Cell adhesion molecules are known to play crucial roles in a variety of developmental processes. The neural cell adhesion molecule N-CAM is strongly implicated in neurulation and neural crest cell (NCC) migration and was thus studied in splotch (Sp) neural tube defect mutant embryos. At the 20 somite-stage of gestation day 9, Sp N-CAM was found to contain polysialic acid (PSA) side chains which are normally only present beginning at gestational day 11. Younger embryos at 12 and 14 somites also showed the presence of PSA on N-CAM, which was absent in controls. Enzymatic removal of PSA from N-CAM resulted in isoforms which migrated identically to PSA-free N-CAM isoforms in SDS-polyacrylamide gels. The post-translational modification of N-CAM appears to be the primary target of the Sp gene. In view of N-CAM's importance during development, an alteration at a critical stage is likely to result in the cascade of abnormalities seen in Sp mutants. / A new genotyping assay was also implemented for examination of N-CAM in Sp and other related wildtype strains.

Immunocytochemical analyses have demonstrated that the Calvin cycle enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is predominantly localized in the pyrenoid region of chloroplasts of evolutionarily diverse algae. That Rubisco remains pyrenoid-localized at photosynthetically-saturating irradiance in the green alga Chlorella pyrenoidosa indicates a catalytic, rather than storage function for pyrenoid-localized Rubisco. This is further supported by the immunolocalization of Rubisco activase to the pyrenoids of two species of green algae. The exclusion of phosphoribulokinase from the pyrenoids of a red and a green alga indicates that pyrenoids do not possess the full complement of Calvin cycle enzymes. / Thylakoid lamellae traverse the pyrenoids of many algae. The absence of light-harvesting phycoerythrin and of photosystem (PS) II activity, but not PSI activity, from the intrapyrenoid thylakoids of the red alga Porphyridium cruentum indicates a structural and functional heterogeneity between these lamellae and those located in the chloroplast stroma. In contrast, the intrapyrenoid thylakoids of cryptomonads, algae whose chloroplast is thought to have evolved from red algae, possess both PSI and PSII protein complexes. These results are discussed with reference to Rubisco being mainly pyrenoid-localized in these algae.

Three distinct K$ sp+$ currents were identified in corn (Zea mays) protoplasts using the whole-cell patch-clamp technique. Inward-rectifying K$ sp+$ currents were evoked at membrane potentials more negative than $-$100 mV. The activation range was sensitive to external K$ sp+$ and shifted in the positive direction as the K$ sp+$ concentration was elevated. The second K$ sp+$ current was voltage-independent and contributed to the resting membrane conductance of the protoplast. Finally, a voltage- and Ca$ sp{2+}$-dependent K$ sp+$ current was observed at potentials positive to $-$60 mV. This current was inhibited by reagents which antagonize plasmalemma Ca$ sp{2+}$ influx (e.g. nitrendipine, verapamil). In contrast, currents were enhanced by increasing the cytosolic free Ca$ sp{2+}$ concentration from 40 to 400 nM. The Ca$ sp{2+}$-dependent K$ sp+$ current was inhibited by tetraethylammonium ions, Cs$ sp+$, Ba$ sp{2+}$, and charybdotoxin which suggested that the channel protein has structural similarities to the high conductance Ca$ sp{2+}$-dependent K$ sp+$ channel observed in animal systems.

No description available.

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 develops image segmentation methods for the application of automated cervical cancer screening. The traditional approach to automating this task has been to emulate the human method of screening, where every one of the hundreds of thousands of cells on each slide is analysed for abnormality. However, due to the complexity of cervical smear images and the low error tolerance imposed upon the segmentation stage, only limited success has previously been found. A different approach is to detect malignancy associated changes (MACs) in a relatively small sample of the total population of cells. Under this paradigm, the requirement to segment every cell is loosened, but delineation accuracy and error checking become essential. Following a review of generic and cervical smear segmentation, it is concluded that prior work on the traditional approach to automation is not suitable for a MACs solution. However, the previously proposed framework of a dual-magnification system is found to be relevant and is therefore adopted. Here, scene images are first captured at low resolution in order to rapidly locate the cells on a slide. Cells that are deemed to be suitable for further analysis are then imaged at high resolution for the more accurate segmentation of their nuclei. A water immersion algorithm is developed for low resolution scene segmentation. This method achieves a rapid and robust initial segmentation of the scene without the requirement of incorporating extensive a priori knowledge of the image objects. A global minimum searching contour is presented as a top-down method for segmenting the high resolution cell nucleus images where the image objects are well characterised by shape and appearance. This latter method is tested upon 20,000 images and found to achieve an accurate segmentation rate of 99.47%. An error checking method, that uses segmentation stability as an indicator of segmentation success, is developed that is capable of detecting 100% of the failures of the nucleus segmenter, at the expense of discarding only 9% of the data. Throughout this work, contemporary issues in the field of generic image segmentation are presented and some of these are addressed for the cervical smear application. Finally, an avenue of future work is proposed which may lead to the much wider proliferation of computer vision solutions to everyday problems.

Thesis (Ph. D.)--Oregon State University, 2005. / Printout. Includes bibliographical references (leaves 100-102). Also available on the World Wide Web.

Thesis (Ph.D.)--Victoria University of Wellington, 2008. / Includes bibliographical references.

Thesis (M.Sc.) - Carleton University, 2002. / Includes bibliographical references (p. 112-124). Also available in electronic format on the Internet.