Protein degradation poses a significant challenge for the efficient production of recombinant proteins in plants, affecting the stability and yield of the recombinant protein. In this study the E. coli-derived enzyme glutathione reductase (GR) was transiently expressed in transgenic tobacco plants constitutively expressing the cysteine protease inhibitor OC-I and non-transgenic plants. A protein resembling the GR was detected in infiltrated leaves. Transiently expressing GR in transgenic N tabacum plants resulted in almost two fold significant increases in GR activity. Transgenic tobacco plants expressing the rice cysteine protease inhibitor OC-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher GR activity and also higher GR amounts in transgenic plants. The study has demonstrated that OC-I is an effective companion protease inhibitor candidate with the potential to protect other high value proteins such as GR, from cysteine protease degradation. / Dissertation (MSc)--University of Pretoria, 2012. / Plant Science / unrestricted

MS is an autoimmune neurodegenerative disease that attacks myelin, a protective sheath that covers neurons within our bodies, which may lead to numbness, tremors, issues with vision, dizziness and more. When researching the efficacy of a therapeutic in neurodegenerative diseases such as multiple sclerosis, it is crucial that the in-vivo model selected for testing allows complete and accurate data collection. Several models attempt to replicate conditions of disease, in which myelin levels have been deliberately reduced in order to study its regrowth. These models (Cuprizone and LPC injection) can be further optimized by validating a new strain of mouse, NG2-creER / Rosa-Optopatch, which will essentially express GFP+ myelin. To validate this mouse line, the following goals were pursued: Confirm NG2+ pre-OLs express GFP in the spinal cord tissue and corpus callosum in our NG2-creER mouse, confirm that myelin formed from NG2+ pre-OLs that have matured into OLs also express GFP and characterize the GFP staining pattern along with other known myelin stains (MBP, Fluoromyelin Red), and in the long run, use the NG2-creER model in MS-related targets for drug candidates as a more efficient option than traditional methods such as electron microscopy (EM). Results show that the NG2-creER mouse was successful (in both CPZ and LPC models) in showing NG2+/GFP+ cells and that these GFP+ pre-OLs matured to form GFP+ myelin, as well as showing the capability of staining myelin at a younger age than other myelin stains. / 2022-06-17T00:00:00Z

No description available.

Alzheimer’s Disease (AD) is the most common form of dementia. It is a fatal neurodegenerative disease that leads to both cognitive decline and altered psychological states. There is currently no cure for AD. The pathology of AD includes the clustering of insoluble amyloid-β (Aβ) plaques, tau tangles, and increased neuroinflammation. These pathological manifestations initially occur in the hippocampus (HPC), then continue to the prefrontal cortex (PFC), and occur throughout the brain as the disease progresses. The heightened neuroinflammatory state in AD is an essential point of focus in AD research. In this study, the novel oral anti-inflammatory tumor necrosis factor-α (TNF-α) inhibitor compound PD2244 was tested to observe its effects on sensorimotor gating using prepulse inhibition (PPI), spatial memory using Barnes Maze, anxiety using an elevated-T maze, and apathy by observing nest building behavior in both female and male 3xTg mice. The 3xTg mice are the only triple-transgenic models of AD that have both Aβ plaques and tau tangles and is also an accelerated mouse model of AD pathology onset. A specialized diet containing variable doses of PD2244 was given to 3xTg mice beginning at 6 months of age. The doses given were 0, 1, 3, 10, and 30 mg/kg of PD2244. Testing was then performed at 9, 12, and 15 months, where 15 months equated to thorough AD pathology. Regarding behavioral improvement, it was observed that all doses of PD2244 were effective to alleviate deficits in PPI at 9, 12, and 15 months of age. On the Barnes Maze, at 9 months of age the 10 mg/kg dose of PD2244 was effective to alleviate deficits, whereas at 12 months of age, 3 and 30 mg/kg dose of PD2244 was effective, and finally, at 15 months of age the 3, 10 and 30 mg/kg doses of PD2244 demonstrated efficacy to alleviate deficits in spatial memory performance. On the elevated T-maze, there were no effects at 9 months of age, but the 3 mg/kg dose of PD2244 resulted in anxiolytic effects at 12 and 15 months of age. Nest building behavior is also being observed in 15-month-old mice to determine effects of PD2244 on apathy since it is a common neuropsychiatric prodrome of AD. Finally, analysis of neurobiological markers has revealed that PD2244 reduced increases in the proinflammatory cytokines TNF-α and interkeukin-1β (IL-1β) at 15 months of age in the HPC. In addition, there is currently a project analyzing immunohistological staining of microglial cells in the HPC and PFC in 15-month-old animals. This project is designed to discover a novel, effective, anti-inflammatory treatment for cognitive deficits and increases in anxiety associate with AD.

The mammary gland of lactating transgenic animals has several advantages for production of heterologous proteins including a high cell density that results in high concentrations of secreted protein and the ability to perform several types of post-translational modifications. Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the Aα, Bβ and γ fibrinogen chains to evaluate the requirements of the transgenic murine mammary gland for high level secretion of fully assembled human fibrinogen. After introducing the constructs into the murine zygotes by microinjection, secretion of fully assembled fibrinogen into milk was measured at concentrations between 10 ug/ml to 200 ug/ml. In one line of mice the total secretion of fibrinogen and unassembled subunits approached 700 ug/ml in milk. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Bβ and γ chains were rate limiting. Also, the subunit complexes γ₂, Aαγ₂ and the individual subunits Aα, Bβ and γ were found as secretion products. This is the first time that secretion of individual Bβ-subunits by any cell type has been reported and suggests the organization of the secretion pathway in mammary epithelia is different from that in liver. Glycosylated forms of individual Bβ-chain contained a complex saccharide with low mannose. Glycosylation of the γ-chain was also observed. These results suggest the 4.1 mWAP promoter can drive expression of fibrinogen cDNAs to high levels and that the amount of fully assembled fibrinogen secreted is equal to the level of the lowest expressing chain. / Master of Science

Pronuclear stage murine embryos received electrical stimulation in 5, 10, or 20 µs pulse lengths, and 0, 100, 200, 250, 300 or 400 voltages. Minimal embryo development occurred with 400 V. Irreversible electroporation occurred in embryos electroporated for 5 µs pulse length at 100 and 400 V, 10 µs pulse length at 400 V, and 20 µs pulse length at 100, 250, 300 and 400 V. Electroporated embryos that underwent reversible electroporation received 100 V for 5 µs, 400 V for 10 µs, and 250 for 20 µs and had similar development (P > 0.05) between the best and worst developed groups. Enhanced green fluorescent protein on a cytomegalovirus promoter (CMV-EGFP) was condensed with MgCl₂ and injected into the cytoplasm of murine zygotes at three concentrations (100, 425 and 625 µg/ml). Zygotes injected with the highest concentration had the highest percentages of fluorescing embryos (44%), fluorescing morula and blastocysts (16.7%), and the lowest percentage of mosaicism after 4 d in culture. Five PCR analyses of tail DNA gave conflicting results between 33.3% positive in two or more analyses to 2.8% positive in all five analyses. Southern Analysis detected 2.8% transgenesis. Cytoplasmic injection of linear CMV-EGFP (625 µg/ml in water) was 3.7% transgenic. Pronuclear injections produced 7.9% transgenesis. This research identified a range of reversible electroporation that could easily be verified <i>in vitro</i> with a selectable dye or marker protein and applied in transgenic as well as preclinical treatment models of research. Furthermore this research identifies the benefits and disadvantages of using Mg²⁺ in DNA condensation and injection buffers. / Master of Science

No description available.

Despite concerted efforts at agricultural development over many years, millions of people in developing countries still suffer from poverty and under-nutrition. New crop varieties, such as those released during the green revolution in Asia, increased farmers' income and reduced the level of under-nutrition. In recent years, while the speed of the development of conventional breeding technology has slowed, biotechnology has developed rapidly. In 2005, about 8.5 million farmers in 21 countries grew transgenic crops. Transgenic rice has not been commercially released on a large scale, but progress has been made in developing varieties with potential to increase yield and reduce input costs. In this context, this research aims to provide empirical evidence on the potential effects of introducing transgenic rice on farm households' income and nutritional well-being in Bangladesh, including the impacts on their current nutritional status and nutritional vulnerability over time. To this end, two econometric models are constructed and estimated. A farm household model is employed to project farm households' production and consumption responses to introducing improved rice varieties such as transgenic rice. The model estimates the profit effect of introducing transgenic rice. The influence of the profit effect on farmers' consumption decisions is then considered. Due to the ex ante nature of this research and data limitations, the effects of transgenic rice are assumed to be similar to that of previous high yielding varieties (HYVs), and the impact of transgenic rice on farm household profit is assumed to be similar to the effect of the percentage of rice area in HYVs and the yield effect of transgenic rice is the same as HYVs. On the production side, the supply of three outputs- rice, all other crops and animal products- and demand of labor and fertilizer were estimated. On the consumption side, both poor and non-poor households' demand for rice, wheat/other food, pulse, oil, vegetables/fruits, meat/egg/ milk, fish, and spices were estimated. Based on the parameter estimates, the calorie intake and protein intake elasticities with respect to introducing transgenic rice were computed. The results indicate that the total profit elasticity with respect to the percentage of rice area in HYVs is 0.08. The calorie elasticity with respect to the percentage of rice area in HYVs ranges from 0.062 in non-poor to 0.074 in poor households, and the protein elasticity ranges from 0.075 in non-poor to 0.084 in poor households. The results indicate that transgenic rice is likely to play a positive role in improving farm households' nutritional status in terms of total calorie/protein intake. The magnitude, however, is likely to be moderate, if only the profit effect is considered. A consumption forecasting model is used to examine farmers' nutritional vulnerability a probabilistic concept defined as having a high probability now of suffering a shortfall in the future. It is assumed that when exposed to risk, farmers' consumption decisions have already considered their risk coping strategies. The effect of transgenic rice is reflected by its impact on farm income. Farm households' calorie intake in the future (hunger season) was predicted by a multivariate regression function with the logarithmic daily per resident calorie intake as the dependent variable. The independent variables include variables that represent households' income, flood exposure, assets, and demographic composition. Farm households' nutritional vulnerability profiles, based on the estimation of ex ante mean and variance, indicate that vulnerability exists among surveyed rice farm households. The model also predicts that the income increase induced by introducing transgenic rice will reduce each individual household's probability of suffering a future consumption shortfall and subsequently will reduce its vulnerability. The overall vulnerability profile of farm households improves in Bangladesh. / Ph. D.

The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the most studied membrane protein models because of its clear clinical significance. Mutations within the CFTR gene lead to cystic fibrosis, the most common autosomal recessive genetic disorder in the Caucasian population. CFTR, a large 160 kDa glycoprotein, is a chloride ion channel in the ABC superfamily of transporter proteins. Due to low natural abundance of CFTR and difficulties producing sufficient amounts in heterologous systems, the exact protein function/structure relationship is unknown. Expression of CFTR in E. coli is lethal and mammalian culture systems are expensive and low yielding. However, successful bioproduction of many complex human proteins has been shown in transgenic plants. Our research objective is to develop tobacco as a model system for expressing human CFTR. Constructs of full-length CFTR fused to the 35S double enhanced promoter could not be propagated in E. coli, suggesting that the CFTR product generated by "leaky" expression was detrimental to bacteria. Two strategies were undertaken to address the problem: 1) a plant intron was introduced into CFTR sequence and 2) a more tightly regulated wound-inducible promoter MeGATM was used. Tobacco was transformed with all constructs. CFTR presence was determined by polymerase chain reaction (PCR). Expression and intron splicing was analyzed by reverse transcriptase-PCR. Splicing did not occur presumably due to intron /exon contexts. In tobacco expressing MeGA:CFTR, however, novel high-molecular-weight membrane-associated proteins were immunodetected using anti-CFTR antibodies suggesting that tobacco may be capable of producing human CFTR. / Master of Science

Amino acids are assimilated by membrane-associated transporters into and out of enterocytes either in their free form or in the form of peptides. The peptide transporter, PepT1, is thought to be the major facilitator of peptide transport in the enterocyte. It is unknown if the peptide transporters and free amino acid transporters operate in a compensatory fashion to regulate the amino acid balance within the enterocyte. Therefore, the objective was to examine the regulatory balance between PepT1 and other peptide and free amino acid transporters in enterocytes. The Mouse Small Intestinal Epithelial (MSIE) cells are conditionally immortalized. It was found that MSIE cells express BoAT1, CAT1, CAT2, LAT1, y+LAT1, and y+LAT2, but not PepT1, EAAT3, Bo,+AT, or LAT2, making this model similar to the basolateral membrane of enterocytes. Growing MSIE cells at high temperatures did not affect the nutrient transporter gene expression profile of these cells. Thus, the human colon carcinoma (Caco-2) cell line was used as a small intestinal in vitro model for this study. These cells express PepT1, HPT1, PTR3 EAAT1, EAAT3, rBAT, Bo,+AT CAT1, LAT1, y+LAT1, y+LAT2, ABCC3, ABCC4, which increased from D0 to D21 post confluency, indicating cell maturation. In Caco-2 cells, PepT1 gene silencing was induced in Caco-2 cells. Despite an reduction of PepT1 gene (82%, P < 0.05) protein (96%), no significant difference in any peptide (HPT1, PTR3, ABCC3, ABCC4) or free amino acid transporters (EAAT1, EAAT3, rBAT, Bo,+AT, BoAT1, CAT1, CAT2, LAT1, LAT2, y+LAT1, y+LAT2) between Caco-2 cells treated with PepT1 siRNA and Caco-2 cells treated with Control siRNA was observed. These results suggest no compensation at the gene expression level of these transporters in response to a reduction of PepT1. To account for the limitations of an in vitro and PepT1 kockout mouse model, transgenic chicken models were pursued. Potential cPepT1 overexpressing, cPepT1 shRNA or control shRNA expressing G0 chickens were generated by embryo injection of pseudolentiviral particles followed by ex ovo egg culture. Overall, 9 potential G0 cPepT1 overexpressing chickens, 15 potential G0 cPepT1 shRNA expressing chickens, and 4 potential G0 control shRNA expressing chickens were generated. / Ph. D.