The molecular basis for the clinical diversity in systemic sclerosis

Clinical heterogeneity is a cardinal feature of systemic sclerosis (SSc). Hallmark SSc autoantibodies are central to diagnosis and associate with distinct patterns of skin and organ-based complications. Understanding molecular differences between patients, which reflect clinical outcomes in a meaningful way, have the potential to benefit clinical practice and research and give insight into pathogenesis of disease. The BIOPSY (BIOlogical Phenotyping in SYstemic sclerosis) dataset provided a platform for the integrated analysis of skin and blood samples, along with detailed clinical phenotyping. Through this I was able to explore key molecular differences in early dcSSc, in the context of the full scleroderma spectrum of patients (established dcSSc and lcSSc). The work presented in this thesis demonstrates that there were molecular differences between autoantibody state in the early dcSSc cohort, in skin and blood in serum protein analytes, histological analysis and skin gene expression. These differences are not appreciated when analysed by skin trajectory over a 12-month period. This could explain the clinical heterogeneity seen in SSc, and emphasized the importance of incorporating known autoantibody states in any clinical trial analysis. This work helps move SSc therapeutic strategies towards a more personalized medicine approach, as autoantibody differences may also explain differing response to therapeutics. The BIOPSY dataset also provided an opportunity to utilise blister fluid as an anchor for identifying plasma proteins that reflect skin severity. Blister fluid provides a unique insight int the local environment of the skin. Integrating skin biopsy transcriptomics and blister fluid allowed for the development of a surrogate skin score, using these modalities to identify key analytes in the plasma, and ensuring the plasma analytes reflected skin severity in a multisystem disease. The findings from this thesis are relevant to understanding disease behaviour and potentially impact future clinical trial design and interpretation

The new technique for accurate estimation of the spinal cord circuitry:recording reflex responses of large motor unit populations

We propose and validate a non-invasive method that enables accurate detection of the discharge times of a relatively large number of motor units during excitatory and inhibitory reflex stimulations. HDsEMG and intramuscular EMG (iEMG) were recorded from the tibialis anterior muscle during ankle dorsiflexions performed at 5%, 10%, and 20% of the maximum voluntary contraction (MVC) force, in 9 healthy subjects. The tibial nerve (inhibitory reflex) and the peroneal nerve (excitatory reflex) were stimulated with constant current stimuli. In total, 416 motor units were identified from the automatic decomposition of the HDsEMG. The iEMG was decomposed using a state-of-the-art decomposition tool and provided 84 motor units (average of two recording sites). The reflex responses of the detected motor units were analyzed using the peri-stimulus time histogram (PSTH) and the peri-stimulus frequencygram (PSF). The reflex responses of the common motor units identified concurrently from the HDsEMG and the iEMG signals showed an average disagreement (the difference between number of observed spikes in each bin relative to the mean) of 8.2±2.2% (5% MVC), 6.8±1.0% (10% MVC), and 7.5±2.2% (20% MVC), for reflex inhibition, and 6.5±4.1%, 12.0±1.8%, 13.9±2.4%, for reflex excitation. There was no significant difference between the characteristics of the reflex responses, such as latency, amplitude and duration, for the motor units identified by both techniques. Finally, reflex responses could be identified at higher force (four of the nine subjects performed contraction up to 50% MVC) using HDsEMG but not iEMG, because of the difficulty in decomposing the iEMG at high forces. In conclusion, single motor unit reflex responses can be estimated accurately and non-invasively in relatively large populations of motor units using HDsEMG. This non-invasive approach may enable a more thorough investigation of the synaptic input distribution on active motor units at various force levels

Genetics of Animal Health and Disease in Livestock

Wood surface attributes can be established by examining its several different physical or chemical properties. Differences in the wood surfaces occur between the manufacturing and post-treatment processes as well. Understanding how their unique anisotropic molecular organization, chemical linkages, branching, and other molecular features govern micro- and macroscale accessibility is essential for coating and complex modification processes. It is therefore important for scientific as well as practical reasons to qualify and quantify the effects of wood surface treatments and modifications. Challenges still exist to fully understanding the effect of the numerous applied chemicals and the wide range of treatment processes on wood surfaces

CLRs and their role during Aspergillus fumigatus infection

CLRs are vital for orchestrating anti-fungal immunity. These receptors are expressed on myeloid immune cells and induce robust anti-A.f. responses including phagocytosis, cytokine and chemokine release, respiratory burst, and inflammasome activation. The role of Dectin-1 has been thoroughly investigated; however, the CLRs requirement during anti-A.f. response is controversial. The impact of Dectin-2, Mcl and Mincle during anti-A.f. immunity is not well understood. A.f. is complex and poses many challenges for the immune system. PRR collaboration is likely required for A.f. clearance. Collaboration between TLRs and CLRs has been identified. Recently, the first CLR:CLR collaboration was demonstrated between Dectin-1 and Dectin-2; however, this was not in response to A.f.. The research in this thesis describes many novel interactions between CLRs and A.f. and seeks to further the understanding of the complex collaborative anti-A.f. immune response. Firstly, a novel role for NE inducing Dectin-1 Isoform A cleavage was described. This impaired A.f. recognition and the anti-A.f. response. CF patients possess high airway NE activity and experience a severe A.f. disease burden. My research suggests blocking the action of NE in CF patient’s airway may restore Dectin-1 expression and improve patient’s anti-A.f. immune response. Secondly, in vivo CLR KO and DKO models were used to elicit alveolar macrophages reliance on CLRs when generating anti-A.f. responses. My research suggests Dectin-1 might exclusively be required during early anti-A.f. responses. Unfortunately, discrepancies between the sex and microbiome of mice restricted the conclusions drawn from in vivo CLR KO and DKO A.f. infection experiments. Finally, I identified novel risk factors that can be used to stratify patients according to their susceptibility to A.f. infection. A.f. disease incidence and mortality rates are unacceptably high in immune-suppressed patients. Patients are often prophylactically treated with inadequate anti-fungal therapeutics. Stratifying patients according to their A.f. disease susceptibility would allow a personalised medicine approach and reduce unnecessary treatment

Characterization of lung tumor-propagating cells reveals a role for CD24 and Yap/Taz in lung cancer progression and metastasis

Lung cancer is the leading cause of cancer deaths worldwide. A large part of this high mortality rate is due to the onset of metastatic disease prior to diagnosis. Advances in treatment for metastatic disease may be achieved by understanding more about the identity of metastatic tumor cells and the mechanisms those cells employ to spread throughout the body. This thesis examined the relationship between cells capable of tumor propagation upon serial transplantation (tumor-propagating cells, or TPCs) and those with metastatic potential