Detection and measurement of stress hormones with mass spectrometry

The purpose of this thesis was to develop and validate a liquid chromatography mass spectrometry (LC-MS) method to measure salivary cortisol and testosterone. Furthermore, to use this method to analyse saliva samples collected during an exercise study measuring the hormonal response to acute exercise. The ongoing contribution to knowledge provided by this research is the application of a developed and validated LC-MS method to analysis of cortisol from pre and post exercise saliva samples, and evaluation of the cortisol and testosterone response to three different training sessions; including an interval session (INT), tempo run (TEMP) and aerobic circuit training (CIR). An LC-MS method was developed to measure cortisol and testosterone. The mass spectrometer used was a triple quadrupole BioQ (Waters/Micromass, UK) coupled with a 1200 series HPLC machine (Agilent Technologies, UK). Optimisation of various parameters was undertaken including: cone voltage and capillary voltage, followed by optimisation of liquid chromatography (LC) parameters such as; mobile phase gradient and flow rate. Saliva samples were collected at rest, and hormones measured with the new LC-MS method and ELISA to validate the developed method. Finally, heart rate and salivary and plasma cortisol and testosterone response to acute exercise were tested, to compare the response to different training protocols in runners. Following this, the MS method underwent further validation against ELISA. Results revealed optimum MS parameters were: cone voltage 25 V; capillary voltage 3.5 kV and LM and HM resolution 8 Da. For the LC method, mobile phase flow rate was optimised at 0.1 mL/min, with a gradient profile ranging from 50 to 95% methanol (MeOH), and a run time of 15 minutes. Sample preparation was also considered (solid phase extraction); and wash phase of 10% MeOH, 10 times sample concentration and reconstitution with 80% MeOH were optimised to improve analyte recovery and detection. Comparison of cortisol measured at rest with LC-MS and ELISA (n=22) revealed a correlation between methods (r=0.83, P<0.001). The exercise study showed the INT elicited a higher peak heart rate (172±11) than CIR (148±10) or TEMP trials (163±10). INT also produced a higher RPE (15 ±2) than CIR (13±1) and TEMP (14±2) trials. Salivary cortisol increased at all time points post exercise in INT and remained higher than rest (1.29 ± 0.51 ng/mL) at 60 minutes recovery (3.13 ±1.59 ng/mL). Salivary testosterone increased pre to post exercise in CIR (119.7±39.5 pg/mL to 150.1 ±51.5 pg/mL), TEMP (142.4±76.5 pg/mL to 185.0±49.1 pg/mL) and INT (117.5±23.5 pg/mL to 176.3±46.0 pg/mL) trials. There was also a correlation between cortisol measured in plasma and saliva (r=0.813, p=0.0001). Additionally, comparison of salivary cortisol measurements obtained with mass spectrometry and ELISA (n=85) showed a correlation (r=0.934, p=00001). In conclusion, validation revealed cortisol could be quantfied accurately and precisely with LC-MS. Additionally, INT elicited a higher mean RPE and cortisol response than TEMP or CIR

Effect of Roadside Vegetation Cutting on Moose Browsing

Moose (Alces americanus ) vehicle collisions (MVCs) are an issue throughout the distribution of moose. Many mitigation strategies have been tested and implemented to reduce the number of MVCs, but there have been few empirical analyses of the effectiveness of roadside vegetation cutting. The goal of this study was to determine if roadside vegetation cutting attracted moose into roadside areas to browse on the vegetation regrowth. We hypothesized that moose would be attracted to roadside areas with cut vegetation. Consequently, we predicted that there would be higher levels of browsing in cut areas compared to uncut areas. To determine if moose were browsing more in cut or uncut areas, we measured the number of plants browsed by moose in paired treatment (cut on or after 2008) and control (not cut since at least 2008) sites, along with a suite of potential environmental covariates. Using a model selection approach, we fit generalized linear mixed-effects models to determine the most parsimonious set of environmental variables to explain variation in the proportion of moose browse among sites. In contrast to our hypothesis, our results show that the proportion of moose browse in the uncut control areas was significantly higher than in the cut treatment areas. The results of this study suggest that recently cut roadside areas (7 years or less based on our work) may create a less attractive foraging habitat for moose. The majority of the variance in the proportion of moose browse among sites was explained by treatment type and nested plot number within site identification (34.16%), with additional variance explained by traffic region (5.00%) and moose density (4.35%). Based on our study, we recommend that vegetation cutting be continued in roadside areas in Newfoundland as recently cut areas may be less attractive browsing sites for moose

Slowly cycling Rho kinase-dependent actomyosin cross-bridge slippage explains intrinsic high compliance of detrusor smooth muscle

Biological soft tissues are viscoelastic because they display timeindependent pseudoelasticity and time-dependent viscosity. However, there is evidence that the bladder may also display plasticity, defined as an increase in strain that is unrecoverable unless work is done by the muscle. In the present study, an electronic lever was used to induce controlled changes in stress and strain to determine whether rabbit detrusor smooth muscle (rDSM) is best described as viscoelastic or viscoelastic plastic. Using sequential ramp loading and unloading cycles, stress-strain and stiffness-stress analyses revealed that rDSM displayed reversible viscoelasticity, and that the viscous component was responsible for establishing a high stiffness at low stresses that increased only modestly with increasing stress compared with the large increase produced when the viscosity was absent and only pseudoelasticity governed tissue behavior. The study also revealed that rDSM underwent softening correlating with plastic deformation and creep that was reversed slowly when tissues were incubated in a Ca2+ -containing solution. Together, the data support a model of DSM as a viscoelastic-plastic material, with the plasticity resulting from motor protein activation. This model explains the mechanism of intrinsic bladder compliance as slipping cross bridges, predicts that wall tension is dependent not only on vesicle pressure and radius but also on actomyosin cross-bridge activity, and identifies a novel molecular target for compliance regulation, both physiologically and therapeutically

Measurement of the hyperfine coupling constants and absolute energies of the 8p 2P1/28p \ ^2P_{1/2} and 8p 2P3/28p \ ^2P_{3/2} levels in atomic cesium

We report measurements of the hyperfine coupling constant for the $8p \ ^2P_{1/2}$level of atomic cesium,$^{133}$Cs, with a relative uncertainty of$\sim$0.019\%. Our result is$A = 42.933 \: (8)$MHz, in good agreement with recent theoretical results. We also examine the hyperfine structure of the$8p \ ^2P_{3/2}$state, and derive new values for the state energies of the$8p \ ^2P_{1/2}$and$8p \ ^2P_{3/2}$ states of cesium.Comment: 8 pages, 7 figures, 5 table

Anillin localization suggests distinct mechanisms of division plane specification in mouse oogenic meiosis I and II

Anillin is a conserved cytokinetic ring protein implicated in actomyosin cytoskeletal organization and cytoskeletal-membrane linkage. Here we explored anillin localization in the highly asymmetric divisions of the mouse oocyte that lead to the extrusion of two polar bodies. The purposes of polar body extrusion are to reduce the chromosome complement within the egg to haploid, and to retain the majority of the egg cytoplasm for embryonic development. Anillin's proposed roles in cytokinetic ring organization suggest that it plays important roles in achieving this asymmetric division. We report that during meiotic maturation, anillin mRNA is expressed and protein levels steadily rise. In meiosis I, anillin localizes to a cortical cap overlying metaphase I spindles, and a broad ring over anaphase spindles that are perpendicular to the cortex. Anillin is excluded from the cortex of the prospective first polar body, and highly enriched in the cytokinetic ring that severs the polar body from the oocyte. In meiosis II, anillin is enriched in a cortical stripe precisely coincident with and overlying the meiotic spindle midzone. These results suggest a model in which this cortical structure contributes to spindle re-alignment in meiosis II. Thus, localization of anillin as a conserved cytokinetic ring marker illustrates that the geometry of the cytokinetic ring is distinct between the two oogenic meiotic cytokineses in mammals

Measurement of the static Stark Shift of the 7s 2S1/27s \ ^2S_{1/2} level in atomic cesium

We report a new precision measurement of the dc Stark shift of the 6s\hspace{1mm} ^2S_{1/2} \rightarrow 7s\hspace{1mm}^2S_{1/2} transition in atomic cesium-133. Our result is 0.72246 (29) $\textrm{Hz}(\textrm{V}/\textrm{cm})^{-2}$. This result differs from a previous measurement of the Stark shift by $\sim$0.5\%. We use this value to recalculate the magnitude of the reduced dipole matrix elements $\langle7s ||r||7p_{j}\rangle$, as well as the vector transition polarizability for the $6s \rightarrow 7s$ transition, $\tilde{\beta} = 27.043 \: (36) \ a_0^3$. This determination helps resolve a critical discrepancy between two techniques for determining the vector polarizability.Comment: 5 pages, 3 figures, 3 table

Experience Improves the Reliability of Subjective Measurements of Temperament in Beef Cattle

Reliability of experienced and inexperienced observers when assessing the behavior of cattle when restrained in a squeeze chute (chute score), and when exiting the chute (exit score), was compared. Overall, experienced observers had higher reliability than inexperienced observers. Increasing the number of individuals scoring an animal decreased the degree of agreement. However, within an acceptable tolerance for difference in scores, such disagreement may be beneficial; it allows for subtlety in interpretations of temperament, which when averaged, may better reflect docility. Reliabilities were higher for exit score than chute score. This may reflect the complexity of the trait being evaluated, with fewer behaviors observed when cattle exit as compared to when restrained in a chute. Producers may profitably use chute and exit score to quantify docility in cattle. However, it may be worthwhile to gain experience in using the scoring system before implementing it for selection decisions