No description available.

No description available.

No description available.

No description available.

No description available.

Currently DNA profiling methods only compare a suspect’s DNA with DNA left at the crime scene. When there is no suspect, it would be useful for the police to be able to predict what the person of interest looks like by analysing the DNA left behind in a crime scene. Determination of the age of the suspect is an important factor in creating an “identikit” (set of drawings of different features that can be put together to form the face of a person). This study investigated if one could use a correlation between telomere length and age, to predict the age of an individual from their DNA. Telomere length, in buccal cells, of 167 individuals aged between 1 and 96 years old was measured using quantitative real time PCR. The causes for the presence of large variation in telomere lengths in the population were further investigated. The age prediction accuracies were low even after dividing samples into non-related Europeans, males and females (5%, 9% and 1% respectively). Mean telomere lengths of eight age groups representing each decade of life showed a non-linear decrease in telomere length with age. There were variations in telomere lengths even among similarly aged individuals aged 26 years old (n = 10) and age 54 years old (n = 9). One of the factors that causes large inter individual variation could be the inheritance of telomere length. If there is a strong paternal or maternal influence, this could be incorporated into the age prediction formula. Parents’ telomere lengths were compared with children’s telomere lengths.

This thesis reports the cell culture establishment and a somatic cell selection system optimized for the isolation of chlorsulfuron-resistant variants in asparagus (Asparagus officinalis L.). The development of this cell selection system benefited the isolation of chlorsulfuron-resistant variants from an elite asparagus genotype. A cell culture system, suitable for somatic cell selection, was established for asparagus genotype CRD 168. Friable callus was initiated from etiolated shoots in darkness and used to produce a high density of single cells in suspension. Cell density was estimated based on a linear relationship with settled cell volume. A mean plating efficiency of 0.19 % was recorded between 1-4x10⁵ cells/Petri dish. In vitro cell selection techniques were developed to identify mutant asparagus cells with resistance to a sulfonylurea herbicide, chlorsulfuron. A few key aspects were important to achieve this: a cell culture system for cell selection was initially established; a toxic concentration for the complete growth inhibition of the wild type asparagus cells was defined; rare, resistant cell colonies were isolated and characterized; and chlorsulfuron-resistant plants were regenerated. From about 50 million cells, 165 cell colonies were isolated in the presence of 8 nM chlorsulfuron. Characterization of these selected cell colonies yielded 24 escapes, 98 unstable variants, and 43 stable-resistant variants. Callus cultures from 34 of these stable variants retained resistance following 11 months growth in the absence of the selection agent. Plants were regenerated from 36 of these stable herbicide-resistant variants. Six of these chlorsulfuron-resistant variants were screened for their degree of resistance to chlorsulfuron, cross resistance to other acetohydroxyacid synthase (AHAS) inhibiting herbicides and AHAS enzyme activity. Cross resistance to imazamox was evident in four of the resistant variants, while lack of cross resistance to metsulfuron methyl was observed in all six resistant variants. A varying degree of resistance to chlorsulfuron was observed among the resistant variants. Both in the original and secondary callus, an uninhibited AHAS enzyme activity in all six resistant variants was recorded in the presence of high chlorsulfuron concentration (70-140 nM), compared to the total inhibition in the wild type. One chlorsulfuron-resistant variant, R-45, was used to compare the biochemical and physiological basis of resistance with the wild type. The AHAS enzyme activity in the tissue culture and greenhouse foliage of R-45 was significantly higher in the presence of up to 280 nM chlorsulfuron compared with the wild type. Chlorsulfuron retention was considerably higher due to the reduction of epicuticular wax deposits on the foliage of R-45, in comparison with the wild type. Consequently, the resistant line absorbed at least 1.6 fold more chlorsulfuron than the wild type plants. Therefore, foliar application of 15 g a.i./ha Glean (commercial formulation of chlorsulfuron) produced typical symptoms of chlorosis in R-45, similar to the wild type, in the greenhouse plants. Somatic cell selection was carried out using two elite asparagus genotypes, CRD 74 and Clone X. Of the 33 rare cell colonies isolated from Clone X, 22 unstable variants and 6 escapes were discarded. All five remaining resistant variants produced plants. One of the stable-resistant variants (Clone X-24) was evaluated for resistance to chlorsulfuron. Both in vitro shoot cultures and greenhouse-grown plants of Clone X-24 showed increased resistance to chlorsulfuron compared with the wild type. The AHAS enzyme activity in the foliar extracts also showed the presence of higher enzyme activity in Clone X-24.

The absolute clarity of the lens of the eye is vital in the visual system. The unique structural and physiological properties of the lens are tightly integrated with highly ordered protein content to allow the lens to remain transparent. Consequently, any alteration or disturbance of these highly ordered proteins can affect the optical properties of the lens. In humans, cataracts are the major cause of blindness, yet the exact aetiology of cataract formation (cataractogenesis) is not fully understood. The purpose of the current research was to investigate whether deregulation of the Ca²⁺-dependent enzyme, calpains, following changes in lens Ca²⁺ homeostasis, is a key mechanism leading to undesired cleavage of a number of proteins that are linked with maintaining lens transparency and contributing to cataractogenesis. An ovine lens culture (in vitro) system and the heritable ovine cataract (in vivo) model were used to test the research hypothesis. The Ca²⁺ ionophore, ionomycin, was used to induce a Ca²⁺ overload and in vitro opacification during lens culture. Opacity in the lens was graded by a computer image analysis program. Protein profile (SDS-PAGE, 2-DE and Western detection), calpain activity (casein zymography), lens structure (microscopic view) and cytotoxicity level (LDH leakage assay) were analysed in Ca²⁺-induced opaque lenses. The involvement of calpain during opacification was further examined by applying synthetic exogenous calpain inhibitors to the in vitro system. Two novel exogenous calpain inhibitors were also assessed for their therapeutic potential in preventing the progression of cataracts in the in vivo cataract model by topical administration of the inhibitor direct to the sheep's eye over a 11 week period. HPLC was used to detect the penetration of these compounds into ocular tissues. Sustained Ca²⁺ influx into cultured lenses caused dense opacification. The opacity was characterised by formation of a turbid fraction and cell death in the outer cortex of the ovine lens. There was increased calpain autolysis associated with the progress of opacification, indicating increased calpain activity. Major degradation of the cytoskeletal proteins, spectrin and vimentin, was observed whilst there was limited degradation of the lens structural soluble proteins, crystallins, in response to a Ca²⁺ flux. Lens proteins were protected from degradation by adding synthetic calpain inhibitors to the culture medium. Topical administration of novel anti-calpain molecules failed to retard the progression of cataractogenesis in the ovine inherited cataract model. Further investigation of drug penetration showed that efficacy of inhibitory compounds was limited by permeability of these molecules across the cornea and the ability of the molecules to reach and penetrate into the lens. The ovine lens Ca²⁺-induced opacification (OLCO) model in this thesis has provided a model to understand the role of Ca²⁺ homeostasis in lens transparency. With sustained intracellular Ca²⁺ level, the degradation of cytoskeletal elements is highly correlated with calpain activity. Cataractogenesis is the pathological response to the loss of lens Ca²⁺ homeostasis in this model. The current results support the hypothesis that the deregulation of calpain activity is a trigger for a series of cascading events, leading to death of the cells in the lens.