Computational strategies to study skeletal muscle transcriptome responses to resistance training

© Yusuf Khan (2021)Livsstilsrelaterte sykdommer er utbredt, og forekomsten øker i en alarmerende hastighet i den menneskelige befolkningen. Disse sykdommene er forbundet med aldring, en stillesittende livsstil og/eller dårlig kosthold, og er vanligvis ledsaget av tap av muskelstyrke, muskelmasse og endringer i muskelbiologi. Sistnevnte kan behandles og forhindres av vektbærende treningsøvelser (motstandstrening), som stimulerer vekst og opprettholder helsen til menneskelig skjelettmuskel, det største organet i kroppen. RNA-seq er en mye brukt metode for å studere cellulære egenskaper og endringer derav (inkludert muskelplastisitet) under forskjellige forhold på et gitt tidspunkt. Den produserer en stor mengde data, og mange verktøy har blitt brukt for å lette analysen. Flere studier har fokusert på å sammenligne forskjellige verktøy for RNA-seq analyse. Imidlertid er det for øyeblikket ingen pipeline spesifikk for å analysere skjelettmuskeldata eller transkriptomrespons på motstandstrening. Derfor er det avgjørende å identifisere verktøy spesielt for motstandstreningsindusert vekst i skjelettmuskulatur. I tillegg er hvert datasett annerledes og trenger grundig evaluering av verktøy for å bestemme hvilket som er best for et gitt prosjekt. Hovedmålet med oppgaven var å utvikle RNA-seq dataanalyse strategier for å analysere transkriptomresponser av skjelettmuskulatur på motstandstrening og å bruke disse til å studere to forskjellige datasett. YOUNG datasett (18 - 40 år) - RNA-seq data fra muskelbiopsier samlet fra deltakere trent i 12 uker med to forskjellige treningsvolumer. For dette datasettet var målet å undersøke virkningene av treningsvolum for transkriptomresponser. OLDER datasett (56 - 77 år) - RNA-seq fra muskelbiopsier samlet fra deltakere, både friske og med kronisk obstruktiv lungesykdomsdiagnose (KOL), som ble trent i 13 uker med to forskjellige treningsbelastninger og utstyrt med enten vitamin D3 eller placebo tilskudd. For dette datasettet var målet å undersøke virkningen av vitamin D3-tilskudd og KOL-diagnose for transkriptomresponser. Det YOUNG datasettet ble brukt til å utvikle den standardiserte RNA-seq pipeline. Bioinformatiske verktøy ble valgt ut fra lesekvalitet, observerte gentall, metodologisk variasjon mellom parede observasjoner og korrelasjoner mellom mRNA-overflod og proteinuttrykk av myosin tungkjede familieproteiner. Basert på å utforske effekten av å bruke forskjellige normaliseringsstrategier, antyder våre resultater at det kan være nødvendig å ta hensyn til biologisk skjevhet forårsaket av globale endringer i den totale RNA (per vevsmasse) for å øke den biologiske relevansen av transkriptomanalyser. Denne utviklede pipeline ble brukt på de YOUNG og OLDER datasettene for å gi en bedre forståelse av muskeltransskriptomresponser. I det YOUNG datasettet var de to 6 forskjellige motstandstreningsvolumene assosiert med differensial uttrykk av 21 gener i uke 2, hovedsakelig relatert til ekstracellulær remodellering. I motsetning ble det ikke observert noen lett forklarbare doseavhengigheter i uke 12. I det OLDER datasettet påvirket verken vitamin D3-tilskudd eller KOL-diagnose transkriptomresponsene på trening, når estimert på enkeltgenivå. Anrikningsanalyser viste imidlertid at begge forholdene var forbundet med små effekter på responsmønstre. For eksempel var vitamin D3 assosiert med økt uttrykk av gensett som er involvert i endotelproliferasjon og blodkarmorfogenese sammenlignet med placebo (uke 13), og KOL var assosiert med mer markante reduksjoner i gensett knyttet til oksidativ fosforylering og myogenese. Resultatene fra denne studien kan potensielt bidra til å forstå transkriptomiske endringer i trening og faktorer som påvirker treningsrespons, noe som kan bidra til å designe bedre treningsprotokoller for sunn aldring og liv.Summary in English Lifestyle-related diseases are widespread, and their incidences are increasing at an alarming rate in the human population. These diseases are associated with ageing, a sedentary lifestyle and/or poor diet and are usually accompanied by loss of muscle strength, muscle mass, and changes in muscle biology. The latter may be treated and prevented by weight-bearing training exercises (resistance training), which stimulates growth and maintain the health of the human skeletal muscle, the largest organ in the body. RNA-seq is a widely used method to study cellular characteristics and changes thereof (including muscle plasticity) under different conditions at a given time point. It produces a large amount of data, and many tools and different pipelines have been used to facilitate analysis. Several studies have focused on comparing different tools for RNA-seq data analysis. However, currently, there is no pipeline specific for analysing skeletal muscle data or transcriptome response to resistance training. Therefore, it is crucial to identify tools specifically for resistance training-induced growth in skeletal muscle. Moreover, each dataset is different and needs careful evaluation of tools to decide which is best for a given project. The main aim of the thesis was to develop RNA-seq data analysis strategies to analyse skeletal muscle transcriptome responses to resistance training and to employ these to study two different datasets. YOUNG dataset (18 – 40 years) – RNA-seq data from muscle biopsies collected from participants trained for 12 weeks with two different training volumes. For this dataset, the aim was to investigate the impacts of training volume for transcriptome responses. OLDER dataset (56 - 77 years) – RNA-seq from muscle biopsies collected from participants, both healthy and with chronic obstructive pulmonary disease diagnosis, which were trained for 13 weeks with two different training loads and provided with either vitamin D3 or placebo supplementation. This dataset aimed to investigate the impact of vitamin D3 supplementation and COPD diagnosis for transcriptome responses. The YOUNG dataset was used to develop the standardised RNA-seq pipeline. Bioinformatic tools were selected based on read quality, observed gene counts, methodological variation between paired observations, and correlations between mRNA abundance and protein expression of myosin heavy chain family proteins. Based on exploring the effects of using different normalisation strategies, our results suggest that it may be necessary to account for biological biasness caused by global changes in the total RNA population (per tissue mass) to increase the biological relevance of transcriptome analyses. This developed pipeline was used on the YOUNG and OLDER datasets to understand better muscle transcriptome responses to the above-mentioned resistance training interventions. In the YOUNG dataset, the two different resistance training volumes were associated with differential expression of 21 genes at week 2, mostly related to extracellular remodelling. In contrast, no readily explainable dose-dependencies were observed at Week 12. In the OLDER dataset, neither vitamin D3 supplementation nor COPD diagnosis affected transcriptome responses to training, as estimated at the single-gene level. However, enrichment analyses revealed that both conditions were associated with slight effects on response patterns. For example, vitamin D3 was associated with increased expression of gene sets involved in endothelial proliferation and blood vessel morphogenesis compared to placebo (Week 13). COPD was associated with more pronounced decreases in gene sets relating to oxidative phosphorylation and myogenesis. The results from this study could potentially help understand transcriptomic changes of training and factors affecting training response, which could help design better training protocols for healthy ageing and life.publishedVersio

Atypical Teratoid Rhabdoid Tumor: two Case Reports and an Analysis of Adult Cases With Implications for Pathophysiology and Treatment

We present the first quantitative analysis of atypical teratoid rhabdoid tumors (ATRT) in adults, including two patients from our own institutions. These are of interest as one occurred during pregnancy and one is a long-term survivor. Our review of pathological findings of 50 reported cases of adult ATRT leads us to propose a solely ectodermal origin for the tumor and that epithelial-mesenchymal transition (EMT) is a defining feature. Thus, the term ATRT may be misleading. Our review of clinical findings shows that ATRT tends to originate in mid-line structures adjacent to the CSF, leading to a high rate of leptomeningeal dissemination. Thus, we hypothesize that residual undifferentiated ectoderm in the circumventricular organs, particularly the pituitary and pineal glands, is the most common origin for these tumors. We note that if growth is not arrested soon after diagnosis, or after the first relapse/progression, death is almost universal. While typically rapidly fatal (as in our first case), long-term remission is possible (as in our second). Significant predictors of prognosis were the extent of resection and the use of chemotherapy. Glial differentiation (GFAP staining) was strongly associated with leptomeningeal metastases (chi-squared p = 0.02) and both predicted markedly worse outcomes. Clinical trials including adults are rare. ATRT is primarily a disease of infancy and radiotherapy is generally avoided in those aged less than 3 years old. Treatment options in adults differ from infants in that cranio-spinal irradiation is a viable adjunct to systemic chemotherapy in the adult population. Given the grave prognosis, this combined approach appears reasonable. As effective chemotherapy is likely to cause myelosuppression, we recommend that stem-cell rescue be available locally

Automated and Standardized Tools for Realistic, Generic Musculoskeletal Model Development

Human movement is an instinctive yet challenging task that involves complex interactions between the neuromusculoskeletal system and its interaction with the surrounding environment. One key obstacle in the understanding of human locomotion is the availability and validity of experimental data or computational models. Corresponding measurements describing the relationships of the nervous and musculoskeletal systems and their dynamics are highly variable. Likewise, computational models and musculoskeletal models in particular are vitally dependent on these measurements to define model behavior and mechanics. These measurements are often sparse and disparate due to unsystematic data collection containing variable methodologies and reporting conventions. To date, there is not a framework to concatenate and manage musculoskeletal data (muscle moment arms and lengths). These morphological measurements need to be assembled to manage, compare, and analyze these data to develop comprehensive musculoskeletal models. Such a framework would enable researchers to select and update the posture-dependent relationships necessary to describe musculoskeletal dynamics, which are essential for simulation of muscle and joint torques in movement. Analogous to all simulations, these models require rigorous validation to ensure their accuracy. This is particularly important for musculoskeletal models that represent high-dimensional, posture-dependent relationships developed from limited and variable datasets. Here, I developed a computational workflow to collect and manage moment arm datasets from available published literature for the development of a human lower-limb musculoskeletal model. The moment arm relationships from multiple datasets were then used to create complete moment arm descriptions for all major leg muscles and were validated within a generic musculoskeletal model. These developments are crucial in advancing musculoskeletal modeling by providing standardized software and workflows for managing high-dimensional and posture-dependent morphological data to creating realistic and robust musculoskeletal models

The importance of a protocol in the recovery and handling of burned human remains in a forensic context

Fire-related fatalities pose many investigative challenges, in part due to their fragility. This can be managed with the creation of protocols, specific to the environment in which they are implemented. Currently, no protocol for the recovery and handling of fire-related fatalities exists in Cape Town, South Africa. Additionally, the challenges, risk factors, and resources present at forensic scenes in the area have not been documented. From April to December of 2015, fire-related death scenes were attended with Salt River Forensic Pathology Laboratory, which serves the West Metropole of Cape Town. Details of the fatal fire scenes were noted, including the challenges faced, and the settings in which the fires occurred. Emphasis was placed on methodologies used to recover, handle, and transport remains, and the availability and utilisation of resources. The affect these methodologies had on the condition of the remains between scene and autopsy was assessed. In total 32 fire-related death scenes were attended, with 48 decedents recovered. Males predominated (64.6%), and the majority were young adults (75%). Accidental deaths were most prevalent (79.2%), however a fire-related suicide and homicides highlighted the importance of thorough investigation. Informal housing constituted 68.8% of the fatal fire scenes and presented unique scene constraints, including no direct road access at 50% of these scenes. Investigative limitations included: inadequate interagency communication, resulting in a lack of collateral information available at autopsy; deficient scene and contextual documentation; non-standardised recovery methodologies; insufficient availability and utilisation of resources (including safety equipment); and no specialised personnel (e.g. forensic pathologists/ anthropologists) conducting scene recovery. The majority of cases (60.4%) were further fragmented or fractured by time of autopsy, illustrating the necessity for improvement of current methodologies and the importance of the involvement of forensic anthropologists in recovery of fragmentary remains