The adaptive response of humans to exercise: Impact of exercise intensity, fibre-type specific responses and molecular patterns of response

In an attempt to improve the current understanding of the adaptive response to exercise in humans, this dissertation performed a series of studies designed to examine the impact of training intensity and mode on aerobic capacity and performance, fibre-type specific adaptations to training, and individual patterns of response across molecular, morphological and genetic factors. Project #1 determined that training intensity, session dose, baseline VO2max and total training volume do not influence the magnitude of change in VO2max by performing a meta-regression, and meta-analysis of 28 different studies. The intensity of training had no effect on the magnitude of increase in maximal oxygen uptake in young healthy participants, but similar adaptations were achieved with lower training doses following high intensity training. Project # 2 determined the acute molecular response, and training-induced adaptations in aerobic performance, aerobic capacity and muscle phenotype following high-intensity interval training (HIT) or endurance exercise (END). The acute molecular response (fibre recruitment and signal activation) and training-induced adaptations in aerobic capacity, aerobic performance, and muscle phenotype were similar following HIT and END. Project # 3 examined the impact of baseline muscle morphology and molecular characteristics on the training response, and if muscle adaptations are coordinated. The muscle phenotype of individuals who experience the largest improvements (high responders) were lower before training for some muscle characteristics and molecular adaptations were coordinated within individual participants. Project # 4 examined the impact of 2 different intensities of HIT on the expression of nuclear and mitochondrial encoded genes targeted by PGC-1α. A systematic upregulation of nuclear and mitochondrial encoded genes was not present in the early recovery period following acute HIT, but the expression of mitochondrial genes were coordinated at an individual level. Collectively, results from the current dissertation contribute to our understanding of the molecular mechanisms influencing skeletal muscle and whole-body adaptive responses to acute exercise and training in humans.

Dynamics of the Developing Chick Chorioallantoic Membrane Assessed by Stereology, Allometry, Immunohistochemistry and Molecular Analysis.

The chick chorioallantoic membrane (CAM) is a widely used model for the study of angiogenesis, tumour growth, as well as drug efficacy. In spite of this, little is known about the developmental alteration from its appearance to the time of hatching. In the current study the CAM has been studied by classical stereology and allometry. Expression levels of selected angiogenesis-related molecules were estimated by RT-PCR and cell dynamics assessed by proliferation and apoptosis assays. Absolute CAM volume increased from a low of 0.47 ± 0.11 cm3 at embryonic day 8 (E8) to a high of 2.05 ± 0.27 cm3 at E18, and then decreased to 1.6 ± 0.47 cm3 at E20. On allometric analysis, three growth phases were identifiable. Between E8-13 (phase I), the CAM grew fastest; moderately in phase II (E13-18) but was regressing in phase III (E18-20). The chorion, the mesenchyme and the allantoic layers grew fastest in phase I, but moderately in phase II. The mesenchyme grew slowly in phase III while the chorion and allantois were regressing. Chorionic cell volume increased fastest in phase I and was regressing in phase III. Chorionic capillaries grew steadily in phase I and II but regressed in phase III. Both the chorion and the allantois grew by intrinsic cell proliferation as well as recruitment of cells from the mesenchyme. Cell proliferation was prominent in the allantois and chorion early during development, declined after E17 and apoptosis started mainly in the chorion from E14. VEGFR2 expression peaked at E11 and declined steadily towards E20, VEGF peaked at E13 and E20 while HIF 1α had a peak at E11 and E20. Studies targeting CAM growth and angiogenesis need to take these growth phases into consideration.

First-principles quantum simulations of dissociation of molecular condensates: Atom correlations in momentum space

We investigate the quantum many-body dynamics of dissociation of a Bose-Einstein condensate of molecular dimers into pairs of constituent bosonic atoms and analyze the resulting atom-atom correlations. The quantum fields of both the molecules and atoms are simulated from first principles in three dimensions using the positive-P representation method. This allows us to provide an exact treatment of the molecular field depletion and s-wave scattering interactions between the particles, as well as to extend the analysis to nonuniform systems. In the simplest uniform case, we find that the major source of atom-atom decorrelation is atom-atom recombination which produces molecules outside the initially occupied condensate mode. The unwanted molecules are formed from dissociated atom pairs with nonopposite momenta. The net effect of this process-which becomes increasingly significant for dissociation durations corresponding to more than about 40% conversion-is to reduce the atom-atom correlations. In addition, for nonuniform systems we find that mode mixing due to inhomogeneity can result in further degradation of the correlation signal. We characterize the correlation strength via the degree of squeezing of particle number-difference fluctuations in a certain momentum-space volume and show that the correlation strength can be increased if the signals are binned into larger counting volumes.

Molecular crowding effects of linear polymers in protein solutions

Measurement of protein-polymer second virial coefficients (B-AP) by sedimentation equilibrium studies of carbonic anhydrase and cytochrome c in the presence of dextrans (T10-T80) has revealed an inverse dependence of B-AP upon dextran molecular mass that conforms well with the behaviour predicted for the excluded-volume interaction between a spherical protein solute A and a random-flight representation of the polymeric cosolute P. That model of the protein-polymer interaction is also shown to provide a reasonable description of published gel chromatographic and equilibrium dialysis data on the effect of polymer molecular mass on BAP for human serum albumin in the presence of polyethylene glycols, a contrary finding from analysis of albumin solubility measurements being rejected on theoretical grounds. Inverse dependence upon polymer chainlength is also the predicted excluded-volume effect on the strength of several types of macromolecular equilibria-protein isomerization, protein dimerization, and 1 : 1 complex formation between dissimilar protein reactants. It is therefore concluded that published experimental observations of the reverse dependence, preferential reaction enhancement within DNA replication complexes by larger polyethylene glycols, must reflect the consequences of cosolute chemical interactions that outweigh those of thermodynamic nonideality arising from excluded-volume effects. (c) 2005 Elsevier B.V. All rights reserved.

A nanoliter volume nuclear magnetic resonance (NMR) system using tunneling magneto-resistive (TMR) sensors to recognize biomolecules

The need to incorporate advanced engineering tools in biology, biochemistry and medicine is in great demand. Many of the existing instruments and tools are usually expensive and require special facilities.^ With the advent of nanotechnology in the past decade, new approaches to develop devices and tools have been generated by academia and industry. ^ One such technology, NMR spectroscopy, has been used by biochemists for more than 2 decades to study the molecular structure of chemical compounds. However, NMR spectrometers are very expensive and require special laboratory rooms for their proper operation. High magnetic fields with strengths in the order of several Tesla make these instruments unaffordable to most research groups.^ This doctoral research proposes a new technology to develop NMR spectrometers that can operate at field strengths of less than 0.5 Tesla using an inexpensive permanent magnet and spin dependent nanoscale magnetic devices. This portable NMR system is intended to analyze samples as small as a few nanoliters.^ The main problem to resolve when downscaling the variables is to obtain an NMR signal with high Signal-To-Noise-Ratio (SNR). A special Tunneling Magneto-Resistive (TMR) sensor design was developed to achieve this goal. The minimum specifications for each component of the proposed NMR system were established. A complete NMR system was designed based on these minimum requirements. The goat was always to find cost effective realistic components. The novel design of the NMR system uses technologies such as Direct Digital Synthesis (DDS), Digital Signal Processing (DSP) and a special Backpropagation Neural Network that finds the best match of the NMR spectrum. The system was designed, calculated and simulated with excellent results.^ In addition, a general method to design TMR Sensors was developed. The technique was automated and a computer program was written to help the designer perform this task interactively.^

The adaptive response of humans to exercise: Impact of exercise intensity, fibre-type specific responses and molecular patterns of response

In an attempt to improve the current understanding of the adaptive response to exercise in humans, this dissertation performed a series of studies designed to examine the impact of training intensity and mode on aerobic capacity and performance, fibre-type specific adaptations to training, and individual patterns of response across molecular, morphological and genetic factors. Project #1 determined that training intensity, session dose, baseline VO2max and total training volume do not influence the magnitude of change in VO2max by performing a meta-regression, and meta-analysis of 28 different studies. The intensity of training had no effect on the magnitude of increase in maximal oxygen uptake in young healthy participants, but similar adaptations were achieved with lower training doses following high intensity training. Project # 2 determined the acute molecular response, and training-induced adaptations in aerobic performance, aerobic capacity and muscle phenotype following high-intensity interval training (HIT) or endurance exercise (END). The acute molecular response (fibre recruitment and signal activation) and training-induced adaptations in aerobic capacity, aerobic performance, and muscle phenotype were similar following HIT and END. Project # 3 examined the impact of baseline muscle morphology and molecular characteristics on the training response, and if muscle adaptations are coordinated. The muscle phenotype of individuals who experience the largest improvements (high responders) were lower before training for some muscle characteristics and molecular adaptations were coordinated within individual participants. Project # 4 examined the impact of 2 different intensities of HIT on the expression of nuclear and mitochondrial encoded genes targeted by PGC-1α. A systematic upregulation of nuclear and mitochondrial encoded genes was not present in the early recovery period following acute HIT, but the expression of mitochondrial genes were coordinated at an individual level. Collectively, results from the current dissertation contribute to our understanding of the molecular mechanisms influencing skeletal muscle and whole-body adaptive responses to acute exercise and training in humans.

The relationship of E. coli and total coliform culture to the quantitative molecular detection of various fecal indicators, sources, and pathogens in private drinking water wells

Water remains a predominant vector for human enteric pathogens not just for developing countries but also developed nations, where numerous infectious disease outbreaks, linked to the contamination of drinking water have been documented. Private drinking water wells are a source of drinking water that is largely unstudied even though a significant percentage of the population in Ontario relies on wells as their primary water source. As there exists little to no systematic surveillance for enteric infections or outbreaks related to well water sources, these individuals may be at higher risk of waterborne infectious diseases. The relationships between various fecal indicators in the water of private drinking water wells, including E. coli, Total Coliforms (TC) and Bacteroides, and enteric pathogens, including Campylobacter jejuni, Salmonella spp., and Shiga toxin producing E. coli, were studied. Convenience private well water samples collected from various regions of interest during the summer of 2014 underwent membrane filtration and culture to determine quantities of E. coli and TC colony forming units. 289 E. coli positive and 230 TC-only waters were successfully analyzed by individual qPCR assays for the aforementioned enteric pathogens. Microbial source tracking methods targeted to specific Bacteroides were used to determine the source of fecal contamination as either human or bovine. The source of fecal contamination varied by geographic region and is thought to be due to such things as differences in septic tank density and underlying geology, among others. Fecal indicators, E. coli and Bacteroides, were significantly correlated. E. coli as measured by qPCR was more strongly correlated to both total and human-specific Bacteroides genetic markers than culturable E. coli. Lastly, 1.9% of samples showed molecular evidence of contamination with enteric pathogens. Although low, this finding is significant given the limited volume of water available for testing, and suggests a potential health risk to consumers. Knowing the extent of contamination, as well as the biologic source, can better inform risk assessment and the development of potential intervention strategies for private well water in specific regions of Ontario.

A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature.

A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature.

Morphological and molecular characterization of a new species of Atlantic stalked barnacle (Scalpelliformes: Pollicipedidae) from the Cape Verde Islands

[EN] The taxonomy of pedunculate cirripedes belonging to the genus Pollicipes has essentially remained unchanged since Charles Darwin described them in his exhaustive work on the Cirripedia. This genus includes three species of stalked barnacles: Pollicipes pollicipes in the north-eastern Atlantic, P. polymerus in the north-eastern Pacific and P. elegans in the central-eastern Pacific. However, a population genetics analysis of P. pollicipes suggested the presence of a putative cryptic species collected from the Cape Verde Islands in the central-eastern Atlantic. This study examines the morphology of these genetically divergent specimens and compares them with that of representative Atlantic samples of the biogeographically closely related P. pollicipes and with the poorly described P. elegans.

Molecular cloning and expression of leptin in gray and harbor seal blubber, bone marrow, and lung and its potential role in marine mammal respiratory physiology

Leptin is a multifunctional hormone, produced predominantly in adipocytes. It regulates energy balance through its impact on appetite and fat metabolism, and its concentration indicates the size of body fat reserves. Leptin also plays a vital role in stretch-induced surfactant production during alveolar development in the fetus. The structure, expression pattern, and role of leptin have not previously been explored in marine mammals. Phocid seals undergo cyclical changes in body composition as a result of prolonged fasting and intensive foraging bouts and experience rapid, dramatic, and repeated changes in lung volume during diving. Here, we report the tissue-specific expression pattern of leptin in these animals. This is the first demonstration of leptin expression in the lung tissue of a mature mammal, in addition to its expression in the blubber and bone marrow, in common with other animals. We propose a role for leptin in seal pulmonary surfactant production, in addition to its likely role in long-term energy balance. We identify substitutions in the phocine leptin sequence in regions normally highly conserved between widely distinct vertebrate groups, and, using a purified seal leptin antiserum, we confirm the presence of the leptin protein in gray seal lung and serum fractions. Finally, we report the substantial inadequacies of using heterologous antibodies to measure leptin in unextracted gray seal serum.

Distribution of neutral prilocaine in a phospholipid bilayer: Insights from molecular dynamics simulations

In this work, we report a 20-ns constant pressure molecular dynamics simulation of prilocaine (PLC), in amine-amide local anesthetic, in a hydrated liquid crystal bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine. The partition of PLC induces the lateral expansion of the bilayer and a concomitant contraction in its thickness. PLC molecules are preferentially found in the hydrophobic acyl chains region, with a maximum probability at similar to 12 angstrom from the center of the bilayer (between the C(4) and C(5) methylene groups). A decrease in the acyl chain segmental order parameter, vertical bar S-CD vertical bar, compared to neat bilayers, is found, in good agreement with experimental H-2-NMR studies. The decrease in vertical bar S-CD vertical bar induced by PLC is attributed to a larger accessible volume per lipid in the acyl chain region. (C) 2008 Wiley Periodicals, Inc.

Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study

Fluorescence spectroscopy andmicroscopy have been utilized as tools in membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Among the latter, 1,6-diphenylhexatriene (DPH) and its trimethylammonium derivative (TMA-DPH) have been extensively used. It is widely believed that, owing to its additional charged group, TMA-DPH is anchored at the lipid/water interface and reports on a bilayer region that is distinct from that of the hydrophobic DPH. In this study, we employ atomistic MD simulations to characterize the behavior of DPH and TMA-DPH in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/cholesterol (4:1) bilayers. We show that although the dynamics of TMA-DPH in thesemembranes is noticeably more hindered than that of DPH, the location of the average fluorophore of TMA-DPH is only ~3–4 Å more shallow than that of DPH. The hindrance observed in the translational and rotational motions of TMA-DPH compared to DPH is mainly not due to significant differences in depth, but to the favorable electrostatic interactions of the former with electronegative lipid atoms instead. By revealing detailed insights on the behavior of these two probes, our results are useful both in the interpretation of past work and in the planning of future experiments using themasmembrane reporters.