Neutrophils are the first immune cells to be recruited to sites of tissue injury or infection. Upon detection of an inflammatory stimulus, neutrophils exit the vasculature and migrate directionally through the interstitial tissue towards the target site. Once at the target site, neutrophils may either focalise and form clusters or may exhibit a more dispersive and exploratory behaviour. Focalisation acts to concentrate local neutrophil effector responses but excess clustering can prove detrimental, resulting in undesirable tissue damage. Neutrophil dispersal promotes the encounter of alternative signals and therefore drives resolution of the response. A fine balance between focalisation and exploration must exist to ensure that the inflammatory response is effective but also transient and self-resolving.
Neutrophils are recruited to target sites by gradients of attractant molecules, a major class of which is chemokines. Chemokines bind to G-protein coupled receptors (GPCRs) and initiate complex intracellular signalling cascades which ultimately result in directional neutrophil migration. Upon ligand binding, GPCRs can undergo multiple trafficking fates which may in turn influence sensitivity to the gradient. However, the functional significance of receptor trafficking during neutrophil responses in vivo remains unknown.
Here, I address this question using zebrafish Cxcl8a (a homologue of human CXCL8) which signals through two G-protein coupled receptors, Cxcr1 and Cxcr2. Through new in vivo biosensors, I show that Cxcr1 and Cxcr2 exhibit differential trafficking in response to endogenous gradients. Cxcr1 is extensively internalised whilst Cxcr2 is sustained on the cell membrane. Live-imaging of receptor knockout neutrophils revealed that Cxcr1 promotes neutrophil clustering at wounds, whilst Cxcr2 drives dispersal. Through receptor mutagenesis I show that neutrophil dispersal relies on Cxcr1 internalisation and membrane sustenance of Cxcr2. Thus, I show that differential trafficking of two receptors balances the rise and fall of neutrophil inflammatory responses. To my knowledge, this is the first study to functionally link receptor dynamics to neutrophil migration behaviour in vivo.
Contributors:Merker, Matthias, Kohl, Thomas A, Barilar, Ivan, Andres, Sönke, Fowler, Philip W, Chryssanthou, Erja, Ängeby, Kristian, Jureen, Pontus, Moradigaravand, Danesh, Parkhill, Julian, Peacock, Sharon J, Schön, Thomas, Maurer, Florian P, Walker, Timothy, Köser, Claudio, Niemann, Stefan
BACKGROUND:A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret whole-genome sequencing data for genotypic drug susceptibility testing (DST). METHODS:We investigated mutations in 92 genes implicated in resistance to 21 anti-tuberculosis drugs using the genomes of 405 phylogenetically diverse MTBC strains. The role of phylogenetically informative mutations was assessed by routine phenotypic DST data for the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide from a separate collection of over 7000 clinical strains. Selected mutations/strains were further investigated by minimum inhibitory concentration (MIC) testing. RESULTS:Out of 547 phylogenetically informative mutations identified, 138 were classified as not correlating with resistance to first-line drugs. MIC testing did not reveal a discernible impact of a Rv1979c deletion shared by M. africanum lineage 5 strains on resistance to clofazimine. Finally, we found molecular evidence that some MTBC subgroups may be hyper-susceptible to bedaquiline and clofazimine by different loss-of-function mutations affecting a drug efflux pump subunit (MmpL5). CONCLUSIONS:Our findings underline that the genetic diversity in MTBC has to be studied more systematically to inform the design of clinical trials and to define sound epidemiologic cut-off values (ECOFFs) for new and repurposed anti-tuberculosis drugs. In that regard, our comprehensive variant catalogue provides a solid basis for the interpretation of mutations in genotypic as well as in phenotypic DST assays.
Contributors:Mühlemann, Barbara Franziska
Next Generation Sequencing (NGS) technology allows researchers to sequence genetic material from a wide range of sources, including patient and environmental samples, and ancient remains. The recovery of viruses from such datasets can provide insights into the diversity and evolution of both novel and already known viruses. This thesis focuses on two aspects of virus discovery in NGS datasets.
In the first part of this thesis, I present ancient viral sequences from hepatitis B virus, human parvovirus B19, and variola virus. The sequences were recovered from NGS datasets from individuals living in Eurasia between ∼150 to ∼31,630 years ago, using standard sequence matching tools. The data show the past existence of viruses similar to variants circulating today. The sequences reveal a complexity of virus evolution that is not evident when considering modern sequences alone, including revised substitution rates and most recent common ancestor dates, as well as geographic movement and extinction of strains.
The identification of viral sequences in NGS datasets relies heavily on sequence-based matching of unknown sequences to a database of known sequences. Comparisons are usually done at the nucleotide or amino acid level. However, those methods only work well on sequences closely related to those already present in the database. With the aim of identifying more diverged viral sequences, in the second part of this thesis, I present an algorithm to compare sequences based on predicted structural features, such as secondary structures and conserved amino acids. The algorithm is modelled after the music-matching algorithm ‘Shazam’. While initial results of the algorithm are somewhat encouraging, problems remain, in particular with the identification of adequate structural features. Identifying highly diverged viral sequences is thus still a challenging problem, hopefully to be solved in the future.
Contributors:Dixon-Woods, Mary, Campbell, Anne, Chang, Trillium, Martin, Graham, Georgiadis, Alexandros, Heney, Veronica, Chew, Sarah, Van Citters, Aricca, Sabadosa, Kathryn A, Nelson, Eugene C
BACKGROUND:New opportunities to record, collate, and analyze routine patient data have prompted optimism about the potential of learning health systems. However, real-life examples of such systems remain rare and few have been exposed to study. We aimed to examine the views of design stakeholders on designing and implementing a US-based registry-enabled care and learning system for cystic fibrosis (RCLS-CF). METHODS:We conducted a two-phase qualitative study with stakeholders involved in designing, implementing, and using the RCLS-CF. First, we conducted semi-structured interviews with 19 program personnels involved in design and delivery of the program. We then undertook 11 follow-up interviews. Analysis of interviews was based on the constant comparative method, supported by NVivo software. RESULTS:The organizing principle for the RCLS-CF was a shift to more partnership-based relationships between patients and clinicians, founded in values of co-production, and facilitated by technology-enabled data sharing. Participants proposed that, for the system to be successful, the data it collects must be both clinically useful and meaningful to patients and clinicians. They suggested that the prerequisites included a technological infrastructure capable of supporting data entry and joint decision-making in an accessible way, and a set of social conditions, including willingness from patients and clinicians alike to work together in new ways that build on the expertise of both parties. Follow-up interviews highlighted some of the obstacles, including technical challenges and practical constraints on refiguring relationships between clinicians and patients. CONCLUSIONS:The values and vision underlying the RCLS-CF were shared and clearly and consistently articulated by design stakeholders. The challenges to realization were often not at the level of principle, but were both practical and social in character. Lessons from this study may be useful to other systems looking to harness the power of "big data" registries, including patient-reported data, for care, research, and quality improvement.
LaserForge is a commercial coating process, that uses a pulsed laser to deposit flat sided wire onto a substrate with minimal heat input. Supersonic Laser Deposition (SLD) is an emerging coating technology that can be used as an alternative to existing thermal spray processes. It has the benefit of low temperature, allowing the deposition of nanostructured and temperature sensitive coatings, which is not currently possible with existing thermal spray.
This main aim of this work was to undertake an experimental study aimed at identifying the process mechanism used in the LaserForge process. The understanding of the process mechanism could then be applied to process improvements for SLD coatings. As part of this study the bonding mechanisms of both LaserForge and SLD were studied.
Initially a laser system to enable the exploration of the LaserForge parameter space was specified and a system set up to enable investigation of LaserForge. The LaserForge process parameter space was characterised using a pulsed laser with Ti-64 on CP aluminium. Successful bonding was achieved with parameters of 10 ms pulse length, 1400 W per pulse and 0.8 mm spot diameter. The process was determined to be a form of welding-based laser cladding, a melt-based process.
Following discovery that LaserForge was a melt-based process, the direction of work was changed to focus on the SLD process mechanisms. Several WC-17Co coatings were deposited as a single layer (0.5 mm thick) on carbon steel. The coating cross section morphology was characterised using an optical microscope and scanning electron microscope. A tensile pull off test was used to measure the coating adhesion, and a four-point bend test with acoustic emission was used to monitor the failure of the coating. Plastic failure of the coating was identified, and a test limited adhesion strength in excess of 70 MPa measured. The coating was shown to have a stress-to fracture of approximately 550 MPa in tension, and a reinforcement effect of approximately 100 MPa when compared to the uncoated substrate. The problems with the deposition of the coatings with SLD were investigated and characterised, with the thermal effect from the laser during deposition found to be significant.
This work has characterised the mechanism behind the commercial LaserForge process and the deposition challenges of depositing WC-17Co using Supersonic Laser Deposition. The benefit of these advancements will provide guidance for the direction of future work into LaserForge, and Supersonic Laser Deposition of nanostructured and advanced materials.
Contributors:Bonaud, Amélie, Clare, Simon, Bisio, Valeria, Sowerby, John M., Yao, Shugang, Ostergaard, Hanne, Balabanian, Karl, Smith, Kenneth G. C., Espéli, Marion
The generation of a potent humoral immune response by B cells relies on the integration of signals induced by the B cell receptor, toll-like receptors and both negative and positive co-receptors. Several reports also suggest that integrin signaling plays an important role in this process. How integrin signaling is regulated in B cells is however still partially understood. Integrin activity and function are controlled by several mechanisms including regulation by molecular adaptors of the paxillin family. In B cells, Leupaxin (Lpxn) is the most expressed member of the family and in vitro studies suggest that it could dampen BCR signaling. Here, we report that Lpxn expression is increased in germinal center B cells compared to naïve B cells. Moreover, Lpxn deficiency leads to decreased B cell differentiation into plasma cells in vitro. However, Lpxn seems dispensable for the generation of a potent B cell immune response in vivo. Altogether our results suggest that Lpxn is dispensable for T-dependent and T-independent B cell immune responses.
Contributors:Mancuso, M, Arnold, M, Bersano, A, Burlina, A, Chabriat, H, Debette, S, Enzinger, C, Federico, A, Filla, A, Finsterer, J, Hunt, D, Lesnik Oberstein, S, Tournier-Lasserve, E, Markus, Hugh
Lipid membranes and lipid vesicles have been studied extensively in the last 50 years in
order to characterise their biological, chemical and physical properties. Such work is of
interest from a fundamental biological perspective, but also due to the applications that their
biocompatibility affords: in biotechnological, pharmaceutical, food science and cosmetic
applications. From this work, it is clear that lipid membranes display a large number of
remarkable traits: they can form a wide range of sizes and morphologies, are deformable and
can be functionalised with a variety of structures.
More recently, multivalent interactions have been exploited to drive self-assembly of
nanoparticles, hard colloids and compliant units including emulsion droplets and lipid
vesicles. By applying this to deformable lipid vesicles, formation of links between two
membranes produces morphological changes unachievable in hard colloidal systems, and the
liquid interface of liquid-phase bilayers allows for the diffusion of the multivalent constructs
across the membrane of the lipid vesicle.
Against this background of membrane science and multivalent interactions, this thesis
develops new experimental approaches to exploit these extra degrees of freedom to develop
novel lipid-based soft responsive materials with potential ’real-world’ applications, such as
in molecular sensing. In Chapter 1, the motivations for this work are introduced, before introducing the requisite background literature and general experimental techniques in Chapters 2
and 3 respectively.
In Chapter 4 we show a system of single lipid vesicles adhering to a flat supported
lipid bilayer through multimeric multivalent interactions, which we study to characterise the
morphological and mechanical changes of the vesicles in response to external ligands. We
show that the mechanical properties of the vesicles, in particular their membrane tension,
change dramatically on adhesion, and that the number of adhering vesicles is dependent on
the concentration of the external ligand due to combinatorial entropy, which we confirm
through consideration of a simple statistical mechanical model.
In Chapter 5 we use Differential Dynamic Microscopy to study the dynamics of a
thermoreversible gel consisting of diffusive attractive soft colloids (large unimlamellar
vesicles functionalised with complementary DNA constructs), and fit the dynamics with
a stretched/compressed exponential model. From the fit parameters, we observe differing
levels of spatial heterogeniety of the dynamics of the sample within different regimes below,
above and around the gel/melting points, as well as differing length scales of the dynamics,
which differ between quenching and melting experiments. From the statics and dynamics,
we see evidence for multiple phenomena, including coarsening as well as ballistic events
corresponding to strand breakages.
In Chapter 6 we propose a method for high-throughput vesicle production. We characterise the method and the vesicles produced, as well as demonstrating novel applications,
most notably the high-throughput production of vesicles encapsulating responsive DNA
circuitry, highlighting the potential of this method in bottom-up synthetic biology and the
design of programmable materials. Furthermore, we demonstrate the possibility of on-chip
functionalisation of membrane constructs into the lipid membranes, in this case cholesterol anchored DNA constructs.
In Chapter 7 we study dense packings of vesicles assembled using multivalent complementary DNA interactions, through passive tracking of diffusive colloidal particles and active
microrheology using magnetic tweezers. We observe changes in the structure in response to
increased temperature, DNA concentration and aging leading to reduced pore sizes. From a
rheological standpoint, we observe strain hardening of the material through repeated creep
tests, with the ability to reset the material by increasing the temperature above the melting
point of the system. The material stiffens and becomes more viscous, which we observe
through the application of a constitutive and fractional rheological model respectively.
In this thesis we demonstrate the responsiveness of these multivalent construct functionalised lipid vesicle based soft materials by showing the ability to tune the structure,
rheology and dynamics of such materials, as well as proposing a method for high throughput,
monodisperse production of functionalised lipid vesicles. These results lead to further potential avenues of research, and demonstrate suitability for and preliminary steps towards
applications of these responsive materials in fields such as molecular sensing.
Inflammatory bowel disease (IBD) is a group of immune-mediated autoinflammatory disorders, primarily manifesting in the gastrointestinal tract. Affecting millions of people around the world, IBD has a severe impact on patients’ quality of life. Several pharmacologic treatments have been available since the 1950s. However, the majority of patients either do not respond to a given therapy or lose response to a previously effective treatment and thus require therapeutic escalation.
In the first research chapter of my thesis, I describe the results of the Personalised Anti-TNF Therapy in Crohn’s disease study. Immunogenicity to anti-TNF therapy is a major cause of loss of response, hypersensitivity reactions, and discontinuation of treatment in patients. Currently, immunogenicity cannot be predicted prior to treatment. My analysis has identified a strong dominant association in the HLA region on chromosome 6 (HLA-DQA1*05, P=5.9e-13; HR=1.90; 95% CI, 1.60 to 2.25). Around 40% of individuals of European ancestry carry HLA-DQA1*05, and the data suggest that around 95% of these would develop immunogenicity within the first year of infliximab monotherapy treatment (a common anti-TNF treatment regime).
In the second research chapter of my thesis, I describe a genome-wide association study of thiopurine-induced liver damage (TILI). Ultimately, the study was underpowered to detect any associations of moderate effect size and did not detect any associations of high effect size amongst the common genetic variants. Interestingly, I was not able to replicate the association in PTPN22, which was reported to be a risk factor for drug-induced liver damage by Cirulli et al. – suggesting that its effect might be heterogeneous depending on the therapy.
Finally, the third research chapter describes the initial analysis of the IBD 15x dataset – a whole-genome sequencing association study of around 7,000 IBD patients paired with 12,000 matching controls. I provide an overview of the sample quality control procedures
and describe some of the novel challenges that sequencing studies bring in comparison to standard GWAS (e.g., sample cross-contamination due to index mismatching). Finally, I also provide the results of the initial meta-analysis of the exome-sequencing dataset produced by the Broad Institute. The results demonstrate that rare coding genetic variants play a role in IBD pathogenesis.
Mental disorders are one of the leading causes of non-communicable disease burden worldwide, with distress emerging as a common factor among such disorders. Most lifetime mental disorders emerge during adolescence and young adulthood, yet no studies have robustly assessed changes in common distress over time during this broad developmental epoch. Common distress is particularly pertinent when assessing mental health treatment effectiveness, as comorbidity rates are higher amongst those who seek treatment. In this dissertation, I seek to redress the paucity of evidence regarding mental health treatment effectiveness in young people. Herein I longitudinally validate a common distress factor, enabling me to use specific and broad mental health outcomes to assess the effectiveness of treatment-as-usual mental health services in two separate samples. In chapter 2, I use a community sample of adolescents to test the relationship of age 14 service contact on depressive symptoms by age 17. I found that 14-year-old adolescents who had contact with mental health services in the past year had a greater decrease in depressive symptoms than those without contact. By age 17 years, the odds of reporting clinical depression were higher in individuals without contact than in service users who had been similarly depressed at baseline. In addition to propensity score weighting to adjust for participants’ initial likelihood to access services, I used a clinically relevant cutoff and adjusted for a wide range of time-varying confounding variables. These adjustments give greater confidence than previous studies to the notion that mental health service contact is related to meaningful improvements in subsequent mental health. Policy implications of these findings are addressed. In chapter 3, I develop a transdiagnostic measure of mental health to be used to assess treatment effectiveness with relevancy across multiple disorders. This transdiagnostic measure is the general factor (common distress or p) from a bifactor model of 118 self-report items from previously validated measures of depression, anxiety, behavioural problems, obsessions, psychotic symptoms, wellbeing, and self-esteem. Longitudinal and gender measurement invariance and reliability of this model was demonstrated in a sample of 14- to 24-year olds assessed annually three times. Predictive validity of the general and specific factors was demonstrated using an extensive set of external variables covering factors such as social environment, personality, and risk behaviour. Accelerated growth modelling revealed developmental changes in the factors from ages 14-27 largely consistent with epidemiological patterns of the associated disorders. I use this validated distress factor in chapter 4 to further test the effectiveness of mental health treatment-as-usual, in a broader age range with a broader outcome than the adolescent sample. Family functioning and friendship support were also explored as potential mechanisms of action. Treatment was related to decreased distress over two years, in unadjusted and adjusted models. In order to propensity weight, I focused analyses on those above the population mean in baseline distress, which contained 85% of young people reporting treatment for a mental disorder. Such individuals were more likely to have required treatment, so treated and untreated groups are more meaningful and comparable. In propensity score weighted models, treatment was related to decreased subsequent distress. Treatment was also related to improved family functioning over two years, but only in adolescents aged 14-18, more likely to be living with their family. In this younger group, the best fitting model revealed distress as a significant mediator: the majority of improvements in family functioning occurred through improvements in distress. However, treatment was not related to a change in friendship support over time. Thus, not only is mental health treatment-as-usual related to a significant decline in young people’s distress, but also improvements in adolescent family functioning. Finally, in chapter 5 I discuss the broader implications of the findings. Analyses in two separate community samples of young people strongly support the notion that treatment-as-usual is related to improvements in both specific and broad measures of mental health. Such treatment-related improvements in mental health appear to also yield benefits in family functioning in adolescents. Findings as a whole argue for increased access to mental health services.