1,412 research outputs found

    Improving rapid pathogen detection: towards a gram-selective lateral flow test

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    The development of rapid detection assays, such as lateral flow tests, have been effective in helping to detect a range of biological targets, most recently in the COVID-19 pandemic. The effective detection and classification of bacteria, such as their Gram status using rapid low-cost lateral flow assay could help diagnose and target treatment of bacterial infections in medical and veterinary applications. This is important as over prescription of broad-spectrum antibiotics is a well-known contributor to the rise in antimicrobial resistance. My work targeted the development of lateral flow assays to achieve the specific detection of Gram-negative bacteria. To make the assay Gram selective, targeting/binding ligands were synthesised based on Polymyxin B, a Gram-negative selective antibiotic. A literature method of selective functionalisation of Polymyxin B was optimised to service material at the gram scale for the development of three Polymyxin conjugates: a Lipoic acid-Polymyxin conjugate and two Biotin-Polymyxin conjugates with and without a spacer. The most effective binding/labelling agent was determined to be a Biotin-Polymyxin conjugate with a six-carbon spacer, which allowed the fluorescently labelling of E. coli by the generation of a Bacteria-Polymyxin-Biotin-Streptavidin-fluorophore sandwich. Most lateral flow tests use gold nanoparticles to label the target analyte as it provides an intense red colour that is easily detectable by eye, and this was replicated here by the attachment of Polymyxin to gold nanoparticles. Through testing a range of methods for the attachment of Polymyxin, a nanoparticle system that could be used to selectively label the surface of bacteria was developed. This Polymyxin-nanoparticle labelling system was characterised via electron microscopy. Nitrocellulose membranes were laser cut to shape and printed with reagents to give a strip assay platform that could analyse the behaviour of the synthesised gold nanoparticles and the Polymyxin targeting agents in flow. 3D printing was used to fabricate an imaging enclosure to enable repeatable imaging that allowed for smartphone capture of the assays and analysis with a custom Python-based image analysis script. This allowed high-throughput testing and optimisation of the assay and flow conditions. Through the optimisation of the lateral flow assay design Gram-negative E. coli were able to be detected in flow, imaged, and analysed. My PhD work thus provides an example of a lateral flow assay functioning without antibodies or aptamers. This work could provide the foundation for further development, including other antibiotics to target different bacteria

    Converging organoids and extracellular matrix::New insights into liver cancer biology

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    Converging organoids and extracellular matrix::New insights into liver cancer biology

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    Primary liver cancer, consisting primarily of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), is a heterogeneous malignancy with a dismal prognosis, resulting in the third leading cause of cancer mortality worldwide [1, 2]. It is characterized by unique histological features, late-stage diagnosis, a highly variable mutational landscape, and high levels of heterogeneity in biology and etiology [3-5]. Treatment options are limited, with surgical intervention the main curative option, although not available for the majority of patients which are diagnosed in an advanced stage. Major contributing factors to the complexity and limited treatment options are the interactions between primary tumor cells, non-neoplastic stromal and immune cells, and the extracellular matrix (ECM). ECM dysregulation plays a prominent role in multiple facets of liver cancer, including initiation and progression [6, 7]. HCC often develops in already damaged environments containing large areas of inflammation and fibrosis, while CCA is commonly characterized by significant desmoplasia, extensive formation of connective tissue surrounding the tumor [8, 9]. Thus, to gain a better understanding of liver cancer biology, sophisticated in vitro tumor models need to incorporate comprehensively the various aspects that together dictate liver cancer progression. Therefore, the aim of this thesis is to create in vitro liver cancer models through organoid technology approaches, allowing for novel insights into liver cancer biology and, in turn, providing potential avenues for therapeutic testing. To model primary epithelial liver cancer cells, organoid technology is employed in part I. To study and characterize the role of ECM in liver cancer, decellularization of tumor tissue, adjacent liver tissue, and distant metastatic organs (i.e. lung and lymph node) is described, characterized, and combined with organoid technology to create improved tissue engineered models for liver cancer in part II of this thesis. Chapter 1 provides a brief introduction into the concepts of liver cancer, cellular heterogeneity, decellularization and organoid technology. It also explains the rationale behind the work presented in this thesis. In-depth analysis of organoid technology and contrasting it to different in vitro cell culture systems employed for liver cancer modeling is done in chapter 2. Reliable establishment of liver cancer organoids is crucial for advancing translational applications of organoids, such as personalized medicine. Therefore, as described in chapter 3, a multi-center analysis was performed on establishment of liver cancer organoids. This revealed a global establishment efficiency rate of 28.2% (19.3% for hepatocellular carcinoma organoids (HCCO) and 36% for cholangiocarcinoma organoids (CCAO)). Additionally, potential solutions and future perspectives for increasing establishment are provided. Liver cancer organoids consist of solely primary epithelial tumor cells. To engineer an in vitro tumor model with the possibility of immunotherapy testing, CCAO were combined with immune cells in chapter 4. Co-culture of CCAO with peripheral blood mononuclear cells and/or allogenic T cells revealed an effective anti-tumor immune response, with distinct interpatient heterogeneity. These cytotoxic effects were mediated by cell-cell contact and release of soluble factors, albeit indirect killing through soluble factors was only observed in one organoid line. Thus, this model provided a first step towards developing immunotherapy for CCA on an individual patient level. Personalized medicine success is dependent on an organoids ability to recapitulate patient tissue faithfully. Therefore, in chapter 5 a novel organoid system was created in which branching morphogenesis was induced in cholangiocyte and CCA organoids. Branching cholangiocyte organoids self-organized into tubular structures, with high similarity to primary cholangiocytes, based on single-cell sequencing and functionality. Similarly, branching CCAO obtain a different morphology in vitro more similar to primary tumors. Moreover, these branching CCAO have a higher correlation to the transcriptomic profile of patient-paired tumor tissue and an increased drug resistance to gemcitabine and cisplatin, the standard chemotherapy regimen for CCA patients in the clinic. As discussed, CCAO represent the epithelial compartment of CCA. Proliferation, invasion, and metastasis of epithelial tumor cells is highly influenced by the interaction with their cellular and extracellular environment. The remodeling of various properties of the extracellular matrix (ECM), including stiffness, composition, alignment, and integrity, influences tumor progression. In chapter 6 the alterations of the ECM in solid tumors and the translational impact of our increased understanding of these alterations is discussed. The success of ECM-related cancer therapy development requires an intimate understanding of the malignancy-induced changes to the ECM. This principle was applied to liver cancer in chapter 7, whereby through a integrative molecular and mechanical approach the dysregulation of liver cancer ECM was characterized. An optimized agitation-based decellularization protocol was established for primary liver cancer (HCC and CCA) and paired adjacent tissue (HCC-ADJ and CCA-ADJ). Novel malignancy-related ECM protein signatures were found, which were previously overlooked in liver cancer transcriptomic data. Additionally, the mechanical characteristics were probed, which revealed divergent macro- and micro-scale mechanical properties and a higher alignment of collagen in CCA. This study provided a better understanding of ECM alterations during liver cancer as well as a potential scaffold for culture of organoids. This was applied to CCA in chapter 8 by combining decellularized CCA tumor ECM and tumor-free liver ECM with CCAO to study cell-matrix interactions. Culture of CCAO in tumor ECM resulted in a transcriptome closely resembling in vivo patient tumor tissue, and was accompanied by an increase in chemo resistance. In tumor-free liver ECM, devoid of desmoplasia, CCAO initiated a desmoplastic reaction through increased collagen production. If desmoplasia was already present, distinct ECM proteins were produced by the organoids. These were tumor-related proteins associated with poor patient survival. To extend this method of studying cell-matrix interactions to a metastatic setting, lung and lymph node tissue was decellularized and recellularized with CCAO in chapter 9, as these are common locations of metastasis in CCA. Decellularization resulted in removal of cells while preserving ECM structure and protein composition, linked to tissue-specific functioning hallmarks. Recellularization revealed that lung and lymph node ECM induced different gene expression profiles in the organoids, related to cancer stem cell phenotype, cell-ECM integrin binding, and epithelial-to-mesenchymal transition. Furthermore, the metabolic activity of CCAO in lung and lymph node was significantly influenced by the metastatic location, the original characteristics of the patient tumor, and the donor of the target organ. The previously described in vitro tumor models utilized decellularized scaffolds with native structure. Decellularized ECM can also be used for creation of tissue-specific hydrogels through digestion and gelation procedures. These hydrogels were created from both porcine and human livers in chapter 10. The liver ECM-based hydrogels were used to initiate and culture healthy cholangiocyte organoids, which maintained cholangiocyte marker expression, thus providing an alternative for initiation of organoids in BME. Building upon this, in chapter 11 human liver ECM-based extracts were used in combination with a one-step microfluidic encapsulation method to produce size standardized CCAO. The established system can facilitate the reduction of size variability conventionally seen in organoid culture by providing uniform scaffolding. Encapsulated CCAO retained their stem cell phenotype and were amendable to drug screening, showing the feasibility of scalable production of CCAO for throughput drug screening approaches. Lastly, Chapter 12 provides a global discussion and future outlook on tumor tissue engineering strategies for liver cancer, using organoid technology and decellularization. Combining multiple aspects of liver cancer, both cellular and extracellular, with tissue engineering strategies provides advanced tumor models that can delineate fundamental mechanistic insights as well as provide a platform for drug screening approaches.<br/

    Silicon-Based Optical Sensors for Fungal Pathogen Diagnostics

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    The last years have witnessed a link between the COVID-19 pandemic with increasing numbers of vulnerable patients and globally emerging incidences of severe drug-resistant fungal infections, thus, calling for rapid, reliable, and sensitive diagnostic tools for fungal infections. However, despite strong warnings from health authorities, such as the World Health Organization, concerning the fatal consequences of the global spread of drug-resistant pathogenic fungi, progress in fungal infection diagnosis and therapy is still limited. Today, gold standard methods for revealing resistance and susceptibility in pathogenic fungi, namely antifungal susceptibility testing (AFST), require several days for completion, and thus this lengthy process can adversely affect antifungal therapy and further promote the spread of resistance. In this work, the use of photonic silicon chips consisting of micropatterned diffraction gratings as sensitive sensors for rapid AFST of clinically relevant fungal pathogens is investigated. These photonic chips provide a surface for the colonization of microbial pathogens at a liquid-solid interface and serve as the optical transducer element for label-free monitoring of fungal growth by detecting real-time changes in the white light reflectance. These sensor elements are used to track morphological changes of fungi in the presence of clinically relevant antifungals at varying concentrations to rapidly determine the minimum inhibitory concentration (MIC) values that help to classify pathogens as resistant or susceptible. We show that by careful design of the chip dimensions, this optical method can extend from bacteria, through yeasts, to filamentous fungi for accelerated AFST, which is at least three times faster than current gold standard methods and can provide same-day results. Moreover, a 3D-printed microfluidic gradient generator was designed to complement the assay and provide an integrated system, which can potentially be employed in point-of-care settings. This gradient generator produces the two-fold dilution series of clinically relevant antimicrobials in an automated manner and is interfaced with the photonic silicon chips to include a complete, on-chip, label-free, and phenotypic assay. Using the bacterial species Escherichia coli and ciprofloxacin as a model pathogen-drug combination, MIC values can be expeditiously determined within 90 minutes compared to current clinical practices, which typically require up to 24 h for bacterial species

    Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

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    Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

    Genomic insights for safety assessment of foodborne bacteria.

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    La sicurezza alimentare e l'accesso ad essa sono fondamentali per sostenere la vita e promuovere una buona salute. Gli alimenti non sicuri, contenenti microrganismi o sostanze chimiche nocive, sono causa di oltre 200 malattie, dalla diarrea al cancro, che colpiscono in particolare i neonati, i bambini piccoli, gli anziani e gli individui immunocompromessi. L'onere globale delle malattie di origine alimentare si ripercuote sulla salute pubblica, sulla società e sull'economia, pertanto è necessaria una buona collaborazione tra governi, produttori e consumatori per contribuire a garantire la sicurezza alimentare e sistemi alimentari più solidi. L'indagine più recente condotta dall'OMS (2015) ha evidenziato una stima di 600 milioni di individui malati e 420.000 decessi annui associati ad alimenti non sicuri. L'impatto economico è dovuto principalmente alla mancanza di alimenti sicuri nei Paesi a basso e medio reddito, con una perdita di 110 miliardi di dollari l'anno in termini di produttività e spese mediche. Le sfide principali per garantire la sicurezza alimentare rimangono legate alla nostra produzione alimentare e alla catena di approvvigionamento, dove fattori come la contaminazione ambientale, le preferenze dei consumatori, il rilevamento tempestivo e la sorveglianza dei focolai giocano un ruolo cruciale. Recentemente, le metodologie basate sul DNA per il rilevamento e l'indagine microbica hanno suscitato particolare interesse, soprattutto grazie allo sviluppo delle tecnologie di sequenziamento. Contrariamente ai metodi tradizionali dipendenti dalla coltura, le tecniche basate sul DNA, come il sequenziamento dell'intero genoma (WGS), mirano a risultati rapidi e sensibili a un prezzo relativamente basso e a tempi di elaborazione brevi. Inoltre, il WGS conferisce un elevato potere discriminatorio che consente di determinare importanti caratteristiche genomiche legate alla sicurezza alimentare, come la tassonomia, il potenziale patogeno, la virulenza e la resistenza antimicrobica e il relativo trasferimento genetico. La comprensione di queste caratteristiche è fondamentale per progettare strategie di rilevamento e mitigazione da applicare lungo l'intera catena alimentare secondo una prospettiva di "One Health", che porta ad acquisire conoscenze sul microbiota che influenza l'uomo, gli animali e l'ambiente. Lo scopo della tesi è quello di approfondire la genomica dei microbi di origine alimentare per la loro caratterizzazione e per creare o migliorare le strategie per la loro individuazione e i metodi di mitigazione. In particolare, questa tesi si concentra sulla valutazione del potenziale patogeno sulla base di analisi genomiche che includono studi di tassonomia, virulenza, resistenza agli antibiotici e mobiloma. Il secondo obiettivo è quello di trarre vantaggio dalle conoscenze genomiche per progettare dispositivi di rilevamento rapidi ed efficaci e metodi di mitigazione affidabili per affrontare i patogeni di origine alimentare. Più in dettaglio, saranno trattati i seguenti argomenti: La presenza di ceppi multiresistenti negli alimenti fermentati pronti al consumo rappresenta un rischio per la salute pubblica per la diffusione di determinanti AMR nella catena alimentare e nel microbiota intestinale dei consumatori. Le analisi genomiche hanno permesso di valutare accuratamente la sicurezza del ceppo UC7251 di E. faecium, in relazione alla sua virulenza e alla co-localizzazione dei geni di resistenza agli antibiotici e ai metalli pesanti in elementi mobili con capacità di coniugazione in diverse matrici. Questo lavoro sottolinea l'importanza di una sorveglianza della presenza di batteri AMR negli alimenti e di incitare lo sviluppo di strategie innovative per la mitigazione del rischio legato alla diffusione della resistenza antimicrobica negli alimenti. L'accuratezza dell'identificazione tassonomica guida le analisi successive e, per questo motivo, un metodo adeguato per identificare le specie è fondamentale. È stata studiata la riclassificazione delle specie di Enterococcus faecium clade B, utilizzando un approccio combinato di filogenomica, tipizzazione di sequenza multilocus, identità nucleotidica media e ibridazione digitale DNA-DNA. L'obiettivo è dimostrare come l'analisi del genoma sia più efficace e fornisca risultati più dettagliati riguardo alla definizione delle specie, rispetto all'analisi della sequenza del 16S rRNA. Ciò ha portato alla proposta di riclassificare tutto il clade B di E. faecium come E. lactis, riconoscendo che i due gruppi sono filogeneticamente separati, per cui è possibile definire una specifica procedura di valutazione della sicurezza, prima del loro utilizzo negli alimenti o come probiotici, compresa la considerazione per l'inclusione nella lista europea QPS. A partire da questa riclassificazione tassonomica, abbiamo sviluppato un metodo basato sulla PCR per la rapida individuazione e differenziazione di queste due specie e per discutere le principali differenze fenotipiche e genotipiche da una prospettiva clinica. A questo scopo, è stato utilizzato un allineamento del core-genoma basato sull'analisi del pangenoma. La differenza allelica tra alcuni geni del core ha permesso la progettazione di primer e l'identificazione della specie mediante PCR con una specificità del 100% e senza reattività crociata. Inoltre, i genomi clinici di E. lactis sono stati classificati come un rischio potenziale a causa della capacità di aumentare la traslocazione batterica. Gli agenti antimicrobici alternativi agli antibiotici sono una delle principali aree di sviluppo e miglioramento dell'attuale catena alimentare. Le nanoparticelle metalliche, come le nanoparticelle di platino (PtNPs), hanno suscitato interesse per le loro potenti attività catalitiche simili alle ossidasi e alle perossidasi che garantiscono forti effetti antimicrobici, e sono state proposte come potenziali candidati per superare gli inconvenienti degli antibiotici come la resistenza ai farmaci. L'obiettivo è studiare la modalità d'azione delle PtNPs in relazione alla capacità di formazione del biofilm, al meccanismo di contrasto delle specie reattive dell'ossigeno (ROS) e al quorum sensing utilizzando batteri di origine alimentare come Enterococcus faecium e Salmonella Typhimurium.Safe food and the access to it is key to sustaining life and promoting good health. Unsafe food containing harmful microorganisms or chemical substances causes more than 200 diseases, ranging from diarrhoea to cancers that particularly affect infants, young children, elderly and immunocompromised individuals. The global burden of foodborne disease affects public health, society, and economy, therefore good collaboration between governments, producers and consumers is needed to help ensure food safety and stronger food systems. The most recent survey conducted by WHO (2015) showed an estimated 600 million ill individuals and 420 000 yearly deaths associated to unsafe food. The economic impact is mainly due to the lack of safe food in low and middle income causing a US$ 110 billion is lost each year in productivity and medical expenses. The main challenges to assure food safety remain tied to our food production and supply chain, where factors like environmental contamination, consumer preferences, timely detection and surveillance of outbreaks play a crucial role. Recently, DNA-based methodologies for microbial detection and investigation have sparked special interest, mainly linked to the development of sequencing technologies. Contrary to the traditional culture-dependent methods, DNA-based techniques such as Whole Genome Sequencing (WGS) that targets fast and sensitive results at a relative low price and short processing time. Moreover, WGS confers high discriminatory power that allows to determine important genomic characteristics linked to food safety like taxonomy, pathogenic potential, virulence and antimicrobial resistance and the genetic transfer thereof. The understanding of these characteristics is fundamental to design detection and mitigation strategies to apply along the entire food-chain following a ‘One Health’ perspective, leading to gain knowledge about the microbiota that affect humans, animals, and environment. The aim of the thesis is to gain insight into the genomics of foodborne microbes for their characterization and to create or improve strategies for their detection and mitigation methods. Particularly, this thesis is focused on the assessment of the pathogenic potential based on genomic analyses including taxonomy, virulence, antibiotic resistance and mobilome studies. The second focus is to profit from the genomic insights to design rapid and time-effective detection devices and reliable mitigation methods to tackle foodborne pathogens. In more detail the following topics will be handled: The presence of multi-drug resistant strains in ready-to-eat fermented food represents a risk of public health for the spread of AMR determinants in the food chain and in the gut microbiota of consumers. Genomic analyses permitted to accurately assess the safety of E. faecium strain UC7251, with respect to its virulence and co-location of antibiotic and heavy metal resistance genes in mobile elements with conjugation capacity in different matrices. This work emphasizes the importance of a surveillance for the presence of AMR bacteria in food and to incite the development of innovative strategies for the mitigation of the risk related to antimicrobial resistance diffusion in food. The accuracy of taxonomic identification drives the subsequent analysis and, for this reason, a suitable method to identify species is crucial. The species re-classification of Enterococcus faecium clade B was investigated, using a combined approach of phylogenomics, multilocus sequence typing, average nucleotide identity and digital DNA–DNA hybridization. The goal is to show how the genome analysis is more effective and give more detailed results concerning the species definition, respect to the analysis of the 16S rRNA sequence. This led to the proposal to reclassify all the E. faecium clade B as E. lactis, recognizing the two groups are phylogenetically separate, where a specific safety assessment procedure can be designed, before their use in food or as probiotics, including the consideration for inclusion in the European QPS list. From this taxonomic re-classification, we developed a PCR-based method for rapid detection and differentiation of these two species and to discuss main phenotypic and genotypic differences from a clinical perspective. To this aim, core-genome alignment base on pangenome analysis was used. Allelic difference between certain core genes allowed primer design and species identification through PCR with 100% specificity and no cross-reactivity. Moreover, clinical E. lactis genomes categorised as a potential risk due to the ability of enhanced bacterial translocation. Antimicrobial agents alternative to antibiotics are one of the main areas of development and improvement in the current food chain. Metallic nanoparticles like Platinum nanoparticles (PtNPs), have awaken interest due to their potent catalytic activities similar to oxidases and peroxidases granting strong antimicrobial effects, have been proposed as potential candidates to overcome the drawbacks of antibiotics like drug resistance. The goal is to study the mode of action of PtNPs related to biofilm formation capacity, reactive oxygen species (ROS) coping mechanism and quorum sensing using foodborne bacteria like Enterococcus faecium and Salmonella Typhimurium

    Examining The Roles of Histone Methyltransferases in Heterochromatin Formation

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    The eukaryotic genome is meticulously stored inside the cell to control gene expression, protect the genome integrity, and facilitate cell division. The storage of genome is often achieved through epigenetic modifications. These modifications divide the genome into euchromatin and heterochromatin, each associated with gene expression and gene repression, respectively. Epigenetic modifications are often covalent modifications onto histone proteins or methylated DNA. On their own, some modifications can alter chromatin states, but most require specific epigenetic machinery that often termed epigenetic reader proteins. The epigenetic reader protein recognizes a specific epigenetic modification to alter chromatin states. For heterochromatin, these modifications often require heterochromatin protein 1 (HP1) to function. Heterochromatin represents a highly condensed form of chromatin that is often devoid of any transcriptional activities. Trimethylation of histone 3 lysine 9 (H3K9me3) and the presence of HP1 are the hallmarks and core players in heterochromatin formation. H3K9me3 recruits HP1, which when bound, can serve as a scaffold protein for more heterochromatin machinery. One key group of proteins HP1 recruits are the histone methyltransferases, the enzymes that catalyze the methylation of H3K9. The recruitment of HP1 often results in more H3K9me3 modifications, which can induce a positive feedback loop that results in a heterochromatin state. This group of histone methyltransferases all belong to the SET-domain family of proteins and demonstrate overlapping roles in heterochromatin formation. The redundancy within this group of enzymes calls for a better understanding in differentiating individual H3K9 methyltransferases. Does the redundancy suggest overlap in functionality or individual methyltransferases contribute to heterochromatin formation differently. The central hypothesis for this project is that different H3K9 methyltransferases can have different impacts on heterochromatin formation. By combining CiA-Oct4 cell line and Molecular biosystem, we have studied the intricacy of heterochromatin formation. Our SETDB1 knockdown cell lines show that SETDB1 contributes heavily to heterochromatin formation. SETDB1 knockdown cell lines have also shown reductions in H3K9me3 accumulation and impaired heterochromatin formation kinetics.Doctor of Philosoph

    Determination of antibiotic susceptibility of the bacteria causing urinary tract infections using a novel lab-on-a-chip design

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    Urinary tract infections (UTIs) are one of the most common types of bacterial infection in the UK, and also are expensive to treat costing the National Health Service ~£54 million between 2016 and 2017. Culture-based antibiotic susceptibility testing (AST) is used to identify an antibiotic to treat drug-resistant urinary tract infections and takes 48 hours to complete. Faster prescription of effective antibiotics should reduce the risk of sepsis and poor clinical outcomes. To address this need, we developed a Lab-on-a-Chip (LOC) based method to conduct electrochemical AST using screen-printed macroelectrodes (SPEs) and antibiotic-loaded hydrogels. SPEs were fabricated using carbon-graphite based inks, with resazurin bulk modified SPEs (R-SPEs) being fabricated through modification of the SPEs WE. Polyvinyl alcohol (PVA) based hydrogels were loaded with the following antibiotics were used; cephalexin, ceftriaxone, colistin, gentamicin, piperacillin, trimethoprim and vancomycin as well as an antibiotic-free control. LOC devices were then designed to encapsulate both the R-SPEs and the antibiotic hydrogels to enable multiplexed electrochemical AST to occur on a single device. In the initial testing of the R-SPEs and the antibiotic hydrogels independently of a LOC device, antibiotic susceptibility could be determined in 90 minutes for E. coli. After the preliminary work, eight chambered LOC devices were spiked with simulated UTI samples. Each chamber contained an R-SPE and an antibiotic hydrogel. After an incubation step, susceptibility of Escherichia coli and Klebsiella pneumoniae could be established in 85 minutes of testing which is significantly faster than the 48 hours required for conventional culture-based AST. The sensitive detection of resazurin afforded by using the electrochemical detection methodology incorporated onto a LOC device described here offers an inexpensive and simple method for the determination of antibiotic susceptibility that is faster than using a culture-based approach

    The Host-Microbiota Axis in Chronic Wound Healing

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    Chronic, non-healing skin wounds represent a substantial area of unmet clinical need, leading to debilitating morbidity and mortality in affected individuals. Due to their high prevalence and recurrence, chronic wounds pose a significant economic burden. Wound infection is a major component of healing pathology, with up to 70% of wound-associated lower limb amputations preceded by infection. Despite this, the wound microbiome remains poorly understood. Studies outlined in this thesis aimed to characterise the wound microbiome and explore the complex interactions that occur in the wound environment. Wound samples were analysed using a novel long-read nanopore sequencing-based approach that delivers quantitative species-level taxonomic identification. Clinical wound specimens were collected at both the point of lower-extremity amputation and via a pilot clinical trial evaluating extracorporeal shockwave therapy (ESWT) for wound healing. Combining microbial community composition, host tissue transcriptional (RNAseq) profiling, with clinical parameters has provided new insight into healing pathology. Specific commensal and pathogenic organisms appear mechanistically linked to healing, eliciting unique host response signatures. Patient- and site-specific shifts in microbial abundance and communitycomposition were observed in individuals with chronic wounds versus healthy skin. Transcriptional profiling (RNAseq) of the wound tissue revealed important insight into functional elements of the host-microbe interaction. Finally, ESWT was shown to confer beneficial effects on both cellular and microbial aspects of healing. High-resolution long-read sequencing offers clinically important genomic insights, including rapid wide-spectrum pathogen identification and antimicrobial resistance profiling, which are not possible using current culture-based diagnostic approaches. Thus, data presented in this thesis provides important new insight into complex host-microbe interactions within the wound microbiome, providing new and exciting future avenues for diagnostic and therapeutic approaches to wound management

    University of Arkansas, Chemistry and Biochemistry Department Research Publications, 2014- November 2023. 107p.

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    This report provides a compilation of the research publications by the Chemistry and Biochemistry faculty for the period: 2014 - November 2023. The information was gathered from major databases in science and technology including Web of Science, SciFinder, Reaxys, PubMed, IEEE Explore and Engineering Index. At least one author in each of the publications has the CHBC department as its affiliation. It includes a table summarizing the research. The listing is organized according to type of publications within specific years
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