research thesis proposal written and oral.

Specific Aim 1: To examine the neurovascular responses to reflex vasoconstriction in essential HTN humans.

Hypothesis 1a : Increases in BP during whole body cooling will be exaggerated in HTN humans.

Hypothesis 1b : Increases in both muscle and skin SNA during whole body cooling will be exaggerated in HTN adults.

Hypothesis 1c : Reductions in skin blood flow during whole body cooling will be greater in HTN adults.

1. State simply the proposed statistical model you will be using for your experiment. You will also want to consider adding in the appropriate assumptions (normally distributed, data will be transformed if necessary, independent observations, etc.)

For all experiments, outcome variables of interest will be reported as mean ± standard error. P-values and 95% confidence intervals for the mean difference estimates will be adjusted using Tukey’s multiple comparison procedure to account for post-hoc multiple comparisons and ensure a family-wise type I error rate <5%. Residual plots will be used to check modeling assumptions, specifically non-constant variances, and non-normality. Log or other transformations of outcomes will be used as needed. Data will be analyzed using SAS PROC MIXED. Subject characteristics will be compared using two-tailed unpaired Student’s t-tests.

Specific Aim 1: A two-way repeated-measures ANOVA [group (NTN vs. HTN) x temperature (skin)] will be used to assess differences in BP (H1a), sympathetic (H1b), and skin blood flow (H1c) variables at thermoneutrality and during whole body cooling-induced decreases in skin temperature (both absolute and delta values). The group represents NTN and HTN subjects and the temperature represents thermoneutrality (baseline, 34°C) and whole-body cooling-induced reductions in skin temperature. Main effects of group and temperature, along with the interaction term, will be assessed. The interaction term will be used to assess whether the degree of change in variables of interest are different between groups. Appropriate post-hoc analyses will be performed when main effects are indicated.

Aim	Hypothesis	Measured Outcomes	Planned Comparisons
SA1	Hyp 1a	BP changes during whole body cooling	Between blood pressure groups
	Hyp 1b	MSNA and SSNA changes during whole body cooling	Between blood pressure groups across the reduction in Tsk during cooling
	Hyp 1c	Changes in CVC during whole body cooling	Between blood pressure groups across the reduction in Tsk

2. Indicate the values you have possibly found from the literature in terms of what potential observed differences you would anticipate.

Study Type Study Cited	δ measured difference in populations means	σ within group SD (or SD of the differences)
BP increases during stress AJP Heart. 2010 299(5):H1318-27	7 mmHg	7 mmHg
Microneurography AJP Heart. 2010 299(5):H1318-27 Circulation. 2003 108(6):729-35	8 bursts/min (MSNA) 6 bursts/min (SSNA)	6 bursts/min (MSNA) 6 bursts/min (SSNA)
Microdialysis AJP Reg. 2005 288(5): R1108-13	15%CVCbase	11%CVCbase

See (Grassi et al., 2003; Thompson et al., 2005; Delaney et al., 2010) for full citations.

3. State your anticipated/desired effect size.

Considering our statistical approach is a mixed-model ANOVA (between factor and within-factor), we have identified omega squared (ω2) as our measure of unbiased effect size that should reasonably predict eta2. Based on previous literature, we have powered this study to have a ω2 of 0.5. This is a medium effect.

Alternatively, we can use Cohen’s f (analogous to Cohen’s d, but for ANOVA). We consider anything over 0.5 to be meaningful. This is also a medium effect.

4. Describe what you would consider/determine a meaningful difference you would consider significant in your field of study.

We classify a meaningful difference in blood pressure as 5-8 mmHg. This difference is what is expected from a standard antihypertensive treatment and/or exercise training. A 5mmHg change in blood pressure is associated with a 15-18% increased risk of cardiovascular events (Blood Pressure Lowering Treatment Trialists, 2014). For the current study, we will perform our sample size calculation to detect a 10mmHg difference between the groups.

Correspondingly, we classify a meaningful difference in MSNA as 10 bursts/min. This difference is what is measured with an ~10mHg change in mean arterial blood pressure during sympathoexcitatory maneuvers (Fonkoue & Carter, 2015). For SSNA, the literature is more sparce. Previously, Grassi et al. found a 6 burst/min difference in SSNA between young and older adults.

For the microdialysis studies, we classify a meaningful difference in skin blood flow as a 15% change in percent baseline. This observed difference is what is measured in direct pharmacological administration of a 10-7 dose of the sympathetic neurotransmitter norepinephrine (Thompson et al., 2005).

5. Describe the variance (variability) you would anticipate in your measurements, and the expected variance in the difference if you are doing an intervention (pre/post measures).

Blood pressure and microneurography studies. Based on previous literature, we assume a within-group standard deviation of 7 mmHg for blood pressure and 6 bursts/min for both MSNA and SSNA.

Microdialysis studies . Based on our previous data, we measure a within group standard deviation of 11% in CVCbase.

6. (if applicable) For studies utilizing regression or multiple regression describe what you are examining in terms of predictors in your model and the anticipation of the type of model (linear, nonlinear).

(example language from a different, unrelated grant)

If all assumptions are met, then our primary hypotheses for specific aims 1 and 2 will be tested using linear regression analysis with average MVPA and sedentary time per day as covariates and moderators of the relation between NA-R and BP reactivity. Given the beneficial effects of light PA on cardiometabolic risk factors and mental health outcomes after adjustment for MVPA (Amagasa et al., 2018) and differential effects of PA intensities on mental and physical health outcomes (Asztalos et al., 2010; Ekblom-Bak et al., 2016), linear regression analysis will also be performed with average light PA per day as a covariate and moderator of the relation in a secondary analysis. Given the potentially large variability in day-to-day PA behaviors (e.g., frequency, intensity, duration), data will be dummy coded based on clinically significant guidelines outlined by the ACSM and Canadian 24-hour Movement Guidelines (Haskell et al., 2007; Ross et al., 2020) as follows: MVPA (≥30 min/day, <5 days/wk or <30 min/day, ≥5 days/wk = 0; ≥30 min/day, ≥5 days/wk = 1), sedentary behavior (≥8 hrs/day, ≥5 days/wk = 0; ≥8 hrs/day, <5 days/wk or <8 hrs/day, ≥5 days/wk = 1).

7. Justify your final sample size to ensure that you will get meaningful data with this design and your anticipated analytical approach.

Blood pressure and microneurography studies. With an alpha error rate of 5%, 9 subjects per group will provide 80% power to detect a difference in the increase in BP during physiological stress of 10 mmHg (2 group ANOVA, power=0.80), Running the same model for microneurography (changes in MSNA/SSNA), we calculate that 10 subjects/group for MSNA and 17 subjects/group for SSNA will provide sufficient power (2 group ANOVA, power=0.80). We will increase the number of subjects to 13 (MSNA) and 20 (SSNA) in each group to account for potential experimental failure rate for microneurography.

Microdialysis studies . We calculate10 subjects per group will be sufficient (2 group ANOVA, power=0.80, α=0.05) to find a 15% difference between the groups in skin blood flow responses. We will increase our sample size to 12 per group to account for the potential for experimental failure with microdialysis perfusion.

REFERENCES

Amagasa S, Machida M, Fukushima N, Kikuchi H, Takamiya T, Odagiri Y & Inoue S. (2018). Is objectively measured light-intensity physical activity associated with health outcomes after adjustment for moderate-to-vigorous physical activity in adults? A systematic review. International Journal of Behavioral Nutrition and Physical Activity 15.

Asztalos M, De Bourdeaudhuij I & Cardon G. (2010). The relationship between physical activity and mental health varies across activity intensity levels and dimensions of mental health among women and men. Public Health Nutrition 13, 1207-1214.

Blood Pressure Lowering Treatment Trialists C. (2014). Blood pressure-lowering treatment based on cardiovascular risk: a meta-analysis of individual patient data. Lancet 384, 591-598.

Delaney EP, Greaney JL, Edwards DG, Rose WC, Fadel PJ & Farquhar WB. (2010). Exaggerated sympathetic and pressor responses to handgrip exercise in older hypertensive humans: role of the muscle metaboreflex. Am J Physiol Heart Circ Physiol 299, H1318-1327.

Ekblom-Bak E, Ekblom Ö, Bergström G & Börjesson M. (2016). Isotemporal substitution of sedentary time by physical activity of different intensities and bout lengths, and its associations with metabolic risk. European Journal of Preventive Cardiology 23, 967-974.

Fonkoue IT & Carter JR. (2015). Sympathetic neural reactivity to mental stress in humans: test-retest reproducibility. Am J Physiol Regul Integr Comp Physiol 309, R1380-1386.

Grassi G, Seravalle G, Turri C, Bertinieri G, Dell'Oro R & Mancia G. (2003). Impairment of thermoregulatory control of skin sympathetic nerve traffic in the elderly. Circulation 108, 729-735.

Haskell WL, Lee I-M, Pate RR, Powell KE, Blair SN, Franklin BA, Macera CA, Heath GW, Thompson PD & Bauman A. (2007). Physical activity and public health: updated recommendation for adults from the American College of Sports Medicine and the American Heart Association. Circulation 116, 1081.

Ross R, Chaput J-P, Giangregorio LM, Janssen I, Saunders TJ, Kho ME, Poitras VJ, Tomasone JR, El-Kotob R, McLaughlin EC, Duggan M, Carrier J, Carson V, Chastin SF, Latimer-Cheung AE, Chulak-Bozzer T, Faulkner G, Flood SM, Gazendam MK, Healy GN, Katzmarzyk PT, Kennedy W, Lane KN, Lorbergs A, Maclaren K, Marr S, Powell KE, Rhodes RE, Ross-White A, Welsh F, Willumsen J & Tremblay MS. (2020). Canadian 24-Hour Movement Guidelines for Adults aged 18–64 years and Adults aged 65 years or older: an integration of physical activity, sedentary behaviour, and sleep. Applied Physiology, Nutrition, and Metabolism 45, S57-S102.

Thompson CS, Holowatz LA & Kenney WL. (2005). Cutaneous vasoconstrictor responses to norepinephrine are attenuated in older humans. Am J Physiol Regul Integr Comp Physiol 288, R1108-1113.