Genomic DNA fingerprinting by restriction fragment end labeling.

A typing method for bacteria was developed and applied to several species, including Escherichia coli and Actinobacillus actinomycetemcomitans. Total genomic DNA was digested with a restriction endonuclease, and fragments were enabled with [alpha-32P]dATP by using the Klenow fragment of DNA polymerase and separated by electrophoresis in 6% polyacrylamide/8 M urea (sequencing gel). Depending on the restriction endonuclease and the bacterium, the method produced approximately 30-50 well-separated fragments in the size range of 100-400 nucleotides. For A. actinomycetemcomitans, all strains had bands in common. Nevertheless, many polymorphisms could be observed, and the 31 strains tested could be classified into 29 distinct types. Furthermore, serotype-specific fragments could be assigned for the three serotypes investigated. The method described is very sensitive, allowing more distinct types to be distinguished than other commonly used typing methods. When the method was applied to 10 other clinically relevant bacterial species, both species-specific bands and strain-specific bands were found. Isolates from different locations of one patient showed indistinguishable patterns. Computer-assisted analysis of the DNA fingerprints allowed the determination of similarity coefficients. It is concluded that genomic fingerprinting by restriction fragment end labeling (RFEL) is a powerful and generally applicable technique to type bacterial species.

Sets of EcoRI fragments containing ribosomal RNA sequences are conserved among different strains of Listeria monocytogenes.

To classify Listeria monocytogenes using taxonomic characters derived from the rRNA operons and their flanking sequences, we studied a sample of 1346 strains within the taxon. DNA from each strain was digested with a restriction endonuclease, EcoRI. The fragments were separated by gel electrophoresis, immobilized on a membrane, and hybridized with a labeled rRNA operon from Escherichia coli. The pattern of bands, positions, and intensities of hybridized fragments were electronically captured. Software was used to normalize the band positions relative to standards, scale the signal intensity, and reduce the background so that each strain was reproducibly represented in a data base as a pattern. With these methods, L. monocytogenes was resolved into 50 pattern types differing in the length of at least one polymorphic fragment. Pattern types representing multiple strains were recorded as the mathematical average of the strain patterns. Pattern types were arranged by size polymorphisms of assigned rRNA regions into subsets, which revealed the branching genetic structure of the species. Subtracting the polymorphic variants of a specific assigned region from the pattern types and averaging the types within each subset resulted in reduced sets of conserved fragments that could be used to recognize strains of the species. Pattern types and reduced sets of conserved fragments were conserved among different strains of L. monocytogenes but were not observed in total among strains of other species.

Dispersion aspects of periodically amplified soliton transmission systems

This thesis presents improvements to optical transmission systems through the use of optical solitons as a digital transmission format, both theoretically and experimentally. An introduction to the main concepts and impairments of optical fibre on pulse transmission is included before introducing the concept of solitons in optically amplified communications and the problems of soliton system design. The theoretical work studies two fibre dispersion profiling schemes and a soliton launch improvement. The first provides superior pulse transmission by optimally tailoring the fibre dispersion to better follow the power, and hence nonlinearity, decay and thus allow soliton transmission for longer amplifier spacings and shorter pulse widths than normally possible. The second profiling scheme examines the use of dispersion compensating fibre in the context of soliton transmission over existing, standard fibre systems. The limits for solitons in uncompensated standard fibre are assessed, before the potential benefits of dispersion compensating fibre included as part of each amplifier are shown. The third theoretical investigation provides a simple improvement to the propagation of solitons in a highly perturbed system. By introducing a section of fibre of the correct length prior to the first system amplifier span, the soliton shape can be better coupled into the system thus providing an improved "average soliton" propagation model. The experimental work covers two areas. An important issue for soliton systems is pulse sources. Three potential lasers are studied, two ring laser configurations and one semiconductor device with external pulse shaping. The second area studies soliton transmission using a recalculating loop, reviewing the advantages and draw-backs of such an experiment in system testing and design. One particular example of employing the recirculating loop is also examined, using a novel method of pulse shape stabilisation over long distances with low jitter. The future for nonlinear optical communications is considered with the thesis conclusions.

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

'We just make the pictures...?' How work is portrayed in children's feature length films

This article explores how adult paid work is portrayed in 'family' feature length films. The study extends previous critical media literature which has overwhelmingly focused on depictions of gender and violence, exploring the visual content of films that is relevant to adult employment. Forty-two G/PG films were analyzed for relevant themes. Consistent with the exploratory nature of the research, themes emerged inductively from the films' content. Results reveal six major themes: males are more visible in adult work roles than women; the division of labour remains gendered; work and home are not mutually exclusive domains; organizational authority and power is wielded in punitive ways; there are avenues to better employment prospects; and status/money is paramount. The findings of the study reflect a range of subject matters related to occupational characteristics and work-related communication and interactions which are typically viewed by children in contemporary society.

Using Trauma Injury Severity Score (TRISS) variables to predict length of hospital stay following trauma in New Zealand

Aim – To develop and assess the predictive capabilities of a statistical model that relates routinely collected Trauma Injury Severity Score (TRISS) variables to length of hospital stay (LOS) in survivors of traumatic injury. Method – Retrospective cohort study of adults who sustained a serious traumatic injury, and who survived until discharge from Auckland City, Middlemore, Waikato, or North Shore Hospitals between 2002 and 2006. Cubic-root transformed LOS was analysed using two-level mixed-effects regression models. Results – 1498 eligible patients were identified, 1446 (97%) injured from a blunt mechanism and 52 (3%) from a penetrating mechanism. For blunt mechanism trauma, 1096 (76%) were male, average age was 37 years (range: 15-94 years), and LOS and TRISS score information was available for 1362 patients. Spearman’s correlation and the median absolute prediction error between LOS and the original TRISS model was ρ=0.31 and 10.8 days, respectively, and between LOS and the final multivariable two-level mixed-effects regression model was ρ=0.38 and 6.0 days, respectively. Insufficient data were available for the analysis of penetrating mechanism models. Conclusions – Neither the original TRISS model nor the refined model has sufficient ability to accurately or reliably predict LOS. Additional predictor variables for LOS and other indicators for morbidity need to be considered.

Using a longitudinal model to estimate the effect of methicillin-resistant staphylococcus aureus infection on length of stay in an intensive care unit

Healthcare-associated methicillin-resistant Staphylococcus aureus(MRSA) infection may cause increased hospital stay or, sometimes, death. Quantifying this effect is complicated because it is a time-dependent exposure: infection may prolong hospital stay, while longer stays increase the risk of infection. We overcome these problems by using a multinomial longitudinal model for estimating the daily probability of death and discharge. We then extend the basic model to estimate how the effect of MRSA infection varies over time, and to quantify the number of excess ICU days due to infection. We find that infection decreases the relative risk of discharge (relative risk ratio = 0.68, 95% credible interval: 0.54, 0.82), but is only indirectly associated with increased mortality. An infection on the first day of admission resulted in a mean extra stay of 0.3 days (95% CI: 0.1, 0.5) for a patient with an APACHE II score of 10, and 1.2 days (95% CI: 0.5, 2.0) for a patient with an APACHE II score of 30. The decrease in the relative risk of discharge remained fairly constant with day of MRSA infection, but was slightly stronger closer to the start of infection. These results confirm the importance of MRSA infection in increasing ICU stay, but suggest that previous work may have systematically overestimated the effect size.

Modelling the effects of bone fragment contact in fracture healing

The fracture healing process is modulated by the mechanical environment created by imposed loads and motion between the bone fragments. Contact between the fragments obviously results in a significantly different stress and strain environment to a uniform fracture gap containing only soft tissue (e.g. haematoma). The assumption of the latter in existing computational models of the healing process will hence exaggerate the inter-fragmentary strain in many clinically-relevant cases. To address this issue, we introduce the concept of a contact zone that represents a variable degree of contact between cortices by the relative proportions of bone and soft tissue present. This is introduced as an initial condition in a two-dimensional iterative finite element model of a healing tibial fracture, in which material properties are defined by the volume fractions of each tissue present. The algorithm governing the formation of cartilage and bone in the fracture callus uses fuzzy logic rules based on strain energy density resulting from axial compression. The model predicts that increasing the degree of initial bone contact reduces the amount of callus formed (periosteal callus thickness 3.1mm without contact, down to 0.5mm with 10% bone in contact zone). This is consistent with the greater effective stiffness in the contact zone and hence, a smaller inter-fragmentary strain. These results demonstrate that the contact zone strategy reasonably simulates the differences in the healing sequence resulting from the closeness of reduction.

The genetic basis of human height : the role of estrogen

Height is a complex physical trait that displays strong heritability. Adult height is related to length of the long bones, which is determined by growth at the epiphyseal growth plate. Longitudinal bone growth occurs via the process of endochondral ossification, where bone forms over the differentiating cartilage template at the growth plate. Estrogen plays a major role in regulating longitudinal bone growth and is responsible for inducing the pubertal growth spurt and fusion of the epiphyseal growth plate. However, the mechanism by which estrogen promotes epiphyseal fusion is poorly understood. It has been hypothesised that estrogen functions to regulate growth plate fusion by stimulating chondrocyte apoptosis, angiogenesis and bone cell invasion in the growth plate. Another theory has suggested that estrogen exposure exhausts the proliferative capacity of growth plate chondrocytes, which accelerates the process of chondrocyte senescence, leading to growth plate fusion. The overall objective of this study was to gain a greater understanding of the molecular mechanisms behind estrogen-mediated growth and height attainment by examining gene regulation in chondrocytes and the role of some of these genes in normal height inheritance. With the heritability of height so well established, the initial hypothesis was that genetic variation in candidate genes associated with longitudinal bone growth would be involved in normal adult height variation. The height-related genes FGFR3, CBFA1, ER and CBFA1 were screened for novel polymorphisms using denaturing HPLC and RFLP analysis. In total, 24 polymorphisms were identified. Two SNPs in ER (rs3757323 C>T and rs1801132 G>C) were strongly associated with adult male height and displayed an 8 cm and 9 cm height difference between homozygous genotypes, respectively. The TC haplotype of these SNPs was associated with a 6 cm decrease in height and remarkably, no homozygous carriers of the TC haplotype were identified in tall subjects. No significant associations with height were found for polymorphisms in the FGFR3, CBFA1 or VDR genes. In the epiphyseal growth plate, chondrocyte proliferation, matrix synthesis and chondrocyte hypertrophy are all major contributors to long bone growth. As estrogen plays such a significant role in both growth and final height attainment, another hypothesis of this study was that estrogen exerted its effects in the growth plate by influencing chondrocyte proliferation and mediating the expression of chondrocyte marker genes. The examination of genes regulated by estrogen in chondrocyte-like cells aimed to identify potential regulators of growth plate fusion, which may further elucidate mechanisms involved in the cessation of linear growth. While estrogen did not dramatically alter the proliferation of the SW1353 cell line, gene expression experiments identified several estrogen regulated genes. Sixteen chondrocyte marker genes were examined in response to estrogen concentrations ranging from 10-12 M to 10-8 M over varying time points. Of the genes analysed, IHH, FGFR3, collagen II and collagen X were not readily detectable and PTHrP, GHR, ER, BMP6, SOX9 and TGF1 mRNAs showed no significant response to estrogen treatments. However, the expression of MMP13, CBFA1, BCL-2 and BAX genes were significantly decreased. Interestingly, the majority of estrogen regulated genes in SW1353 cells are expressed in the hypertrophic zone of the growth plate. Estrogen is also known to regulate systemic GH secretion and local GH action. At the molecular level, estrogen functions to inhibit GH action by negatively regulating GH signalling. GH treated SW1353 cells displayed increases in MMP9 mRNA expression (4.4-fold) and MMP13 mRNA expression (64-fold) in SW1353 cells. Increases were also detected in their respective proteins. Treatment with AG490, an established JAK2 inhibitor, blocked the GH mediated stimulation of both MMP9 and MMP13 mRNA expression. The application of estrogen and GH to SW1353 cells attenuated GH-stimulated MMP13 levels, but did not affect MMP9 levels. Investigation of GH signalling revealed that SW1353 cells have high levels of activated JAK2 and exposure to GH, estrogen, AG490 and other signalling inhibitors did not affect JAK2 phosphorylation. Interestingly, AG490 treatment dramatically decreased ERK2 signalling, although GH did stimulate ERK2 phosphorylation above control levels. AG490 also decreased CBFA1 expression, a transcription factor known to activate MMP9 and MMP13. Finally, GH and estrogen treatment increased expression of SOCS3 mRNA, suggesting that SOCS3 may regulate JAK/STAT signalling in SW1353 cells. The modulation of GH-mediated MMP expression by estrogen in SW1353 cells represents a potentially novel mechanism by which estrogen may regulate longitudinal bone growth. However, further investigation is required in order to elucidate the precise mechanisms behind estrogen and GH regulation of MMP13 expression in SW1353 cells. This study has provided additional evidence that estrogen and the ER gene are major factors in the regulation of growth and the determination of adult height. Newly identified polymorphisms in the ER gene not only contribute to our understanding of the genetic basis of human height, but may also be useful in association studies examining other complex traits. This study also identified several estrogen regulated genes and indicated that estrogen modifies the expression of genes which are primarily expressed in the hypertrophic region of the epiphyseal growth plate. Furthermore, synergistic studies incorporating GH and estrogen have revealed the ability of estrogen to attenuate the effects of GH on MMP13 expression, revealing potential pathways by which estrogen may modulate growth plate fusion, longitudinal bone growth and even arthritis.