Probing the protein homeostasis mechanisms in long-lived naked mole-rats

The naked mole-rat (NMR) is a fascinating animal which has unique biological features including eusociality, strict subterranean inhabitation and poikilothermy. It is the longest-living rodent, showing negligible senescence over the majority of its lifespan and high resistance to diseases such as cancer and neurodegeneration. This animal is therefore a compelling system for understanding ageing and age-related diseases. Recent evidence suggests that protein homeostasis (proteostasis) mechanisms may play a vital role in mediating the resistance to multiple forms of stress and diseases and, subsequently, contribute to the exceptional longevity of the NMR. However, this view has been predominately based on protein-level and/or cell viability analyses, and our knowledge is still limited about the modulation of proteotoxic stress responses at the transcriptional level due to a lack of reliable and validated molecular tools. This thesis sets out to develop, optimise and apply new methods to investigate two important and complex proteostatic mechanisms, namely the unfolded protein response (UPR) in the endoplasmic reticulum (ER) and macroautophagy (autophagy), in the NMR. Using these methodologies, the effects of pharmacologically induced in vitro stress and the effects of disease-related neurotoxic protein species on the UPR and autophagy in NMR fibroblasts were investigated. In Chapter 3, RNA-based methods, including an Xbp1 splicing assay and quantitative PCR (RT-qPCR) assays were successfully established to probe the activation and outputs of the UPR in a NMR kidney fibroblast cell line in response to tunicamycin (TU) and thapsigargin (TG)-induced ER stress. In Chapter 4, differences between the UPR of NMR kidney fibroblasts and mouse homologues were identified, where a notably higher threshold of pharmacologically induced UPR activation was observed in the NMR under conditions of mild ER stress. In Chapter 5, LC3B turnover and transcriptional changes of autophagy markers under rapamycin (RA) and chloroquine (CQ)-treated conditions were monitored in an NMR skin fibroblast cell line, where the sensitivity of the NMR skin fibroblasts to CQ, when compared to NMR kidney fibroblasts and mouse NIH3T3 embryonic cells, seemed to be partly attributed to the downregulation of TFEB, a master transcription factor of autophagy. In Chapter 6, the effects of amyloid-beta (Aβ) and α-synuclein oligomers, which are believed to be the major pathogenic species of Alzheimer’s and Parkinson’s diseases, respectively, on the UPR and/or autophagy were investigated in NMR and mouse cells using a combination of molecular and cellular tools. Although no significant changes of UPR markers were observed under Aβ oligomer-treated conditions, the chronic toxicity of wild-type α-synuclein oligomers seemed to be associated with downregulation of genes encoding ER chaperones and autophagy proteins. In Chapter 7, we demonstrate the utility of rational design to create a protein-specific binding probe for the NMR LC3B protein by introducing a peptide derived from a LC3-interacting region (LIR) motif into the inter-repeat loop of a consensus-designed tetratricopeptide repeat protein (CTPR). The results provide proof-of-concept validation of using CTPR-based probes to detect proteins in emerging animal models. Having established a set of reliable methods to investigate the molecular details of the UPR and autophagy in the NMR, we have demonstrated unique features of the NMR, at the transcriptional level, when different forms of in vitro stress are employed. Exploiting these assays to measure the UPR and autophagy, as well as other proteostastic mechanisms, in the NMR under more disease-relevant conditions, may ultimately shed light on therapeutic developments to combat age-related neurodegenerative diseases

Elastic-Plastic Fracture Mechanics Analysis of a Tubular T-Joint in Offshore Structures

A tubular welded T-joint containing a series of semi-elliptical cracks of increasing depth located near the chord-brace intersection under both elastic and elastic-plastic conditions, has been analysed using shell elements with the cracks modelled by line springs. The same problem has also been modelled with 20 noded bricks allowing the stress intensity factor and J integral to be determined by virtual crack extension. The direction of crack growth has been determined both using off-axis virtual crack extension, and solutions for kinked cracks, to determine the orientation which maximise either the strain energy release rate or the mode I stress intensity factor. The calculated crack path agrees with reported experiments. The stress intensity factors for straight and curved cracks in simple welded joints have been compared in terms of their effective depth. Finally, the stress field in single edge bars under mixed mode elastic-plastic loading condition relevant to tubular joints has been analysed, and the size requirement for J dominance is discussed

A linear method to extract diode model parameters of solar panels from a single I–V curve

The I-V characteristic curve is very important for solar cells/modules being a direct indicator of performance. But the reverse derivation of the diode model parameters from the I-V curve is a big challenge due to the strong nonlinear relationship between the model parameters. It seems impossible to solve such a nonlinear problem accurately using linear identification methods, which is proved wrong in this paper. By changing the viewpoint from conventional static curve fitting to dynamic system identification, the integral-based linear least square identification method is proposed to extract all diode model parameters simultaneously from a single I-V curve. No iterative searching or approximation is required in the proposed method. Examples illustrating the accuracy and effectiveness of the proposed method, as compared to the existing approaches, are presented in this paper. The possibility of real-time monitoring of model parameters versus environmental factors (irradiance and/or temperatures) is also discussed

Study on the weight coefficient influence of surface water on the stability of open-pit dump. Comparative analysis of 6 degree seismic simulation

Taking Baorixile open-pit coal mine as an example, the regional hydrogeological conditions and the mining area hydrogeological conditions are briefly introduced. Analyzing the factors influencing the stability of open pit dump, discussing the classification and proportion of influencing factors, the numerical simulation of the influence of surface water and seismic vibrations on the stability of open-pit dump is carried out. Results show: Surface water is an important factor affecting the stability of open-pit dump, extremely sensitive to the safety factor, the influence of slope instability is similar to that of intensity 6 earthquake. The weight coefficient of surface water to slope stability is in the range of 0.4 to 0.9