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Implications of stress-induced dsRNA in melanoma in the context of activating MAPK mutations in vitro/ex vivo

Mon, 01 Jan 2024 00:00:00 GMT

Title: Implications of stress-induced dsRNA in melanoma in the context of activating MAPK mutations in vitro/ex vivo Authors: Sadeq, Shaymaa Abstract: Many studies in the past 10 years have drawn attention to the presence of endogenous RNA structures that can adopt double strand form (Dhir et al., 2018). These dsRNA have been proposed to play important biological roles in the pathogenesis of many inflammatory and autoimmune diseases as well as cancers (Hur, 2019a). The exact trigger for the forma4on of these dsRNA has yet to be identfied. Moreover, there is no direct method to quantify and locate dsRNA inside cells. This thesis will discuss the dsRNA stress response in melanoma in vitro/ex vivo. DsRNA derived from repetitive DNA elements has the potential to induce an interferon response that renders resistant melanoma sensitive to immunotherapy. As a consequence, dsRNA has the potential to add a new approach to treating melanoma. Tissue culture and molecular biology were used to induce dsRNA formation in melanoma cell lines using hypomethylating drugs and other stressors. The impact was measured by quantifying cytosolic dsRNA sensors, also known as pattern recognition receptors PRRs (pPKR, MDA5, RIG1, and ADAR1) using RT-qPCR and western bloVng. Immunofluorescence and high- resolution microscopy were then applied to investigate the co-localisation of dsRNA and its sensors in melanoma cell lines and melanoma patient skin biopsy samples. Moreover, a novel method of labelling dsRNA by flow cytometry to investigate its effect on the cell cycle and proliferation in BRAF mutant and WT cell lines was performed. Analysis revealed that inducing endogenous dsRNA significantly activates PRRs and promotes subcellular co-localisation with dsRNA and the mitochondria. FACS experiments indicated a potential role of dsRNA and PKR in regulating the cell cycle and proliferation. To summarize, our data support the hypothesis that inducing endogenous dsRNA activates dsRNA sensors and triggers innate immune signalling. Hence, dsRNA signalling may be explored as a potential therapeutic strategy to treat resistant melanoma. Description: Ph. D. Thesis.

Building a 3D model of the human keratoconic cornea

Wed, 01 Jan 2020 00:00:00 GMT

Title: Building a 3D model of the human keratoconic cornea Authors: Volatier, Thomas Abstract: Keratoconus is a corneal disease characterized by the affected tissue adopting a conical shape, leading to loss of vision and compromised structural integrity. The disease is manageable in its early stage with lenses or glasses and can be treated in its later stages with surgery or crosslinking. The underlying cause of the disease is still poorly understood, a model would aid the exploration of this disease. In this study, primary stromal cells harvested from healthy and keratoconic corneas using a variety of extraction methods were cultured in compressed collagen gels and serum-free medium containing retinoic acid to simulate the cornea’s natural environment. The survival of stromal cells and their gene expression was assayed, paying special attention to genes related to differentiation and ECM maintenance. Among healthy cells, differences were identified between limbal and central population while central keratoconic cells more closely resembled healthy limbal cells. Use of retinoic acid as a medium supplement allowed for the culture of cells in serum-free conditions and caused gene expression that resembled in vivo behaviour. The final addition of ECM in the form of curved, compressed collagen gels to this culture system caused the stromal cells to radically change their behaviour and reaction to retinoic acid. Overall, this study identified a stromal tissue model as well as the multiple considerations in the construction of such a model. The use of this model in keratoconus research may lead to breakthroughs in attempts to better understand the disease. Description: PhD Thesis

Benzene exposure from automobiles fuelled with petrol

Tue, 01 Jan 2002 00:00:00 GMT

Title: Benzene exposure from automobiles fuelled with petrol Authors: Al-Khulaifi, Nabeel Abstract: Benzene is a leukaemogenic and mutagenic agent, which may pose a risk to the general public even at low levels of exposure. Since petrol fuel contains a high concentration (1-5%) of benzene, there is the potential for exposure to man during car journeys. The main aim of this study was to develop and validate a sensitive method to detect urinary t,t-muconic acid (uMA) following low level environmental exposures to benzene. Subjects potentially exposed to benzene were divided into petrol (n= 9) and diesel groups (n= 7). The control group (n=14) consisted of individuals who were not exposed to benzene inside the car. The uMA method developed during this study involved butanol extraction instead of the traditional solid phase extraction followed by DV (259nm) detection. The method was reasonably precise (CV=1.5%) with >80% recovery from urIne. Air samples were collected on charcoal tubes and analysed for benzene, toluene, ethylbenzene, and xylenes by GCMS following extraction with purified carbon disulphide. The benzene concentration of ambient air samples taken from inside the cabins of petrol fuelled cars (7.5 ppb) was about triple that found from diesel-fuelled cars (2.6 ppb)(P=O.Ol). The uMA of volunteers exposed to petrol increased (p<0.01) post-sample in compared to pre-exposure level (0.66mgMA/gCr and 0.38mgMA/gCr, respectively). There was no increase in uMA for volunteers exposed to diesel. The uMA level of samples collected from individuals 2h-7h after exposure to petrol showed a significant association with the air benzene (p=0.012) and toluene (p=0.042) concentrations taken inside the car cabins. Half of the 24h-profiles of individuals exposed to petrol had at least one urine with 1 mgMA/gCr or higher, while all of the profiles of controls were below 1 mgMA/gCr. The technique developed in this study for the determination uMA showed promise as a tool for monitoring levels of benzene arising from low-level environmental exposures to petrol. Description: PhD Thesis

Adjuncts to pre-hospital resuscitation strategies for haemorrhagic shock and blast injury : supplemental oxygen and recombinant activated factor VII

Tue, 01 Jan 2013 00:00:00 GMT

Title: Adjuncts to pre-hospital resuscitation strategies for haemorrhagic shock and blast injury : supplemental oxygen and recombinant activated factor VII Authors: Granville-Chapman, Jeremy Abstract: Explosion is responsible for almost 80% of Coalition injuries in today’s conflicts. Haemorrhage is the leading cause of death and blast lung injury is evident in 11% of Coalition casualties surviving to reach the (UK) Field Hospital. Military prehospital evacuation times can be prolonged and the combined insults of haemorrhage and blast injury present a ‘double hit’ to oxygen delivery. Resuscitation strategies must be capable of preserving life from such trauma for several hours. Alongside fluid therapy, adjuncts to resuscitation might improve battlefield survival. This randomized controlled animal trial assessed two adjuncts: supplemental inspired oxygen and recombinant activated Factor VII (rFVIIa). Neither adjunct is currently available in the far-forward military echelon, but with modern technology, both are potentially deployable. 18 terminally anaesthetized swine were exposed to blast, controlled haemorrhage and grade IV liver laceration (uncontrolled haemorrhage). Animals were allocated randomly into three treatment groups. All animals were resuscitated with normal saline to a hypotensive systolic target (80mmHg), which continued until the 8hr end point. Thirty minutes after the onset of resuscitation each group received one of the following: single (180mcg/kg) dose of rFVIIa; supplemental oxygen (min FiO2 0.3 to maintain SaO2>95%) or the control group (breathed air throughout and received saline placebo 0.18ml/kg). 5/6 control animals died within 4 hours. Supplemental oxygen improved survival (4/6 survival to 8h endpoint, P=0.014). Single dose rFVIIa did not prolong survival compared to control (2/6 survived, p=0.65). Oxygen arrested physiological decline while control and rFVIIa animals continued to decline until death. Supplemental oxygen is a useful adjunct to fluid resuscitation in the context of haemorrhage and blast injury. Delivery of oxygen support capability to forward echelon units is recommended. By contrast, a single intravenous (pre-hospital) dose of rFVIIa was not an effective treatment for blast lung based on our model of complex battlefield injury. Description: M.D. Thesis