Introduction Titanium-45, a candidate PET isotope, is under-employed largely because of the challenging aqueous chemistry of Ti(IV). The propensity for hydrolysis of Ti(IV) compounds makes radio-labeling difficult and excludes 45Ti from use in bio-conjugate chemistry. This is unfortunate because the physical characteristics are extremely desirable: 45Ti has a 3 hour half-life, a positron branching ratio of 85 %, a low Eβmax of 1.04 MeV, and negligible secondary gamma emission. In terms of isotope production, 45Ti is transmuted from naturally mono-isotopic 45Sc by low energy proton irradiation. The high cross-section and production rates on an unenriched metal foil target contribute to make 45Ti an ideal PET radionuclide. In order to bring 45Ti to even a preclinical plat-form, the hydrolytic instability of aqueous Ti(IV) needs to be addressed. Recently, the groups of Edit Tshuva (Hebrew University of Jerusalem) and Thomas Huhn (University of Konstanz) have synthesized several stable Ti(IV) compounds based upon the salan ligand [1,2]. Additionally, these compounds have shown heightened cyto-toxicity against HT-29 (human colorectal cancer) cells, amongst others, as compared to traditional metal-based chemotherapeutics such as cisplatin. The aim of our work has been to produce the radioactive analogue of one of these Ti(IV)-salan compounds, Ti-salan-dipic [2], which has hydro-lytic stability on the order of weeks. Not only will this allow us to shed some light on the still un-known mechanism of antiproliferative action of titanium-based chemotherapeutics, but it will also make progress toward bioconjugate 45Ti PET tracers. In the current abstract, we present some of the methods we are using to separate 45Ti from irradiated Sc, and subsequent labeling conditions. Material and Methods 45Ti was produced by proton irradiation of 250μm scandium foils at currents ranging from 10-20μA on a GE PETTrace. In order to increase production rate in the thin foil, an 800μm aluminum degrader was used to take the proton energy down from the nominal 16 MeV. The scandium was cooled by contact to a water-cooled silver plate. The activated foil was dissolved in 4M HCl, dried under argon at 120 oC, and taken back up in 12M HCl. Here, four (i-iv below) different approaches to removing the Ti from the Sc and labeling were taken with varying success. Briefly: i. 45Ti was separated on hydroxamate resin, as presented by K. Gagnon [3], only at 12M acid concentration followed by on-column radiolabeling. ii. 45Ti was extracted into 1-octanol [4], stripped with 12M HCl, and used directly for labeling from the organic phase. iii. 45Ti was trapped on a C-18 cartridge that had been pre-loaded with 1-octanol, similar to ion-pairing, and eluted with isopropanol. iv. 45Ti was extracted onto a polystyrene based 1,3 diol resin (RAPP polymers) and labeling commenced on the column. Radiolabeling was slightly different in each condition, but in general the salan and dipic ligands were added to the 45Ti in pyridine and reacted at elevated temperature (60–100 oC) for several (10–30) minutes. Reaction progression and radiochemical purity were assessed with silica TLC in chloroform : ethyl acetate (1 : 1). Results and Conclusion The trap, release, and yields for the four methods listed above are shown in TABLE 1. The best result was with the 1,3 diol resin which had the added advantage of reacting on-column. Further optimization is underway including a test of a solid supported 1,2 diol, and preclinical imaging with HT-29 xenografts. We conclude that hydrolytically stable 45Ti com-pounds can be synthesized in high yield, and hope that this advances the radiochemistry and use of 45Ti toward more widespread applications.

Sarcocystis neurona is an apicomplexan parasite that is a major cause of equine protozoal myeloencephalitis (EPM). During intracellular development of S. neurona, many genes are temporally regulated. To better understand gene regulation, it is important to identify and characterize regulatory elements controlling gene expression in S. neurona. To perform this study, it was essential to establish transfection system for this parasite. Hence, the 5 flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules -galactosidase (-gal) and yellow fluorescent protein (YFP) were expressed by electroporated S. neurona, thereby confirming the feasibility of performing molecular genetic experiments in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of T. gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express -gal and YFP. These transgenic clones were shown to be useful for analyzing growth rate of parasites in-vitro and for assessing drug sensitivities. To uncover possible sequence elements involved in promoter activity, the 5 flanking regions of five S. neurona genes were subjected to comparative analysis. This revealed the presence of a 7-base conserved motif GCGTCTC. Using a dual luciferase assay system, the SnSAG1 promoter was subjected to functional analysis. The motif GAGACGC located between -136 and -129 upstream of the transcription start site was found to be essential for SnSAG1 expression. This motif functions in an orientation dependent manner and was shown to play a role in binding nuclear proteins of S. neurona.

Recent observed changes in bird distributions provide an unprecedented opportunity to gain a deeper understanding of the processes that influence species' persistence. By modelling presence-absence data from the North American Breeding Bird Survey, we found evidence that the breeding range of the western burrowing owl has contracted at its northern, western, and eastern boundaries since 1967. We suggest that the species' breeding distribution is also expanding southwards to former wintering grounds into northern Mexico, facilitated by the appearance of new breeding habitat created by irrigated agriculture in the arid areas of southwestern United States and northwestern Mexico. This dissertation explores the hypothesis that burrowing owls from northern migratory populations have become resident breeders in areas of northwestern Mexico that were formerly used only by migratory owls during winter, contributing to both population declines near the northern extent of the species' breeding range and population increases in the southern half of the species' range. We used novel DNA microsatellite markers to test patterns of gene flow predicted by this migration-mediated range-shift hypothesis. We genotyped 1,560 owls from 36 study locations in Canada, Mexico, and the United States. Analyses of molecular variance provided evidence that burrowing owl populations in both northwestern Mexico and Canada are genetically different from the rest of the populations in the breeding range, lending some support to the migration-mediated range-shift hypothesis. We found evidence of subtle genetic differentiation associated with subtropical irrigated agricultural areas in southern Sonora and Sinaloa, demonstrating that land use can produce location-specific population dynamics leading to genetic structure even in the absence of dispersal barriers. We also used stable isotopes 2/H, 13/C, and 15/N in feathers to test philopatry and breeding dispersal patterns predicted by this migration-mediated range-shift hypothesis. Burrowing owl populations near the northern edge of the species' breeding range had a high proportion of immigrants compared to interior populations, while other populations had high levels of philopatry. Stable isotopes also provided evidence of breeding dispersal events from Canadian populations to northwestern Mexico in support of the migration-mediated range-shift hypothesis, but similar isotope signatures in nestling feathers between these two regions prevent stronger inferences.

Many tropical trees do not form reliable annual growth rings, making it a challenge to develop tree-ring width chronologies for application to paleoclimatology in these regions. Here, I seek to establish high-resolution proxy climate records from trees without rings from the Monteverde Cloud Forest in Costa Rica using stable isotope dendroclimatology. Neotropical cloud forest ecosystems are associated with a relatively narrow range of geographic and hydroclimatic conditions, and are potentially sensitive to climate variability and change at time scales from annual to centennial and longer. My approach takes advantage of seasonal changes in the d18O of water sources used by trees over a year, a signature that is imparted to the radial growth and provides the necessary chronological control. A rapid wood extraction technique is evaluated and found to produce cellulose with d18O values indistinguishable from conventional approaches, although its application to radiocarbon requires a statistical correction. Analyses of plantation-grown Ocotea tenera reveal coherent annual d18O cycles up to 9 permil. The width of these cycles corresponds to observed basal growth increments. Interannual variability in d18O at this site is correlated with wet season precipitation anomalies. At higher elevations within the orographic cloud bank, year-to-year changes in the amplitude of oxygen isotope cycles show a relationship with dry season climate. Longer d18O chronologies from mature Pouteria (Sapotacae) reveal that dry season hydroclimatology is controlled at interannual time scales by variability in the eastern equatorial Pacific (ENSO) and the Western Hemisphere Warm Pool (WHWP), which are correlated with trade wind strength and local air temperature. A change in the late 1960s toward enhanced annual d18O amplitude may reflect low frequency changes in the Atlantic and Pacific ocean-atmosphere system. This study establishes the basis for cloud forest isotope dendroclimatology and demonstrates that the local climate of neotropical cloud forests is sensitive to interannual, and perhaps, multidecadal changes in important large-scale modes of climate variability.

The spatial ecology of a species underpins an array of important questions about the ecology and evolution of a species, relating for instance to sociality, population dynamics, geographical distribution or genetic partitioning. Furthermore, the understanding of the spatial ecology of a species also has important implications for conservation and for disease management. It is in this context that I have examined the spatial ecology of the serotine bat (Eptesicus serotinus), in Europe, and especially at the northern limit of its distribution in England, using population genetic and stable isotope analyses. Data from nuclear microsatellite markers indicated higher levels of gene flow on the continent than in England. Consistent with this, England was separated into three genetically distinct populations, inter-connected by male-driven gene flow. Substantial asymmetric gene movement over the English Channel was inferred, possibly indicating a northward and westward range expansion. Mitochondrial DNA revealed fast population expansion and strong female philopatry. Moving to finer scale, δ13C in wing tissue and fur revealed that the association of individuals at roosts is flexible and dynamic, possibly reflecting their membership to communities operating at larger geographic scales. Communities of E. serotinus seem therefore to create a network of roosts with individuals continually re-assorting among themselves both within and across breeding seasons. The integration of the genetic and isotopic data highlighted complex spatial, social and temporal interactions in this species. While individual associations within communities seem to be flexible, communities were found to be strongly segregated over the breeding season. In contrast, genetic connectivity operates on a larger geographical scale than the local summer landscape. Together, these findings help explain the apparent absence in the UK of the European Bat Lyssavirus 1 (EBLV-1) for which this species is the main host. Hence, the relative fragmentation of this low-density population may inhibit the virus’ spread. However, with regular immigration from the continent, EBLV-1 could eventually enter the UK.

Lakes play an important role in the global carbon (C) cycle as they process carbon from terrestrial (allochthonous) and within lake (autochthonous) sources and may store C over long periods of time. Some arctic lakes contain high concentrations of dissolved organic carbon (DOC) that does not absorb light and thus remains colourless. The origin of this DOC remains unknown, but the sediment of these lakes have been suggested to accumulate primarily autochthonous (algal) C. I developed an experimental chamber for hydrogen (H) isotope pre-treatments and applied a novel H isotope tracing approach to determine the origin of the DOC and sediment C of a lake on SW Greenland known to contain colourless DOC. I hypothesized that autochthonous C was the prime source of DOC and sediment C, in line with previous theories. Analyses of algae and soil samples from the catchment revealed that local allochthonous and autochthonous C sources had a δ2H composition of -139 ‰ and -209 ‰, respectively. In contrast to my hypothesis, the analysed DOC had a mean δ2H isotopic composition of -147 ‰ indicating a dominance (ca 80-90 %) of allochthonous C. Similarly, the sediment had a mean δ2H isotopic composition of -155 ‰, suggesting that about 84 % of the C accumulating in the sediment was derived from terrestrial sources. The terrestrial origin was supported by field observations of high DOC seepage water (up to 70 mg L-1) with uncharacteristically low light absorption values entering the lake during high precipitation events. My results indicate that terrestrial processes are fundamental C sources for arctic lakes, even in regions with very low precipitation.

Delineating spatial patterns of precipitation isotopes (“isoscapes”) is important for studies including the hydrology of terrestrial systems, present and past interpretations of climate, and tracer-aided hydrological modelling, among others. However, the extent to which precipitation isoscapes can be predicted across Canada has not been fully articulated. This thesis combines isotopes in precipitation (δ18Oppt) observations from two regional and one global network to create long term and time series precipitation isoscapes for Canada and the northern United States. Multi-linear regressions of a small suite of geographic and climate variables generate the best performing long-term and seasonal models of δ18Oppt. These models are used to develop long term isoscapes for Canada, which capture the general spatial and seasonal trends in δ18Oppt, showing an improvement upon results from previous studies using global models. Building upon long-term δ18Oppt prediction, δ18Oppt observations alongside climatological and geographic predictors are used to create empirical time series prediction models. Five regionalization approaches are used to separate the study domain into isotope zones to explore the effect of spatial grouping on simulations. Generally, the models capture the timing and magnitude of intra-annual (seasonal) δ18Oppt cycles across the study domain while simulating moderate inter-annual variation; however often fail to capture the anomalies in observed δ18Oppt. Uncertainty in predictions is quantified spatially and temporally, and the Köppen-Geiger (Kpn) regionalization is selected as the preferred regionalization scheme for future applications due to adequate model performance and lack of border issues at regional boundaries. Finally, estimates of monthly δ18Oppt from Kpn models, long term annual averages, and daily REMOiso output are used to force an isotope-enabled hydrological model, isoWATFLOOD, in the Fort Simpson Basin, NWT, Canada. Results show streamflow simulations are not significantly impacted by choice of δ18Oppt input; however, oxygen-18 in streamflow and the internal apportionment of water (and model parameterizations) are impacted, particularly during large precipitation and snowmelt events. This work shows how isoWATFLOOD can be used in regions with limited δ18Oppt observations, and that the model can be of value in such regions. This study reinforces that a tracer-aided modelling approach works towards diagnosing issues surrounding model equifinality. / February 2017

To evaluate the niche of coastal fish populations in the Beaufort Sea, stable isotopes (SI) and fatty acids (FA) were used to characterize species-specific niches, niche overlaps and resource partitioning (nicheROVER) of the Shingle Point fish populations. Fishes were grouped into three isotopic groups: marine, coastal, and freshwater (Ward’s clustering analysis), and five dietary groupings (using FA), where benthic feeding strategies were prevalent (correspondence analysis). Niche metrics were used to evaluate if total mercury (THg) could contribute complementary trophic information (residual permutation procedure (RPP)). Three THg groups (high, intermediate, low) were identified (boxplot analysis). High THg was identified in high trophic and benthic feeders, high THg ranges were observed in species with large niche sizes, high trophic feeding, and freshwater influences (RPP). The bioavailability of freshwater introduced THg to marine biota was assessed, however further research needs to be performed. Combining dietary indicators SI, FA, and THg, allowed for the characterization of the diet and habitat use of coastal fish populations, better understanding of the niches of these species, and developed baseline information for future monitoring in an MPA, as climate change continues to effect the Beaufort coastal environment. / February 2017

Capelin (Mallotus villosus (Müller, 1776)) is an important marine forage fish species that typically inhabits sub-Arctic and temperate circumpolar waters. Capelin have recently been reported in greater abundance in Arctic regions, and are considered an indicator for warming climate in the northern marine ecosystem. The goal of this thesis is to examine intrinsic factors among capelin populations, and whether niche overlap among sympatric capelin and Arctic cod (Boreogadus saida (Lepechin, 1774)) will affect the abundance and persistence of capelin in the Arctic as temperatures continue to rise. Life history comparisons of capelin from two Arctic regions (Western Beaufort Sea, Cumberland Sound) relative to a sub-Arctic population (Newfoundland) show that body size, body condition, growth rate and age-at-maturity vary among these regions. Life history characteristics that are adapted to northern environments and increasing temperatures will allow the presence and persistence of this species to increase in the Arctic. The consequence of increased abundance of capelin in the Arctic environment could be competition between capelin and Arctic cod, an Arctic species within a similar dietary niche. Stomach contents indicated that both species feed primarily on calanoid copepods and this result was corroborated with high dietary overlap in isotopic bivariate space (carbon and nitrogen stable isotopes). The occurrence of capelin is expected to increase in the Arctic with rising temperatures, thus adaptation in life history traits in capelin specific to each region may facilitate increased abundance and persistence, and possibly contribute to competitive pressure on Arctic cod. Potential competition will be particularly important in nearshore and shelf habitats where shifts in availability of intermediate trophic level taxa will influence diet and distribution of key predators such as beluga, sea birds, and anadromous fishes. As the Arctic environment changes with climate shifts, newly adapted or dispersed species from sub-Arctic systems are expected to drive shifts in ecosystem structure and function in the marine environment. / October 2016

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