Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Arthur Landy. Includes bibliographical references.

Profound deafness affects approximately one in a thousand infants at birth and two in a thousand children by the age of 6 years. In about half of these children hearing loss is hereditary in origin. In order to elucidate genetic factors in profound childhood deafness in South Africa, a diagnostic screening survey of 1060 hearing impaired children attending five special schools for the deaf was performed and the aetiology of hearing impairment in each child was classified into one of four categories: syndromal genetic deafness (11 % of children), familial non-syndromal deafness (10%), acquired deafness (38%), and deafness of unknown cause (41 %). Selected families with genetic deafness were then appraised clinically, otologically and genealogically for purposes of syndromal delineation. In this way, affected kindreds with four distinct genetic deafness disorders were investigated and a variety of molecular techniques were used in order to localise the causative genes to a chromosomal map position. Where possible, genetic markers that could be used for pre-symptomatic and pre-natal diagnosis were identified. The four disorders studied were sclerosteosis (SCL), autosomal dominant craniometaphyseal dysplasia (AD CMD), familial streptomycin ototoxicity (FSO) and a private syndrome comprising deafness, microtia and facial palsy (DMF). Sclerosteosis is an autosomal recessive disorder of bone overgrowth with a high gene frequency among the Afrikaner population of South Africa. Gene mapping of this disorder has important implications for affected families in terms of pre-natal diagnosis and the detection of asymptomatic gene carriers. In the major theme of the project, twenty-two affected families with sclerosteosis were ascertained and the SCL gene was localised by homozygosity mapping to the long arm of chromosome 1 7 at 17q12-17q21 (maximum LOD score of 6.21 at a recombination fraction [8] of Oat marker locus D17S806). Following localisation of the gene, a bone densitometry study of SCL was undertaken in order to determine whether heterozygous gene carriers manifest significant changes in bone mineral density. The results show that the SCL gene is expressed in the heterozygous state. This finding has positive implications for carrier detection as well as for development of research into treatment of disorders of decreased bone density, such as osteoporosis. The gene for AD CMD in an affected family was linked to a locus on the short arm of chromosome 5 at 5p 15 (maximum LOD score 4.08 at 8 = 0 at marker locus D5S 1963) providing evidence for molecular homogeneity of the condition. Fine mapping of the AD CMD region enabled the candidate gene interval to be narrowed to a distance of 11 cM, between the marker loci D5S2004 and D5S486. A family with a maternally inherited genetic predisposition to streptomycin-induced ototoxicity was found to have an adenine to guanine point mutation at position 1555 of the mitochondrial (mt) genome. This mutation was identified by mtDNA sequencing in a single affected individual and it was then established in the rest of the family by restriction enzyme PCR analysis. Three generation kindred with a unique branchial arch disorder (DMF syndrome), was ascertained and genome screening was initiated in an attempt to map the gene. Positive gene linkage in this family was not established but a large proportion of the genome and several candidate gene loci were excluded. The investigation of the four disorders studied in this project has provided a major step forward in the elucidation of genetic deafness at the molecular level. More importantly, for the affected families concerned, it will enable the implementation of improved management in the form of accurate genetic counselling and presymptomatic and pre-natal diagnosis.

Lamb adipose tissue, representing 4 breeds, 3 sex-types and 3 weight categories, were studied to measure the variation in the flavor profile between individual animals, based on breed, sex- type and carcass weight. The flavor concentrates were prepared by steam distillation, ether extraction and fractionation. Pooled samples were prepared in the same way to measure any variations due to methodology. Gas chromatographic separation and statistical analysis, based on analysis of variance, indicated that 21 peaks out of 124 peaks showed significant differences (at 5% level or 1% level). Among these 21 peaks, 5 were affected by breed, 4 by sex-type, 8 by carcass weight and 7 peaks were influenced by the interaction of breed, sex-type and carcass weight. Variations due to breed revealed that Rambouillet lambs possessed the highest proportion of flavor peaks which showed significant differences, with Targhee lambs the second highest, Columbia the third and Suffolk- cross the lowest proportion of flavor compounds. The effect of sex-type suggested that was contributed to the highest concentration of peaks which might influence the expression of flavor. However, there was no evidence showing that the concentration of peaks for ewes was greater than that of wethers or vice versa. Differences associated with weight indicated that light animals yielded a higher concentration of flavor volatiles, medium weight lambs the second and heavy lambs the lowest concentration of volatiles. The influence of the interaction of three variables was found to be more prevalent in the combination of Suffolk-cross ewe heavy and Suffolk- cross ewe medium . Several reasons are employed to explain the deviations of some peaks present in the chromatograms of pooled samples.

No description available.

The effect of Mg status on the behavior of rats, as determined by nonspecific excitability level and audio genic seizure susceptibility, was investigated. Also, selected biochemical and neurological mechanisms mediating the chain of events from dietary magnesium deficit to the hyper excitability symptoms were examined. Weanling rats fed a low magnesium (10 ppm) diet for 14 days had reduced serum, cerebrospinal fluid and brain magnesium concentrations, and increased brain (Na+ K+)-ATPase activity. They exhibited increased NEL and became highly susceptible to audio genic seizures. Through dietary manipulation and intraperitoneal and cerebral intraventricular injections, it was possible selectively to alter either serum or cerebrospinal fluid magnesium concentrations. Both nonspecific excitability level and audio genic seizure susceptibility responded inversely to cerebrospinal fluid magnesium concentration, but not to serum magnesium, unless the latter was very high. In this instance, nonspecific excitability level but not seizure activity was depressed. Brain serotonin concentration was elevated in 150- 200 g rats fed a low magnesium diet for 21 days, compared with rats fed a control diet (0.73 μg/g fresh brain cf. 0.56 μg/g) . The magnesium deficient rat may serve as an excellent model for epilepsy research. The animal can often be revived following seizure, thus enabling the study of drug interactions while using a small number of subjects. Preliminary findings indicate that the most promising subjects are female rats between 3 and 5 weeks old at the start of feeding and who are fed a low magnesium diet 17 to 21 days. A procedure for determining (Na+ K+)-ATPase activity in rat brain homogenates, without requiring isolation or purification, is described. Computer modeling techniques have yielded expressions for equating enzyme activity to sodium, potassium, magnesium or calcium concentrations in the reaction media. A thorough statistical treatment of the data is presented.

Advanced prostate cancer (PrC) is the second leading cause of death from cancer in US males. Advanced PrC cells are initially androgen-sensitive and thus androgen ablation therapy causes tumors to undergo regression and fall into a remission phase where residual cells remain dormant while androgen levels remain very low. Unfortunately, this phase usually lasts 3 to 5 years prior to tumor relapse, where the tumor cells re-grow in the absence of androgens. This form of the disease is aggressive and invariably fatal. In this study, we investigated the effects of a combination of herbal extracts on various stages of PrC, androgen-dependent and castrate-resistant, using CWR22 and CWR22Rv1 cells, respectively. Zyflamend, a commercially available product consisting of 10 different herbal extracts, had been shown to reduce pre-malignant forms of PrC in clinical trials. We expanded these earlier experiments by using Zyflamend in a model of advanced PrC. Our initial results indicated that Zyflamend could repress androgen-sensitive and castrate-resistant (androgen-insensitive) prostate tumor growth. Using a cell model for castrate-resistant PrC, Zyflamend inhibited the growth of CWR22Rv1 cells by increasing the expression of the cell cycle inhibitors p21 and p27. These effects were mediated via hyperacetylation of histone 3 through the suppression of class I and II histone deacetylases (HDACs) and an induction of CBP/p300 histone acetyl transferase activity. The latter effect was mediated by the upregulation/activation of Erk-1/-2 and Elk-1. Zyflamend also inhibited androgen receptor expression, its downstream gene target, prostate specific antigen (PSA), and increased cell death by inducing apoptosis as indicated by caspase 3 activity and PARP cleavage. The reduction of androgen receptor was confirmed in CWR22Rv1 xenograft tissues. Our results suggest the extracts of this herbal combination inhibits castrate-resistant prostate cancer cell growth epigenetically and by coordinately affecting androgen receptor signaling pathways involved in cell growth/death. In an androgen-dependent PrC tumor xenograft model, Zyflamend reduced the growth of CWR22-derived tumors and enhanced the responsiveness of tumor cells to hormone ablation. Zyflamend potentiated the regression of PrC cells and their sensitivity to androgen deprivation. These results suggest that Zyflamend may be an effective adjuvant when used with hormone ablation therapy.

This study explored angiotensin IV’s (Ang IV) affects on the signaling pathways involved in the development of insulin resistance in 3T3-L1 adipocytes. Ang IV, working through the AT4 receptor, interferes with insulin signaling through the blockade of the phosphatidylinositol 3-kinases (PI3K)/Akt pathway and through activating mitogen activated protein kinases (MAPK): extracellular signal regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) which are known to impair insulin receptor substrate-1 (IRS-1) signaling. The expression of AT4 receptors was confirmed by reverse transcription polymerase chain reaction. Ang IV’s effects were found by treating adipocytes with combinations of Ang IV, AT4 receptor inhibitor Norleual, and insulin. Cell lysates were resolved by SDS-PAGE electrophoresis and immunoblotted. Ang IV down-regulated the PI3K/Akt pathway. Insulin exerts its effects on adipocytes by activating this pathway, phosphorylating Akt (S473 and T308) residues. Pre-treatment with Ang IV blocked insulin’s effects, reversing Akt activation. Addition of Norleual blocked Ang IV’s inhibitory actions, leading to the phosphorylation of Akt residues. Studies show elevated MAPK levels produced by angiotensin peptides catalyze the phosphorylation of serine residues on IRS-1, impairing insulin signal transduction. Ang IV increased the activation of ERK 1/2 and JNK. This was reversed by pretreatment with Norleual. Ang IV’s effects on IRS-1 residues were found by immunoprecipitation of IRS-1 followed by SDS-PAGE immunoblotting. Ang IV increased the phosphorylation of IRS-1(S307 and S612). Pre-incubation with Norleual attenuated Ang IV’s effects. Ang IV’s stimulation of adipocytes quickly caused the phosphorylation of MAPK corresponding to serine residue phosphorylation on IRS-1, which may implicate Ang IV activation of MAPK in the development of insulin resistance. Ang IV is involved in the down-regulation of the insulin-signaling cascade by inhibiting insulin’s phosphorylation on Akt (S473 and T308). Ang IV increased phosphorylation of ERK 1/2 and JNK, corresponding with increases in serine phosphorylation of IRS-1. Pre-treatment with Norleual inhibited Ang IV’s negative effects on insulin signaling. This study elucidates a new mechanism that may lead to the development of insulin resistance in adipose tissue.

Forty-nine female rats initially weighing about 95 grams were used in this experiment to determine the effect of contraceptive hormones on body composition in zinc deficient and zinc supplemented rats. Four treatment groups were: 2 ppm of dietary zinc with daily contraceptive steroid injections, or with placebo injection, and 140 ppm of dietary zinc with contraceptive steroid injection or with placebo. The daily injected steroids consisted of 0.lug of Ethynyl Estradiol and 0.lmg of Medroxyprogesterone in 0.lml of corn oil. Non-hormone treated groups received O.lml of plain corn oil. All injections were administered into the Gluteus Maximus muscle. Food intake, weight gain and serum zinc level were determined weekly. After eight weeks of experimental treatment, animals were killed by decapitation. Livers, adrenals and ovaries were removed, weighed and frozen until cholesterol determinations could be made. The remaining carcass minus the intestinal tract was autoclaved and homogenized. Carcass moisture, ash, total lipid and cholesterol ester levels were determined. Carcass zinc and calcium levels were also determined. The results indicate that zinc deficiency causes a marked loss of appetite with a reduced growth rate and a reduced feed efficiency in the growing female rats. Serum zinc level was also reduced. Rats fed the zinc deficient diet had smaller ovaries than the controls, but their adrenal weights were unaffected. Total cholesterol content of the adrenals was, however, increased. Zinc deficient diet caused a significant elevation of carcass cholesterol esters and carcass water content, and tended to decrease liver cholesterol level. Carcass ash concentration was increased in zinc deficient rats on the basis of fat free samples. Hormone injection caused a slight reduction in growth rate, irrespective of zinc intake, although it slightly improved appetite in the zinc deficient rats, and which caused a slight further decrease in serum zinc level. The relative weight of adrenals and ovaries were significantly decreased by hormone administration, having markedly increased total cholesterol levels. Hormone treatment tended to increase carcass cholesterol ester and liver cholesterol levels. In zinc supplemented rats, carcass total lipids was reduced, while carcass lipid was elevated in zinc deficient rats by hormone injection. Hormone treatment elevated carcass ash content with an increase in its calcium and zinc content which was statistically significant. This increase indicates the anabolic effect of contraceptive steroids on mineral in general.

Whey proteins have restricted use in many food applications because of limited functional properties. Whey proteins' relatively high content of disulfide bonds may be responsible for their lack of functionality, especially in extrusion applications. To determine the effect of disulfide bond content on functional properties and extrudate performance, whey protein concentrate was treated with sodium sulfite to achieve four levels of disulfide bond sulfonation (0, 31, 54, and 71%). Sulfonated whey protein functional properties, extrusion-expanded snack properties (32% total protein), and extrusion-textured fibrous product properties (48% protein) were determined. Correlation analysis was performed to determine relationships between functional properties and extrudate performance. Sulfonation of whey protein concentrate (80% protein) increased foaming and emulsion properties and decreased melt temperatures. These changes were largely attributed to increased protein unfolding and flexibility. Sulfonation decreased gel strength and increased resolubilization after heat treatment. These changes were likely the result of increased electric charge on the proteins, limiting protein-protein interactions during heating. Snack products extruded from the 31 and 71% sulfonated samples were less expanded and released less protein and carbohydrate during extrudate solubilization. Sulfonation may have promoted protein unfolding, thereby exposing interaction sites and increasing the formation of insoluble protein-starch aggregates . In support of this suggestion, negative correlation s were found between extrusion performance and protein functional properties related to flexibility , including emulsification activity index, foam stability, and melt onset temperature. The anomalous behavior of the 54% sulfonated sample may be the result of significant structural and functional changes of a-Lb that are predicted to occur at approximately 50% sulfonation. Although the textured extrudate produced from all levels of sulfonation (including the control) did not possess typical fibrous texture, sulfonation at 31% and higher decreased stability after hydration . Decreased stability and fibrous texture may have resulted from decreased protein-protein interactions caused by the repulsion of electric charges contributed by sulfite groups. In conclusion, sulfonated whey protein functional and extrudate properties were influenced by disulfide bond content. Changes in these properties were attributed primarily to increased protein unfolding and flexibility. Increased electric charge on proteins also played a role where protein-protein interactions were important.

Molecular genetic protocols have been developed to provide evidence in infractions of wildlife statutes in Canada. We have utilized DNA marker systems to address specific questions in wildlife investigations based on their different levels of genetic variability. Multilocus DNA fingerprinting has been applied to poaching infractions to determine if tissue samples associated with a suspected poacher originated from the remains of an animal at a known illegal kill site. The hypervariability of the variable number of tandem repeat (VNTR) loci detected by multilocus DNA fingerprinting allows the individual identification of samples. Highly repetitive satellite DNA markers have been applied to determining the species of origin of unknown tissue samples based on their species-specificity. Satellite DNA profiling have provided evidence in illegal commercialization investigations involving species such as moose (Alcesalces) and white-tailed deer (Odocoileus virginianus), including the illegal addition of game meat in processed meat products. A sex-specific DNA locus, the sex-determining region on the Y-chromosome (Sry), has been utilized to determine the sex of cervid samples that have had gender-specific physical characteristics, antlers and genitalia removed in violation of the validation tag system. Finally, a polymerase chain reaction (PCR) based protocol has been established for the species identification of samples that produce minute amounts of DNA or degraded DNA. Cytochrome b sequences demonstrate low intra-specific levels of sequence divergence and higher inter-specific levels of sequence divergence. Cytochrome b sequence analysis has been applied to fish, game and domestic species commonly involved in wildlife investigations and to the identification o fa number of species, mostly seal species, involved in the trade of animal parts. / Thesis / Master of Science (MSc)