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.

The microflora of two acidic environments was investigated using analysis of 16S rDNA amplified by the polymerase chain reaction (PCR) from environmental DNA. These environments had different chemical characteristics from most of the acidic environments studied by others. The first sample site, a coal spoil (Birch Coppice, Warwickshire), might have been expected to produce niches enriched in humic matter. The second, comprising geothermal vents on the Island of Vulcano, was unusual for natural acidic environments since it was saline. Three vent regions of different temperatures (30°C, 45°C and 80°C) were examined. Prior to the 16S rDNA analysis of the sites, a brief investigation into selection of a suitable method of DNA extraction was carried out. A bead-beating method and a chemical lysis/freeze-thaw method were compared. With regard to clone types found via each method, there was little qualitative difference. DNA was extracted from the two sites and 16S rRNA genes were amplified by PCR. PCR products were ligated and competent E. coli cells were transformed to produce clone libraries. Restriction fragment length polymorphisms (RFLPs) were examined and representatives of each RFLP type were sequenced and analysed with reference to RNA gene sequence data bases. The coal spoil clone library was dominated by sequences related to those from uncultured actinobacteria, particularly those found previously in peat bogs and various soils. Representatives of some well-known acidophiles were also found (e.g. Leptospirillum species). The clone bank from the saline, geothermal site DNA comprised sequences from acidophiles capable of growth at the respective temperatures of different samples. The lowest temperature samples produced sequences from a novel Acidithiobacillus species and also indicated a novel species probably related to Thiobacillus prosperus (which was isolated previously from Vulcano). A high temperature sample gave sequences from archaeal acidophiles, Acidianus brierleyi and, previously isolated from Vulcano, Acidianus infernus and Thermoplasma volcanium. Where the clone banks revealed the presence of novel organisms, attempts were made to isolate and characterise them. The novel actinobacteria did not appear to grow in laboratory enrichment cultures. The novel Acidithiobacillus species and two novel Thiobacillus prosperus-like species were characterised.

The rotaviruses are a recently defined ubiquitous group of viruses responsible for causing acute-gastroenteritis in human infants and young animals. Biochemical studies have shown that the rotavirus genome consists of 11 segments of double-stranded RNA (dsRNA). This thesis concerns an investigation of the nature and extent of genomic variation in rotaviruses. A rapid and sensitive method for analyzing the genome profiles of rotavirus field isolates was developed. This is based on the direct extraction of dsRNA from faecal samples followed by radiolabelling with [³²P] pCp using T4 RNA ligase. This procedure has been further developed to produce a method for generating diagnostic fingerprints from individual species of dsRNA. A detailed structural study making use of this fingerprinting method has been undertaken on bovine, porcine and human rotavirus isolates. These analyses show that genome segment mobility variations are always associated with detectable changes in nucleotide sequence. They also show that corresponding genome segments with no mobility variation can have sequence-changes at least as great as those found in segments showing electrophoretic mobility variation. These results also revealed evidence for genome segment specific regions of terminal sequence conservation. Evidence for the occurrence of genome segment reassortment between viruses in the field was obtained. Finally evidence for the existence of a 'new' porcine rotavirus which is antigenically unrelated to previously described rotaviruses and has an unusual pattern for its 11 genome segments is presented.

Studies on the molecular organisation and properties of the first three enzymes of pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPTase), aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase), in various organisms have been reviewed. The molecular organisation of these three enzymes has been investigated in wheat using gel filtration chromatography. CPSase activity could not be detected in gel filtered extracts and in crude extracts from wheat seedlings was shown to be highly labile. ATCase and DHOase activity was detected and the molecular weights of these enzymes were estimated to be 1.03 x 105 ( 1.4 x 10^4) and 8.6 x 10^4 (6 x 103), respectively. At no time during these investigations were high molecular weight species (consistent with the presence of a multifunctional complex containing these enzymes) detected. During the course of these investigations, a protease was detected which was shown to co-migrate with ATCase and DHOase activities. This protease was shown to be insensitive to the serine protease inhibitor PMSF, but was partially inactivated by iodoacetamide, consistent with the protease being a member of the cysteinyl protease family. Inclusion of iodoacetamide during chromatography also failed to reveal high molecular weight species of these enzymes. Antisera were raised against purified wheat ATCase and were characterised by their ability to inactivate the enzyme. These antisera were then used to probe western blots of crude extracts from wheat seedlings and screen a wheat cDNA expression library to ATCase sequences. Western blotting failed to show any immunoreactive species in extracts prepared under conditions which suppressed protease activity (SDS, -mercaptoethanol), although a low molecular weight (approximately 3.7 x 10^4) ATCase could be detected in samples obtained after gel filtration chromatography. Antisera also showed very little cross-reactivity with the ATCase from E.coli a result consistent with studies on the enzyme from B. subtilis.

Thesis (M.A.)--University of Nevada, Reno, 2007. / "May, 2007." Includes bibliographical references (leaves 53-60). Online version available on the World Wide Web.

Thesis (Ph.D.) - Carleton University, 2007. / Includes bibliographical references (p. 102-109). Also available in electronic format on the Internet.

Thesis (Ph. D.)--University of Chicago, Committee on Deveopmental Biology, August 2002. / Includes bibliographical references. Also available on the Internet.

Endosomal trafficking and autophagy are two fundamental processes of eukaryotic cell biology, from unicellular organisms such as yeast to multicellular metazoans such as C.elegans and Humans. Both processes are involved in a diverse number of physiological processes and implicated in a number of pathologies. A recent study has exhibited a mutation on the SM protein Vps45 as a cause of severe congenital neutropenia in humans. The same mutation in yeast causes defects in endosome to vacuole trafficking in S.cerevisiae as well as a temperature sensitive lethality at the non-permissive temperature. A null allele of vps-45 in C.elegans results in developmental arrest during the highly secretory phase of moulting in a similar temperature conditional manner to yeast and defects in yolk protein trafficking. The work presented in this thesis aims to provide basic understanding in an animal model of the impact of loss of Vps45 function that might be informative of the reason for the death of the highly secretory neutrophil cells under the absence of a functional Vps45 protein. The vps-45 and unc-51 mutants as well as a novel unc-51 vps-45 double mutant where possible, were characterised for lifespan, duration of post- embryonic development as well as moulting duration. Reduced embryonic viability, reduced lifespan as well as delays in the moulting process were identified. Data suggested that both autophagy and protein trafficking play a role in C.elegans development through unc-51 and vps-45 respectively. In addition to this, the seam cells of both the vps-45 and unc-51 defective C.elegans were observed during the moult using an autophagy marker. An increase in autophagic activity during the moult was observed, which was more pronounced in the case of the vps-45 mutant. As such the obtained data suggest autophagy and endosomal trafficking play an important role in the moulting process. Following up to previous work conducted in our lab in yeast defective for Vps45 trafficking which exhibited increased sensitivity to oxidative stress, the redox state of the vps-45 and unc-51 animals as well as their sensitivity to oxidative stress was assessed using a set of ER and cytosolic GFP markers and killing assays. Both the vps-45 and unc-51 mutants showed a higher sensitivity to oxidative stress, with the unc-51 exhibiting the more pronounced phenotype overall. These results came in agreement with the shorter lifespan phenotypes exhibited by both mutants in the previous experiments, possibly as a result of accumulation of ROS, as well as the severe defects of the double mutant. Finally, a suppressor identified for the moulting death of the vps-45 mutant was characterized for a set of phenotypes, in order to exclude suppression of any of the other phenotypes identified for the vps-45 mutant. Furthermore, the suppressor was identified as being autosomal and recessive and as thus an SNP full genome sequencing technique was employed, which gave rise to two suppression loci in two different chromosomes, along with two different subpopulations corresponding to these loci which exhibited different growing patterns.

The effects of pupal cold shock on the life cycle and wing morphology of the Speckled Wood butterfly are examined and their genetic assimilation is investigated. Metamorphosis is modelled in terms of changes in stability, and the mediation of cold shock effects by hormones is considered. Current theories of pattern formation are evaluated for the species, and pattern is analysed using manual, photographic and digital methods. The development of wing morphology is modelled, and cold shock effects understood by comparison with normal development. Developmental canalisation is estimated as variability and fluctuating asymmetry. An index is developed that predicts the extent of assimilation. Likely modes of inheritance are suggested, and the possibility of natural cold shock and assimilation in the species is considered. Recent trends in biology indicate that neo-Darwinian concepts cannot adequately account for certain developmental and hereditary phenomena and that a new paradigm is emerging. The two schools are compared with particular reference to Weismann and Waddington, and the phenomenology is re-examined in the light of the new findings.