Anharmonic Helmholtz free energy to O(lambdaâ ´) in the non- leading term approximation /|nby Leslie Wilk. -- 260 St. Catharines, Ont. : [s. n.],

Expressions for the anharmonic Helmholtz free energy contributions up to o( f ) ,valid for all temperatures, have been obtained using perturbation theory for a c r ystal in which every atom is on a site of inversion symmetry. Numerical calculations have been carried out in the high temperature limit and in the non-leading term approximation for a monatomic facecentred cubic crystal with nearest neighbour c entralforce interactions. The numbers obtained were seen to vary by a s much as 47% from thos e obtai.ned in the leading term approximati.on,indicating that the latter approximati on is not in general very good. The convergence to oct) of the perturbation series in the high temperature limit appears satisfactory.

Localization of the mechanism of pH-dependent non- photochemical fluorescence quenching in thylakoid membranes

Higher plants have evolved a well-conserved set of photoprotective mechanisms, collectively designated Non-Photochemical Quenching of chlorophyll fluorescence (qN), to deal with the inhibitory absorption of excess light energy by the photosystems. Their main contribution originates from safe thermal deactivation of excited states promoted by a highly-energized thylakoid membrane, detected via lumen acidification. The precise origins of this energy- or LlpH-dependent quenching (qE), arising from either decreased energy transfer efficiency in PSII antennae (~ Young & Frank, 1996; Gilmore & Yamamoto, 1992; Ruban et aI., 1992), from alternative electron transfer pathways in PSII reaction centres (~ Schreiber & Neubauer, 1990; Thompson &Brudvig, 1988; Klimov et aI., 1977), or from both (Wagner et aI., 1996; Walters & Horton, 1993), are a source of considerable controversy. In this study, the origins of qE were investigated in spinach thylakoids using a combination of fluorescence spectroscopic techniques: Pulse Amplitude Modulated (PAM) fluorimetry, pump-probe fluorimetry for the measurement of PSII absorption crosssections, and picosecond fluorescence decay curves fit to a kinetic model for PSII. Quenching by qE (,..,600/0 of maximal fluorescence, Fm) was light-induced in circulating samples and the resulting pH gradient maintained during a dark delay by the lumenacidifying capabilities of thylakoid membrane H+ ATPases. Results for qE were compared to those for the addition of a known antenna quencher, 5-hydroxy-1,4naphthoquinone (5-0H-NQ), titrated to achieve the same degree of Fm quenching as for qE. Quenching of the minimal fluorescence yield, F0' was clear (8 to 130/0) during formation of qE, indicative of classical antenna quenching (Butler, 1984), although the degree was significantly less than that achieved by addition of 5-0H-NQ. Although qE induction resulted in an overall increase in absorption cross-section, unlike the decrease expected for antenna quenchers like the quinone, a larger increase in crosssection was observed when qE induction was attempted in thylakoids with collapsed pH gradients (uncoupled by nigericin), in the absence of xanthophyll cycle operation (inhibited by DTT), or in the absence of quenching (LlpH not maintained in the dark due to omission of ATP). Fluorescence decay curves exhibited a similar disparity between qE-quenched and 5-0H-NQ-quenched thylakoids, although both sets showed accelerated kinetics in the fastest decay components at both F0 and Fm. In addition, the kinetics of dark-adapted thylakoids were nearly identical to those in qEquenched samples at F0' both accelerated in comparison with thylakoids in which the redox poise of the Oxygen-Evolving Complex was randomized by exposure to low levels of background light (which allowed appropriate comparison with F0 yields from quenched samples). When modelled with the Reversible Radical Pair model for PSII (Schatz et aI., 1988), quinone quenching could be sufficiently described by increasing only the rate constant for decay in the antenna (as in Vasil'ev et aI., 1998), whereas modelling of data from qE-quenched thylakoids required changes in both the antenna rate constant and in rate constants for the reaction centre. The clear differences between qE and 5-0H-NQ quenching demonstrated that qE could not have its origins in the antenna alone, but is rather accompanied by reaction centre quenching. Defined mechanisms of reaction centre quenching are discussed, also in relation to the observed post-quenching depression in Fm associated with photoinhibition.

Orientation of Non-Native 2-Monosubstituted and 2,3-Disubstituted 1,4-Naphthoquinones in the A1 binding site of PSI and the effect on the rate of electron transfer : an electron paramagnetic resonance spectroscopy study

The proce-ss ofoxygenic photosynthesis is vital to life on Earth. the central event in photosynthesis is light induced electron transfer that converts light into energy for growth. Ofparticular significance is the membrane bound multisubunit protein known as Photosystem I (PSI). PSI is a reaction centre that is responsible for the transfer of electrons across the membrane to reduce NADP+ to NADPH. The recent publication ofa high resolution X-ray structure of PSI has shown new information about the structure, in particular the electron transfer cofactors, which allows us to study it in more detail. In PSI, the secondary acceptor is crucial for forward electron transfer. In this thesis, the effect of removing the native acceptor phylloquinone and replacing it with a series of structurally related quinones was investigated via transient electron paramagnetic resonance (EPR) experiments. The orientation of non native quinones in the binding site and their ability to function in the electron transfer process was determined. It was found that PSI will readily accept alkyl naphthoquinones and anthraquinone. Q band EPR experiments revealed that the non-native quinones are incorporated into the binding site with the same orientation of the headgroup as in the native system. X band EPR spectra and deuteration experiments indicate that monosubstituted naphthoquinones are bound to the Al site with their side group in the position occupied by the methyl group in native PSI (meta to the hydrogen bonded carbonyl oxygen). X band EPR experiments show that 2, 3- disubstituted methyl naphthoquinones are also incorporated into the Al site in the same orientation as phylloquinone, even with the presence of a halogen- or sulfur-containing side chain in the position normally occupied by the phytyl tail ofphylloquinone. The exception to this is 2-bromo-3-methyl --.- _. -. - -- - - 4 _._ _ _ - _ _ naphthoquinone which has a poorly resolved spectrum, making determination of the orientation difficuh. All of the non-native quinones studied act as efficient electron acceptors. However, forward electron transfer past the quinone could only be demonstrated for anthraquinone, which has a more negative midpoint potential than phylloquinone. In the case of anthraquinone, an increased rate of forward electron transfer compared to native PSI was found. From these results we can conclude that the rate ofelectron transfer from Al to Fx in native PSI lies in the normal region ofthe Marcus Curve.

Effect of pH on non-photochemical quenching in spinach photsystem 2 particles as measured by picosecond fluorescence decay kinetics

Single photon timing was used to study picosecond chlorophyll a fluorescence decay kinetics of pH induced non-photochemical quenching in spinach photosystem 2 particles. The characteristics of this quenching are a decrease in chlorophyll a fluorescence yield as well as a decrease in photochemistry at low pH. Picosecond kinetics of room temperature fluorescence temporally resolve the individual components of the steady state fluorescence yield into components that are related to primary energy conversion processes in photosystem 2. Four components were resolved for dark adapted (Fo), light saturated (Fm), and chemically reduced (Nadithionite) photosystem 2 reaction centres. The fastest and slowest components, indicative of energy transfer to and energy capture by the photosystem 2 reaction centre and uncoupled ("dead") chlorophyll, respectively, were not affected by changing pH from 6.5 to 4.0. The two intermediate components, indicative of electron transfer processes within the reaction centre of photosystem 2, were affected by the pH change. Results indicate that the decrease in the steady state fluorescence yield at low pH was primarily due to the decrease in lifetime and amplitude of the slower of the intermediate components. These results imply that the decrease in steady state fluorescence yield at low pH is not due to changes in energy transfer to and energy capture by the photosystem 2 reaction centre, but is related to changes in charge stabilization and charge recombination in the photosystem 2 reaction centre.

The Fast Regulation of Photosynthesis in Diatoms: an inquiry into the physiological and physical origins of non-photochemical chlorophyll fluorescence quenching.

Diatoms are renowned for their robust ability to perform NPQ (Non-Photochemical Quenching of chlorophyll fluorescence) as a dissipative response to heightened light stress on photosystem II, plausibly explaining their dominance over other algal groups in turbulent light environs. Their NPQ mechanism has been principally attributed to a xanthophyll cycle involving the lumenal pH regulated reversible de-epoxidation of diadinoxanthin. The principal goal of this dissertation is to reveal the physiological and physical origins and consequences of the NPQ response in diatoms during short-term transitions to excessive irradiation. The investigation involves diatom species from different originating light environs to highlight the diversity of diatom NPQ and to facilitate the detection of core mechanisms common among the diatoms as a group. A chiefly spectroscopic approach was used to investigate NPQ in diatom cells. Prime methodologies include: the real time monitoring of PSII excitation and de-excitation pathways via PAM fluorometry and pigment interconversion via transient absorbance measurements, the collection of cryogenic absorbance spectra to measure pigment energy levels, and the collection of cryogenic fluorescence spectra and room temperature picosecond time resolved fluorescence decay spectra to study excitation energy transfer and dissipation. Chemical inhibitors that target the trans-thylakoid pH gradient, the enzyme responsible for diadinoxanthin de-epoxidation, and photosynthetic electron flow were additionally used to experimentally manipulate the NPQ response. Multifaceted analyses of the NPQ responses from two previously un-photosynthetically characterised species, Nitzschia curvilineata and Navicula sp., were used to identify an excitation pressure relief ‘strategy’ for each species. Three key areas of NPQ were examined: (i) the NPQ activation/deactivation processes, (ii) how NPQ affects the collection, dissipation, and usage of absorbed light energy, and (iii) the interdependence of NPQ and photosynthetic electron flow. It was found that Nitzschia cells regulate excitation pressure via performing a high amplitude, reversible antenna based quenching which is dependent on the de-epoxidation of diadinoxanthin. In Navicula cells excitation pressure could be effectively regulated solely within the PSII reaction centre, whilst antenna based, diadinoxanthin de-epoxidation dependent quenching was implicated to be used as a supplemental, long-lasting source of excitation energy dissipation. These strategies for excitation balance were discussed in the context of resource partitioning under these species’ originating light climates. A more detailed investigation of the NPQ response in Nitzschia was used to develop a comprehensive model describing the mechanism for antenna centred non-photochemical quenching in this species. The experimental evidence was strongly supportive of a mechanism whereby: an acidic lumen triggers the diadinoxanthin de-epoxidation and protonation mediated aggregation of light harvesting complexes leading to the formation of quencher chlorophyll a-chlorophyll a dimers with short-lived excited states; quenching relaxes when a rise in lumen pH triggers the dispersal of light harvesting complex aggregates via deprotonation events and the input of diadinoxanthin. This model may also be applicable for describing antenna based NPQ in other diatom species.

The Association of Energy Intake with Body Mass in Children With and Without Probable Developmental Coordination Disorder

Objective To determine if there is an association between energy intake (EI) and overweight or obesity status (OWOB) in children with and without probable developmental coordination disorder (p-DCD). Methods 1905 children were included. The Bruininks-Oseretsky Test of Motor Proficiency was used to assess p-DCD, body mass index for OWOB, and the Harvard Food Frequency Questionnaire for EI. Comparative tests and logistic regressions were performed. Results Reported EI was similar between p-DCD and non-DCD children among boys (2291 vs. 2281 kcal/day, p=0.917), but much lower in p-DCD compared to non-DCD girls (1745 vs.. 2068 kcal/day, p=0.007). EI was negatively associated with OWOB in girls only (OR: 0.82 (0.68, 0.98)). Conclusions Girls with p-DCD have a lower reported EI compared to their non-DCD peers. EI is negatively associated with OWOB in girls with p-DCD. Future research is needed to assess longitudinally the potential impact of EI on OWOB in this population.

Infrared thermography: A non-invasive window into thermal physiology

Infrared thermography is a non-invasive technique that measures mid to long-wave infrared radiation emanating from all objects and converts this to temperature. As an imaging technique, the value of modern infrared thermography is its ability to produce a digitized image or high speed video rendering a thermal map of the scene in false colour. Since temperature is an important environmental parameter influencing animal physiology and metabolic heat production an energetically expensive process, measuring temperature and energy exchange in animals is critical to understanding physiology, especially under field conditions. As a non-contact approach, infrared thermography provides a non-invasive complement to physiological data gathering. One caveat, however, is that only surface temperatures are measured, which guides much research to those thermal events occurring at the skin and insulating regions of the body. As an imaging technique, infrared thermal imaging is also subject to certain uncertainties that require physical modeling, which is typically done via built-in software approaches. Infrared thermal imaging has enabled different insights into the comparative physiology of phenomena ranging from thermogenesis, peripheral blood flow adjustments, evaporative cooling, and to respiratory physiology. In this review, I provide background and guidelines for the use of thermal imaging, primarily aimed at field physiologists and biologists interested in thermal biology. I also discuss some of the better known approaches and discoveries revealed from using thermal imaging with the objective of encouraging more quantitative assessment.