Global climate change is identified as a major threat to the survival of natural ecosystems. The variations in global climate have gained the attention of researchers worldwide, as these changes negatively affect agriculture by reducing crop productivity and food security.  Projects related to abiotic stress tolerance are significant because they address important challenges facing agriculture and food security, contribute to more sustainable agricultural practices, and advance our understanding of fundamental plant biology. Some plants have defense mechanisms that are activated upon receiving stress stimuli to increase systemic tolerance to abiotic stresses such as heat, light, and cold. Salicylic acid-binding protein 2 (SABP2) from tobacco exhibits a high affinity for salicylic acid (SA) and is an important component in the SA-signaling pathway. SABP2 interacts with other cellular proteins to initiate downstream signaling and activate responses leading to resistance. Several SABP2-interacting proteins (SIP), including SIP-428, have been identified. The main goal of this proposed research is to determine the role of SIP-428 in mediating environmental stresses. SIP-428 is a SIR2-type non-histone deacetylase enzyme. De/acetylation is a common post-translational modification of proteins in eukaryotes. Since SIP-428 is a SABP2-interacting protein, it is involved in plant immune signaling. To determine the role of SIP-428 in plant physiology, it was biochemically characterized, and transgenic tobacco plants silenced in SIP-428 expression were previously generated and analyzed. Transgenic tobacco plants overexpressing SIP-428 were also generated. These lines expressed SIP428 at higher levels upon treatment with estradiol. Transgenic tobacco that overexpresses SIP-428 has been used in this study. To test the role of SIP428 in abiotic stress, the transgenic plants will be treated with abiotic stress-inducing chemicals, e.g. NaCl (salinity stress), mannitol (osmotic stress), and PEG6000 (drought stress). The treated seedlings will be allowed to grow for a specific time (1-2 weeks). The expression of SIP-428 will be monitored by western blotting (using anti-myc antibodies). The effects of SIP-428 expression on abiotic stress tolerance will be investigated biochemically by examining the activities of antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX). Additionally, gene expression analysis will also be conducted to determine the expression of antioxidant genes.

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Rotavirus is the leading cause of severe infantile diarrhea worldwide. Most related deaths occur in infants from developing countries. Current vaccines are expensive and not readily available throughout the world. Chloroplast transformation technology can be utilized to generate genetically modified plants that produce large quantities of therapeutic proteins and vaccine antigens within their leaves. Plants that are used as bioreactors for vaccine antigens are economically advantageous because they eliminate the need for purification steps and are cheaper to transport. A genetically modified crop could potentially be grown near an endemic area and harvested as needed. There are many influencing factors for transgene expression levels within plant leaves that must be taken into account prior to their harvest. In this work, we seek to determine the optimal expression of CTB-NSP4 in two different cultivars of tobacco plant that have been previously generated by the Daniell lab. The fusion protein, CTBNSP4, is hoped to confer resistance to both rotavirus and cholera. We will determine how the expression of the protein is affected by different variables such as the lighting conditions during harvest and the relative age of leaf at the time of harvest. This knowledge can be used to raise the productivity of the genetically modified plants, further decreasing the cost. Additionally, as unprocessed leaf cannot be used directly for oral delivery due to an unknown concentration of the vaccine antigen, quantification is an important barrier to overcome. Low cost vaccines can be prepared after optimization of dosage and stability. This project seeks to substantiate and quantify genetically modified tobacco plants producing the rotavirus and cholera vaccine antigens.

<p> Tissue plasminogen activator (tPA), is commonly used as thrombolytic agent for the treatment of various cardiovascular diseases. This thesis constitutes the first report on cloning and expression of tPA in the methylotrphic yeast Pichia pastoris. </p> <p> The tPA gene was first cloned into an E. coli/Pichia shuttle plasmid and then integrated into the Pichia genome. The recombinant Pichia was able to express tPA intracellularly, under methanol induction. The tPA produced by the Pichia had a similar molecular weight as native tPA and it had serine protease activity. At the shake flask scale, the recombinant Pichia strain was able to produce twice as much tPA as reported for E. coli. </p> <p> Elastin-like polypeptides (ELP) are proteins that have a peculiar characteristic: they are able to undergo a reversible inverse phase transition temperature within a very narrow temperature range. On a second aspect of heterologous protein, a construct composed of thioredoxin-intein-ELP was used to provide direct evidence, for the first time, that protein folding and activity, in this case the intein, was maintained when the tripartite fusion was present in the aggregated state. These results are important, since they provide the necessary degree of confidence to stimulate future work directed towards expression and maintenance of proper folding of aggregation-prone proteins when expressed in-vivo E. Coli as ELP directed inclusion bodies. It is also shown that the intein-ELP system may be a very interesting system to be used as a drug delivery vehicle. </p> / Thesis / Master of Applied Science (MASc)

The integrins play a crucial role in cancer cell proliferation, migration, differentiation, survival and angiogenesis. It has been shown that integrin expression is positively correlated to cancer dissemination, this suggests targeting selected integrins as an anti-metastatic strategy. The aim of this study is to investigate the effect of novel antagonists of α5β1, αvβ3 and αvβ5 integrins on cancer cell migration, a key process in tumour cell dissemination. Immunohistochemistry was used to evaluate the expression of α5, αv, β3 and β5 integrin subunits in prostate cancer tissues. Furthermore the expression of these integrin subunits in tumour and normal human head and neck tissues was compared. The expression profile of these integrin subunits in established human cancer cell lines was subsequently evaluated using immunodetection methods in cells and xenograft tumour samples. The effect of integrin inhibition on cell migration was then assessed using neutralizing antibodies against αvβ3, αvβ5, and α5β1 integrins in the scratch-wound healing assay. This assay was then used to evaluate the potential of novel small molecule integrin antagonists in preventing tumour cell migration. In H & N tissues, αvβ3, αvβ5 and α5β1 integrins are extensively expressed in tumour tissues but weakly expressed in normal tissue from the same patient. Further, prostate cancer tissues expressed variable levels of αvβ3, αvβ5 and α5β1 integrins. αvβ3 and αvβ5 integrins were expressed in variable levels in OSC-19, PC-3, DU145, DLD-1, HT-29, HUVEC, MCF-7, MCF-7ADR and M14 human tumour cell lines and in OSC-19, PC-3, HT-29 and MCF-7 xenografts. α5β1 integrin was expressed in all cell lines and xenografts except in MCF-7 cell line and HT-29 cell line and xenograft. Overall, the expression was elevated in xenografts compared to the corresponding cultured cells. Based on the expression profile and ability of cells to migrate, three cell lines (DLD-1 colon, DU145 prostate and OSC-19 HNSCC) were selected as models to further evaluate the potential of novel small molecule integrin antagonists to inhibit cell migration. The cell lines were characterized by using neutralizing antibodies against αvβ3, αvβ5, and α5β1 integrins to determine which of these three integrins were primarily involved in tumour cell migration. In DLD-1 and DU145, blocking αvβ5 and αvβ3 significantly inhibited migration, whilst the migration of OSC-19 was 50% inhibited by a multi-integrin inhibitor combination. Among the antagonists, ICT9055 and ICT9072 significantly decreased DLD-1 cell migration by 70% and 60% respectively while ICT9023, ICT9024, and ICT9026 significantly decreased DU145 cell migration by 60%, 60% and 50% respectively. The findings suggest that single integrin inhibition is not sufficient to prevent cell migration whereas dual or multiple inhibition is more effective. Two novel anti-migratory agents were identified in colon cancer and three in prostate cancer which would warrant further investigation. / Princess Nora Bint Abdul Rahman University

Expressing foreign proteins in E.coli is a major challenge because they often tend to develop into unsolvable and inactive proteins. They aggregate into so called  inclusion bodies which prevent expression of the protein. This problem might be avoided by fusing the gene of the foreign protein with a soluble protein called solubility tags, which  function is to enhance the solubility of the foreign protein. This report investigates whether Sterol Carrier Protein-2 (SCP-2) could function as a solubility tag. The experiment was carried out by fusing SCP-2 to two recombinant proteins, Green fluorescent protein (GFP) and a form of chloroamphenicol acetyl transferase (CATΔ9). The gene fusion was then inserted into a pET-15 vector and transformed into  the E.coli strain BL21(DE3) to be expressed. The results obtained from Western blot and PageBlue staining indicates that SCP-2 does not enhance the solubility of GFP or CATΔ9 since neither of them was expressed.  Furthermore, previous studies have shown that GFP can in fact be expressed  usingmaltose binding protein (MBP) as a solubility tag. Unfortunately, no success has been made regarding CATΔ9. In conclusion, regarding the results from this report, SCP-2 does not function as a solubility tag. However, further studies should be carried out on SCP-2 with more experiments before rejecting the possibility to use SCP-2 as a solubility tag.

African horsesickness (AHS) is one of the most deadly diseases of horses, with a mortality rate of over 90% in horses that have not been exposed to any African horsesickness virus (AHSV) serotype previously (Howell, 1960; Darpel et al., 2011). The Orbiviruses, African horsesickness virus (AHSV) and Bluetongue virus (BTV), are primarily transmitted to their mammalian hosts through certain haematophagous midge vectors (Culicoides spp.) (Erasmus, 1973). The selective cleavage of BTV and AHSV VP2 by trypsin-like serine proteases (Marchi et al., 1995) resulted in the generation of subsequent infectious sub-viral particles (ISVP) (Marchi et al., 1995; van Dijk & Huismans, 1982). It is believed that this cleavage affects the ability of the virus to infect cells of the mammalian and vector host (Darpel et al., 2011). Darpel et al (2011) identified a trypsinlike serine protease in the saliva of Culicoides sonorensis (C. sonorensis), which also cleaves the serotype determinant viral protein 2 (VP2) of BTV. And, a similar cleavage pattern was also observed by van Dijk & Huismans (1982) and Marchi et al (1995) with the use of trypsin and chymotrypsin. Manole et al (2012) recently determined the structure of a naturally occurring African horsesickness virus serotype 7 (AHSV7) strain with a truncated VP2. Upon further investigation, this strain was also shown to be more infective than the AHSV4 HS32/62 strain, since it outgrew AHSV4 in culture (Manole et al., 2012). Therefore, through proteolytic cleavage of these viral particles, the ability of the adult Culicoides to transmit the virus might be significantly increased (Dimmock, 1982; Darpel et al., 2011). Based on these findings, it is important to investigate the factors that influence the capability of arthropod-borne viruses to infect their insect vectors, mammalian hosts and their known reservoirs. In this study, we postulated that one of the vectors for AHSV, Culicoides imicola (C. imicola), has a protease similar to the 29 kDa C. sonorensis trypsin-like serine protease identified by Darpel et al (2011). Proteins in the total homogenate of C. imicola were separated on SDS-PAGE and yielded several protein bands, one of which also had a molecular mass of around 29 kDa. Furthermore, proteolytic activity was observed on a gelatin-based sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) gel. The activity of the protein of interest was also confirmed to be a trypsin-like serine protease with the use of class-specific protease inhibitors. A recombinant trypsin-like serine protease of C. sonorensis was generated using the pColdIII bacterial expression vector. The expressed protein was partially purified with nickel ion affinity chromatography. Zymography also confirmed proteolytic activity. With the use of the protease substrates containing fluorescent tags and class specific protease inhibitors, the expressed protein was classified as a serine protease. It was also proposed that incubation of purified AHSV4 with the recombinant protease would result in the cleavage of AHSV4 VP2, resulting in similar VP2 digestion patterns as observed in BTV by Darpel et al (2011) or the truncated VP2 of AHSV7 by Manole et al (2012). BHK-21 cell cultured AHSV4 was partially purified through Caesium chloride gradient ultracentrifugation after which the virus was incubated with the recombinant protease. Since not enough virus sample was obtained, the outcome of VP2 digestion was undetermined. In the last part of this study, it was postulated that C. imicola and C. sonorensis have the same trypsin-like serine protease responsible for the cleavage of VP2 based on the protease activity visualised in the whole midge homogenate. Since the genome of C. imicola is not yet sequenced, the sequence of this likely protease is still unknown. Therefore, we attempted to identify this C. imicola protease through polymerase chain reaction (PCR) amplification. Total isolated ribonucleic acid (RNA) of C. imicola was used to synthesize complementary deoxyribonucleic acid (cDNA). The cDNA was subjected to PCR using C. sonorensis trypsin-like serine protease-based primers. An 830 bp DNA fragment was amplified. However, sequence alignment and the basic local alignment software tool (BLAST), revealed that DNA did not encode with any other known proteins or proteases. From the literature it seems that there is a correlation between the proteases in the vector and the mammalian species that succumb to AHS (Darpel et al., 2011, Wilson et al., 2009, Marchi et al., 1995). Based on the work performed in the study, a proteolytically active protein similar to the 29 kDa protein of C. sonorensis is present in C. imicola. The 29 kDa protease of C. sonorensis can also be expressed in bacteria which could aid in future investigations on how proteolytic viral modifications affect infectivity between different host species. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Winter wheat has several advantages over spring varieties, higher (25 % more) yield, efficient use of spring moisture, reduction of soil erosion by providing ground cover during the fall and early spring, rapid initial spring growth to out - compete weeds and circumvent the peak of Fusarium head blight infections by flowering early. Winter wheat is planted in early autumn when it germinates and developing seedlings acclimate to cold. The crown survives under snow cover and in spring rapidly grows into a vigorously growing plant for grain to be harvested in summer. However, the harsh Canadian prairie winters require that winter wheat has increased cold hardiness and improved winter survival to reduce losses from sudden cold snaps during winter and spring. Low temperature (LT) tolerance is one of the major components of cold hardiness. Genetic mapping studies have revealed a major quantitative trait locus (Fr-A2) at wheat chromosome 5A which can explain at least 50 % of LT tolerance in wheat. Physical mapping of 5A LT QTL in a hardy winter wheat cv Norstar revealed a cluster of at least 23 C - repeat binding factors (CBF) coinciding with peak of Fr-A2 QTL. The objective of this study is biochemical, and molecular characterization of CBF co - located at Fr-A2 to identify key CBF participating in conferring LT tolerance in winter wheat. A comparative analysis of CBF gene cluster at the Fr-A2 collinear region among Poaceae members showed an expansion in the number of CBF genes with increased LT tolerance. Rice, a cold sensitive member, had only three CBF genes, whereas cold hardy winter wheat cv Norstar has 23 CBF genes. Amino acid sequence - based cluster analysis of complete CBF genes, or their major functional components such as the AP2 - DNA binding domain and C - terminal trans - activation domain, divide Norstar CBF into Pooideae specific clades. However, analyses of Norstar CBF amino acid sequences of different functional groups revealed a shift in clade members. These results suggest divergence of CBF functions which could lead to possible differences / similarity in the regulon activated by a CBF in a specific group. The 15 CBF genes from winter wheat cv Norstar were expressed in E. coli to produce recombinant TrxHisS - CBF fusion proteins in adequate quantities for structural and functional assays. All CBF fusion proteins could be recovered in the E. coli soluble phase of cell extract, except that the CBF17.0 fusion protein could only be recovered with 6 M urea extraction. Eleven of the 15 CBF fusion proteins were very stable in heat (98 oC), 10 % SDS and 6 M urea treatment. The five other CBF members were very labile under native conditions, but were stable in E. coli cell extracts or when extracted under denaturing conditions. Most of the CBF recombinant proteins in denaturing gel electrophoresis migrated slower than expected from their predicted molecular mass, based on amino acid sequence. The slow migration could be associated to their elongated protein structure as determined by dynamic light scattering (DLS). CBF 12.2 and CBF 17.0 were highly resistant to denaturation and retained their secondary structure in these conditions as determined by circular dichroism (CD) spectra. The high stability of these two CBF proteins may be important for cold acclimation or maintenance of cold hardiness in wheat. CBF proteins are transcription factors that bind to the dehydration-responsive element / C-repeat element (DRE / CRT) motif (CCGAC). Ten of the 15 Norstar recombinant CBFs whether purified under native or denaturing conditions showed in vitro binding to the CRT motif. Within hours of cold exposure (4 oC) the native CBF increased their affinity to CRT interaction which could be due to changes in the CBF secondary structures. Some of the CBF for binding preferred the core GGCCGAC motif while others preferred TGCCGAC. Similarly binding assays with truncated CBF revealed that for some CBF proteins, the second signature motif (DSAWR) and remaining C - terminal were not needed, while for others a considerable portion of the C -terminal region was needed for binding. Norstar CBF 12.1 has a memory of cold experience, and upon exposure to cold, has a high and immediate affinity to CRT elements. A homolog CBF12.2 in less cold - hardy winter wheat cv Cappelle - Desprez had a non - functional protein due to a R → Q substitution in a highly conserved residue within the AP2 domain. Several of the cv Norstar CBFs showed increased activity under LT and denaturing conditions, which may be the reason for the greater cold hardiness in Norstar. In conclusion, detailed and extensive analyses of CBF in this study characterized their structure and function relationships, which are important for understanding and improving LT tolerance in plants. The identification of specific CRT binding motifs and two CBFs which were very stable under adverse conditions may be prime candidates for further study to improve LT tolerance in plants.

The enzyme 11β-Hydroxysteroid Dehydrogenase 1 (11β-HSD1) catalyses the intracellular biosynthesis of the active glucocorticoid cortisol. Tissue specific dysregulation of the enzyme has been implicated in the development of metabolic syndrome and other associated diseases. Experiments with transgenic mice and prototype inhibitors show that inhibition of 11β-HSD1 in visceral adipose tissue and liver leads to a resistance of diet-induced hyperglycemia and a favourable lipid and lipoprotein profile as compared to controls. 11β-HSD1 inhibition has thus been proposed as an effective strategy to decrease intracellular glucocorticoid levels without affecting circulating glucocorticoid levels that are essential for stress responses. The clinical development of selective and potent drugs has therefore become a priority. In this research, a process of virtual screening employing the novel algorithm UFSRAT (Ultra Fast Shape Recognition with Atom Types) was used to discover compounds which had specific physicochemical and spatial atomic parameters deemed essential for inhibition of 11β-HSD1. The top scoring compounds were assayed for inhibitory activity against recombinant human and mouse enzyme, using a fluorescence spectroscopy approach. In addition, HEK-293 cell based assays with either human, mouse or rat enzymes were carried out using a scintillation proximity assay (SPA). The most potent compound competitively inhibited human 11β-HSD1 with a Kiapp value of 51 nM. Recombinant mouse and human enzyme were expressed, purified and characterised and used in a series of ligand binding assays. Further to this, an X-ray crystal structure of mouse 11β-HSD1 in complex with a tight binding inhibitor – carbenoxolone was solved.

Protozoan parasites of the genus Plasmodium are the causative agent of malaria. The most severe form of human malaria is caused by P. falciparum, responsible for approximately three quarters of a million deaths each year. One major problem in the treatment of malaria is resistance to existing chemotherapies. Consequently, there is an urgent need to identify and validate novel drug targets. A possible recently identified drug target is the PfNitA protein of P. falciparum which contains orthologues in other Plasmodium species but is absent from humans. The gene is annotated as a putative formate-nitrite transporter and orthologues are found in a range of prokaryotes as well as the lower eukaryotes algae and fungi. To determine the biological function of the protein, pfnita was expressed in Escherichia coli strains lacking the endogenous formate and nitrite transporters. In order to analyse the essentiality of the gene a reverse genetics approach was taken and the data discussed. Results indicate that the PfNitA protein is located in the plasma membrane and digestive vacuole of intraerythrocytic parasites suggesting a role in the uptake or excretion of metabolites. A second complexity with regard to treatment is the lack of a vaccine. A problem in crating a vaccine is antigenic variation. The PIR family of proteins contain a so-called hypervariable domain that has led to the suggestion that the family may play a role in antigenic variation. The objective of the work carried out in this thesis was to investigate the topology and structure of the PcCir2 protein of Plasmodium chabaudi, using E. coli as the expression host. The topology of Cir2 has been examined by means of reporter fusions and overexpression/purification studies undertaken towards crystallisation. As the PcCir2 amino acid sequence does not show significant homology to other proteins, structural data may provide insights into potential functional or binding domains.