Identification and molecular mechanisms of the rapid tonicity-induced relocalization of the aquaporin 4 channel

The aquaporin family of integral membrane proteins is comprised of channels that mediate cellular water flow. Aquaporin 4 (AQP4) is highly expressed in the glial cells of the central nervous system and facilitates the osmotically-driven pathological brain swelling associated with stroke and traumatic brain injury. Here we show that AQP4 cell surface expression can be rapidly and reversibly regulated in response to changes of tonicity in primary cortical rat astrocytes and in transfected HEK293 cells. The translocation mechanism involves protein kinase A (PKA) activation, influx of extracellular calcium and activation of calmodulin. We identify five putative PKA phosphorylation sites and use site-directed mutagenesis to show that only phosphorylation at one of these sites, serine- 276, is necessary for the translocation response. We discuss our findings in the context of the identification of new therapeutic approaches to treating brain oedema.

Retinal arteriolar diameter response to flicker light provocation - A useful marker for risk stratification in cardiovascular disease?

Purpose: To to evaluate the benefit of bilinear and linear fitting to characterize the retinal vessel dilation to flicker light stimulation for the purpose of risk stratification in cardiovascular disease. Methods: Forty-five patients (15 with coronary artery disease (CAD), 15 with Diabetes Mellitus (DM) and 15 with CAD and DM) all underwent contact tonometry, digital blood pressure measurement, fundus photography, retinal vessel oximetry, static retinal vessel analysis and continous retinal diameter assessment using the retinal vessel analyser (and flicker light provocation). In addition we measured blood glucose (HbA1c) and keratinin levels in DM patients. Results: With increased severity of cardiovascular disease a more linear reaction profile of retinal arteriolar diameter to flicker light provocation can be observed. Conclusion: Absolute values of vessel dilation provide only limited information on the state of retinal arteriolar dilatory response to flicker light. The approach of bilinear fitting takes into account the immediate response to flicker light provocation as well as the maintained dilatory capacity during prolonged stimulation. Individuals with cardiovascular disease however show a largely linear reaction profile indicating an impairment of the initial rapid dilatory response as usually observed in healty individuals

Rapid tonicity induced re-localisation of endogenous aquaporin 4 in primary rat astrocytes - a therapeutic target for cytotoxic brain oedema?

It is estimated that 69-75 million people worldwide will suffer a traumatic brain injury (TBI) or stroke each year. Brain oedema caused by TBI or following a stroke, together with other disorders of the brain cost Europe €770 billion in 2014. Aquaporins (AQP) are transmembrane water channels involved in many physiologies and are responsible for the maintenance of water homeostasis. They react rapidly to changes in osmolarity by transporting water through their highly selective central pore to maintain tonicity and aid in cell volume regulation. We have previously shown that recombinant AQP1-GFP trafficking occurs in a proteinkinase C-microtubule dependant manner in HEK-293 cells in response to hypotonicity. This trafficking mechanism is also reliant on the presence of calcium and its messenger-binding protein calmodulin and results in increased cell surface expression of AQP1 in a time-scale of ~30 seconds. There is currently very little research into the trafficking mechanisms of endogenous AQPs in primary cells. AQP4 is the most abundantly expressed AQP within the brain, it is localised to the astrocytic end-feet, in contact with the blood vessels at the blood-brain-barrier. In situations where the exquisitely-tuned osmotic balance is disturbed, high water permeability can become detrimental. AQP4-mediated water influx causes rapid brain swelling, resulting in death or long term brain damage. Previous research has shown that AQP4 knock-out mice were protected from the formation of cytotoxic brain oedema in a stroke model, highlighting AQP4 as a key drug target for this pathology. As there are currently no treatments available to restrict the flow of water through AQP4 as all known inhibitors are either cytotoxic or non-specific, controlling the mechanisms involved in the regulation of AQP4 in the brain could provide a therapeutic solution to such diseases. Using cell surface biontinylation of endogenous AQP4 in primary rat astrocytes followed by neutraavidin based ELISA we have shown that AQP4 cell surface localisation increases by 2.7 fold after 5 minutes hypotonic treatment at around 85 mOsm/kg H2O. We have also shown that this rapid relocalisation of AQP4 is regulated by PKA, calmodulin, extra-cellular calcium and actin. In summary we have shown that rapid translocation of endogenous AQP4 occurs in primary rat astrocytes in response to hypotonic stimuli; this mechanism is PKA, calcium, actin and calmodulin dependant. AQP4 has the potential to provide a treatment for the development of brain oedema.

Adaptation in knowledge-based teams: An examination of team composition, leader sensegiving, and cognitive, behavioral, and motivational mechanisms

Organizations are increasingly relying on teams to do the work that has traditionally been done by individuals. At the same time, the environments in which these organizations and teams operate have been becoming progressively more complex and uncertain. These trends raise important questions about the factors that enable teams to adapt. In response to these questions, the current study sought to identify the cognitive, behavioral, and motivational processes and emergent states that promote a team's adaptation to unforeseen changes and novel events, and the team compositional characteristics and leadership processes that enabled these processes and emergent states. Two hundred twenty two undergraduate students from a large Southeastern University composed 74 3-person teams, and participated in a computerized decision-making simulation where each team formed the governing body (i.e., Mayor's cabinet) for two separate simulated cities, and made strategic decisions about city operations. Participants were randomly assigned to one of three roles, distributing expertise and creating mutual interdependence. External team leader sensegiving was manipulated through video recorded communications from an external team leader. Results indicate that team cognitive ability, achievement striving, and psychological collectivism, as well as external team leader sensegiving, were all related to the similarity and quality of team members' strategy-focused mental models (cognitive emergent states), and to the amount of information sharing among members (behavioral process). In turn, teams with more similar and higher quality mental models, and who shared greater levels of information, were found to have a greater ability to react and adapt to environmental changes, and to have greater levels of decision-making effectiveness. Results indicate a pattern of relationships consistent with hypotheses, and have important implications for organizations and knowledge-based teams charged with management responsibilities. Organizations should staff teams with the compositional characteristics that enable the development of similar and high quality mental models, and that promote information sharing among teammates. Similarly, organizations which train and develop leaders to engage in sensegiving behaviors enable team adaptability and promote enhanced decision-making effectiveness when faced with unforeseen changes and novel situations.

Effect of arginine and oscillatory calcium ion (II) on vascular response mediated via nitric oxide signaling in normal and salt sensitive hypertensive rat mesenteric arterioles

Hypertension, a major risk factor in the cardiovascular system, is characterized by an increase in the arterial blood pressure. High dietary sodium is linked to multiple cardiovascular disorders including hypertension. Salt sensitivity, a measure of how the blood pressure responds to salt intake is observed in more than 50% of the hypertension cases. Nitric Oxide (NO), as an endogenous vasodilator serves many important biological roles in the cardiovascular physiology including blood pressure regulation. The physiological concentrations for NO bioactivity are reported to be in 0-500 nM range. Notably, the vascular response to NO is highly regulated within a small concentration spectrum. Hence, much uncertainty surrounds how NO modulates diverse signaling mechanisms to initiate vascular relaxation and alleviate hypertension. Regulating the availability of NO in the vasculature has demonstrated vasoprotective effects. In addition, modulating the NO release by different means has proved to restore endothelial function. In this study we addressed parameters that regulated NO release in the vasculature, in physiology and pathophysiology such as salt sensitive hypertension. We showed that, in the rat mesenteric arterioles, Ca2+ induced rapid relaxation (time constants 20.8 ± 2.2 sec) followed with a much slower constriction after subsequent removal of the stimulus (time constants 104.8 ± 10.0 sec). An interesting observation was that a fourfold increase in the Ca 2+ frequency improved the efficacy of arteriolar relaxation by 61.1%. Our results suggested that, Ca2+ frequency-dependent transient release of NO from the endothelium carried encoded information; which could be translated into different steady state vascular tone. Further, Agmatine, a metabolite of L-arginine, as a ligand, was observed to relax the mesenteric arterioles. These relaxations were NO-dependent and occurred via &agr;-2 receptor activity. The observed potency of agmatine (EC50, 138.7 ± 12.1 ± μM; n=22), was 40 fold higher than L-arginine itself (EC50, 18.3 ± 1.3 mM; n = 5). This suggested us to propose alternative parallel mechanism for L-arginine mediated vascular relaxation via arginine decarboxylase activity. In addition, the biomechanics of rat mesentery is important in regulation of vascular tone. We developed 2D finite element models that described the vascular mechanics of rat mesentery. With an inverse estimation approach, we identified the elasticity parameters characterizing alterations in normotensive and hypertensive Dahl rats. Our efforts were towards guiding current studies that optimized cardiovascular intervention and assisted in the development of new therapeutic strategies. These observations may have significant implications towards alternatives to present methods for NO delivery as a therapeutic target. Our work shall prove to be beneficial in assisting the delivery of NO in the vasculature thus minimizing the cardiovascular risk in handling abnormalities, such as hypertension.

Multifunctional nanoparticles in cancer: In vitro characterization, in vivo distribution, and cellular response after laser-NIR dye-induced heating

A novel biocompatible and biodegradable polymer, termed poly(Glycerol malate co-dodecanedioate) (PGMD), was prepared by thermal condensation method and used for fabrication of nanoparticles (NPs). PGMD NPs were prepared using the single oil emulsion technique and loaded with an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The size of the void PGMD NPs, IR820-PGMD NPs and DOX-IR820-PGMD NPs were approximately 90 nm, 110 nm, and 125 nm respectively. An acidic environment (pH=5.0) induced higher DOX and IR820 release compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of DOX-IR820-PGMD NPs was comparable in MES-SA but was higher in Dx5 cells compared to free DOX plus IR820 (p<0.05). The combination of hyperthermia (HT) and chemotherapy improved cytotoxicity in both cell lines. We also explored the cellular response after rapid, short-term and low thermal dose (laser/Dye/NP) induced-heating, and compared it to slow, long-term and high thermal dose cell incubator heating by investigating the reactive oxygen species (ROS) level, hypoxia-inducible factor-1&agr; (HIF-1&agr;) and vascular endothelial growth factor (VEGF) expression. The cytotoxicity of IR820-PGMD NPs after laser/Dye/NP HT resulted in higher cancer cell killing compared to incubator HT. ROS level, HIF-1&agr; and VEGF expression were elevated under incubator HT, while maintained at the baseline level under the laser/Dye/NP HT. In vivo mouse studies showed that NP formulation significantly improved the plasma half-life of IR820 after tail vein injection. Significant lower IR820 content was observed in kidney in DOX-IR820-PGMD NP treatment as compared to free IR820 treatment in our biodistribution studies (p<0.05). In conclusion, both IR820-PGMD NPs and DOX-IR820-PGMD NPs were successfully developed and used for both imaging and therapeutic purposes. Rapid and short-term laser/Dye/NP HT, with a low thermal dose, did not up-regulate HIF-1&agr; and VEGF expression, whereas slow and long-term incubator HT, with a high thermal dose, can enhance expression of both HIF-1&agr; and VEGF.^

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.

Development of calix[4]arene-functionalized microcantilever array sensing system for the rapid, sensitive and simultaneous detection of metal ions in aqueous solutions

The work described in this thesis was conducted with the aim of: 1) investigating the binding capabilities of calix[4]arene-functionalized microcantilevers towards specific metal ions and 2) developing a new16-microcantilever array sensing system for the rapid, and simultaneous detection of metal ions in fresh water. Part I of this thesis reports on the use of three new bimodal calix[4]arenes (methoxy, ethoxy and crown) as potential host/guest sensing layers for detecting selected ions in dilute aqueous solutions using single microcantilever experimental system. In this work it was shown that modifying the upper rim of the calix[4]arenes with a thioacetate end group allow calix[4]arenes to self-assemble on Au(111) forming complete highly ordered monolayers. It was also found that incubating the microcantilevers coated with 5 nm of Inconel and 40 nm of Au for 1 h in a 1.0 M solution of calix[4]arene produced the highest sensitivity. Methoxy-functionalized microcantilevers showed a definite preference for Ca²⁺ ions over other cationic guests and were able to detect trace concentration as low as 10⁻¹² M in aqueous solutions. Microcantilevers modified with ethoxy calix[4]arene displayed their highest sensitivity towards Sr²⁺ and to a lesser extent Ca²⁺ ions. Crown calix[4]arene-modified microcantilevers were however found to bind selectively towards Cs⁺ ions. In addition, the counter anion was also found to contribute to the deflection. For example methoxy calix[4]arene-modified microcantilever was found to be more sensitive to CaCl₂ over other water-soluble calcium salts such as Ca(NO₃)₂ , CaBr₂ and CaI₂. These findings suggest that the response of calix[4]arene-modified microcantilevers should be attributed to the target ionic species as a whole instead of only considering the specific cation and/or anion. Part II presents the development of a 16-microcantilever sensor setup. The implementation of this system involved the creation of data analysis software that incorporates data from the motorized actuator and a two-axis photosensitive detector to obtain the deflection signal originating from each individual microcantilever in the array. The system was shown to be capable of simultaneous measurements of multiple microcantilevers with different coatings. A functionalization unit was also developed that allows four microcantilevers in the array to be coated with an individual sensing layer one at the time. Because of the variability of the spring constants of different cantilevers within the array, results presented were quoted in units of surface stress unit in order to compare values between the microcantilevers in the array.

Individual Differences in the Neural Response to Personal Goal Pursuit Success and Failure: A Developmental Analysis

Regulatory focus theory (RFT) proposes two different social-cognitive motivational systems for goal pursuit: a promotion system, which is organized around strategic approach behaviors and "making good things happen," and a prevention system, which is organized around strategic avoidance and "keeping bad things from happening." The promotion and prevention systems have been extensively studied in behavioral paradigms, and RFT posits that prolonged perceived failure to make progress in pursuing promotion or prevention goals can lead to ineffective goal pursuit and chronic distress (Higgins, 1997).

Research has begun to focus on uncovering the neural correlates of the promotion and prevention systems in an attempt to differentiate them at the neurobiological level. Preliminary research suggests that the promotion and prevention systems have both distinct and overlapping neural correlates (Eddington, Dolcos, Cabeza, Krishnan, & Strauman, 2007; Strauman et al., 2013). However, little research has examined how individual differences in regulatory focus develop and manifest. The development of individual differences in regulatory focus is particularly salient during adolescence, a crucial topic to explore given the dramatic neurodevelopmental and psychosocial changes that take place during this time, especially with regard to self-regulatory abilities. A number of questions remain unexplored, including the potential for goal-related neural activation to be modulated by (a) perceived proximity to goal attainment, (b) individual differences in regulatory orientation, specifically general beliefs about one's success or failure in attaining the two kinds of goals, (c) age, with a particular focus on adolescence, and (d) homozygosity for the Met allele of the catechol-O-methyltransferase (COMT) Val158Met polymorphism, a naturally occurring genotype which has been shown to impact prefrontal cortex activation patterns associated with goal pursuit behaviors.

This study explored the neural correlates of the promotion and prevention systems through the use of a priming paradigm involving rapid, brief, masked presentation of individually selected promotion and prevention goals to each participant while being scanned. The goals used as priming stimuli varied with regard to whether participants reported that they were close to or far away from achieving them (i.e. a "match" versus a "mismatch" representing perceived success or failure in personal goal pursuit). The study also assessed participants' overall beliefs regarding their relative success or failure in attaining promotion and prevention goals, and all participants were genotyped for the COMT Val158Met polymorphism.

A number of significant findings emerged. Both promotion and prevention priming were associated with activation in regions associated with self-referential cognition, including the left medial prefrontal cortex, cuneus, and lingual gyrus. Promotion and prevention priming were also associated with distinct patterns of neural activation; specifically, left middle temporal gyrus activation was found to be significantly greater during prevention priming. Activation in response to promotion and prevention goals was found to be modulated by self-reports of both perceived proximity to goal achievement and goal orientation. Age also had a significant effect on activation, such that activation in response to goal priming became more robust in the prefrontal cortex and in default mode network regions as a function of increasing age. Finally, COMT genotype also modulated the neural response to goal priming both alone and through interactions with regulatory focus and age. Overall, these findings provide further clarification of the neural underpinnings of the promotion and prevention systems as well as provide information about the role of development and individual differences at the personality and genetic level on activity in these neural systems.

Nutrient availability affects the response of the calcifying chlorophyte Halimeda opuntia (L.) J.V. Lamouroux to low pH

Atmospheric carbon dioxide emissions cause a decrease in the pH and aragonite saturation state of surface ocean water. As a result, calcifying organisms are expected to suffer under future ocean conditions, but their physiological responses may depend on their nutrient status. Because many coral reefs experience high inorganic nutrient loads or seasonal changes in nutrient availability, reef organisms in localized areas will have to cope with elevated carbon dioxide and changes in inorganic nutrients. Halimeda opuntia is a dominant calcifying primary producer on coral reefs that contributes to coral reef accretion. Therefore, we investigated the carbon and nutrient balance of H. opuntia exposed to elevated carbon dioxide and inorganic nutrients. We measured tissue nitrogen, phosphorus and carbon content as well as the activity of enzymes involved in inorganic carbon uptake and nitrogen assimilation (external carbonic anhydrase and nitrate reductase, respectively). Inorganic carbon content was lower in algae exposed to high CO2, but calcification rates were not significantly affected by CO2 or inorganic nutrients. Organic carbon was positively correlated to external carbonic anhydrase activity, while inorganic carbon showed the opposite correlation. Carbon dioxide had a significant effect on tissue nitrogen and organic carbon content, while inorganic nutrients affected tissue phosphorus and N:P ratios. Nitrate reductase activity was highest in algae grown under elevated CO2 and inorganic nutrient conditions and lowest when phosphate was limiting. In general, we found that enzymatic responses were strongly influenced by nutrient availability, indicating its important role in dictating the local responses of the calcifying primary producer H. opuntia to ocean acidification.

Genomic and biochemical investigation of soybean antioxidant metabolism in response to growth at elevated carbon dioxide and elevated ozone

Metabolism in an environment containing of 21% oxygen has a high risk of oxidative damage due to the formation of reactive oxygen species. Therefore, plants have evolved an antioxidant system consisting of metabolites and enzymes that either directly scavenge ROS or recycle the antioxidant metabolites. Ozone is a temporally dynamic molecule that is both naturally occurring as well as an environmental pollutant that is predicted to increase in concentration in the future as anthropogenic precursor emissions rise. It has been hypothesized that any elevation in ozone concentration will cause increased oxidative stress in plants and therefore enhanced subsequent antioxidant metabolism, but evidence for this response is variable. Along with increasing atmospheric ozone concentrations, atmospheric carbon dioxide concentration is also rising and is predicted to continue rising in the future. The effect of elevated carbon dioxide concentrations on antioxidant metabolism varies among different studies in the literature. Therefore, the question of how antioxidant metabolism will be affected in the most realistic future atmosphere, with increased carbon dioxide concentration and increased ozone concentration, has yet to be answered, and is the subject of my thesis research. First, in order to capture as much of the variability in the antioxidant system as possible, I developed a suite of high-throughput quantitative assays for a variety of antioxidant metabolites and enzymes. I optimized these assays for Glycine max (soybean), one of the most important food crops in the world. These assays provide accurate, rapid and high-throughput measures of both the general and specific antioxidant action of plant tissue extracts. Second, I investigated how growth at either elevated carbon dioxide concentration or chronic elevated ozone concentration altered antioxidant metabolism, and the ability of soybean to respond to an acute oxidative stress in a controlled environment study. I found that growth at chronic elevated ozone concentration increased the antioxidant capacity of leaves, but was unchanged or only slightly increased following an acute oxidative stress, suggesting that growth at chronic elevated ozone concentration primed the antioxidant system. Growth at high carbon dioxide concentration decreased the antioxidant capacity of leaves, increased the response of the existing antioxidant enzymes to an acute oxidative stress, but dampened and delayed the transcriptional response, suggesting an entirely different regulation of the antioxidant system. Third, I tested the findings from the controlled environment study in a field setting by investigating the response of the soybean antioxidant system to growth at elevated carbon dioxide concentration, chronic elevated ozone concentration and the combination of elevated carbon dioxide concentration and elevated ozone concentration. In this study, I confirmed that growth at elevated carbon dioxide concentration decreased specific components of antioxidant metabolism in the field. I also verified that increasing ozone concentration is highly correlated with increases in the metabolic and genomic components of antioxidant metabolism, regardless of carbon dioxide concentration environment, but that the response to increasing ozone concentration was dampened at elevated carbon dioxide concentration. In addition, I found evidence suggesting an up regulation of respiratory metabolism at higher ozone concentration, which would supply energy and carbon for detoxification and repair of cellular damage. These results consistently support the conclusion that growth at elevated carbon dioxide concentration decreases antioxidant metabolism while growth at elevated ozone concentration increases antioxidant metabolism.

Calcium response of KCl-excited populations of ventricular myocytes from the European sea bass (Dicentrarchus labrax): a promising approach to integrate cell-to-cell heterogeneity in studying the cellular basis of fish cardiac performance

Climate change challenges the capacity of fishes to thrive in their habitat. However, through phenotypic diversity, they demonstrate remarkable resilience to deteriorating conditions. In fish populations, inter-individual variation in a number of fitness-determining physiological traits, including cardiac performance, is classically observed. Information about the cellular bases of inter-individual variability in cardiac performance is scarce including the possible contribution of excitation-contraction (EC) coupling. This study aimed at providing insight into EC coupling-related Ca2+ response and thermal plasticity in the European sea bass (Dicentrarchus labrax). A cell population approach was used to lay the methodological basis for identifying the cellular determinants of cardiac performance. Fish were acclimated at 12 and 22 A degrees C and changes in intracellular calcium concentration ([Ca2+](i)) following KCl stimulation were measured using Fura-2, at 12 or 22 A degrees C-test. The increase in [Ca2+](i) resulted primarily from extracellular Ca2+ entry but sarcoplasmic reticulum stores were also shown to be involved. As previously reported in sea bass, a modest effect of adrenaline was observed. Moreover, although the response appeared relatively insensitive to an acute temperature change, a difference in Ca2+ response was observed between 12- and 22 A degrees C-acclimated fish. In particular, a greater increase in [Ca2+](i) at a high level of adrenaline was observed in 22 A degrees C-acclimated fish that may be related to an improved efficiency of adrenaline under these conditions. In conclusion, this method allows a rapid screening of cellular characteristics. It represents a promising tool to identify the cellular determinants of inter-individual variability in fishes' capacity for environmental adaptation.

Pasture production and composition response after killing Eucalypt trees with herbicides in central Queensland

Clearing woodlands is practised world-wide to increase crop and livestock production, but can result in unintended consequences including woody regrowth and land degradation. The pasture response of 2 eucalypt woodlands in the central Queensland rangelands to killing trees with herbicides, in the presence or absence of grazing and regular spring burning, was recorded over 7 or 8 years to determine the long-term sustainability of these common practices. Herbage mass and species composition plus tree dynamics were monitored in 2 replicated experiments at each site. For 8 years following herbicide application, killing Eucalyptus populnea F. Muell. (poplar box) trees resulted in a doubling of native pasture herbage mass from that of the pre-existing woodland, with a tree basal area of 8.7 m2 ha-1. Conversely, over 7 years with a similar range of seasons, killing E. melanophloia F. Muell. (silver-leaved ironbark) trees of a similar tree basal area had little impact on herbage mass grown or on pasture composition for the first 4 years before production then increased. Few consistent changes in pasture composition were recorded after killing the trees, although there was an increase in the desirable grasses Dichanthium sericeum (R. Br.) A. Camus (Queensland bluegrass) and Themeda triandra Forssk. (kangaroo grass) when grazed conservatively. Excluding grazing allowed more palatable species of the major grasses to enhance their prominence, but seasonal conditions still had a major influence on their production in particular years. Pasture crown basal area was significantly higher where trees had been killed, especially in the poplar box woodland. Removing tree competition did not have a major effect on pasture composition that was independent of other management impositions or seasons, and it did not result in a rapid increase in herbage mass in both eucalypt communities. The slow pasture response to tree removal at one site indicates that regional models and economic projections relating to tree clearing require community-specific inputs.

Applied evolution: An integrated approach to studying life history traits in response to drug selection

The use of chemical control measures to reduce the impact of parasite and pest species has frequently resulted in the development of resistance. Thus, resistance management has become a key concern in human and veterinary medicine, and in agricultural production. Although it is known that factors such as gene flow between susceptible and resistant populations, drug type, application methods, and costs of resistance can affect the rate of resistance evolution, less is known about the impacts of density-dependent eco-evolutionary processes that could be altered by drug-induced mortality. The overall aim of this thesis was to take an experimental evolution approach to assess how life history traits respond to drug selection, using a free-living dioecious worm (Caenorhabditis remanei) as a model. In Chapter 2, I defined the relationship between C. remanei survival and Ivermectin dose over a range of concentrations, in order to control the intensity of selection used in the selection experiment described in Chapter 4. The dose-response data were also used to appraise curve-fitting methods, using Akaike Information Criterion (AIC) model selection to compare a series of nonlinear models. The type of model fitted to the dose response data had a significant effect on the estimates of LD50 and LD99, suggesting that failure to fit an appropriate model could give misleading estimates of resistance status. In addition, simulated data were used to establish that a potential cost of resistance could be predicted by comparing survival at the upper asymptote of dose-response curves for resistant and susceptible populations, even when differences were as low as 4%. This approach to dose-response modeling ensures that the maximum amount of useful information relating to resistance is gathered in one study. In Chapter 3, I asked how simulations could be used to inform important design choices used in selection experiments. Specifically, I focused on the effects of both within- and between-line variation on estimated power, when detecting small, medium and large effect sizes. Using mixed-effect models on simulated data, I demonstrated that commonly used designs with realistic levels of variation could be underpowered for substantial effect sizes. Thus, use of simulation-based power analysis provides an effective way to avoid under or overpowering a study designs incorporating variation due to random effects. In Chapter 4, I 3 investigated how Ivermectin dosage and changes in population density affect the rate of resistance evolution. I exposed replicate lines of C. remanei to two doses of Ivermectin (high and low) to assess relative survival of lines selected in drug-treated environments compared to untreated controls over 10 generations. Additionally, I maintained lines where mortality was imposed randomly to control for differences in density between drug treatments and to distinguish between the evolutionary consequences of drug treatment versus ecological processes affected by changes in density-dependent feedback. Intriguingly, both drug-selected and random-mortality lines showed an increase in survivorship when challenged with Ivermectin; the magnitude of this increase varied with the intensity of selection and life-history stage. The results suggest that interactions between density-dependent processes and life history may mediate evolved changes in susceptibility to control measures, which could result in misleading conclusions about the evolution of heritable resistance following drug treatment. In Chapter 5, I investigated whether the apparent changes in drug susceptibility found in Chapter 4 were related to evolved changes in life-history of C. remanei populations after selection in drug-treated and random-mortality environments. Rapid passage of lines in the drug-free environment had no effect on the measured life-history traits. In the drug-free environment, adult size and fecundity of drug-selected lines increased compared to the controls but drug selection did not affect lifespan. In the treated environment, drug-selected lines showed increased lifespan and fecundity relative to controls. Adult size of randomly culled lines responded in a similar way to drug-selected lines in the drug-free environment, but no change in fecundity or lifespan was observed in either environment. The results suggest that life histories of nematodes can respond to selection as a result of the application of control measures. Failure to take these responses into account when applying control measures could result in adverse outcomes, such as larger and more fecund parasites, as well as over-estimation of the development of genetically controlled resistance. In conclusion, my thesis shows that there may be a complex relationship between drug selection, density-dependent regulatory processes and life history of populations challenged with control measures. This relationship could have implications for how resistance is monitored and managed if life histories of parasitic species show such eco-evolutionary responses to drug application.

A genetic perspective on rapid evolution in cane toads (Rhinella marina)

The process of biological invasion exposes a species to novel pressures, in terms of both the environments it encounters and the evolutionary consequences of range expansion. Several invaders have been shown to exhibit rapid evolutionary changes in response to those pressures, thus providing robust opportunities to clarify the processes at work during rapid phenotypic transitions. The accelerating pace of invasion of cane toads (Rhinella marina) in tropical Australia during its 80-year history has been well characterized at the phenotypic level, including common-garden experiments that demonstrate heritability of several dispersal-relevant traits. Individuals from the invasion front (and their progeny) show distinctive changes in morphology, physiology and behaviour that, in combination, result in far more rapid dispersal than is true of conspecifics from long-colonized areas. The extensive body of work on cane toad ecology enables us to place into context studies of the genetic basis of these traits. Our analyses of differential gene expression from toads from both ends of this invasion-history transect reveal substantial upregulation of many genes, notably those involved in metabolism and cellular repair. Clearly, then, the dramatically rapid phenotypic evolution of cane toads in Australia has been accompanied by substantial shifts in gene expression, suggesting that this system is well suited to investigating the genetic underpinnings of invasiveness.