Proteomic analysis of diurnal variation in human skeletal muscle performance

Phenotyping of human muscle based on its profile of myosin heavy chain isoforms is commonly used to help understand changes in muscle function. However, in many instances, measureable changes in force output or contractility occur in the absence of any change in myosin heavy chain profile. Therefore, more sophisticated analysis is required. Proteomic techniques including 2-dimensional gel electrophoresis, high- performance liquid chromatography and peptide mass spectrometry can be used to investigate changes in the abundance of hundreds of proteins simultaneously. To date, such techniques have not been used to specifically characterise the human myofibrillar proteome, or study how the myofibrillar proteome relates to muscle outputs such as peak isometric force or the velocity of contraction. This thesis presents a series of studies that develop proteomic techniques for the analysis of myofibrillar proteins as well as optimisation of techniques for measuring the range of muscle output from isometric through to velocity maximum of the human knee extensor muscles in vivo. After optimisation, the proteomic and muscle function measurement were employed to study diurnal variation. Time-of-day differences in sports performance and muscle function are widely reported, and typically, performance is ~10 % greater in the evening compared to the morning. This is consistent with our result in Chapter 3; we investigated this chapter by conducting a battery of muscle performance tests in a population of well-familiarised participants. Our data show that RFD exhibits the greatest diurnal variation (18 %) followed by isometric force (10.2 %). The diurnal variation in IKD data was less robust (range 8.1 - 9.8 %), which may have been due to the lesser precision of this technique compared to MVC and RFD. Therefore MVC and RFD were used in the final study. In final study, this thesis reports significantly (P<0.05) greater peak isometric force (11 %) and rate of force development (16 %) of knee extensor muscles of young strength-trained males in the evening compared to morning. Proteomic analysis of biopsy samples of the vastus lateralis profiled more than 100 myofibrillar protein species and detected 8 significant differences in protein abundance between morning and evening samples. The greatest difference was in the abundance of the slow isoform of myosin binding protein C (MyBPC1), which is known to modulate the activity of actin-bound myosin ATPases. MyBPC1 was resolved to 6 species; therefore the difference in abundance of one species reported here likely represents a change in post-translational modification. Therefore, this thesis provides associational evidence that post-translational modification of MyBPC1 contributes to the diurnal variation in muscle function

Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1.

Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. Our study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function

Security lies in obedience - Voices of young women of a slum in Pakistan

<p>Abstract</p> <p>Background</p> <p>Existing literature shows that young people, especially women, have poor knowledge about sexuality and reproductive health. Many of the difficulties young women experience are related to beliefs and expectations in society making them more vulnerable to reproductive ill health. The objective of this study was to explore how young women living in a slum in Islamabad are prepared for marriage and how they understand and perceive their transition to marriage and the start of sexual and childbearing activity.</p> <p>Methods</p> <p>Twenty qualitative interviews and three focus group discussions were conducted with young women residing in a slum of Islamabad. Content analysis was used to explore how the participants represented and explained their situation and how decisions about their marriage were made.</p> <p>Results</p> <p>The main theme identified was <it>security lies in obedience</it>. The two sub-themes contributing to the main theme were <it>socialization into submissiveness </it>and <it>transition into adulthood in silence</it>. The theme and the sub-themes illustrate the situation of young women in a poor setting in Pakistan.</p> <p>Conclusion</p> <p>The study demonstrates how, in a culture of silence around sexuality, young women's socialization into submissiveness lays the foundation for the lack of control over the future reproductive health that they experience.</p

Multiple micronutrient supplementation improves vitamin B12 and folate concentrations of HIV infected children in Uganda: a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>The effect of multiple micronutrient supplementation on vitamin B₁₂and folate has hither to not been reported in African HIV infected children. This paper describes vitamin B₁₂and folate status of Ugandan HIV infected children aged 1-5 years and reports the effect of multiple micronutrient supplementation on serum vitamin B₁₂and folate concentrations.</p> <p>Methods</p> <p>Of 847 children who participated in a multiple micronutrient supplementation trial, 214 were assessed for vitamin B₁₂and folate concentrations pre and post supplementation. One hundred and four children were randomised to two times the recommended dietary allowance (RDA) of a 14 multiple micronutrient supplement (MMS) and 114 to a 'standard of care' supplement of 6 multivitamins (MV). Serum vitamin B₁₂was measured by an electrochemiluminescence immunoassay and folate by a competitive protein-binding assay using Modular E (Roche) automatic analyzer. Vitamin B₁₂concentrations were considered low if less than 221picomoles per litre (pmol/L) and folate if < 13.4 nanomoles per litre (nmol/L). The Wilcoxon Signed Ranks test was used to measure the difference between pre and post supplementation concentrations.</p> <p>Results</p> <p>Vitamin B₁₂was low in 60/214 (28%) and folate in 62/214 (29.0%) children. In the MMS group, the median concentration (IQR) of vitamin B₁₂at 6 months was 401.5 (264.3 - 518.8) pmol/L compared to the baseline of 285.5 (216.5 - 371.8) pmol/L, p < 0.001. The median (IQR) folate concentrations increased from 17.3 (13.5 - 26.6) nmol/L to 27.7 (21.1 - 33.4) nmol/L, p < 0.001. In the 'standard of care' MV supplemented group, the median concentration (IQR) of vitamin B₁₂at 6 months was 288.5 (198.8 - 391.0) pmol/L compared to the baseline of 280.0 (211.5 - 386.3) pmol/L while the median (IQR) folate concentrations at 6 months were 16.5 (11.7 - 22.1) nmol/L compared to 15.7 (11.9 - 22.1) nmol/L at baseline. There was a significant difference in the MMS group in both vitamin B₁₂and folate concentrations but no difference in the MV group.</p> <p>Conclusions</p> <p>Almost a third of the HIV infected Ugandan children aged 1-5 years had low serum concentrations of vitamin B₁₂and folate. Multiple micronutrient supplementation compared to the 'standard of care' supplement of 6 multivitamins improved the vitamin B₁₂and folate status of HIV infected children in Uganda.</p> <p>Trial registration</p> <p><url>http://ClinicalTrials.gov</url><a href="http://www.clinicaltrials.gov/ct2/show/NCT00122941">NCT00122941</a>)</p

Substitutions in the Amino-Terminal Tail of Neurospora Histone H3 Have Varied Effects on DNA Methylation

Eukaryotic genomes are partitioned into active and inactive domains called euchromatin and heterochromatin, respectively. In Neurospora crassa, heterochromatin formation requires methylation of histone H3 at lysine 9 (H3K9) by the SET domain protein DIM-5. Heterochromatin protein 1 (HP1) reads this mark and directly recruits the DNA methyltransferase, DIM-2. An ectopic H3 gene carrying a substitution at K9 (hH3K9L or hH3K9R) causes global loss of DNA methylation in the presence of wild-type hH3 (hH3WT). We investigated whether other residues in the N-terminal tail of H3 are important for methylation of DNA and of H3K9. Mutations in the N-terminal tail of H3 were generated and tested for effects in vitro and in vivo, in the presence or absence of the wild-type allele. Substitutions at K4, K9, T11, G12, G13, K14, K27, S28, and K36 were lethal in the absence of a wild-type allele. In contrast, mutants bearing substitutions of R2, A7, R8, S10, A15, P16, R17, K18, and K23 were viable. The effect of substitutions on DNA methylation were variable; some were recessive and others caused a semi-dominant loss of DNA methylation. Substitutions of R2, A7, R8, S10, T11, G12, G13, K14, and P16 caused partial or complete loss of DNA methylation in vivo. Only residues R8-G12 were required for DIM-5 activity in vitro. DIM-5 activity was inhibited by dimethylation of H3K4 and by phosphorylation of H3S10, but not by acetylation of H3K14. We conclude that the H3 tail acts as an integrating platform for signals that influence DNA methylation, in part through methylation of H3K9

Monosodium urate crystals promote innate anti-mycobacterial immunity and improve BCG efficacy as a vaccine against tuberculosis

A safer and more effective anti-Tuberculosis vaccine is still an urgent need. We probed the effects of monosodium urate crystals (MSU) on innate immunity to improve the Bacille Calmette-Guerin (BCG) vaccination. Results showed that in vitro MSU cause an enduring macrophage stimulation of the anti-mycobacterial response, measured as intracellular killing, ROS production and phagolysosome maturation. The contribution of MSU to anti-mycobacterial activity was also shown in vivo. Mice vaccinated in the presence of MSU showed a lower number of BCG in lymph nodes draining the vaccine inoculation site, in comparison to mice vaccinated without MSU. Lastly, we showed that MSU improved the efficacy of BCG vaccination in mice infected with Mycobacterium tuberculosis (MTB), measured in terms of lung and spleen MTB burden. These results demonstrate that the use of MSU as adjuvant may represent a novel strategy to enhance the efficacy of BCG vaccination

Coexistence of static magnetism and superconductivity in SmFeAsO1-xFx as revealed by muon spin rotation

The recent observation of superconductivity with critical temperatures up to 55 K in the FeAs based pnictide compounds marks the first discovery of a non copper-oxide based layered high-Tc superconductor (HTSC) [1-3]. It has raised the suspicion that these new materials share a similar pairing mechanism to the cuprates, since both exhibit superconductivity following charge doping of a magnetic parent material. Here we present a muon spin rotation study on SmFeAsO1-xFx (x=0-0.30), which shows that static magnetism persists well into the superconducting regime. The analogy with the cuprates is quite surprising since the parent compounds appear to have different magnetic ground states: itinerant spin density wave for the pnictides contrasted with the Mott-Hubbard insulator in the cuprates. Our findings suggest that proximity to magnetic order and associated soft magnetic fluctuations, rather than the strong electronic correlations in the vicinity of a Mott-Hubbard-metal-to-insulator transition, may be the key ingredients of HTSC.Comment: Accepted in Nature Material