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Abstract Winter energy management in small passerines living year-round in boreal or alpine areas presumably results in strong selective pressure since they need to find food, at a time when natural resources diminish and become less available, and energy requirements increase dramatically. In this thesis energy management during the non-breeding season was studied in three species of tits (Parus spp.), from three different populations: Coll de Pal (Spanish Pyrenees), Lund (Southern Sweden) and Oulu (Northern Finland). Energy management strategies vary significantly between species and among populations and individuals of the same species. Such differences may depend on several environmental factors, food predictability and individual characteristics. Birds from the studied populations appear to react to energetic challenges on a short-term basis and in a highly flexible way. The coal tit (Parus ater) in Coll de Pal and the willow tit (Parus montanus) in Oulu, both hoarding species, relied mostly on short-term management of energy for winter survival. Social and residence status appeared to be the most important factors in determining the level of energy reserves, underlining the importance of food predictability for energy management in wintering tits. Further studies were carried out on two distinct populations of great tit (Parus major) exposed to different winter hardiness. Birds from both populations increased their resting metabolic rate (MR) with experimentally decreasing ambient temperatures. Birds from Oulu maintained higher expenditures than birds from Lund in all cases, but also experienced higher energetic cost of thermoregulation at the lowest temperatures. The differences probably did not arise from a differential insulation capacity between populations, despite the differences in plumage structure found, but from a differential metabolic acclimatization. Birds from Lund probably became hypothermic at the lowest temperatures, which may have exceeded the levels they were acclimatized for. The observed differences in basal MR in laboratory conditions were consistent in wild birds throughout the non-breeding season. Birds from both populations experienced similar patterns of variation in basal MR, with expenditures increasing with mass but decreasing with day length, size and age. Great tits modulate their energy expenditure in a flexible way as a means for surviving the non-breeding season. Further, despite such flexibility, populations appear to be locally adapted for such metabolic acclimatization. These results may have important implications on their life-history and distribution. Winter acclimatization appears to be a complex set of entangled strategies that are based on a metabolic adjustment to cope with changing energy requirements. Other mechanisms that apparently play a secondary role, for example the long term management of reserves through fattening or hoarding, or conserving heat through hypothermia and by developing a better insulative plumage, are certainly important emergency strategies that in natural conditions may explain how some populations can endure winter conditions.

In recent years, the subsets of mammalian CD4+ T cells and their repertoire of effector cytokines has expanded beyond the original Th1/Th2 paradigm, to include natural (n) and inducible (i) regulatory T cells (Treg), Th17, Th9, Th22 and follicular T helper (Tfh) cells. Whilst Th1, Th2 and nTreg immune responses have been described in the chicken, the existence of other Th cell subsets is yet to be determined. To investigate Th17 immune responses in the chicken, the mammalian components of these responses currently unannotated in the chicken genome, IL-23 p19 and the IL-23R, were identified and cDNAs cloned. A chicken IL-23 flexiconstruct, containing IL-23 p19 and p40 joined by a linker, was designed. Recombinant chicken IL-23 protein (rchIL-23) was expressed and purified. Bioactivity of rchIL-23 was demonstrated by increased mRNA expression of chIL- 17F and chIL-22 in rchIL-23-stimulated splenocytes. Monoclonal antibodies which identify chIL-12/chIL-23 p40 also recognised purified rchIL-23. Further, chIL-23 p19 mRNA levels were measured and detected in a wide range of tissues but was not up-regulated in stimulated splenocytes, thymocytes or bursal cells. Messenger RNA (mRNA) expression levels of Th17 cytokines (chIL-17A, chIL-17F, chIL-21, chIL-22 and chIL-23) were measured in a chicken tissue panel, in stimulated splenocytes, thymocytes and bursal cells, as well as during infections previously described as initiating typical Th1 or Th2 adaptive immune responses in the chicken. Chicken IL-17A mRNA expression levels were up-regulated in susceptible chickens during infection with Marek’s disease virus (a disease which typically drives a Th1 immune response), but were down-regulated in resistant birds. Chicken CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS) and recombinant Th17-associated cytokines used to attempt to drive the cells towards a Th17 phenotype, as measured by expression of mRNA for chIL-17A and chIL-23R. The sorted chicken CD4+ cells failed to proliferate or respond to Th17 cytokine stimulation. ChIL-23R was also correctly identified and cloned as cDNA, and its mRNA expression measured in a panel of unstimulated and stimulated tissues and cells. The chIL-23R mRNA levels were detected in a wide range of tissues as well as stimulated splenocytes, thymocytes and bursal cells. Future work would seek to positively identify Th17 cells in the chicken and determine the role of Th17 immune responses against avian diseases.

Climatic variation is an important driver of avian life history and population dynamics. Climate change models predict increased variability for many regions and to predict the effects on species, we need to examine how their life history characteristics influence their response to climate. I studied how environmental conditions influenced the ecology of white-tailed (Lagopus leucura) and rock ptarmigan (L. mutus) in tundra habitats of the southern Yukon Territory. Although sympatric in the study area, breeding territories were generally segregated, with white-tailed ptarmigan selecting steep, rocky slopes at higher elevations and rock ptarmigan preferring lower elevation sedge meadows. For both species, cold spring temperatures delayed the onset of breeding, resulting in smaller clutch sizes and fewer hatched young per female. However, delayed breeding led to a stronger reduction in these rates for rock ptarmigan, suggesting a lower resilience to extend reproductive effort in colder years. White-tailed ptarmigan were also more likely to re-nest following failure and had higher daily nest survival, both of which contributed to greater annual productivity compared to rock ptarmigan. Annual adult survival showed the opposite pattern to productivity as rock ptarmigan survival was 24 percent higher than white-tailed ptarmigan. This finding suggested a reproduction-survival trade-off exists for the two species, which may be driven by differing susceptibility to environmental factors in the region. Life history theory predicts that if the likelihood of future breeding opportunities is low, individuals should increase current reproductive effort, which may explain why white-tailed ptarmigan have longer breeding seasons and higher reproductive effort under unfavourable climatic conditions. Population models showed that growth rates (λ)were approximately stable for rock ptarmigan (λ=1.01), but declining for white-tailed ptarmigan (λ=0.96). Simulations showed that warmer spring temperatures over the next few decades would elevate λ by ~0.05 for both species, but the extent of increase in λ may be reduced with more variable spring conditions. Population growth will also depend on how changing winter conditions influence survival for each species. Model simulations suggest that if juvenile and adult survival are positively correlated, rock ptarmigan would be more resilient to severe years that simultaneously depress reproduction and survival. / Forestry, Faculty of / Graduate

There is a growing interest in assessing the neurotoxic potential and endocrine disrupting properties of perfluoroalkyl compounds (PFCs). Several studies have reported in vitro and in vivo effects related to neuronal development, neural cell differentiation, pre- and post- natal development and behaviour. PFC exposure altered hormone levels (e.g. thyroid hormone, estrogen, and testosterone) and the expression of hormone-responsive genes in mammalian and aquatic species. Hormone-mediated events are critical in central nervous system development and function, especially those controlled by thyroid hormones (THs). The studies presented in this thesis are the first to assess the effects of PFCs on primary cultures of neuronal cells in two avian species; the domestic chicken (Gallus domesticus) and herring gull (Larus argentatus). The following TH-responsive genes were examined using real-time RT-PCR: type II iodothyronine 5’-deiodinase (D2), D3, transthyretin (TTR), neurogranin (RC3), octamer motif binding factor (Oct-1), and myelin basic protein (MBP). Several PFCs were shown to alter mRNA expression levels of genes associated with the TH pathway in avian neuronal cells. It was determined that short-chained PFCs (<8 carbons) altered the expression of TH-responsive genes to a greater extent than long-chained PFCs (≥8 carbons). Although several significant changes in mRNA expression were observed in TH-responsive genes following PFC exposure in chicken embryonic neuronal (CEN) cells (Chapter 2), there were fewer changes in herring gull embryonic neuronal (HGEN) cells (Chapter 3). The mRNA levels of D2, D3, TTR, and RC3 were altered following treatment with several short-chained PFCs in CEN cells. Oct-1 and RC3 expression were induced following treatment with several short-chained PFCs in HGEN cells. These studies are the first to report that PFC exposure alters mRNA expression in primary cultures of avian neuronal cells and provide insight into the possible mechanisms of action of PFCs in the avian brain.

Perfluoroalkyl acids (PFAAs) are a family of synthetic substances used in a wide variety of consumer and industrial applications, including non-stick and stain-resistant products. PFAAs, specifically perfluorinated sulfonates and carboxylates, are chemically stable and virtually non-biodegradable in the environment. In recent years, PFAAs have been detected in tissues and blood of humans and wildlife. Furthermore, PFAAs have a tendency to bioaccumulate and biomagnify in biota. Perfluorooctane sulfonate and perfluorooctanoate are known to be toxic when animals are exposed to environmentally-relevant levels, but scientists and regulators are challenged with determining and predicting their modes of action. There is some evidence to suggest that PFAAs can impact the thyroid hormone (TH) pathway and neurodevelopment. The studies presented in this thesis investigated the developmental effects and potential modes of action of newer PFAAs that are being introduced into the global market place. Egg injection experiments were performed in domestic chicken (Gallus gallus domesticus) embryos to assess the in ovo toxicity of perfluorohexane sulfonate (PFHxS) and perfluorohexanoate (PFHxA) during development. Real-time RT-PCR was used to measure the transcription of candidate genes in the liver and cerebral hemisphere of day 21-22 embryos. Candidate genes were selected based on their responsiveness to PFAA exposure in an in vitro screening assay conducted previously. In ovo exposure to PFHxS decreased embryo pipping success and overall growth at 38,000 ng/g; several orders of magnitude higher than concentrations reported in wild bird eggs. The expression of TH-responsive genes, including type II and III 5'-deiodinase, neurogranin, and octamer motif binding factor 1, were induced. In addition, PFHxS diminished free thyroxine (T4) levels in plasma. PFHxA had no affect on pipping success, gene expression or T4 levels in chicken embryos at the doses assessed. The transcriptional profiles in the cerebral hemisphere of chicken embryos exposed to 890 and 38,000 ng/g PFHxS were compared to a solvent control using microarray technology. The expression of 78 different genes were significantly altered (fold change > 1.5, p < 0.001) by PFHxS. Functional analysis showed that PFHxS affected genes involved in tissue development and morphology and cellular assembly and organization. Pathway and interactome analysis suggested that gene expression may be affected through integrin receptors and signaling pathways via TH–dependent and –independent modes of action. It is expected that the findings presented in this thesis will be of general relevance and importance to regulatory agencies and of interest to research scientists and risk assessors.

Analytical models of avian flight have previously been used to predict mechanical and metabolic power consumption during cruise. These models are limited, in that they neglect details of wing kinematics, and model power by assuming a fixed or rotary wing (actuator disk) weight support mechanism. Theoretical methods that incorporate wing kinematics potentially offer more accurate predictions of power consumption by calculating instantaneous aerodynamic loads on the wing. However, the success of these models inherently depends on the availability and accuracy of experimental kinematic data. The predictive simulation approach offers an alternative strategy, whereby kinematics are neither neglected nor measured experimentally, but calculated as part of the solution procedure. This thesis describes the development of a predictive tool for simulating avian wingbeat kinematics and wakes. The tool is designed in a modular format, in order to be extensible for future research in the biomechanics community. The primary simulation module is an inverse dynamic avian wing model that predicts aerodynamic forces and mechanical power consumption for given wing kinematics. The model is constructed from previous experimental studies of avian wing biomechanics. Wing motion is defined through joint kinematic time histories, and aerodynamic forces are predicted using blade element momentum theory. Mechanical power consumption at the shoulder joint is derived from both aerodynamic and inertial torque components associated with the shoulder joint rotation rate. An optimisation module is developed to determine wing kinematics that generate aerodynamic loads for propulsion and weight support in given flight conditions, while minimising mechanical power consumption. For minimum power cruise, optimisation reveals numerous local minima solutions that exhibit large variations in wing kinematics. Validation of the model against wind tunnel data shows that optimised solutions capture qualitative trends in wing kinematics with varying cruise speed. Sensitivity analyses show that the model outputs are most affected by the defined maximum lift coefficient and wing length, whereby perturbations in these parameters lead to significant changes in the predicted amount of upstroke wing retraction. Optimised solutions for allometrically scaled bird models show only small differences in predicted advance ratio, which is consistent with field study observations. Accelerating and climbing flight solutions also show similar qualitative trends in wing kinematics to experimental measurements, including a reduction in stroke plane inclination for increasing acceleration or climb angle. The model predicts that both climb angle and climb speed should be greater for birds with more available instantaneous mechanical power. Simulations of the wake using a discrete vortex model capture fundamental features of the wake geometry that have been observed experimentally. Reconstruction of the velocity field shows that this method overpredicts induced velocity in retracting-wing wakes, and should therefore only be applied to extended-wing phases of an avian wingbeat.

Please read the abstract in the secton 00front of this document. / Thesis (PhD (Zoology))--University of Pretoria, 2007. / Zoology and Entomology / unrestricted

abstract: This work advances structural and biophysical studies of three proteins important in disease. First protein of interest is the Francisella tularensis outer membrane protein A (FopA), which is a virulence determinant of tularemia. This work describes recombinant expression in Escherichia coli and successful purification of membrane translocated FopA. The purified protein was dimeric as shown by native polyacrylamide gel electrophoresis and small angle X-ray scattering (SAXS) analysis, with an abundance of β-strands based on circular dichroism spectroscopy. SAXS data supports the presence of a pore. Furthermore, protein crystals of membrane translocated FopA were obtained with preliminary X-ray diffraction data. The identified crystallization condition provides the means towards FopA structure determination; a valuable tool for structure-based design of anti-tularemia therapeutics. Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination. Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020