Human skeletal muscle pyruvate dehydrogenase phosphatase activity and expression : the effect of aerobic capacity

Activation of pyruvate dehydrogenase (PDH), which converts pyruvate into acetyl-CoA, is accomplished by a pair of specific phosphatases (PDP 1 & 2). A cross-sectional study investigating the effect of aerobic capacity on PDP activity and expression found that: 1) PDP activity and PDP! protein expression were positively correlated with most aerobic capacity measures in males (n=lS), but not females (n=12); 2) only males showed a positive correlation between PDP activity and PDPl protein expression (r=0.47; p=O.05), indicating that the increase in PDP activity in males is largely explained by increased PDPl protein expression, but that females rely on another level for PDP activity regulation; and 3) PDP} and Ela protein expression increase in unison when expressed relative to the E2 core. These data suggest that with increased aerobic capacity there is an increased capacity for carbohydrate oxidation through PDH, via El a, and an increased ability to activate PDH, via PDP, when exercising maximally.

Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.

Protein subcellular targeting inTrichoderma aggressivum f. aggressivum

Trichoderma aggressivum f. aggressivum is a filamentous soil fungus. Green mold disease of commercial mushrooms caused by this species in North America has resulted in millions of dollars in lost revenue within the mushroom growing industry. Research on the molecular level of T aggressivum have jus t begun with the goal of understanding the functions of each gene and protein, and their expression control. Protein targeting has not been well studied in this species yet. Therefore, the intent of this study was to test the protein localization and production levels in T aggressivum with green fluorescent protein (GFP) with an intron and tagged with either nuclear localization signal (NLS) or an endoplasmic reticulum retention signal (KDEL). Two GFP constructs (with and without the intron) were used as controls in this study. All four constructs were successfully transferred into T aggressivum and all modified strains showed similar growth characteristics as the wild type non-transformed isolate. GFP expression was detected from all modified T aggressivum with confocal microscopy and the expression was similar in all four strains. The intron tested in this study had no or very minor effects as GFP expression was similar with or without it. The GFP signal increased over a 5 day period for all transformants, while the GFP to total protein ratio decreased over the same period for all transformants. The GFP-KDEL transformant showed similar protein expression level and localization as did the control transformant lacking the KDEL retention signal. The GFP-NLS transformant similarly failed to localize GFP into nucleus as fluorescence with this strain was virtually identical to the GFP transformant lacking the NLS. Thus, future research is required to find effective localization signals for T aggressivum.

Investigation of the Expression of Glucose Transporter Proteins in Human Cancer Cells

Cancer cells are known to display increased glucose uptake and consumption. The glucose transporter (GLUT) proteins facilitate glucose uptake, however, their exact role in cancer metabolism remains unclear. The present study examined mRNA and protein expression of GLUT1, GLUT3, GLUT4 and GLUT12 in lung, breast and prostate cancer cells and corresponding noncancerous cells. Additionally, GLUT expression was determined in tumours from mice xenografted with human cancer cells. Differences in the mRNA and protein expression of GLUTs were found between cancerous and corresponding noncancerous cells. These findings demonstrate abundant expression of GLUT1 in cancer and highlight the importance of GLUT3 as it was expressed in several cancer cells and tumours. GLUT expression patterns in vitro were supported by the in vivo findings. The study of GLUT protein expression in cancer is important for understanding cancer metabolism and may lead to identification of biomarkers of cancer progression and development of target therapies.

Potential Urinary Protein Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients

Globally, Prostate cancer (PCa) is the most frequently occurring non-cutaneous cancer, and is the second highest cause of cancer mortality in men. Serum prostate specific antigen (PSA) has been the standard in PCa screening since its approval by the American Food & Drug Administration (FDA) in 1994. Currently, PSA is used as an indicator for PCa - patients with a serum PSA level above 4ng/mL will often undergo prostate biopsy to confirm cancer. Unfortunately fewer than similar to 30% of these men will biopsy positive for cancer, meaning that the majority of men undergo invasive biopsy with little benefit. Despite PSA's notoriously poor specificity (33%), there is still a significant lack of credible alternatives. Therefore an ideal biomarker that can specifically detect PCa at an early stage is urgently required. The aim of this study was to investigate the potential of using deregulation of urinary proteins in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the protein signatures specific for PCa, protein expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using LC-MS/MS. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR. This approach revealed that significant the down-regulation of Fibronectin and TP53INP2 was a characteristic event among PCa patients. Fibronectin mRNA down-regulation, was identified as offering improved specificity (50%) over PSA, albeit with a slightly lower although still acceptable sensitivity (75%) for detecting PCa. As for TP53INP2 on the other hand, its down-regulation was moderately sensitive (75%), identifying many patients with PCa, but was entirely non-specific (7%), designating many of the benign samples as malignant and being unable to accurately identify more than one negative.

The cloning and reconstitution of a bovine adenovirus type 2 E3 deletion mutant and the sequencing and analysis of the early 4 region

Recombinant Adenoviruses (Ads) have been shown to have potential applications in three areas: gene therapy, high level protein expression and recombinant vaccines.' At least three different locations within the Ad genome can be deleted and subsequently used for the insertion of foreign sequences. These include the Early 3 (E3), Early 1 (E1) and Early 4 (E4) regions. Viral vectors of this type have been well studied in Human Ads 2 and 5, however one has not yet been constructed for Bovine Adenovirus Type 2 (BAV2). The E3 region is located between 76.6 and 86 m.u. on the r-strand and is transcribed in a rightward direction. The gene products of the Early 3 region (E3) have been shown to be non-essential for viral replication, in vitro, but are required for host immunosurveillance. This study represents the cloning and reconstitution of a BAV2 E3 deletion mutant. A deletion of 1800bp was made within the E3 region of BAV2 and the thymidine kinase gene was subsequently inserted in the deleted area . . The plasmid pdlE3-4tk1 (23.4Kbp) was constructed and used to to facilitate homologous recombination with the wild type BAV2 to produce a mutant. Southern Blotting and Hybridization results suggest the presence of a BAV2 E3 deletion mutant with thymidine kinase sequences present. The E4 region of Human Adenovirus types 2 and 5 is located at the extreme right end of the genome (91.3 map units - 99.1 map units) and is transcribed in a leftward direction giving rise to a complicated set of differentially spliced mRNAs. Essentially there are 7 open reading frames (ORFs) encoding for at least 7 polypeptides. The gene products encoded by the E4 region have been shown to be essential for the expression of late viral genes, host cell shutoff and normal viral growth. We have cloned and sequenced the right end segment between 90.5 map units and 100 map units of the BAV2 genome. The results show several open reading frames which encode polypeptides exhibiting homology to three polypeptides encoded by the E4 region of human adenovirus type 2. These include the 14kDa protein encoded by ORF1, the 34kDa protein encoded by ORF6 and the 13kDa protein encoded by ORF3. The nucleotide sequence, restriction enzyme map, and ORF map of the E4 region could be very useful in future molecular manipulation of this region and could possibly explain the slow growth rate of BAV2 in MDBK cells.

Evolutionary synthesis of stochastic gene network models using feature-based search spaces

A feature-based fitness function is applied in a genetic programming system to synthesize stochastic gene regulatory network models whose behaviour is defined by a time course of protein expression levels. Typically, when targeting time series data, the fitness function is based on a sum-of-errors involving the values of the fluctuating signal. While this approach is successful in many instances, its performance can deteriorate in the presence of noise. This thesis explores a fitness measure determined from a set of statistical features characterizing the time series' sequence of values, rather than the actual values themselves. Through a series of experiments involving symbolic regression with added noise and gene regulatory network models based on the stochastic 'if-calculus, it is shown to successfully target oscillating and non-oscillating signals. This practical and versatile fitness function offers an alternate approach, worthy of consideration for use in algorithms that evaluate noisy or stochastic behaviour.

The Structural and Functional Changes of Blood Vessels during Aging

The vascular adventitia is recognized as a dynamic mediator of vascular structure and function, yet its role in aging is not understood. The purpose of this thesis was to examine the age-related changes of the vascular adventitia and determine the underlying mediators responsible. Male Sprague-Dawley rats were aged to 15, 30, 50 and 80 weeks before being anesthetised and euthanized by exsanguination. Thoracic aortas, mesenteric and pudental arteries were isolated, formalin fixed, and embedded in paraffin then sectioned at 5μm. Vessels were examined by microscopy and protein expression was determined by indirect immunofluorescence. The thickness of the adventitia increased dramatically with age. Immunofluorescence revealed a robust expression of endothelin system proteins in the adventitia. Additionally, extracellular matrix proteins collagen and fibronectin, and the proliferation marker Ki67 showed strong adventitial origin. The changes observed in the vascular adventitia with aging clearly demonstrate an important role in the process of vascular aging.

The structural and functional changes of blood vessels during aging

The vascular adventitia is recognized as a dynamic mediator of vascular structure and function, yet its role in aging is not understood. The purpose of this thesis was to examine the age-related changes of the vascular adventitia and determine the underlying mediators responsible. Male Sprague-Dawley rats were aged to 15,30,50 and 80 weeks before being anesthetised and euthanized by exsanguination. Thoracic aortas, mesenteric and pudental arteries were isolated, formalin fixed, and embedded in paraffin then sectioned at 51lm. Vessels were examined by microscopy and protein expression was determined by indirect immunofluorescence. The thickness of the adventitia increased dramatically with age. Immunofluorescence revealed a robust expression of endothelin system proteins in the adventitia. Additionally, extracellular matrix proteins collagen and fibronectin, and the proliferation marker Ki67 showed strong adventitial origin. The changes observed in the vascular adventitia with aging clearly demonstrate an important role in the process of vascular aging.

Localization of monoterpenoid indole alkaloid (MIA) biosynthesis by in situ hybridization (ISH)

Monoterpenoid indole alkaloids (MIA) are among the largest and most complex group of nitrogen containing secondary metabolites that are characteristic of the Apocynaceae plant family including the most notable Catharanthus roseus. These compounds have demonstrated activity as successful drugs for treating various cancers, neurological disorders and cardiovascular conditions. Due to the low yields of these compounds and high pharmacological value, their biosynthesis is a major topic of study. Previous work highlighting the leaf epidermis and leaf surface as a highly active area in MIA biosynthesis and MIA accumulation has made the epidermis a major focus of this thesis. This thesis provides an in-depth analysis of the valuable technique of RNA in situ hybridization (ISH) and demonstrates the application of the technique to analyze the location of the biosynthetic steps involved in the production of MIAs. The work presented in this thesis demonstrates that most of the MIAs of Eurasian Vinca minor, African Tabernaemontana e/egans and five Amsonia species, including North American Amsonia hubrichitii and Mediterranean A. orienta/is, accumulate in leaf wax exudates, while the rest of the leaf is almost devoid of alkaloids. Biochemical studies on Vinca minor displayed high tryptophan decarboxylase (TOe) enzyme activity and protein expression in the leaf epidermis compared to whole leaves. ISH studies aimed at localizing TOe and strictosidine synthase suggest the upper and lower epidermis of V. minor and T. e/egans as probable significant production sites for MIAs that will accumulate on the leaf surface, however the results don't eliminate the possibility of the involvement of other cell types. The monoterpenoid precursor to all MIAs, secologanin, is produced through the MEP pathway occurring in two cell types, the IPAP cells (Gl0H) and epidermal cells (LAMT and SLS). The work presented in this thesis, localizes a novel enzymatic step, UDPG-7-deoxyloganetic acid glucosyltransferase (UGT8) to the IPAP cells of Catharanthus longifolius. These results enable the suggestion that all steps from Gl0H up to and including UGT8 occur in the IPAP cells of the leaf, making the IPAP cells the main site for the majority of secologanin biosynthesis. It also makes the IPAP cells a likely cell type to begin searching for the gene of the uncharacterized steps between Gl0H and UGT8. It also narrows the compound to be transported from the IPAP cells to either 7-deoxyloganic acid or loganic acid, which aids in the identification of the transportation mechanism.

Methoxypyrazine removal in grape juice: Development of a removal system using odorant and pheromone binding proteins coupled with bentonite fining

Multicoloured Asian Lady Beetles (MALB) and 7-spot Lady Beetles that infect vineyards can secrete alkyl-methoxypyrazines when they are processed with the grapes, resulting in wines containing a taint. The main methoxypyrazine associated with this taint is 3-isopropyl-2-methoxypyrazine (IPMP). The wines are described as having aroma and flavours of peanut butter, peanut shells, asparagus and earthy which collectively, have become known as “ladybug taint”. To date, there are no known fining agents used commercially added to juice or wine that are effective in removing this taint. The goal of this project was to use previously identified proteins with an ability to bind to methoxypyrazines at low pH, and subsequently develop a binding assay to test the ability of these proteins to bind to and remove methoxypyrazines from grape juice. The piglet odorant binding protein (plOBP) and mouse major urinary protein (mMUP) were identified, cloned and expressed in the Pichia pastoris expression system. Protein expression was induced using methanol and the proteins were subsequently purified from the induction media using anion exchange chromatography. The purified proteins were freeze-dried and rehydrated prior to use in the methoxypyrazine removal assay. The expression and purification system resulted in yields of approximately 78% of purified plOBP and 62% of purified mMUP from expression to rehydration. Purified protein values were 87 mg of purified plOPB per litre of induction media and 19 mg of purified mMUP per litre of induction medium. In order to test the ability of the protein to bind to the MPs, an MP removal assay was developed. In the assay, the purified protein is incubated with either IPMP or 3-isobutyl-2-methoxypyrazine (IBMP) for two hours in either buffer or grape juice. Bentonite is then used to capture the protein-MP complex and the bentonite-protein-MP complex is then removed from solution by filtration. Residual MP is measured in solution following the MP removal assay and compared to that in the starting solution by Gas Chromatography Mass Spectrometry (GC/MS). GC/MS results indicated that the mMUP was capable of removing IBMP and IPMP from 300 ng/L in buffer pH 4.0, buffer pH 3.5 and Riesling Juice pH 3.5 down to the limit of quantification of the instrument, which is 6ng/L and 2ng/L for IBMP and IPMP, respectively. The results for the plOBP showed that although it could remove some IBMP, it was only approximately 50-70 ng/L more than bentonite treatment followed by filtration, resulting in approximately 100 ng/L of the MPs being left in solution. pIOBP was not able to remove IPMP in buffer pH 3.5 using this system above that removed by bentonite alone. As well, the pIOBP was not able to remove any additional MPs from Chardonnay juice pH 3.5 above that already removed by the bentonite and filtration alone. The mouse MUP was shown to be a better candidate protein for removal of MPs from juice using this system.