In vitro models for investigating keratinocyte responses to ultraviolet B radiation

This thesis describes the use of 2- and 3-dimensional cell-based models for studying how skin cells respond to ultraviolet radiation. These methods were used to investigate skin damage and repair after exposure to radiation in the context of skin cancer development. Interactions between different skin cell types were demonstrated as being significant in protecting against ultraviolet radiation-induced skin damage. This has important implications in understanding how skin cancers occur, as well as in the development of new strategies to prevent and treat them.

Vibrational spectroscopy of the borate mineral olshanskyite Ca3[B(OH)4]4(OH)2

The mineral olshanskyite is one of many calcium borate minerals which has never been studied using vibrational spectroscopy. The mineral is unstable and decomposes upon exposure to an electron beam. This makes the elemental analysis using EDX techniques difficult. Both the Raman and infrared spectra show complexity due to the complexity of the structure. Intense Raman bands are found at 989, 1,003, 1,025 and 1,069 cm-1 with a shoulder at 961 cm-1 and are assigned to trigonal borate units. The Raman bands at 1,141, 1,206 and 1,365 cm-1 are assigned to OH in-plane bending of BOH units. A series of Raman bands are observed in the 2,900–3,621cm-1 spectral range and are assigned to the stretching vibrations of OH and water. This complexity is also reflected in the infrared spectra. Vibrational spectroscopy enables aspects of the structure of olshanskyite to be elucidated.

Genetic diversity of cultured and wild populations of the giant freshwater prawn Macrobrachium rosenbergii (de Man, 1879) based on microsatellite analysis

Freshwater prawn (Macrobrachium rosenbergii) culture in the Western Hemisphere is primarily, if not entirely, derived from 36 individual prawns originally introduced to Hawaii from Malaysia in 1965 and 1966. Little information is available regarding genetic variation within and among cultured prawn stocks worldwide. The goal of the current study was to characterize genetic diversity in various prawn populations with emphasis on those cultured in North America. Five microsatellite loci were screened to estimate genetic diversity in two wild (Myanmar and India-wild) and seven cultured (Hawaii-1, Hawaii-2, India-cultured, Israel, Kentucky, Mississippi and Texas) populations. Average allelic richness ranged from 3.96 (Israel) to 20.45 (Myanmar). Average expected heterozygosity ranged from 0.580 (Israel) to 0.935 (Myanmar). Many of the cultured populations exhibited reduced genetic diversity when compared with the Myanmar and the India-cultured populations. Significant deficiency in heterozygotes was detected in the India-cultured, Mississippi and Kentucky populations (overall Fis estimated of 0.053, 0.067 and 0.108 respectively) reflecting moderate levels of inbreeding. Overall estimate of fixation index (Fst = 0.1569) revealed moderately high levels of differentiation among the populations. Outcome of this study provide a baseline assessment of genetic diversity in some available strains that will be useful for the development of breeding programmes.

Climate variability over the last 35,000 years recorded in marine and terrestrial archives in the Australian region : an OZ-INTIMATE compilation

The Australian region spans some 60° of latitude and 50° of longitude and displays considerable regional climate variability both today and during the Late Quaternary. A synthesis of marine and terrestrial climate records, combining findings from the Southern Ocean, temperate, tropical and arid zones, identifies a complex response of climate proxies to a background of changing boundary conditions over the last 35,000 years. Climate drivers include the seasonal timing of insolation, greenhouse gas content of the atmosphere, sea level rise and ocean and atmospheric circulation changes. Our compilation finds few climatic events that could be used to construct a climate event stratigraphy for the entire region, limiting the usefulness of this approach. Instead we have taken a spatial approach, looking to discern the patterns of change across the continent. The data identify the clearest and most synchronous climatic response at the time of the Last Glacial Maximum (LGM) (21 ± 3 ka), with unambiguous cooling recorded in the ocean, and evidence of glaciation in the highlands of tropical New Guinea, southeast Australia and Tasmania. Many terrestrial records suggest drier conditions, but with the timing of inferred snowmelt, and changes to the rainfall/runoff relationships, driving higher river discharge at the LGM. In contrast, the deglaciation is a time of considerable south-east to north-west variation across the region. Warming was underway in all regions by 17 ka. Post-glacial sea level rise and its associated regional impacts have played an important role in determining the magnitude and timing of climate response in the north-west of the continent in contrast to the southern latitudes. No evidence for cooling during the Younger Dryas chronozone is evident in the region, but the Antarctic cold reversal clearly occurs south of Australia. The Holocene period is a time of considerable climate variability associated with an intense monsoon in the tropics early in the Holocene, giving way to a weakened monsoon and an increasingly El Niño-dominated ENSO to the present. The influence of ENSO is evident throughout the southeast of Australia, but not the southwest. This climate history provides a template from which to assess the regionality of climate events across Australia and make comparisons beyond our region. The data identify the clearest and most synchronous climatic response at the time of the Last Glacial Maximum (LGM) (21 ± 3 ka), with unambiguous cooling recorded in the ocean, and evidence of glaciation in the highlands of tropical New Guinea, southeast Australia and Tasmania. Many terrestrial records suggest drier conditions, but with the timing of inferred snowmelt, and changes to the rainfall/runoff relationships, driving higher river discharge at the LGM. In contrast, the deglaciation is a time of considerable south-east to north-west variation across the region. Warming was underway in all regions by 17 ka. Post-glacial sea level rise and its associated regional impacts have played an important role in determining the magnitude and timing of climate response in the north-west of the continent in contrast to the southern latitudes. No evidence for cooling during the Younger Dryas chronozone is evident in the region, but the Antarctic cold reversal clearly occurs south of Australia. The Holocene period is a time of considerable climate variability associated with an intense monsoon in the tropics early in the Holocene, giving way to a weakened monsoon and an increasingly El Niño-dominated ENSO to the present. The influence of ENSO is evident throughout the southeast of Australia, but not the southwest. This climate history provides a template from which to assess the regionality of climate events across Australia and make comparisons beyond our region.

A class B switch-mode assisted linear amplifier

A switch-mode assisted linear amplifier (SMALA) combining a linear (Class B) and a switch-mode (Class D) amplifier is presented. The usual single hysteretic controlled half-bridge current dumping stage is replaced by two parallel buck converter stages, in a parallel voltage controlled topology. These operate independently: one buck converter sources current to assist the upper Class B output device, and a complementary converter sinks current to assist the lower device. This topology lends itself to a novel control approach of a dead-band at low power levels where neither class D amplifier assists, allowing the class B amplifier to supply the load without interference, ensuring high fidelity. A 20 W implementation demonstrates 85% efficiency, with distortion below 0.08% measured across the full audio bandwidth at 15 W. The class D amplifier begins assisting at 2 W, and below this value, the distortion was below 0.03%. Complete circuitry is given, showing the simplicity of the additional class D amplifier and its corresponding control circuitry.

Relative abundance of full-length and truncated FOXP1 isoforms is associated with differential NFκB activity in Follicular Lymphoma

FOXP1 is a transcriptional repressor that has been proposed to repress the expression of some NFκB-responsive genes. Furthermore, truncated forms of FOXP1 have been associated with a subtype of Diffuse Large B-cell Lymphoma characterised by constitutive NFκB activity, indicating that they may inhibit this repression. We have shown that FL tumors have increased relative abundance of truncated FOXP1 isoforms and this is associated with increased expression of NFκB-associated genes. Our results provide strong evidence that relative FOXP1 isoform abundance is associated with NFκB activity in FL, and could potentially be used as a marker for this gene signature.

Pyrolysing poultry litter reduces N2O and CO2 fluxes

Application of poultry litter (PL) to soil can lead to substantial nitrous oxide (N2O) emissions due to the co-application of labile carbon (C) and nitrogen (N). Slow pyrolysis of PL to produce biochar may mitigate N2O emissions from this source, whilst still providing agronomic benefits. In a corn crop on ferrosol with similarly matched available N inputs of ca. 116 kg N/ha, PL-biochar plus urea emitted significantly less N2O (1.5 kg N2O-N/ha) compared to raw PL at 4.9 kg N2O-N/ha. Urea amendment without the PL-biochar emitted 1.2 kg N2O-N/ha, and the PL-biochar alone emitted only 0.35 kg N2O-N/ha. Both PL and PL-biochar resulted in similar corn yields and total N uptake which was significantly greater than for urea alone. Using stable isotope methodology, the majority (~ 80%) of N2O emissions were shown to be from non-urea sources. Amendment with raw PL significantly increased C mineralisation and the quantity of permanganate oxidisable organic C. The low molar H/C (0.49) and O/C (0.16) ratios of the PL-biochar suggest its higher stability in soil than raw PL. The PL-biochar also had higher P and K fertiliser value than raw PL. This study suggests that PL-biochar is a valuable soil amendment with the potential to significantly reduce emissions of soil greenhouse gases compared to the raw product. Contrary to other studies, PL-biochar incorporated to 100 mm did not reduce N2O emissions from surface applied urea, which suggests that further field evaluation of biochar impacts, and methods of application of both biochar and fertiliser, are needed.

Genetic polymorphisms in DPF3 associated with risk of breast cancer and lymph node metastases

Background Several studies have identified rare genetic variations responsible for many cases of familial breast cancer but their contribution to total breast cancer incidence is relatively small. More common genetic variations with low penetrance have been postulated to account for a higher proportion of the population risk of breast cancer. Methods and Results In an effort to identify genes that influence non-familial breast cancer risk, we tested over 25,000 single nucleotide polymorphisms (SNPs) located within approximately 14,000 genes in a large-scale case-control study in 254 German women with breast cancer and 268 age-matched women without malignant disease. We identified a marker on chromosome 14q24.3-q31.1 that was marginally associated with breast cancer status (OR = 1.5, P = 0.07). Genotypes for this SNP were also significantly associated with indicators of breast cancer severity, including presence of lymph node metastases ( P = 0.006) and earlier age of onset ( P = 0.01). The association with breast cancer status was replicated in two independent samples (OR = 1.35, P = 0.05). High-density association fine mapping showed that the association spanned about 80 kb of the zinc-finger gene DPF3 (also known as CERD4 ). One SNP in intron 1 was found to be more strongly associated with breast cancer status in all three sample collections (OR = 1.6, P = 0.003) as well as with increased lymph node metastases ( P = 0.01) and tumor size ( P = 0.01). Conclusion Polymorphisms in the 5' region of DPF3 were associated with increased risk of breast cancer development, lymph node metastases, age of onset, and tumor size in women of European ancestry. This large-scale association study suggests that genetic variation in DPF3 contributes to breast cancer susceptibility and severity.

A molecular genetic approach for forensic animal species identification

This study investigated potential markers within chromosomal, mitochondrial DNA (mtDNA) and ribosomal RNA (rRNA) with the aim of developing a DNA based method to allow differentiation between animal species. Such discrimination tests may have important applications in the forensic science, agriculture, quarantine and customs fields. DNA samples from five different animal individuals within the same species for 10 species of animal (including human) were analysed. DNA extraction and quantitation followed by PCR amplification and GeneScan visualisation formed the basis of the experimental analysis. Five gene markers from three different types of genes were investigated. These included genomic markers for the β-actin and TP53 tumor suppressor gene. Mitochondrial DNA markers, designed by Bataille et al. [Forensic Sci. Int. 99 (1999) 165], examined the Cytochrome b gene and Hypervariable Displacement Loop (D-Loop) region. Finally, a ribosomal RNA marker for the 28S rRNA gene optimised by Naito et al. [J. Forensic Sci. 37 (1992) 396] was used as a possible marker for speciation. Results showed a difference of only several base pairs between all species for the β-actin and 28S markers, with the exception of Sus scrofa (pig) β-actin fragment length, which produced a significantly smaller fragment. Multiplexing of Cytochrome b and D-Loop markers gave limited species information, although positive discrimination of human DNA was evident. The most specific and discriminatory results were shown using the TP53 gene since this marker produced greatest fragment size differences between animal species studied. Sample differentiation for all species was possible following TP53 amplification, suggesting that this gene could be used as a potential animal species identifier.

The molecular structure of the borate mineral inderite Mg(H4B3O7)(OH)⋅5H2O – A vibrational spectroscopic study

We have undertaken a study of the mineral inderite Mg(H4B3O7)(OH)⋅5H2O a hydrated hydroxy borate mineral of magnesium using scanning electron microscopy, thermogravimetry and vibrational spectroscopic techniques. The structure consists of [B3O3(OH)5]2-[B3O3(OH)5]2- soroborate groups and Mg(OH)2(H2O)4 octahedra interconnected into discrete molecules by the sharing of two OH groups. Thermogravimetry shows a mass loss of 47.2% at 137.5 °C, proving the mineral is thermally unstable. Raman bands at 954, 1047 and 1116 cm−1 are assigned to the trigonal symmetric stretching mode. The two bands at 880 and 916 cm−1 are attributed to the symmetric stretching mode of the tetrahedral boron. Both the Raman and infrared spectra of inderite show complexity. Raman bands are observed at 3052, 3233, 3330, 3392 attributed to water stretching vibrations and 3459 cm−1 with sharper bands at 3459, 3530 and 3562 cm−1 assigned to OH stretching vibrations. Vibrational spectroscopy is used to assess the molecular structure of inderite.

Reproducibility and clinical utility of tendon palpation to detect patellar tendinopathy in young basketball players

Background—Palpation is an important clinical test for jumper's knee. Objectives—To (a) test the reproducibility of palpation tenderness, (b) evaluate the sensitivity and specificity of palpation in subjects with clinical symptoms of jumper's knee, and (c) determine whether tenderness to palpation may serve as a useful screening test for patellar tendinopathy. The yardstick for diagnosis of patellar tendinopathy was ultrasonographic abnormality. Methods—In 326 junior symptomatic and asymptomatic athletes' tendons, palpation was performed by a single examiner before ultrasonographic examination by a certified ultrasound radiologist. In 58 tendons, palpation was performed twice to test reliability. Tenderness to palpation was scored on a scale from 0 to 3 where 0 represented no pain, and 1, 2, and 3 represented mild, moderate, and severe tenderness respectively. Results—Patellar tendon palpation was a reliable examination for a single examiner (Pearson r = 0.82). In symptomatic tendons, the positive predictive value of palpation was 68%. As a screening examination in asymptomatic subjects, the positive predictive value of tendon palpation was 36–38%. Moderate and severe palpation tenderness were better predictors of ultrasonographic tendon pathology than absent or mild tenderness (p<0.001). Tender and symptomatic tendons were more likely to have ultrasound abnormality than tenderness alone (p<0.01). Conclusions—In this age group, palpation is a reliable test but it is not cost effective in detecting patellar tendinopathy in a preparticipation examination. In symptomatic tendons, palpation is a moderately sensitive but not specific test. Mild tenderness in the patellar tendons in asymptomatic jumping athletes should be considered normal.

The B subunit of coagulation factor XIII is linked to renin and the Duffy blood group to α-spectrin on human chromosome 1

Family linkage studies were used to detect two linkage relationships on human chromosome 1. The B subunit of coagulation factor XIII showed significant linkage to renin with a maximum lod score of 5.071 at a distance of 10 cM. Significant linkage was also shown between the Duffy blood group and α-spectrin with linkage results giving a combined lod score of 3.194 at 5 cM.

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental E. faecalis and E. faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E and esp were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.79% of E. faecalis and 70.37 % of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linozolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.18% of E. faecalis and 70.3 % of E. faecium) compared to water isolates (only 5.66 % E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.

A linkage and cross-sectional study of hypertension and obesity using a poly(A) Alu-repeat polymorphism at the glucagon receptor gene locus (17q25)

1. Previous glucagon receptor gene (GCGR) studies have shown a Gly40Ser mutation to be more prevalent in essential hypertension and to affect glucagon binding affinity to its receptor. An Alu-repeat poly(A) polymorphism colocalized to GCGR was used in the present study to test for association and linkage in hypertension as well as association in obesity development. 2. Using a cross-sectional approach, 85 hypertensives and 95 normotensives were genotyped using polymerase chain reaction primers flanking the Alu-repeat. Both hypertensive and normotensive populations were subdivided into lean and obese categories based on body mass index (BMI) to determine involvement of this variant in obesity. For the linkage study, 89 Australian Caucasian hypertension affected sibships (174 sibpairs) were genotyped and the results were analysed using GENE-HUNTER, Mapmaker Sibs, ERPA and SPLINK (all freely available from http://linlkage.rockefeller. edu/soft/list.html). 3. Cross-sectional results for both hypertension and obesity were analysed using Chi-squared and Monte Carlo analyses. Results did not show an association of this variant with either hypertension (χ2 = 6.9, P = 0.14; Monte Carlo χ2 = 7.0, P = 0.11; n = 5000) or obesity (χ2 = 3.3, P = 0.35; Monte Carlo χ2 = 3.26, P = 0.34; n = 5000). In addition, results from the linkage study using hypertensive sib-pairs did not indicate linkage of the poly(A) repent with hypertension. Hence, results did not indicate a role far the Alu-repeat in either hypertension or obesity. However, as the heterozygosity of this poly(A) repeat is low (35%), a larger number of hypertensive sib-pairs may be required to draw definitive conclusions.