gave 90 % sensitivity and specificity.
44

Development and evaluation of a "molecular biopsy" for improved diagnosis of prostate cancer

Nair, Sabarinath Balachandran January 2013 (has links)
Prostate cancer is the most commonly diagnosed cancer in men in United Kingdom. Current diagnosis involves measurement of serum PSA levels and prostate biopsy. However, these tests can give false positive or negative results. Furthermore, they do not indicate disease staging, the behaviour and development of the cancer and hence do not lend to optimal management. Using blood samples, quantitative, real-time RT-PCR (LightCycler™) was used to evaluate molecular markers for inclusion in an alternative minimally invasive diagnostic and prognostic test. Expression levels of Clusterin, Caveolin-1 (Cav-1), E-cadherin (ECAD), Alpha-Methylacyl-CoenzymeA Racemase (AMACR), Enhance of zeste homologue 2 (EZH2) and Matrix metalloproteinases 2 and 24 (MMP2, MMP24) were measured in patient groups and correlated with known clinical details collected on a prostate cancer database. Normal males and patients with negative biopsy results were used as control groups. GAPDH was chosen as the housekeeping gene to normalize gene expression levels. Results and subsequent statistical analysis indicate that Clusterin, Cav-1, and ECAD are potentially strong markers for prostate cancer diagnosis and staging of the disease, and therefore in patient management. The strength of these markers can be increased by combining the results with other similarly evaluated markers. In this way, a combination of diagnostically and prognostically strong markers may be identified forming a panel of biomarkers for inclusion in a potential test. The development of further products now allows transportation of blood at ambient temperature with guaranteed mRNA stability. This will allow blood samples to be taken in a local surgery and transported to a routine laboratory for testing. This would help encourage men to seek prostate disease screening, enabling early diagnosis and intervention while avoiding unnecessary prostate biopsies and treatment.
45

The functional role of ADAMTS-1 and -15 in prostate cancer progression

Molokwu, Chidiebele N. January 2011 (has links)
Introduction: Prostate cancer is a leading cause of cancer death in men. Death is usually a consequence of castration resistant tumour progression. Some metalloproteinases are implicated in the process of cancer progression. ADAMTS proteinases (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) are metalloproteinases that play diverse roles in tissues. Prostate cancer cells express ADAMTS-l and -15 but the role played by these proteinases in prostate cancer progression is unknown. This study was designed to determine the role of ADAMTS-l and 15 in prostate cancer progression. Materials & Methods: Prostate cancer and stromal cell tumour spheroids were grown in 3-dimensional culture in ECM gel containing a quenched-fluorescent substrate. The tumour spheroids were observed for evidence of proteolytic activity. Prostate cancer cells were treated with DHT and TNF. Changes in expression of ADAMTS-I and -15 were analysed. ADAMTS-l and -15 expression was knocked-down in PC3 prostate cancer cells and the effect of knock-down on proliferation, migration and invasion was analysed. Results: Tumour spheroids emitted fluorescence after approximately 24 hours in culture, indicating proteolytic activity. DHT and TNF down-regulated ADAMTS-15 expression in LNCaP cells and stromal cells respectively. The validated anti-ADAMTS- 15 antibody detected 50kDa bands, suggesting a novel cleavage site within the disintegrin-like domain of ADAMTS-15. ADAMTS-l and 15 knock-down had no effect on proliferation, migration or invasion ofPC3 prostate cancer cells. Conclusions: Prostate cancer and stromal cells degrade components of the surrounding ECM. ADAMTS-15 but not ADAMTS-I expression is androgen and TNF-regulated. ADAMTS-l and 15 expression do not affect the proliferation, migration or invasive potential of PC3 cells in vitro. Cleavage of ADAMTS-15 in the disintegrin-like domain results in the release of a C-terminal fragment with potential anti-angiogenic properties. Down-regulation by DHT in prostate cancer cells suggests that ADAMTS-15 could be playing an anti-tumour role in prostate cancer progression.
46

Evaluation of Morphological and Other Aspects of male Infertility in Testicilar Neoplasia using Lectin Histochemistry and Semen Analysis

Ghasemi, Behrooz January 2008 (has links)
It has been estimated that approximately 15% of couples attempting to attain pregnancy are unable to do so. A female factor is the primary aetiology in nearly 40% of these couples and in another 30% the factors are purely male. A combmation of male and female factors has been implicated for the remaining 30% of cases. Infertility is a common problem among men with testicular cancer. Such men are especially affected by infertility and they often decide to undergo sperm banking (the collection and freezing of sperm) before beginning chemo/radiotherapy.
47

Structure-property relationships in biological tissues

Yang, Teo Heng Jimmy January 2007 (has links)
No description available.
48

Functional analysis of TSPY and its role in prostate carcinogenesis

Omar, Mahmoud Mustafa January 2005 (has links)
Testis specific protein Y chromosome encoded (TSPY) is a multicopy gene located on the human Y chromosome. It has been reported that the human genome harbours between 20 to 40 copies of the gene. Mammalian homologues of human TSPY were also found to be repetitive. The gene is expressed in human foetal and adult testis. The precise function of the expressed TSPY gene product is not fully defined, but it has been postulated to regulate proliferation of testicular spermatogonia. Up-regulation of TSPY expression has been detected in gonadoblastoma, testicular cancer and prostate cancer. The contribution of abnormal expression of TSPY to prostate carcinogenesis has not been investigated. Prostate cancer (CaP) and benign prostate hyperplasia (BPH) are the most common diseases of the human prostate. Prostate cancer is a disease of the elderly and is the second most common cancer and second most common cause of death from cancer among men in UK. In this thesis, the role of TSPY in prostate carcinogenesis was studied in four related aspects. First, TSPY protein and transcript expression pattern in CaP compared to Benign Prostate Hyperplasia (BPH) was studied using a combination of immunohistochemistry and mRNA in situ hybridisation. Second, the ability of TSPY to regulate cellular proliferation was investigated by transfection experiments of the prostate cancer LNCaP cell line. Third, TSPY genomic copy number in prostate cancer was characterised and compared to BPH. Finally, the downstream genes regulated by TSPY were investigated using high density microarray gene profiling method. An anti-human TSPY polyclonal antibody was developed for immunodetection of TSPY expression level in resected prostate tissues. In total, 72 cases of patients with prostate cancer and 20 cases of patients affected with BPH were studied by immunohistochemistry. TSPY was predominantly detected in the prostatic epithelium. In the benign gland, TSPY expression was limited to the basal cells compartment. TSPY expression was significantly up-regulated in prostate cancer when compared to BPH (P<0.0001). Furthermore, increased TSPY expression level was associated with aggressive disease (tumour with high Gleason score; P<0.02) and the presence of bone metastasis at the time of diagnosis (P<0.028). To address the functional role of 3 TSPY in prostate cancer, FLAG-TSPY was cloned and stably transfected into LNCaP cells. The presence of transfected TSPY increased LNCaP proliferation by two fold compared to empty vector control, consistent with a mitogenic function in CaP (P<0.0001). An absolute quantitative real time PCR based on Taqman assay was established and validated. TSPY genomic copy number was determined from comparing 161 samples: CaP-serum (n=47), resected tumour (n=31); BPH-serum (n=27), resected prostate (n=13) and control-serum (n=45). Of the clinical samples analysed, interpersonal variability of TSPY copy numbers was observed with the majority of cases containing between 20 to 50 TSPY copies per genome. Although, there were variability in TSPY copy numbers among individuals, there was no statistically significant correlation between TSPY copy number (serum and prostatic tissue) and the development of prostate cancer. Studying LNCaP stably transfected with TSPY and empty vector control, the key genes mediating the functional effect of TSPY were identified using Affymetrix oligonucleotide microarray method. In total, 332 genes have been altered 1.5 to 90 fold due to the effect of TSPY over-expression. Ten genes were selected and the gene expression levels were confirmed using semi-quantitative RT-PCR method. Gene clustering analysis has indicated changes in genes that regulate cellular differentiation (NRDG1, NF2, C-MAF and BMP11), apoptotic gene (BAX), cell cycle gene (cyclin G), detoxification gene (GST-2A) and genes linked to adhesion (PCDH7a, PLOD2 and IRS 1). In summary, TSPY is over-expressed in prostate cancer. This abnormal expression is linked to prostate cancer progression and metastasis. The up-regulation of TSPY expression is unlikely to be due to increased copy number. Expression of exogenous TSPY in prostate cancer cells enhanced cellular proliferation. The gene meditates its effect by down-regulating the expression pattern of selected differentiation, apoptosis and adhesion genes. Hence, over-expression of TSPY contributes to prostate carcinogenesis.
49

An investigation into the effects of protease activated receptors on biological aspects of prostate cancer progression

Gallagher, Sandra January 2006 (has links)
No description available.
50

A putative role for TMPRSS2 in prostate cancer progression

Wilson, S. R. January 2004 (has links)
No description available.

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