Optimising and applying RNA based approaches to identify active nitrifiers in coastal sediments

Nitrogen is an essential element for all forms of life on earth. Since the beginning of the industrial revolution, nitrogen has become a major pollutant of marine and coastal ecosystems due to the huge rise in the use of inorganic fertiliser. Like many other nutrients, the transformations of the nitrogen cycle are mainly controlled by the activity of microorganisms. Understanding the factors influencing the activity of microbes involved in the biochemical transformation of nitrogen in the environment is therefore crucial. The aim of this thesis is to establish a robust workflow for the study of microbial activity in coastal sediment using transcriptomics. In particular, this work focuses on nitrification, the aerobic chemo-litho-autotrophic oxidation of ammonia to nitrite carried out by ammonia oxidizing bacteria and archaea (AOB and AOA respectively). The First part of the thesis (Chapter I) will consist of a review of the literature on the nitrogen cycle, with a particular focus on nitrification. A review of the techniques used to measure microbial activity in natural environment will also be presented and the knowledge gap that exist in transcriptomic workflow in environmental microbiology identified alongside current understanding of active nitrifiers in coastal sediments. The second part of the thesis (Chapters II and III) will present the first experimental work package which consists of the optimisation of reverse-transcription (RT)- based protocol for the study of microbial activity via transcriptomics. First, a new technique to evaluate RNA integrity, extracted from environmental samples, based mRNA will be developed and tested in a controlled-RNA degradation experiment. We show that this technique can provide a useful complement to the commercial approaches that evaluate RNA integrity mainly through the 16S/23S rRNA ratio. Then, the effect of the RT protocol itself on RT-Q-PCR and RT-PCR- sequencing results will be evaluated by testing a combination of four different RT enzymes and two priming strategies. We show that the choice of the correct protocol can greatly improve accuracy and precision of RT-based results. The third part (Chapters IV) will present the application of the optimised protocol to study the effects of sedimentary structures (ridge/runnel) on microbial nitrification activity measured via reverse-transcriptase quantitative PCR (RT-Q-PCR) and reverse-transcriptase PCR- sequencing. Here, the work developed in part two to ensure RNA integrity and optimal RT-PCR protocols will be applied to ensure robust and reliable measure of nitrifier mRNA from within coastal sediments to inform ecological understanding of the active organisms and controls of nitrification. The study site chosen was the Montportail-Brouage intertidal mudflat, located along the French Atlantic coast. This site has been shown to display interesting characteristics in term of microbial dynamics, with the sedimentary structures (ridges/runnels) significantly influencing microbial nitrification rates. The hypothesis proposed previously to explain the differences in nitrification rates is that AOB are more abundant in the runnels, where the higher nitrification rates had been measured. Here, we will show that these differences are explained by the presence of low abundance but highly active AOB groups that drive ammonia oxidation. Furthermore, we show the inadequacy of DNA studies as stand alone methods to explore nitrification activity, with a negative correlation between abundance of AOB amoA genes and nitrification rates, due to the presence of a highly abundant but inactive AOB cluster

The culture of teenage and young adult cancer care in England: A multiple-case study across four networks of care

Background It has long been recognised that young people have unique needs which differ from those of children or adults. Yet within healthcare, they have spent years without being recognised as a speciality within their own right. Teenage and young adult cancer networks in the United Kingdom have however evolved over the past twenty years. Services have been shaped by national guidance, released in 2005, and with much input from the charitable sector. These services in England are structured around complex networks, with secondary and tertiary care delivered in either a specialist Principal Treatment Centre, or within an associated ‘designated’ or ‘shared care’ hospital. Aims The aim of this research was to look closely at these services, to specifically examine the culture of care for young people receiving cancer care in England. The current economic climate of the National Health Service (NHS) presents an ongoing challenge for specialist health services to demonstrate their value and cost-effectiveness: teenage and young adult cancer care being one of these specialist services. Evidence to underpin specialist health services is crucial to make a difference and sustain change. Methods Adopting a case study approach through a critical realist lens, this multiple-case study was conducted across four teenage and young adult cancer networks in England. A conceptual framework consisting of three core concepts of culture was used to guide study design, conduct and reporting. A qualitative, multi-method approach was used across 24 NHS hospitals, and included: semi-structured interviews with healthcare professionals (n=41), and young people (n=29); participant observation, walking tours and shadowing techniques. Thematic analysis was used to identify themes between, within and across the four cases; deconstructing and reconstructing the components of the culture of care that emerged thereby enabling synthesis and contextualisation of data. Findings The findings of this study are presented in three parts, framed around the three core concepts of culture: the dynamic contexts of the culture; the visible processes and less visible values which underpin the culture; and the way culture was learned and shared. In terms of the context of care, the impact of the physical environment on young people’s experiences of socialising and accessing peer support were highlighted. There were key features of the culture which were clear and visible. Underpinning these features were three values at the core of delivering young person-centred care: recognising individuality, empowering and promoting normality for young people. To develop a culture which embraces and promotes these core values, it was found to be particularly important to have effective communication and leadership, healthcare professionals with the right attitude, and a sufficient number of young people using a service. Conclusion and recommendations The importance of the core values that underpin care, and the need for education, effective leadership and multi-disciplinary team working, should all be recognised and prioritised when developing and evaluating interventions that contribute to the delivery of care to this unique patient group. Care delivered in an environment which promotes normality through facilitating socialisation with peers is optimal for the creation of a culture of care which is holistic and young person-centred. The findings of this study provide qualitative evidence of care delivery. This data will be further considered alongside the quantitative data generated in other workstreams of the BRIGHTLIGHT programme of research. However, taken alone, these findings provide evidence to assist all healthcare professionals and services to grow and nurture a culture of care that meets the unique needs of young people with cancer, and to improve their experiences of care; and further progress healthcare organisations towards a future where specialist, holistic care is accessible for all young people

Development of RT-PCR based method for genome amplification of human picornaviruses and rescue of viable viruses

Picornaviruses (family Picornaviridae), causing diseases form respiratory illness to life-threatening CNS disease, are among the most common human pathogens. More than 300 human picornavirus types have been currently identified, yet there are no antivirals or vaccines against the common picornavirus types. Picornaviruses are RNA viruses and thus prone to mutations, which alter the virus tropism and pathogenesis. Viral cDNA clones or vectors are essential in the studies of picornaviruses. The standard cDNA clone production methods, however are cumbersome and old fashioned. The aim of the thesis was to create a streamlined, robust method for amplification of picornaviral RNA genome to enable easy mutagenesis and generation of functional viral vectors. To amplify viral RNA and cDNA clones with the backbone of coxsackievirus A9 and human parechovirus 1, four RT enzymes and five PCR enzymes, respectively, were used. PCR parameters (primer and nucleotide concentrations, number of cycles, performance based on copy numbers) were assessed to compile an optimal protocol. Further plans were made to follow virus rescue after cell transfection of viral genomes cloned under T7 promoter. Finally, HuTu80 cell line was evaluated for its suitability for parechovirus propagation and for reverse genetics experiments. Platinum SuperFi II DNA polymerase was the most robust and sensitive PCR enzyme and allowed amplification from 102 viral cDNA genome copies. It performed consistently and worked on a wide range of nucleotide and primer concentrations. LunaScript and SuperScript IV produced the best results in RT step and were also the most straightforward in use. Parechovirus growth in HuTu80 cell line was observed based on cytopathic effect, suggesting that HuTu80 cells are suitable for further parechovirus studies. In all, novel findings were made to develop a sensitive and robust protocol for amplification of full-length picornaviral genomes. Together with further optimization and optimal regulatory primers, it should allow efficient and sensitive viral genome amplification from small RNA amounts. This study paves way for development of modified viruses for pathogenicity studies and for oncolytic virotherapy

The Role of Attachment, Self-Esteem and Impulsivity on Intimate Partner Violence in Same- and Opposite-Sex Relationships

Intimate partner violence (IPV) is a pervasive problem that impacts individuals in both same and opposite sex relationships. As such, understanding risk factors for the perpetration of this type of violence within each population are important for intervention efforts. The present study examined the interactive effects of attachment, self-esteem, and impulsivity on men and women’s perpetration of intimate partner violence in same- and opposite- sex relationships. Participants were 417 individuals recruited from an undergraduate and community population, who completed measures of adult attachment, self-esteem, impulsivity, as well as physical and psychological aggression against intimate partners within the past 12 months. Tests of mediation, moderation, moderated mediation, and moderated moderated mediation were conducted. Results revealed that self-esteem mediated the relationship between problematic attachment and psychological aggression. In addition, (lack of) perseverance moderated the indirect effect of problematic attachment on psychological aggression through self-esteem. Finally, results revealed that among heterosexual participants, sensation seeking moderated the indirect effect of problematic attachment on psychological aggression through self-esteem. However, those findings were not significant among gay and lesbian participants. Limitations and future directions are discussed

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection

Brouwer et al. present preclinical evidence in support of a COVID-19 vaccine candidate, designed as a self-assembling two-component protein nanoparticle displaying multiple copies of the SARS-CoV-2 spike protein, which induces strong neutralizing antibody responses and protects from high-dose SARS-CoV-2 challenge.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication i

Hidden stitches: RNA cryptic splicing and its role in human disease

A great majority of human genes contain introns: tracts of mostly non-functional sequence that intervene the functional exons. When intron-bearing genes are transcribed into RNA, the introns are removed from the transcript via splicing, a process controlled by a multimolecular assembly called the spliceosome. Although splicing is generally well-regulated, the spliceosome sometimes splices RNA transcripts at sites other than their canonical exon boundaries. This “cryptic” splicing can be a random event, part of an unidentified regulatory process, the effect of a mutation, or the result of other perturbances to the spliceosome’s normal behaviour. In this thesis, I present four reports on the mechanisms underlying certain forms of cryptic splicing. In the first report, an analysis of pathogenic pseudoexons in the DMD gene reveals that each causative mutation falls into a distinct category defined by its proximity to the pseudoexon, and that many DMD pseudoexon splice sites are actively spliced in non-mutant cells. The second report builds on this by constructing a catalogue of over 400 pseudoexon variants from across the human transcriptome and uses this dataset to propose new and revised pseudoexon mutation categories. Like the first report, this second report also finds substantial congruence between pseudoexons and active deep intronic splice sites – including several recursive splice sites – suggesting a causal link between these phenomena. A third report explores how some cryptic exons may provide an explanatory mechanism to connect common genetic variants with their associated population phenotypes and outlines a simple method for discovering new examples. The fourth and final report uses RNA secondary structure modelling to explain why some antisense oligonucleotides can induce partial exon skipping through cryptic splice-site activation. Collectively, these reports present several novel insights into the causes of cryptic splicing and offer suggestions for how future research may build upon these insights

Adventures in software engineering : plugging HCI & acessibility gaps with open source solutions

There has been a great deal of research undertaken in the field of Human-Computer Interfaces (HCI), input devices, and output modalities in recent years. From touch-based and voice control input mechanisms such as those found on modern smart-devices to the use of touch-free input through video-stream/image analysis (including depth streams and skeletal mapping) and the inclusion of gaze tracking, head tracking, virtual reality and beyond - the availability and variety of these I/O (Input/Output) mechanisms has increased tremendously and progressed both into our living rooms and into our lives in general. With regard to modern desktop computers and videogame consoles, at present many of these technologies are at a relatively immature stage of development - their use often limited to simple adjuncts to the staple input mechanisms of mouse, keyboard, or joystick / joypad inputs. In effect, we have these new input devices - but we're not quite sure how best to use them yet; that is, where their various strengths and weaknesses lie, and how or if they can be used to conveniently and reliably drive or augment applications in our everyday lives. In addition, much of this technology is provided by proprietary hardware and software, providing limited options for customisation or adaptation to better meet the needs of specific users. Therefore, this project investigated the development of open source software solutions to address various aspects of innovative user I/O in a flexible manner. Towards this end, a number of original software applications have been developed which incorporate functionality aimed at enhancing the current state of the art in these areas and making that software freely available for use by any who may find it beneficial.Doctor of Philosoph