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"Supermice" (TRrGH mice) contain multiple copies of rat growth hormone genes incorporated into a single chromosome. This results in double normal growth rates reaching adult body sizes twice that of normal mice. To determine how exposure to constant light (LL) affects various physiological processes, reproduction, feeding, and behaviour were examined in LL-reared TRrGH mice. Fertility, organ allometries, feeding rates, behavioural time budgets, and circadian feeding and sleep rhythms were compared for both LL and standard 12h dark: 12h light (LD). Both TRrGH and normal females exhibited a significant decrease in fertility in LL. On a mass-specific basis, TRrGH females showed increased combined ovary mass and a reduction in thymus and heart size in LL. TRrGH males demonstrated increased testes mass in LL. When adrenal size was compared between males and females, both TRrGH and normal females exhibited larger adrenals than their male counterparts in both light treatments. The fertility decrease observed in LL may have been associated with reduced food intake. LL-reared TRrGH females ate less than those in LD, although significantly more than TRrGH males in both LL and LD. When compared to normal mice, both sexes of TRrGH mice ate less in both photoperiods. The feeding rates of transgenic GLUT -4 mice were also examined. GLUT -4 mice contain double the amount of insulin responsive GLUT -4 glucose transporters which results in an increased blood glucose clearance rate. These mice, like TRrGH mice, ate less than normals, although a different age-related feeding pattern was observed. TRrGH mice in LL are behaviourally more lethargic than those reared in LD, and spend less time feeding and drinking. Circadian feeding and sleep patterns were shifted in LL by approximately 12 hours, and exhibited reduced peak amplitudes. Ultradian patterns appeared to survive the breakdown of circadian organization. TRrGH mice demonstrate a hormonal imbalance due to the excess allocation of energy into growth. It appears that, in LL, hormonal systems are further altered resulting in an increase in reproductive impairment associated with reduced feeding. One of these altered hormones may be estrogen. Hormones involved in hypothalamic-pituitary-adrenal axis (stress axis) are also implicated. It is concluded that photoperiod is important in regulating physiological processes, and TRrGH mice are more susceptible to environmental alterations due to their altered endocrinological state. / Thesis / Master of Science (MS)

Hemophilia B is a debilitating and life-threatening disorder caused by a deficiency in or dysfunction of factor IX (FIX), a complex plasma glycoprotein required for the formation and maintenance of blood clots. Treatment of hemophilia B involves infusion of replacement FIX currently derived from two sources: FIX purified from pools of human plasma (pd-FIX) and a single recombinant FIX product generated in genetically engineered Chinese hamster ovary (CHO) cells. Both of these FIX products are prohibitively expensive, limiting of the treatment options of hemophiliacs worldwide. As a result, a more abundant and affordable FIX product would greatly improve the life prospects for hemophiliacs. The biological activity of FIX is dependent upon its numerous post-translational modifications (PTMs), including gamma-carboxylation, proteolytic maturation, phosphorylation, sulfation, and glycosylation. Of these PTMs, those known to be vital for activity are gamma-carboxylation of multiple glutamate residues near the N-terminus and proteolytic cleavage of the FIX propeptide. When expressed at a high rate in exogenous expression systems, however, the ability of current systems to effect the necessary PTMs is severely rate limited, restricting the production of active FIX. The transgenic pig bioreactor represents a promising source for the production of large quantities biologically active FIX due to its demonstrated ability to perform the required FIX PTMs. It was the goal of this study to characterize the PTM structure and the resulting function of recombinant FIX when expressed at 1-3 mg/ml in the transgenic pig mammary epithelium (tg-FIX). It was found that the expressed tg-FIX is comprised of a heterogeneous mixture of FIX PTM isoforms. This mixture represents a spectrum of tg-FIX molecules of varying gamma-carboxyglutamic acid (Gla) and propeptide content, indicating that rate limitations in effecting these PTMs are present. A purification process was developed utilizing heparin-affinity chromatography to purify the total population of tg-FIX from pig milk, a complex multi-phase feedstock. Subsequently, a process was developed to fractionate the total population of tg-FIX into subpopulations based upon the extent of post-translational modification. Q ion-exchange chromatography was utilized to fractionate tg-FIX based upon molecular acidity which was found to be correlated to both biological activity and Gla content. The resulting biologically active tg-FIX population contained an average of 7 of the 12 Gla residues found in pd-FIX. Immuno-affinity chromatography was subsequently utilized to further fractionate tg-FIX into mature tg-FIX and propeptide-containing tg-FIX populations. The isolated FIX PTM populations were subjected to functional analysis by investigating in vitro clotting activity, activation by factor XIa, and in vivo pharmacokinetics. From this analysis it was found that mature tg-FIX with an average 7 Gla residues, representing approximately 9% of the total tg-FIX produced, exhibits wild-type in vitro clotting activity and normal activation by factor XIa. The remainder of the tg-FIX produced, characterized by either a lower Gla content or the presence of the propeptide, was found to be inactive and displayed less efficient activation by factor IXa. In an in vivo pharmacokinetic study in the hemophilia B mouse model, biologically active tg-FIX was found to possess altered circulating properties. Tg-FIX was characterized by a lower recovery, approximately one-sixth that of pd-FIX, but an extended circulation half-life. From this study it was found that the mean residence time of tg-FIX after injections is approximately twice that observed for pd-FIX. These altered pharmacokinetic properties are likely linked to the unique tg-FIX PTM structure, perhaps through altered endothelial cell binding characteristics caused by the reduced Gla content. / Ph. D.

This dissertation is comprised of three essays that generate methods to measure the ex-ante economic impacts of agriculture technology innovations. The first essay entitled 'Valuing Intellectual Property Rights in an Imperfectly Competitive Market: A Biopharming Application' presents a method for valuing the intellectual property rights (IPRs) for an innovation that lowers product production costs below those associated with the patented process of a monopolist. The application to Glucocerebrosidase enzyme from transgenic tobacco suggests an intellectual property rights (IPRs) value of about $1.75 billion. Despite the innovator's market power, significant surplus gains also accrue to consumers. Further, U.S. antitrust laws that prohibit IPRs acquisition by the current monopolist increase consumer welfare by almost 50 percent. The second essay entitled 'Ex-Ante Analysis of the Benefits of Transgenic Drought Tolerance Research on Cereal Crops in Low-Income Countries' develops a framework to examine the ex-ante benefits of transgenic research on drought in eight low-income countries, including the benefits to producers and consumers from farm income stabilization and the potential magnitude of private sector profits from IPRs. The framework employs country-specific agroecological-drought risk zones and considers both yield increases and yield variance reductions when estimating producer and consumer benefits from research. Benefits from yield variance reductions are shown to be an important component of aggregate drought research benefits, representing 40 percent of total benefits across the eight countries. Further, estimated annual private sector benefits of $US 178 million suggest that significant incentives exist for private sector participation in transgenic drought tolerance research. The third essay entitled 'Ex-Ante Evaluation of Alternative Strategies to Increase the Stability of Cropping Systems in Eastern and Central Africa' examines the ex-ante economic impact of transgenic drought resistance maize breeding and of conventional maize, millet and sorghum drought resistance breeding in Kenya, Uganda, and the Amhara region in Ethiopia. An expected utility framework is combined with a partial equilibrium model and a spatial drought risk zonation scheme to estimate benefits from mean yield increases and yield variance reductions at the market level as well as at the household level for maize, millet and sorghum producers in the administrative regions of each country. Results suggest that annual ex-ante benefits of $87 million, $6.8 million and $4.8 million can be generated from public sector conventional breeding research on maize, sorghum and millet, respectively. Private sector transgenic drought tolerance research may also generate substantial benefits of $97 million for maize producers and consumers, particularly through the reduction of yield variance arising from drought, and an additional $21 million as profits from intellectual property rights protection. / Ph. D.

Tobacco has been studied as a host for producing recombinant therapeutic proteins on a large-scale, commercial basis. However, the proteins expressed in tobacco usually need to be purified to high yield and purity from large amounts of biomass in order for their production to be commercially viable. The methods needed to purify proteins from tobacco are very challenging and not well studied. The objective of this research was to develop a process for the purification of the acidic model protein, recombinant β-glucuronidase (rGUS), from transgenic tobacco leaf tissue to high yield and purity. Polyelectrolyte precipitation with polyethyleneimine (PEI) was identified as an initial purification step for purifying acidic recombinant proteins from tobacco. Polyethyleneimine precipitation allowed for high recovery and concentration of the target protein while removing large amounts of impurities from the initial extract. At dosages of 700-800 mg PEI/g total protein, nearly 100% of the rGUS activity was precipitated with generally more than 90% recovered from the pellet. In addition, more than 60% of the native tobacco proteins were removed in the process, resulting in a purification factor near 4. Recombinant GUS was further purified by a step of hydrophobic interaction chromatography (HIC) followed by a step of hydroxyapatite chromatography (HAC). The HIC step served to remove PEI and other contaminants such as nucleic acids that were accumulated during the precipitation step, while the HAC step served to separate rGUS from the remaining native tobacco proteins, most notably ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco). Nearly 40% of the initial rGUS activity was recovered as a near homogeneous fraction based on SDS-PAGE analysis after the three step process. The main steps used in this process are anticipated to be scalable and do not rely on affinity separations, making the process potentially applicable to a wide variety of acidic recombinant proteins expressed in tobacco as well as other leafy crops. / Master of Science

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