Importance: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the highest cause of mortality by a single infectious agent and kills 1.5 million people annually. Treatment of TB in humans is with lengthy, toxic, multidrug regimens and the increasing emergence of multidrug resistance (MDR) is a global concern. Mtb is able to evade T-cell clearance and enter a physiological persistent state which is phenotypically resistant to antimicrobials. The ability of Mtb to enter into/exit from the persistent state compounds the problem of antimicrobial resistance. Background: Persistent states are not restricted to Mtb, many bacterial species the small colony variant (SCV) is associated with persistence. The clinical importance of SCVs is highlighted by their isolation from patients with persistent, recurrent infections. SCVs show delayed growth anddefects in community behaviours such as biofilm formation and cell-cell signalling. Deficiencies in electron transport components such as haem are consistently described in SCVs suggesting an involvement for alterations in membrane potential. There has been a single description of an SCV in Mtb which was observed from clinical samples and associated with antimicrobial resistance. An association between persistence, the occurrence of SCVs and antimicrobial resistance in mycobacteria is an understudied phenomenon. As part of an RVC PhD studentship (Faulkner 2017-2020) we silenced a putative haem synthase (HAS) in a non-pathogenic model of Mtb (Mycobacterium smegmatis) and observed a SCV phenotype. In this project we explore this phenotype using a combination of molecular microbiology (Kendall, RVC), electrophysiology (Karlikowska and Stratford, Cytecom) and innovative mathematical modelling (Chang, RVC).

Strategic Research Priority: Bioscience for Health Protein quality control is vital to cellular health. Defects in protein quality control underlie a broad range of diseases, and are closely linked to the ageing process. The aim of this study is to describe the processes that regulate and modulate protein quality control at a systems-level and to identify new chemical modulators. The project will combine the genetic tractability of the yeast micromammal Schizosaccharomyces pombe with mammalian cell biology and a vertebrate zebrafish model, providing a highly detailed understanding of the networks and pathways that regulate this process, and new tools for its study.

Mitochondria reside inside all cells in the body (except red blood cells), and function to generate cellular energy needed for healthy function and survival. Because they are present in all organs of the body, when they become dysfunctional, a wide variety of diseases, injuries and impairments can occur, leading to life long consequences for health and healthy ageing. This proposal requests funding to purchase a Seahorse XF Pro Analyser. This equipment is capable of measuring tiny fluctuations in oxygen usage and pH in a variety of samples, including isolated mitochondria, cells, tissue samples and whole organisms (e.g. zebrafish larvae). This generates a snapshot of mitochondrial function, a cellular bioenergetic profile, which can then be monitored for impairments after injury or be used to report when therapeutic intervention is beneficial. The Seahorse XF Pro Analyser uses a 96-well plate format which means that many samples can be compared rapidly. Research carried out at the RVC and elsewhere has identified mitochondrial dysfunction in the development of neurodegeneration (e.g. Parkinson's disease, Alzheimer's disease), brain injury (e.g. following oxygen deprivation during birth), cardiovascular disease, kidney disease and musculoskeletal impairment. Acquiring a Seahorse XF Pro Analyser would enable researchers to determine how target proteins implicated in these conditions perturb the cellular bioenergetic profile and how potential new therapies might restore cellular health.

Organs-on-chips (OoC) are systems containing engineered or natural miniature tissues grown inside microfluidic chips. To better mimic species physiology, the chips are designed to control cell microenvironments and maintain tissue-specific functions. Indeed, compared to traditional 2D cell culturing, such as primary cell cultures or tissue slices , organ-on-a-chip systems allow the controlled co-culture of different cells to mimic various structures and functions of tissues and organs, such as blood-brain barrier, lung and heart. More importantly, OoC reduce the sample volume substantially, reduce the cost of reagents and maximize information gleaned from precious samples by real time analysis, provide gains in scalability for screening applications and batch sample processing analogous to multi-well plates. OoC technology allows researchers to replicate the function of tissues and organs, bridging the gap between animals and human systems, but also reducing the need for large animal numbers, thus being in-line with the 3Rs. OoC are seen as an exciting in vitro alternative to assess new systems for regenerative system as well as vaccinology. This proposal requests funding to purchase an Emulate Zoe device. This culture module provides dynamic culture conditions for up to 12 Organ-Chips. Users can set a range of automated flow and cyclic stretch parameters depending on study needs As an open platform, Zoë enables researchers to build a wide variety of organ models for myriad applications-from disease modeling, to target validation, to drug from disease modeling, to target validation, to drug candidate safety and efficacy evaluation. Indeed, research carried out at the RVC and elsewhere has identified multiple possibilities to use such a system, in addition to classical primary cell cultures/tissue slices to assess the development of new vaccines/vaccine approaches by better understanding host-pathogen interaction in a 3d tissue complex, the development of regenerative medicine therapies (e.g. stem cell therapy for tendon, heart and eye), kidney failures and cancer in a multi-cell system, allowing for the interaction of tissue with other cell types/treatment strategies to be analysed. Acquiring this Zoe Culture module would not only enable researchers to determine how target proteins implicated in these conditions perturb the cellular bioenergetic profile and how potential new therapies might restore cellular health.

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