Effect of creatine supplementation on housekeeping genes in human skeletal muscle using real-time RT-PCR

The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (C_T) values were established for ß-actin, ß2-microglobulin (ß₂M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between C_T values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw C_T values and the linear value of 2^-C_T, respectively. Interassay variability was 2.3% for raw C_T values and 34% for the linear value of 2^-C_T. We also examined the expression of various housekeeping genes in human skeletal muscle at days 0, 1, and 5 following oral supplementation with either creatine or a placebo employing a double-blind crossover study design. Treatments were separated by a 5-wk washout period. Immediately following each muscle sampling, subjects performed two 30-s all-out bouts on a cycle ergometer. Creatine supplementation increased (P < 0.05) muscle total creatine content above placebo levels; however, there were no changes (P > 0.05) in C_T values across the supplementation periods for any of the genes. Nevertheless, 95% confidence intervals showed that GAPDH was variable, whereas ß-actin, ß₂M, and CYC were the least varying genes. Normalization of the data to these housekeeping genes revealed variable behavior for ß₂M with more stable expressions for both ß-actin and CYC. We conclude that, using real-time RT-PCR, ß-actin or CYC may be used as housekeeping genes to study gene expression in human muscle in experiments employing short-term creatine supplementation combined with high-intensity exercise.

Periodic variability in cetacean strandings: links to large-scale climate events

Cetacean strandings elicit much community and scientific interest, but few quantitative analyses have successfully identified environmental correlates to these phenomena. Data spanning 1920–2002, involving a total of 639 stranding events and 39 taxa groups from southeast Australia, were found to demonstrate a clear 11–13- year periodicity in the number of events through time. These data positively correlated with the regional persistence of both zonal (westerly) and meridional (southerly) winds, reflecting general long-term and large-scale shifts in sea-level pressure gradients. Periods of persistent zonal and meridional winds result in colder and presumably nutrient-rich waters being driven closer to southern Australia, resulting in increased biological activity in the water column during the spring months. These observations suggest that large-scale climatic events provide a powerful distal influence on the propensity for whales to strand in this region. These patterns provide a powerful quantitative framework for testing hypotheses regarding environmental links to strandings and provide managers with a potential predictive tool to prepare for years of peak stranding activity.

Distribution of an alien aquatic snail in relation to flow variability, human activities and water quality

1. Disturbance and anthropogenic land use changes are usually considered to be key factors facilitating biological invasions. However, specific comparisons of invasion success between sites affected to different degrees by these factors are rare.

2. In this study we related the large-scale distribution of the invading New Zealand mud snail (Potamopyrgus antipodarum) in southern Victorian streams, Australia, to anthropogenic land use, flow variability, water quality and distance from the site to the sea along the stream channel.

3. The presence of P. antipodarum was positively related to an index of flow-driven disturbance, the coefficient of variability of mean daily flows for the year prior to the study.

4. Furthermore, we found that the invader was more likely to occur at sites with multiple land uses in the catchment, in the forms of grazing, forestry and anthropogenic developments (e.g. towns and dams), compared with sites with low-impact activities in the catchment. However, this relationship was confounded by a higher likelihood of finding this snail in lowland sites close to the sea.

5. We conclude that P. antipodarum could potentially be found worldwide at sites with similar ecological characteristics. We hypothesise that its success as an invader may be related to an ability to quickly re-colonise denuded areas and that population abundances may respond to increased food resources. Disturbances could facilitate this invader by creating spaces for colonisation (e.g. a possible consequence of floods) or changing resource levels (e.g. increased nutrient levels in streams with intense human land use in their catchments).

Exposure estimation, uncertainty and variability in occupational hygiene retrospective assessment

This thesis reports on a quantitative exposure assessment and on an analysis of the attributes of the data used in the estimations, in particular distinguishing between its uncertainty and variability. A retrospective assessment of exposure to benzene was carried out for a case control study of leukaemia in the Australian petroleum industry. The study used the mean of personal task-based measurements (Base Estimates) in a deterministic algorithm and applied factors to model back to places, times etc for which no exposure measurements were available. Mean daily exposures were estimated, on an individual subject basis, by summing the task-based exposures. These mean exposures were multiplied by the years spent on each job to provide exposure estimates in ppm-years. These were summed to provide a Cumulative Estimate for each subject. Validation was completed for the model and key inputs. Exposures were low, most jobs were below TWA of 5 ppm benzene. Exposures in terminals were generally higher than at refineries. Cumulative Estimates ranged from 0.005 to 50.9 ppm-years, with 84 percent less than 10 ppm-years. Exposure probability distributions were developed for tanker drivers using Monte Carlo simulation of the exposure estimation algorithm. The outcome was a lognormal distribution of exposure for each driver. These provide the basis for alternative risk assessment metrics e.g. the frequency of short but intense exposures which provided only a minimal contribution to the long-term average exposure but may increase risk of leukaemia. The effect of different inputs to the model were examined and their significance assessed using Monte Carlo simulation. The Base Estimates were the most important determinant of exposure in the model. The sources of variability in the measured data were examined, including the effect of having censored data and the between and within-worker variability. The sources of uncertainty in the exposure estimates were analysed and consequential improvements in exposure assessment identified. Monte Carlo sampling was also used to examine the uncertainties and variability associated with the tanker drivers' exposure assessment, to derive an estimate of the range and to put confidence intervals on the daily mean exposures. The identified uncertainty was less than the variability associated with the estimates. The traditional approach to exposure estimation typically derives only point estimates of mean exposure. The approach developed here allows a range of exposure estimates to be made and provides a more flexible and improved basis for risk assessment.

Towards powerful experimental and statistical approaches to study intraindividual variability in labile traits

There is a long-standing interest in behavioural ecology, exploring the causes and correlates of consistent individual differences in mean behavioural traits ('personality') and the response to the environment ('plasticity'). Recently, it has been observed that individuals also consistently differ in their residual intraindividual variability (rIIV). This variation will probably have broad biological and methodological implications to the study of trait variation in labile traits, such as behaviour and physiology, though we currently need studies to quantify variation in rIIV, using more standardized and powerful methodology. Focusing on activity rates in guppies (Poecilia reticulata), we provide a model example, from sampling design to data analysis, in how to quantify rIIV in labile traits. Building on the doubly hierarchical generalized linear model recently used to quantify individual differences in rIIV, we extend the model to evaluate the covariance between individual mean values and their rIIV. After accounting for time-related change in behaviour, our guppies substantially differed in rIIV, and it was the active individuals that tended to be more consistent (lower rIIV). We provide annotated data analysis code to implement these complex models, and discuss how to further generalize the model to evaluate covariances with other aspects of phenotypic variation.