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Recognizing how climate change will impact populations can aid in making decisions about approaches for conservation of endangered species. The Blunt-nosed Leopard Lizard (Gambelia sila) is a federally endangered species that, despite protection, remains in extremely arid, hot areas and may be at risk of extirpation due to climate change. We collected data on the field-active body temperatures, preferred body temperatures, and upper thermal tolerance of G. sila. We then described available thermal habitat using biophysical models, which allowed us to (1) describe patterns in lizard body temperatures, microhabitat temperatures, and lizard microhabitat use, (2) quantify the lizards’ thermoregulatory accuracy, (3) calculate the number of hours they are currently thermally restricted in microhabitat use, (4) project how the number of restricted hours will change in the future as ambient temperatures rise, and (5) assess the importance of Giant Kangaroo Rat burrows and shade-providing shrubs in the current and projected future thermal ecology of G. sila. Lizards maintained fairly consistent daytime body temperatures over the course of the active season, and use of burrows and shrubs increased as the season progressed and ambient temperatures rose. During the hottest part of the year, lizards shuttled among kangaroo rat burrows, shrubs, and open habitat to maintain body temperatures below their upper thermal tolerance, but occasionally, higher than their preferred body temperature range. Lizards are restricted from staying in the open habitat for 75% of daylight hours and are forced to seek refuge under shrubs or burrows to avoid surpassing their upper thermal threshold. After applying climatic projections of 1 and 2˚C increases to 2018 ambient temperatures, G. sila will lose additional hours of activity time that could compound stressors faced by this population, potentially leading to extirpation. Finally, temperature-based activity estimation (TBAE) is an automated method for predicting surface activity and microhabitat use based on the temperature of an organism and its habitat. In an attempt to lessen impacts on sensitive species and costs, we assessed continuously logged field active body temperatures as a tool to predict the surface activity and microhabitat use of an endangered lizard (Blunt-nosed Leopard Lizard, Gambelia sila). We found that TBAE accurately predicts whether a lizard is above or below ground 75.7% of the time when calculated using air temperature, and 60.5% of the time when calculated using biophysical models. While surface activity was correctly predicted about 93% of the time using either method, accuracy in predicting below ground (burrow) occupancy was 62% for air temperature and 51% for biophysical models. Using biophysical model data, TBAE accurately predicts microhabitat use in 79% of observations in which lizards are in the sun, 47% in the shade, and 51% in burrows. Heliotherms bask in the sun, and thus body temperatures can shift rapidly when the animal moves to a new microhabitat. This sensitivity, makes TBAE a promising means of remotely monitoring animal activity, particularly for specific variables like emergence time and surface activity.

The capabilities of a number of remote-sensing techniques for biophysical mapping in the subarctic have been examined at Pen Island in northwestern Ontario. After a two week field reconnaissance, colour infrared aerial photography was studied and a detailed biophysical map of the area was produced. Using this knowledge LANDSAT satellite data of the site were investigated. In a visual analysis of the data, the majority of the units identified in the airphoto interpretation were detected, and these were distinguished primarily by their spectral characteristics. Digital analysis of the satellite data using the Bendix MAD system allowed many of the classes of the earlier studies to be delineated and also permitted the classification to be readily extended beyond the original site. In both LANDSAT analyses specific biophysical units could be mapped from the satellite data but could not be identified without the airphoto interpretation. / Thesis / Master of Science (MSc)

Focused ultrasound (FUS) mediated neuromodulation has become a trending topic due to its promising attributes that enable precise and transcranial neuromodulation. Despite multiple reports of FUS effects on neurons, nervous systems, and the human brain, the mechanisms underlying such excitation or inhibition remain controversial. In our previous study, we showed that 2.1 MHz FUS induced membrane depolarizations on single crayfish motor axons in the presence of voltage-gated channel blockers, which led to a nanopore hypothesis: FUS triggered lipid molecule reconfiguration and form ion-permeable nanopores on the axonal membrane. Based on this hypothesis, stretching of the axonal membrane due to swelling in low osmolarity should increase the probability of nanopore formation under FUS. As predicted, exposure to 75% hypotonic saline induced significant increases in amplitude and frequency of occurrence of those FUS-induced depolarizations (FUSD) while the onset latency of the FUSD showed a significant decrease. Those results support the hypothesis that FUSD can be modulated by mechanically altering membrane properties. Since FUS inevitably perturbs cell membranes, we examined the role of mechanosensitive K2P channels at the crayfish opener neuromuscular junction. At ultrasound intensity lower than those used to evoke FUSD, FUS consistently induced membrane hyperpolarization (FUSH) in motor axons but not muscle fibers, which may lack K2P. Since K2P channels are also thermosensitive, we varied the temperature from 12 to 32 °C. However, there was no significant correlation between FUSH amplitudes and temperature. Furthermore, FUSH was not inhibited by the K2P channel blockers, although the presence of the channels was confirmed by K2P blockers which increased input resistance and depolarized axonal resting membrane potential. Thus, it is unlikely that K2P channels underlie FUSH. We also studied the impact of FUS on propagating action potentials (APs) in the crayfish motor axons. APs recorded during FUS took off from a hyperpolarized membrane potential and exhibited larger amplitudes and shorter duration. Three hypotheses were examined and eliminated. The US modulated AP shape changes cannot be due to: (1) alterations in microelectrode characteristics, (2) the increase in the fraction of sodium channels in the closed and not-inactivated state due to the hyperpolarization and (3) US activation of K2P channels which in turn altered AP shapes. One potential mechanism that requires further investigation is that FUS may accelerate the activation of sodium channel opening. Other factors that may indirectly modulate AP shapes are discussed. In summary, results presented in this thesis suggest that FUS-mediated membrane responses in a single cell could vary depending on the FUS intensity and the type of ion channel a given cell expresses. Furthermore, ultrasound not only evokes changes membrane potential but also modulates action potentials. Collectively, these results represent significant contribution to the understanding of mechanisms underlying ultrasound neuromodulation at the cellular level.

Transcription factors (TFs) have been a focal point of molecular biology research for decades, with evolving methodologies offering progressively deeper insights into their critical roles in gene regulation. Recent advancements in experimental and computational techniques have significantly enhanced our understanding of TF functionality, yet this depth of knowledge varies widely across the spectrum of known TFs — from extensively characterized ones with quantitative binding affinity data to those scarcely studied or understood. In this work, we systematically carried out binding and expression experiments on all Escherichia coli TFs using a standardized computational pipeline to identify direct and indirect regulatory targets. We further leveraged our binding data to develop a novel biophysically motivated neural network capable of predicting TF-DNA binding affinity from DNA sequence. This approach allowed us to design binding sites with specified affinities, including those stronger than any sequence observed in nature, which we validate experimentally using an in vitro binding assay. We further optimized this assay to provide insight into complex TF binding regimes, where chemical signals can modulate TF binding affinity. Finally, we demonstrate the utility of systematically mapping TF binding sites through a case study on a previously thought dormant TF acquired from viral infection, revealing an unexpected phenotype where it can hijack the host cell. This work not only offers broad insights into the determinants of TF binding and regulation, but also provides a means to predictively engineer binding sites with desired affinity, while demonstrating the power of efficient data processing in uncovering intricate biological processes. / 2025-05-23T00:00:00Z

Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for obtaining structural information about chemical centres with unpaired electrons. In complex biological systems, EPR spectroscopy can be used to probe these paramagnetic centres and the long-range interactions between them. This thesis investigates two important types of enzymes, and in particular the role of the iron-sulfur electron-transfer centres they contain, with a variety of EPR techniques. Complex I (NADH:Ubiquinone Oxidoreductase) plays a key role in the electron transfer chain essential to the formation of ATP, and its malfunction has been related to numerous human diseases. It is a giant enzyme that contains the longest relay of iron-sulfur clusters known. EPR experiments conducted on complex I from bovine mitochondria yield crucial insight into the mechanism of efficient long-range electron transfer and bring us a step closer to understanding the functioning of this important complex. Hydrogenases are produced by micro-organisms and catalyse the reversible oxidation of H2. Most hydrogenases, including Hyd-2 from Escherichia coli, are very air-sensitive, but some, including E. coli Hyd-1 and Salmonella Hyd-5, are able to function in the presence of atmospheric levels of O2. Understanding the origins of this 'O2-tolerance' is of paramount importance if hydrogenases are to be exploited in future energy technologies. In this thesis, native E. coli Hyd-1 and Hyd-2, Salmonella Hyd-5, as well as O2-tolerant and O2-sensitive variants of E. coli Hyd-1 are characterised using EPR. The EPR investigations elucidate properties of the active site and the electron-transfer relay and, in conjunction with other techniques, reveal structural and mechanistic details of how a highly unusual iron-sulfur cluster in the electron-transfer chain enables some hydrogenases to sustain catalytic activity in the presence of O2.

The research described in this thesis employs a variety of Electron Paramagnetic Resonance (EPR) techniques for the study of the electronic and structural properties of artificial supramolecular porphyrin systems and of protein complexes of biological relevance. The electron delocalisation in the cationic radical and photoexcited triplet states of linear and cyclic Π-conjugated multiporphyrin arrays was investigated. In the radical cations, information on the extent of delocalisation can be inferred from the measurement of hyperfine couplings, either indirectly from the continuous wave EPR spectrum or directly using pulsed hyperfine EPR techniques. The results of room temperature EPR experiments showed complete delocalisation of the electron on the timescale of the EPR experiments, but frozen solution EPR measurements revealed localisation onto mainly two to three porphyrin units in the larger porphyrin systems. Information on the delocalisation of the triplet state in the same porphyrin systems is contained both in the hyperfine couplings and in the zero-field splitting (ZFS) interaction. The results outlined in this thesis show that the hyperfine couplings provide a much more accurate estimate of the extent of delocalisation. The trends in proton and nitrogen hyperfine couplings with the size of the porphyrin systems indicate uneven spin density distributions over the linear arrays, but complete delocalisation in the cyclic systems. Time-resolved EPR and magnetophotoselection experiments have shown a reorientation of the zero-field splitting tensor between a single porphyrin unit and longer linear arrays, resulting in alignment of the main optical transition moment and the Z axis of the ZFS tensor. Continuous wave and pulsed dipolar EPR techniques were employed for the determination of the structure of two different protein complexes, the homomultimeric twin-arginine translocase A (TatA) protein channel and the ferredoxin-P450 complex of the electron transport chain in Novosphingobium aromaticivorans. The interaction between nitroxide spin labels introduced at different positions of the TatA monomer was investigated in the complex reconstituted in detergent micelles by analysing the dipolar broadening of the EPR spectra and the results of three- and four-pulse Double Electron-Electron Resonance (DEER) measurements. In combination with results from NMR and molecular dynamics calculations, a structure for the channel complex was proposed. The structure of the ferredoxin-cytochrome P450 complex was investigated by orientation-selective DEER between nitroxide labels introduced on the cytochrome P450 protein and the iron-sulfur cluster of the ferredoxin. The distance and orientation information contained in the experimental DEER data was interpreted in terms of a structural model of the protein complex by orientation-selective DEER simulations combined with a modelling approach based on protein-protein docking.

Macromolecular transitions such as conformational changes and protein-protein association underlie many biological processes. Conformational changes in the N-terminal domain of the transmembrane protein DsbD (nDsbD) were studied by NMR and molecular dynamics (MD) simulations. nDsbD supplies reductant to biosynthetic pathways in the oxidising periplasm of Gram-negative bacteria after receiving reductant from the C-terminal domain of DsbD (cDsbD). Reductant transfer in the DsbD pathway happens via protein-protein association and subsequent thiol-disulphide exchange reactions. The cap loop shields the active-site cysteines in nDsbD from non-cognate oxidation, but needs to open when nDsbD bind its interaction partners. The loop was rigid in MD simulations of reduced nDsbD. More complicated dynamics were observed for oxidised nDsbD, as the disulphide bond introduces frustration which led to loop opening in some trajectories. The simulations of oxidised and reduced nDsbD agreed well with previous NMR spin-relaxation and residual dipolar coupling measurements as well as chemical shift-based torsion angle predictions. NMR relaxation dispersion experiments revealed that the cap loop of oxidised nDsbD exchanges between a major and a minor conformation. The differences in their conformational dynamics may explain why oxidised nDsbD binds its physiological partner cDsbD much tighter than reduced nDsbD. The redox-state dependent interaction between cDsbD and nDsbD is thought to enhance turnover. NMR relaxation dispersion experiments gave insight into the kinetics of the redox-state dependent interaction. MD simulations identified dynamic encounter complexes in the association of nDsbD with cDsbD. The mechanism of the conformational changes in the transport cycle of LacY were also investigated. LacY switches between periplasmic open and cytoplasmic open conformations to transport sugars across the cell membrane. Two mechanisms have been proposed for the conformational change, a rocker-switch mechanism based on rigid body motions and an “airlock” like mechanism in which the transporter would switch conformation via a fully occluded structure. In MD simulations using the novel dynamics importance sampling approach such a fully occluded structure was found. The simulations argued against a strict “rocker-switch” mechanism.

The uniquely predictable and controllable binding mechanism of DNA strands has been exploited to construct a vast range of synthetic nanodevices, capable of autonomous motion and computation. This thesis proposes novel ideas for the control and observation of such devices. The first of these proposals hinges on introducing mismatched base pairs into toehold-mediated strand displacement – a fundamental primitive in most dynamic DNA devices and reaction networks. Previous findings that such mismatches can impede strand displacement are extended insofar as it is shown that this impediment is highly dependent on mismatch position. This discovery is examined in detail, both experimentally and through simulations created with a coarse-grained model of DNA. It is shown that this effect allows for kinetic control of strand displacement decoupled from reaction thermodynamics. The second proposal improves upon a previously presented strand displacement scheme, in which two strands perform displacement cooperatively. This scheme is extended to be cascadable, so that the output of one such reaction serves as input to the next. This scheme is implemented in reaction networks capable of performing fundamental calculations on directed graphs. The third proposal is exclusively concerned with a novel observation methodology. This method is based on single-molecule fluorescence microscopy, and uses quantum dots, a fluorescent type of semiconductor nanocrystal, as a label. These quantum dots display a set of characteristics particularly promising for single-molecule studies on the time- and length scales most commonly encountered in DNA nanotechnology. This method is shown to allow for highly precise measurements on static DNA devices. Preliminary data for the observation of a complex dynamic device is also presented.

(3R,5R)-Clavulanic acid is a clinically important inhibitor of Class A β-lactamases. Progress has been made in to establishing the steps of clavulanic acid biosynthesis leading to (3S,5S)-clavaminic acid. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde remains elusive. It is believed that the products of the later genes (orf10-orf18) of the clavulanic acid biosynthesis gene cluster are probably involved in this conversion. Part I of this thesis describes biochemical and structural studies carried out on OAT2, a member of N-terminal nucleophile (Ntn) hydrolase superfamily of enzymes. OAT2 has been characterised to be an ornithine acetyl transferase (OAT) and is involved in clavulanic acid biosynthesis. OAT2 catalyses the reversible transfer of the acetyl group between N-acetyl-L-ornithine and L-glutamate. It was found that this reaction is catalysed via the formation of an acyl-enzyme intermediate. Subsequent studies including mass spectrometry, 13C NMR spectroscopy, infrared spectroscopy, X-ray crystallography and molecular dynamics simulations, further confirmed the viability of the intermediate. This acyl-enzyme intermediate of OAT2 was found to be exceptionally stable at physiological pH, as compared to the acyl-enzyme intermediates involved in catalysis by hydrolytic enzymes including proteases, Ntn hydrolases and β-lactamses. The X-ray studies revealed possible reason for this unusual stability. The infrared studies revealed two conformations for the acyl-enzyme. Modeling (MDS) studies assigned one of these to the structure observed by X-ray and proposed the other one to result from a hydroxyl hydrogen 'flip' involving the oxyanion hole component Thr-111 resulting in a singly hydrogen bonded acyl-enzyme intermediate. α, β Subunit co-expression studies with OAT2 were used to investigate the autocatalytic cleavage step. In one case an interesting N-acyl enzyme species was observed. Part II of this thesis describes efforts carried out to characterise the ORF10 and ORF15 proteins of clavulanic acid biosynthesis. ORF10 was characterised to be an 'active' cytochrome P450 and ORF10 crystals were obtained in the presence spinach ferredoxin, highlighting the role of the ferredoxin interaction in assisting ORF10 crystallisation. ORF15 was shown to be a probable peptide transporter, which binds bradykinin as observed in the crystal structure.