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Plant growth regulators have a vital role in plant growth and development. The cellular response to these regulators depends on the presence and the action of specific receptors. The plant growth regulators and their receptors act together in complexes which determine the final effects of the plant growth regulators. In the research reported here, emphasis has been given to the regulation of the activity of the receptors themselves. The regulation of the N-l-naphthylphthalamic acid (N~A) receptor through phosphorylation and dephosphorylation and the regulation of the auxin binding protein (ABP) through gene manipulation have been investigated. NPA, an auxin transport inhibitor, was found to bind specifically to a crude membrane preparation from sugar beet seedling leaf cell suspension cultures. The in vitro binding was optimal at pH 4.5 and 4?C. Binding parameters for NP A binding were determined by Scatchard analysis. The dissociation constant (Kd) and binding protein concentration were found to be 1.71 x 10-7 mol dm-3 and 220 pmoles g-I membrane protein respectively. It was found that the amount of specific 3H-NPA binding was significantly increased by adding Mg2+ A TP to the binding assay solution; treatment of membrane preparations with acid phosphatase, prior to the NP A binding assay, resulted in lower specific binding. A TP activation and phosphatase inactivation were culture stage dependent. Although a considerable effect could be detected when using cells from day 8 (representing the linear phase), the same treatment did not alter the binding if cells from day I (representing lag phase) or day 14 (representing the stationary phase) were used. These observations have strongly highlighted the possible involvement of a phosphorylation and dephosphorylation mechanism in vivo in the regulation of the activity of the NP A receptor. High phosphatase activity was found in the supernatant, but not in the membrane pellet, after 50,000 g centrifugation. The presence of a membrane-bound auxin receptor, ABP, was demonstrated by Scatchard analysis in sugar beet seedlings. The Kd value and the receptor concentration were found to be 2.15 x 10-6 mol dm-3 and 68 pmoles g-I membrane protein. The protein could be solubilised either with the detergent Triton X-I 00 or by acetone-washing, with a recovery of about 40%. An acetone-solubilised ABP preparation could be partially purified by DEAE-Sephacel ion exchange chromatography, NAA-linked AH-Sepharose 4B affmity chromatography or Sephadex G-200 gel filtration. The recovery after any of these chromatographic treatments was very low so that successive chromatography for further purification was unsuccessful. The low level of detectable binding after purification resulted mainly from the low abundance of ABP in the plant material. Non-radioactive labelling and detection techniques were used to show that an ABP-probe hybridized to sugar beet genomic DNA during dot blotting. The present study has indicated that receptor activity could be regulated by a phosphorylation and dephosphorylation mechanism in plants. The investigation has also suggested that the effect of plant growth regulators on plant development could be regulated through the manipulation of the expression of their receptor genes.

Cancer cachexia is characterised by selective depletion of skeletal muscle protein reserves. The ubiquitin-proteasome proteolytic pathway has been shown to be responsible for muscle wasting in a range of cachectic conditions including cancer cachexia. To establish the importance of this pathway in muscle wasting during cancer (and sepsis), a quantitative competitive RT-PCR (QcRT-PCR) method was developed to measure the mRNA levels of the proteasome sub units C2a and C5ß and the ubiquitin-conjugating enzyme E214k. Western blotting was also used to measure the 20S proteasome and E214k protein expression. In vivo studies in mice bearing a cachexia inducing murine colon adenocarcinoma (MAC16) demonstrated the effect of progressive weight loss on the mRNA and protein expression for 20S proteasome subunits, as well as the ubiquitin-conjugating enzyme, E214k, in gastrocnemius and pectoral muscles. QcRT-PCR measurements showed a good correlation between expression of the proteasome subunits (C2 and CS) and the E214k enzyme mRNA and weight loss in gastrocnemius muscle, where expression increased with increasing weight loss followed by a decrease in expression at higher weight losses (25-27%). Similar results were obtained in pectoral muscles, but with the expression being several fold lower in comparison to that in gastrocnemius muscle, reflecting the different degrees of protein degradation in the two muscles during the process of cancer cachexia. Western blot analysis of 20S and E214k protein expression followed a similar pattern with respect to weight loss as that found with mRNA. In addition, mRNA and protein expression of the 20S proteasome subunits and E214k enzyme was measured in biopsies from cachectic cancer patients, which also showed a good correlation between weight loss and proteasome expression, demonstrating a progressive increase in expression of the proteasome subunits and E214k mRNA and protein in cachectic patients with progressively increasing weight loss. The effect of the cachexia-inducing tumour product PIF (proteolysis inducing factor) and 15-hydroxyeicosatetraenoic acid (15-HETE), the arachidoinic acid metabolite (thought to be the intracellular transducer of PIF action) has also been determined. Using a surrogate model system for skeletal muscle, C2C12 myotubes in vitro, it was shown that both PIF and 15-HETE increased proteasome subunit expression (C2a and C5ß) as well as the E214k enzyme. This increase gene expression was attenuated by preincubation with EPA or the 15-lipoxygenase inhibitor CV-6504; immunoblotting also confirmed these findings. Similarly, in sepsis-induced cachexia in NMRI mice there was increased mRNA and protein expression of the 20S proteasome subunits and the E214k enzyme, which was inhibited by EPA treatment. These results suggest that 15-HETE is the intracellular mediator for PIF induced protein degradation in skeletal muscle, and that elevated muscle catabolism is accomplished through upregulation of the ubiquitin-proteasome-proteolytic pathway. Furthermore, both EPA and CV -6504 have shown anti-cachectic properties, which could be used in the future for the treatment of cancer cachexia and other similar catabolic conditions.

Head and neck squamous cell carcinoma (HNSCC) is the sixth commonest cancer worldwide with an increasing incidence in developing countries. Despite numerous advances in surgery, radiotherapy and chemotherapy over the past few decades the overall survival rate for patients with HNSCC has changed little. Currently, the management of HNSCC patients is based on the assessment of a variety of clinical and pathological parameters. However, in many instances, these factors fail to accurately predict the clinical behaviour of an individual patients tumour. HNSCC therefore, is a tumour entity that would benefit from a greater insight into the chromosomal alterations underlying the disease. Knowledge of such alterations would be expected to provide many benefits to the HNSCC clinician in terms of diagnostic and prognostic markers and may eventually identify novel molecular targets for therapeutic intervention. This thesis was aimed at characterising the chromosomal abnormalities involved in the tumourigenesis of HNSCC, principally using the powerful molecular cytogenetic technique of comparative genomic hybridisation (CGH), and the clinical applications of such data. Firstly, the technique was optimised and initially applied to specimens of primary HNSCC and surrounding uninvolved mucosa from 19 patients in order to investigate the phenomenon of 'field cancerization'. Specimens of primary HNSCC and histologically normal mucosa taken from 1cm and 5cm distant to the primary site were analysed from each patient in order to characterise the chromosomal abnormalities associated with malignant tissue and attempt to identify aberrations underlying the `field change'. CGH of the primary tumour specimens revealed numerous chromosomal aberrations with a relatively consistent pattern. Frequent deletions of DNA were identified on chromosome 3p, 4p, 8p, 9p, 11 q, 13q and 18q and frequent gains on chromosomes 2q, 3q, 5p, 7q, 8q, 9q and 11q. The histologically normal mucosa did not show chromosomal abnormalities within the cells analysed. Therefore, if molecular abnormalities were present in the mucosa surrounding a primary HNSCC they would be below the resolution of CGH, such as subtle point mutations, or only present in a minority of cells. In order to investigate the genetic relationship between primary HNSCC and lymph node metastases, matched pairs of primary and metastatic tumours were obtained from 18 patients and analysed by CGH. Whilst the overall frequency of genetic alterations was similar between primary and metastatic tumours, a surprising degree of discordance was identified between each individual's matched pair of tumours. At least one common aberration was identified in all cases studied, however the percentage of aberrations detected in the lymph node metastases that were shared with the primary tumour varied greatly, ranging from 100% - 8.3%. Several chromosomal regions were found to be altered at similar frequencies in both the primary and metastatic tumours. Most interestingly, regions of the genome found to be altered at a higher frequency in the population of metastatic HNSCC included deletion of 4p15.3-pter and 17q22-qter and gain of 6gcen-q15 and 13q21-22. In addition, both gains and deletions of material from chromosome 22 were found at a higher frequency in the metastatic tumours. These chromosomal regions may contain genes important in the process of metastasis in HNSCC. The level of discordance identified between matched pairs of tumours also suggests that a linear progression model may not satisfactorily explain the progression to metastases in all HNSCC. This thesis also addressed the important clinical question of resistance to radiotherapy demonstrated by a significant fraction of laryngeal tumours. No markers that reliable predict the response of an individual tumour to radiotherapy are currently available. The expression of a panel of markers involved in DNA damage recognition, cell cycle arrest, DNA repair and apoptosis were evaluated in 23 glottic laryngeal tumours (8 radio-resistant and 13 radio-sensitive). Of these, the expression of bcl-2, an anti-apoptotic marker, was specifically associated with the resistant phenotype. This statistically significant association provides preliminary evidence for the dysregulation of apoptosis as a mechanism by which resistant tumours can evade radiotherapy induced tumour regression. Overall, CGH analysis of primary HNSCC identified a relatively consistent pattern of DNA alterations with several distinct regions of DNA deletion and gain identified. Frequent deletions of DNA were identified on chromosomes 1p, 2q, 3p, 4p, 4q, 5q, 7q, 8p, 9q, 10q, 11p, 11q, 13q, 17p, 18q, 19 and 21 and frequent gains of DNA on chromosomes 1q, 2q, 3q, 4q, 5p, 6q, 7p, 7q, 8q, llq, 12p, 13q, 18p and 18q. Chromosome 3 was the most frequent site of both deletions and gains. Follow up data was obtained for all patients analysed by CGH and Kaplan-Meier survival analysis demonstrated a significant correlation between gain of DNA on 3q25-27 and reduced overall survival. This finding highlights the necessity for further, high resolution, characterisation of this region in order that the specific genetic marker can be identified. This thesis demonstrates that molecular analysis of tumours is able to offer new, and valuable information for the understanding of HNSCC carcinogenesis and that these data can be used to compliment existing methodology. Further work is required to isolate specific genes and to understand their interactions.

The mitochondrial DNA from the ciliate protozoan Tetrahymena furgasoni str. W/ATCC. was isolated and mapped using six restriction enzymes. The linear molecule was seen to be approximately 35 Md. in size, slightly larger than the mtDNA seen in other Yetrahymena strains. The T. furgasoni mtDNA molecule also showed a heterogeneity in the length of its terminal regions, a characteristic of Tetrahymena mtDNA.The position of the mitochondrial rRNA genes were mapped on the molecule by hybridization studies using isolated mitochondrial rRNA's. The T. furgasoni mtDNA molecule possesses two large rRNA genes, one at each end of the molecule in sub-terminal locations, and a single small rRNA gene. A third, incomplete large rRNA gene was also found. The presence of this extra, incomplete rRNA gene may indicate why T. furgasoni mtDNA is slightly larger than the mtDNA from other Tetrahymena strains.The terminal Hind III restriction fragment from the mtDNA of T. pyriformis was cloned using the vector pJB 8. Three copies of the fragment were cloned. These three recombinant molecules were different in size, a difference which was associated with the size of the original terminus of the mtDNA molecule. DNA sequencing studies showed the difference in length to be associated with a variation in the number of copies of a 31 bp repeat sequence present at the original terminus of the mtDNA molecule.The significance of this mtDNA terminal structure is discussed with respect to the completion of replication of the linear mtDNA molecule, and to the generation of the terminal length heterogeneity of the linear mtDNA molecule. The structural characteristics of the Tetrahymena mtDNA terminus are compared with the structures seen at the termini of other linear genetic elements from a variety of sources.

This thesis examined genetic and phenotypic aspects of production of farmed Red deer in the UK. Heritabilities for weight traits tended to be moderate to high. Selection on weight at a given age will tend to lead to a correlated increase in weight and all ages and has implications for increased calving difficulty and higher maternal overheads. Animals of Wapiti and Eastern European parentage tended to have higher liveweights than those of British parentage pointing to their possible use as 'terminal' sires. Care is needed when selecting hinds to cross with these stags. Older dams were more likely to have a successful pregnancy and calve earlier. Calving traits tended to have low genetic variation. A central performance test was set up to improve across herd linkages. It is concluded that in future the test should start earlier and a lower limit on the weight of animals going on test should be set. The traits that were included in the economic breeding objective for Red deer included number of calves weaned, hind and offspring food consumption, stag calf and hind carcass weight and hind calf liveweight at 15 months. It was concluded that antlercharacteristics should be excluded from the breeding objective as they have no monetary value in the UK deer industry, but they may be included in selection criteria if they can be shown to improve the accuracy of breeding value prediction. Sources of variation in carcass traits and weight traits were investigated using linear body measurements and photographic techniques. Heights and girths were found to be the best predictors of weight traits. Weight was found to be the best predictor of carcass composition. Recommendations are made for future research. These include the setting up of cross breeding and selection experiments for more accurate parameter estimation and the heterotic effects of using Wapiti and animals of European parentage. Farmers are encouraged to use artificial insemination and the BDFA and MAFF are advisedto set up a performance recording scheme.

The localisation of the prokaryotic metallothionein (MT) divergon smt (which includes the MT gene smtA and a divergently transcribed gene smtB] was examined, and smt deficient mutants of Synechococcus PCC 7942 (strain R2-PIM8) have been generated by insertional inactivation/partial gene deletion mediated by homologous recombination. The structure and homozygosity (of the smt region) of these mutants, designated R2-PIM8(smt), was confirmed by Southern analyses and plasmid recovery in Escherichia coli (involving the generation of a ca. 7.8 kb plasmid from Soil digested R2-PIM8(smt) DNA). Furthermore, smtA transcripts were not detected in R2-PIM8(smt) RNA. Viability of R2-PIM8(smt) reveals that smt performs no essential role in Synechococcus under these culture conditions. R2-PIM8(smt) has reduced tolerance to Zn(^2+) and Cd(^2+), and short term reduced resistance to Ag(^+). Restoration of Zn(^2+) tolerance was used as a phenotypic selection to isolate recombinants derived from R2-PIM8(smt) after reintroduction of a linear DNA fragment containing an uninterrupted smt divergon. These smt-restored cells also exhibited restored Cd(^2+) tolerance. Hypersensitivity to Cu(^2+) or Hg(^2+)was not detected in R2-PIM8(smt) indicating independence of Cu(^2+) and Hg(^2+) resistance to smt-mediated metal tolerance. Sequences upstream of smtA (Including smtB and/or the smt operator-promoter) fused to a promoterless locZ, conferred metal-dependent β-galactosidase expression in R2-PIM8. At maximum permissive concentrations for growth, β-galactosidase assays revealed Zn(^2+) to be a more potent elicitor of metal-dependent expression from the smtA operator-promoter than Cd(^2+). Equivalent experiments, in R2-PIM8(smQ and R2-PIM8(smtA+/B-) (containing functional chromosomal smtA and non-functional chromosomal smtB), revealed that smtB encodes a repressor of smtA transcription. In addition, it is demonstrated that SmtB can act in trans. It is proposed that Zn(^2+) is the most potent (metal ion) inducer of SmtB mediated derepression of smtA transcription. Furthermore, β-galactosidase assays indicated that, in addition to SmtB, other regulatory elements (including a transcriptional activator) are involved in the regulation of expression from the smt operator-promoter. Restoration of Zn(^2+) tolerance was also used as a phenotypic selection to isolate recombinants derived from R2-PIM8(smt) after reintroduction of a linear DNA fragment, containing functional smtA and non-functional smtB. The resulting transformants, R2-PIM8(smtA+/B-), exhibited increased (early) tolerance to Zn(^2+) and Cd(^2+) as compared to R2-PIM8(smt-. reintroduced ) (equivalent to R2-PIM8).The work presented in this thesis proposes a role for SmtA in Zn(^2+) homoeostasis/metabolism and Cd(^2+) detoxification. SmtB is confirmed to be a trans-acting inducer- (metal ion) responsive negative regulator of smtA. The phenotype of R2-PIM8(sm(A+/B-) (with respect to metal tolerance) has significance regarding previous work (Gupta et al., 1993. Molecular Microbiology 7, 189-195), in which analysis of the smt region of Synechococcus PCC 6301 cells selected for Cd(^2+) resistance, by stepwise adaptation, revealed the functional deletion of smtB. It was proposed that loss of smtB may be beneficial for continuously metal challenged cells. Loss of smtB, now shown to encode a repressor of smtA transcription, is shown to confer constitutive derepressed expression from the smtA operator- promoter and determine an (early) increase in metal (Zn(2+)/Cd(^2+)) tolerance.

Arsenic is an acute poison and its contamination in soil and water is widespread. Crambe abyssinica accumulates significantly higher levels of arsenic as compared to other species of the Brassicaceae. Being a non-food, high biomass crop that is naturally tolerant to heavy metals, crambe has significant potential for phytoremediation of arsenic. In order to identify the pathways involved in arsenic metabolism and detoxification in C. abyssinica, differentially expressed genes in response to arsenic exposure were isolated employing a PCR-Select Suppression Subtraction Hybridization approach. A total of 105 differentially expressed subtracted cDNAs were sequenced which were found to represent 38 genes. Those genes encode proteins functioning as antioxidants, metal transporters, reductases, enzymes involved in the protein degradation pathway, and several novel uncharacterized proteins. The differential expression of transcripts corresponding to the subtracted cDNAs was confirmed by the semi-quantitative RT-PCR. ^ Arabidopsis homologs of two uncharacterized proteins from this subtracted cDNA library were further characterized for their role in As detoxification in plants. One of these two genes, AtChaC2-1 functions as a gamma-glutamyl cyclotransferase as evident from in vivo studies in yeast as well as in Arabidopsis. It plays a significant role in glutathione homeostasis and participates in gamma-glutamyl cycle to recycle Glu. T-DNA insertion AtChaC2-1 mutant plants were tolerant to arsenic toxicity due to the elevated glutathione contents. AtChaC2-1 over-expression lines were also tolerant to As presumably due to more active gamma-glutamyl cycle and an efficient Glu recycling. Furthermore, AtChaC2-1 overexpression increased the N utilization efficiency as it decreased the de novo synthesis of Glu and thereby N assimilation. ^ A second gene, AtMATE21, is an efflux protein of MATE family of secondary transporters. Heterologous expression in yeast RM1 mutant strain decreased the As accumulation in yeast presumably by efficient effluxing of As from yeast cells. Arabidopsis plants with T-DNA insertional mutation in the AtMATE21 locus were sensitive to arsenate. The AtMATE21 over-expression lines were more tolerant to arsenate and accumulated a significantly higher amount of arsenic in the aboveground parts. Both AtChaC2-1 and AtMATE21 genes have significant potential to be utilized for developing plant-based strategies for arsenic mitigation in the environment.^

The mechanisms controlling the transmission of bluetongue virus (DTV) by vector Culicoides species were studied using immunohistochemistry, virus titration assays, in vitro transmission tests, viral binding protein analyses and transmission electron microscopy. After infection with BTV by intrathoracic (IT) inoculation, 100% of C. variipennis individuals from a susceptible colony developed a fully disseminated infection and transmitted the virus through their saliva. However only 35.4% of midges were . persistently infected after ingestion of an infectious blood meal, while only 12.1 % of persistently infected midges transmitted the virus through their saliva. The titres of BTV were about 10,·oTCIDsJmidge [Standard error of means (SEM) of log-transformed data=0.15, n=1400] in IT inoculated midges and varied from 0.32 to lQs.oTCIDsJmidge in orally infected individuals. Only those midges containing ~1 03.oTCIDso of BTV could transmit the virus through their saliva. The following patterns were observed in orally (persistently) infected individuals: 1) virus was restricted to the anterior and posterior midgut, and the foregut-midgut junction; 2) virus replicated in the gut cells, disseminated into the haemocoel but could only be detected in a few sporadic fat body cells beyond the gut; 3) virus escaped from the gut cells into the haemocoel and replicated in some secondary organs/tissues but at low levels; 4) a fully disseminated infection was observed and virus replicated in the haemocoel and secondary organs/tissues, including the salivary glands, at high levels. The infection of the gut can be divided into two main types: 1) virus replication in gut cells ranging from very low to higher levels but with virus spread throughout the cytoplasm of the infected cells; 2) virus positive reaction restricted to endosome-like structures in the cytoplasm of some gut cells. BTV was detected in the anterior and posterior midgut, foregut-midgut junction, fat body, ganglia, salivary glands and ommatidia of the compound eyes of some infected midges. No virus was ever found in the hindgut cells, muscles, Malpighian tubes and oocytes/nurse cells of the ovaries. BTV infection of the salivary glands of C. l'ariipcnnis was shown to follow a typical pattern. Virus entered the acinar cells from the haemococl passing through the basement membrane, then localised and replicated in virus inclusion bodies (VIBs) in the cytoplasm of acinar cells. Mature progeny virus particles were released into acini, then transported through intermediate ducts and accumulated in crystalline arrays in the lumen of the major secretory ducts. No virus was released back into the haemocoel through the basement membrane; nor was virus released back into acinar cells from the acini. Nervous tissue of C. l'ariipennis is one of the most susceptible tissues to BTV. Ultrastructural observation showed characteristics ofBTV replication, including formation of VIBs, large amounts of progeny virus particles and tubules, in infected thoracic ganglia. A 60-kD viral protein adhered to both BHK-21 (mammalian) cells and a Culicoides cell line, KC cells. A 44-kD BTV viral protein, co-migrating with non structural protein NS2, adsorbed to BHK-21 cells but not to KC cells, while a 39.6 kD viral protein, co-migrating with major inner capsid protein VP7, adhered only to KC cells but not to BHK-21 cells.