APLICACIÓN DE ENCUESTAS DIRIGIDAS AL DOCENTE

 A fundamental weakness of the research presented in this thesis is that it is primarily descriptive in nature. While mechanisms for upregulation of the various acid/base transporters were proposed, without direct measurement of, for example, muscle La− and H+ during and after exercise, these remain speculative. Measuring changes in substrates and metabolites following a representative training session, or estimating the maximal accumulated O2 deficit from the V̇O2 versus power output relationship, or perhaps

comparing the first and last sessions, could have provided some invaluable data to support more robust conclusions. Inferences have been made on the basis that there were contrasting bioenergetic costs of different intensities of HIT, or through having differing rest interval durations during SIT, but no direct measurements were made to support these assumptions. In addition, calculation of La−/H+ production and subsequent flux, through measurement of arteriovenous differences (13), and using microdialysis to measure interstitial changes at the exercising muscle (453), may have helped support or falsify some of the suppositions contained in this thesis.

 This thesis adds to the body of research showing a dissociation between changes in pHi

regulation and changes in exercise performance, in particular in the more highly trained (18, 150, 186, 187, 232, 468). However, the functional relevance of the acid/base transporters and muscle buffer capacity for exercise capacity, or their potential role in mitigating fatigue, is uncertain, and has not been addressed by the current research. While this can be estimated by correlating changes in protein content with changes in performance, for high precision of estimation, sample sizes of 30 or more are probably needed (cf. Figure 2.10).

 Four weeks of HIT or SIT elicited only modest performance gains in the two training studies and may have provided insufficient training stimulus to thoroughly investigate adaptations in pHi regulation. Without a larger effect of training on performance, it was

not possible to relate reversal of adaptations in protein content to potential reductions in performance. Similarly, the modest improvements in performance, in particular aerobic capacity, indicate that longer interventions were needed to observe larger effects of training in these active populations.

 Fractionation of muscle prior to immunoblotting may have introduced error to measurement of protein content in this thesis, and indeed all previous research into the acid/base transport proteins. If cellular distribution of proteins varies at different timepoints following exercise, measuring protein content in crude cytosolic and, especially, membrane fractions, risks discarding some of the target protein. Recent novel techniques for measuring protein content in unspun whole muscle homogenate, or in single muscle fibres (336), are recommended in future.

 A limitation of study 1 was the failure to take a resting muscle biopsy on the same day as the high-intensity exercise bout. The initial rationale was to minimise the number of biopsies taken, of which, there were already nine. It was reasoned that the response of the H+ transporters over a 72-h period following exercise was of primary interest, and therefore a same day baseline measurement of protein content was not essential. In hindsight, this was a poor decision. A resting biopsy on the same day as exercise could have answered whether MCT1 and NHE1 protein content decreased following exercise, and subsequently returned to pre-exercise levels 24–72 h later, or if there was a delayed increase in protein synthesis in response to a single exercise bout. Furthermore, without a pre-exercise biopsy it is unknown whether βmin vitro increased immediately post-exercise

and decreased thereafter. One might assume that the highest βmin vitro post-exercise in

study 1 was because of greater phosphate buffering, but study 3 showed no change in βmin vitro immediately after a bout of SIT. It is therefore unclear how the post-exercise

βmin vitro data fits in with the sample-to-sample variability of the titration assay. Finally, it

would also be essential to have a same-day resting biopsy for potential future measurement of changes in mRNA content post-exercise.

 Short-term changes in protein content post-exercise in the first study may have been an artefact of post-translational modification, such that antibodies may not have bound to target proteins that had been modified by the cellular stress of exercise. NHE1 can be both phosphorylated and glycosylated, and although the MCTs are not glycosylated (188), they do form heterodimers with glycosylated basigin, the expression of which has been shown to decrease 6 h after SIT (129). Therefore, it is unclear whether the cause of reduced protein abundance was, for example, inhibited transcription (285), or increased protein carbonylation (129), or was an artefact of lower antibody binding affinity for post-translationally modified protein sequences.

 Measuring target protein content in homogenised wet muscle potentially leads to contamination by erythrocyte content of the same protein, notably the MCTs and CAs (70). While samples were carefully blotted free of blood prior to snap-freezing in liquid nitrogen, it is possible that erythrocyte contamination affected results. Freeze-drying muscle and dissecting samples free of blood prior to preparation for immunoblotting, as others have done for some of these target proteins, e.g., Bangsbo et al. (18), may be advisable.

 Despite demonstrating in the literature review that existing NBC protein evidence in human muscle cannot be confidently determined to be the NBCe1 isoform without further validation, this thesis relied on apparent molecular mass to identify NBCe1. Unfortunately, full length recombinant NBCe1 protein could not be sourced, nor could a reliable overexpression lysate. The antibody used in this thesis was raised against the C-

terminus of the NBCe1 protein and deemed by Cell Signaling to be isoform-specific, unlike the previously used N-terminus antibody, which is described by Merck Millipore as being raised against a sequence common to most NBC isoforms. And the N-terminus shows greater sequence similarity between SLC4 proteins. Nevertheless, while it is probable that NBCe1 was the isoform detected here, without further validation it is not certain.

 The first study has raised doubts about the ecological validity of the βmin vitro technique

because of the large between-sample variability compared to the typical effect of training. In addition, because of the failure to observe group mean changes in either training study, it was not possible to confidently determine the effects of different training modalities on actual muscle buffer capacity, or the subsequent effects of training cessation or reduced training volume.