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Asthma is a heterogeneous disease characterised by frequent airway obstruction in the lung resulting from allergic (atopic) or non-allergic (non-atopic) triggers. Allergic asthma is associated with airway inflammation and is characterised by the infiltration of inflammatory cells including eosinophils, mast cells and T-helper 2 cells (TH2). Several soluble mediators have been shown to play a critical role in the pathogenicity of allergic asthma. These mediators include the hallmark TH2 cytokines interleukin (IL)-4, IL-5 and IL-13 and the epithelial-derived cytokines IL-25, IL-33 and Thymic Stromal Lymphopoietin (TSLP). Until recently, it was believed that allergic inflammation is linked to the CD4+ TH2 cells and mast cells. However, the recently discovered group 2 innate lymphoid cells (ILC2s) have been found to play an important role in mucosal surfaces including airway tracts, as they have the capacity to produce TH2 cytokines including IL-4, IL-5 and IL-13 upon stimulation with IL-25, IL-33 and TSLP. Interleukin 1 receptor-like 1 (IL1RL1), also known as ST2, with its ligand IL33, induces allergic inflammation due to the release of type 2 cytokines. The biology of ST2 is complex because it is expressed in two forms, the soluble protein sST2, which down regulates TH2 responses, and the transmembrane form ST2 (ST2 or ST2L). Several studies have demonstrated that ST2 is expressed by ILC2s in mice; however, it is unclear from the literature whether human ILC2s express ST2. Therefore, the aims of this project were to validate antibodies to ST2 and to use validated antibodies to examine ST2 expression on human peripheral blood samples, in particular ILC2s. We hypothesised that ST2 may be upregulated on human ILC2s from asthmatic subjects compared to healthy controls. The results demonstrated that most of the commonly used ST2 antibodies were not specific to ST2, calling into question several of the published reports in the literature. Despite identifying an appropriate antibody for use in flow cytometry, it was not possible to complete the assessment of ST2 expression in asthma due to the COVID19 pandemic.

Introduction: Regenerative endodontic procedures have been practiced approximately over the past twenty years. These regenerate pulp-like tissue within the root canals of immature permanent teeth with pulpal necrosis which may also have periapical disease. The procedure involves disinfecting the root canal first, followed by conditioning the root surface dentine before a scaffold (such as a blood clot) is introduced into the canal space, along with mesenchymal stem cells (MSCs). Removal of the smear layer is usually undertaken with ethylenediaminetetraacetic acid (EDTA), which is also able to liberate extracellular matrix proteins embedded within dentine that help support the differentiation of MSCs. Maleic acid (MA) is another chelator which has been introduced to endodontics approximately ten years ago. It's ability to extract dentine matrix proteins (DMP) and their subsequent influence on MSCs is unknown. AIMS: Characterise the DMP extracted by EDTA (EDMP) or MA (MDMP) from dentine and evaluate their effects on odontoblastic differentiation of dental pulp stem cells (DPSC). Methods: Dentine powder was prepared from human healthy caries-free third molar teeth and exposed to EDTA or MA and total soluble proteins recovered were measured. The constituent proteins in each sample were also analysed using mass spectrometry (MS) & transforming growth factor 1 (TGF1) concentration was estimated using Enzyme-linked immunosorbent assay (ELISA). The proteins were then added to culture medium and exposed to DPSC which had also been characterised. DPSC grown in osteogenic medium (OM) served as the positive control & MDMP+OM and EDMP+OM as additional experimental groups. The influence of DMPs on odontoblastic differentiation of DPSCs was evaluated by using reverse transcription-quantitative polymerase chain reaction assessing relative gene expression of COL1A1, RUNX2, MSX2, DLX5, NAT10, OCN, OPN, DMP1 and DSPP and Alizarin red assay to quantify mineralisation. Results & Conclusions: Even though the total protein quantified in MDMP was lower than EDMP, proteomic profiles differed, with more proteins predicted in MDMP. TGF1 as a fraction of the total protein quantum was about 2-3 times higher in MDMP than EDMP. These findings may indicate that while dentine structure may be better preserved with MDMP, biologically relevant proteins may be extracted better by MA than EDTA. The DPSCs were not true stem cells, but were at least unipotent and displayed all canonical stem cell markers. DPSCs exposed to both MDMP and EDMP did not differentiate into odontoblasts and genes associated with mineralisation were generally suppressed. This may have relevance in formulating novel pulp regeneration strategies.

The development of flow cytometry data analysis computational tools in recent yearshas the potential to reduce the variation arising from manual gating and improve the quality of cell characterisations performed within academic, clinical and biomanufacturing settings. However, there is a need to understand the uncertainty of measurements from automated tools, alongside a need for benchmarking datasets with ground truth that enable systematic comparisons to be made between these tools. This thesis investigates the cell identification outputs of software that utilise different classes of clustering algorithms, with a focus on implementing highly controlled synthetic datasets for performance evaluation. A literature survey was conducted to identify the most cited tools, enabling the selection of the most relevant ones representative of different unsupervised clustering techniques: Flock2, flowMeans, FlowSOM, PhenoGraph, SPADE1, SPADE3 and SWIFT. Synthetic flow cytometry datasets were designed and generated with specific data characteristics of separation, normal/skew distributions, rarity and noise elements, and demonstrated to be credible substitutes for real cell data. These synthetic datasets were applied to the different software tools to determine the accuracy and repeatability of absolute cell counts. The results demonstrated how outputs from software analysing the same reference synthetic dataset vary considerably with accuracy deteriorating as the clusters overlapped and the separation index fell below zero. Moreover, SWIFT was found to be more negatively affected than other software in the presence of skewed cell populations. Assessment of rare cell detection revealed most software failed to consistently achieve a limit of detection of 100 cells in 106 events (0.01%). The addition of noise events resulted in a decrease in performance from all software, most significantly for FlowSOM. Furthermore, an automated versus manual comparison study carried out using a CD34+ stem cell dataset revealed higher variability from automated outputs compared to manual ones (mean coefficient of variations of 96% and 54%, respectively), and a weak correlation between the two methods (r=0.33) when analysing less well-separated cell populations. This work has illustrated how the generation of novel synthetic flow cytometry datasets, and their application in comparison studies, has allowed the performance limitations of different automated software tools to be uncovered. The synthetic datasets benefit from having known ground truth not obtainable from real world datasets, therefore have potential utility as digital reference materials, possibly leading to enhanced measurement confidence in automated cell characterisations and enumerations in fields such as diagnostics and cell therapy productions.

Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy or vascular-targeted photodynamic therapy with radiotherapy fractions used to treat prostate cancer. Firstly, the aim of this study was to investigate the tumour immune microenvironment and tumour vascular responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, the effects of combining fractionated radiotherapy with anti-PD-L1 immune checkpoint blockade or vascular-targeted photodynamic therapy on tumour growth delay was investigated. 3 x 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8+ T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post 3 x 5Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8+, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 x 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy. Fractionated radiotherapy induced 'vascular normalisation' changes in prostate cancer flank tumour allografts, improving vascular function as demonstrated using dynamic contrast-enhanced magnetic resonance imaging. Fractionated radiotherapy followed by vascular-targeted photodynamic therapy significantly delayed tumour growth in flank prostate cancer allograft pre-clinical models, compared with monotherapy with fractionated radiotherapy or vascular-targeted photodynamic therapy, and improved overall survival. This research suggests that multi-modal therapies for prostate cancer beyond immunotherapy may be necessary to achieve radiotherapy-induced anti-tumour responses. Combining fractionated radiotherapy with vascular-targeted photodynamic therapy may be a promising multimodal approach in prostate cancer therapy. This research provides proof-of-concept for this multimodality treatment to inform early phase clinical trials.

Glioblastoma multiforme (GBM) is one of the most aggressive human cancers, with a poor prognosis. Despite recent advances in the understanding of GBM's molecular biology, patient outcome continues to be poor due to the disease's incurable, aggressive, and recurrent nature. As a result, novel therapeutic strategies for the management of GBM patients are urgently needed. Itraconazole (ITC), a systemic antifungal, is a key regulator of hedgehog (HH) signalling, which is frequently deregulated in GBM. Because of its inhibitory effects on cell proliferation and tumour angiogenesis, ITC has been identified as a novel potential anticancer agent and is herein investigated in the context of GBM. In the present study, cell viability and reproductive integrity of GBM cells were assessed by AlamarBlue cell viability and clonogenic assays. Spheroid growth and relative colony cell growth were assessed by spheroid and associated absorbance reading assays. DNA damage levels and cell cycle distribution of the GBM cells were assessed by single cell gel electrophoresis (COMET) and flow cytometry, respectively, and O6-methylguanine DNA methyltransferase (MGMT) and HH protein levels were assessed by western blot analysis. Using these techniques we show that ITC, both alone and in combination treatments, inhibits GBM cell and spheroid growth in vitro, increased IR-induced DNA damage and caused G1 cell cycle arrest. Furthermore, ITC treatment decreased the levels of HH signalling pathway proteins sonic hedgehog (SHH) and smoothened (SMO). Together, these findings indicate that ITC inhibition of HH signalling pathway may increase TMZ, radiation and radiochemotherapy (RCT) responses and so serve as a potential adjunct to RCT in the management of GBM.

Acanthamoeba is a small free-living amoeba found in tap water and soil with two life stages: the trophozoite and cyst. Acanthamoeba species are opportunistic pathogens of humans that cause two main diseases including a potentially blinding infection of the cornea called Acanthamoeba keratitis (AK) in immunocompetent individuals, and a fatal granulomatous encephalitis in the immunocompromised. In this study, an ex vivo model of AK was developed to better understand the pathophysiological processes that occur in this disease. The model has several applications such as studying the interaction of Acanthamoeba with cells of innate immunity, investigating the efficacy of different pharmaceutical products in stopping the progression of the disease beside using the model to correlate between in vivo and ex vivo confocal microscopy findings of various morphologies of Acanthamoeba cysts and trophozoites. Furthermore, the study evaluated the phenotypic similarities between Acanthamoeba and cells of the innate immune system mainly macrophages using flow cytometry analysis to enhance the understanding of how immune cells interact with Acanthamoeba Porcine corneas were used to establish a reproducible ex vivo model which was maintained for four weeks and optimised by the supplement of CO2 and the use of air - liquid interface rocking system that mimics natural eye blinking. Once the model was established, Acanthamoeba trophozoites and cysts were added to the corneal model to evaluate the pathogenesis of Acanthamoeba keratitis, and the study successfully demonstrated the development of the infection in the model. This study was also able to demonstrate that the addition of macrophages and neutrophils to the AK model did not limit the process of the infection as these cells were phagocytosed by Acanthamoeba. The model was also used to investigate the efficacy of doxycycline and polyhexamethylene biguanide (PHMB) in stopping the disease progression and the results demonstrated the ability of PHMB to inactivate Acanthamoeba trophozoites with minimal toxicity to the corneal epithelium. Doxycycline was not found to have any major antimicrobial effect on the viability of Acanthamoeba. The model was also utilized to study the ex vivo confocal microscopy (EVCM) features of various forms of Acanthamoeba and correlate these findings to in vivo confocal microscopy (IVCM) images from culture positive AK cases. The study demonstrated similarity in the morphological features of Acanthamoeba in both ex vivo and in vivo confocal microscopy images, which makes EVCM images a reliable reference to validate IVCM findings. Finally, this study evaluated the phenotypic similarities between Acanthamoeba and macrophages using flow cytometry analysis which identified various degrees of positive reactivity of amoebic cell surface to a limited number of anti-human monoclonal antibodies. This suggests some structural and functional similarities in protein surfaces between amoeba and macrophages which can potentially offer a future tool for screening.

Mental health represents one of the most significant and increasing burdens to global public health. Depression and schizophrenia, among other mental illnesses, constitute strong risk factors for suicidality which results in over 800,000 deaths every year. The majority of suicides worldwide are indeed related to psychiatric diseases. A growing body of genetic, epigenetic and epidemiological evidence suggests that psychiatric disorders are highly complex phenotypes originating from the multilevel interplay between the strong genetic component and a range of environmental and psychosocial factors. Deeper understanding about the biology of the genome has led to increased interest for the role of non-sequence-based variation in the etiology of neuropsychiatric phenotypes, including suicidality. Epigenetic alterations and gene expression dysregulation have been repetitively reported in post-mortem brain of individuals who died by suicide. To date, however, studies characterizing disease-associated methylomic and transcriptomic variation in the brain have been limited by screening performed in bulk tissue and by the assessment of a single marker at a time. The main aim of this thesis was to investigate DNA methylation and miRNA expression differences in post-mortem brain associated with suicidality and unravel the complexity of epigenetic signals in a heterogeneous tissue like the human brain by developing a method to profile genomic variation at the resolution of individual neural cell types. The results here reported, provide further support for a suicide-specific epigenetic signature, independent from comorbidity with other psychiatric phenotypes, as well as confirming the strong bias perpetrated by bulk tissue studies hence the need to examine genomic variations in purified cell types. In summary, this thesis has identified a) a suicide-specific signal in two different epigenetic markers (DNA methylation and miRNA expression) and b) a protocol to simultaneously profile DNA methylation levels across three purified cell types in the healthy brain highlighting the utility of cell sorting for identifying cell type-driven epigenetic differences associated with etiological variation in complex psychiatric phenotypes.

The widespread and non-regulated use of antibiotics has resulted in pathogens developing resistance against them. Mis-prescribing of antibiotics is partly responsible for the current crisis of antimicrobial resistance, and it would be ideal for a patient to know the correct antibiotic against the causative organism before leaving a GP's surgery. To enable this strategy, a rapid antimicrobial susceptibility test needs to be performed with clinical samples. Flow cytometry was used to detect changes in bacterial cell size, viability, membrane energisation and DNA distribution. 3,3'-dipropylthiadicarbocyanine iodide (di-S-C3(5)), a red fluorescent membrane energisation dye, enabled assessment of bacterial cell numbers, and could be used to assess those capable of proliferating. E. coli were inoculated in 'terrific broth (TB)' at a concentration of 105 cells.mL-1 (representing bacteriuria), and bacterial proliferation was observed within 20 minutes by counting the live stained E. coli cells using a flow cytometer. Initially, this assay was used to see the effects of antibiotics (commonly used in UTIs), on lab-grown sensitive strains. The time point for the earliest detection of antimicrobial susceptibility ranged between 20-40 minutes, depending on the antibiotic used. A new growth medium was developed, from TB, to improve the period of the assay to a maximum of 30 minutes. The newly developed assay was cross-validated with real, UTIsuspected clinical samples. The results from the above assay could help in the development of a single-laser-based benchtop flow cytometer to be placed in a GP's surgery. While developing the rapid antimicrobial susceptibility assay, heterogeneous uptake of the cationic dye (di-S-C3(5)) was observed by the individual cells in a bacterial population. It was intriguing to understand how the dyes entered into the cells. The assumption still persists that most xenobiotics cross phospholipid bilayer by diffusion. Alternatively, different transporters are involved in the uptake of xenobiotics and the directed unfacilitated transport through phospholipid bilayers is negligible. High-throughput flow cytometry was used in this study to measure the uptake of xenobiotics (the fluorescent dyes di-S-C3(5) and SYBR Green I were used here) in individual cells. A subset of the single-gene knockout E. coli Keio collection was used to analyse the contribution of each transporter to the dye uptake. With di-S-C3(5), the range of steady-state uptake was observed to be 36-fold. Many knockouts with lower uptake than the wild-type consisted of y-genes (unknown function). Four of the five most fluorescent strains were knockouts of known major efflux transporters. Knockout of ATP synthase subunits a- and b- decreased the ATP production and also the dye uptake signifying that the uptake was ATP-driven. About half of the knockout strains analysed showed more than a 50% change (up and down) in the uptake of the dye as compared to the wild-type. Overexpression of some of these genes (ASKA collection) showed opposite effects as compared to their knockout counterparts. The uptake range with SYBR Green I was even higher than di-S-C3(5) at 69-fold. There was no correlation found between the uptake distributions of SYBR Green I and the DNA content of the cells in different strains. The results strengthen the belief that multiple transporters could be responsible for the uptake of these dyes. The broad range of uptake also signifies that diffusion through phospholipid bilayer is negligible, and emphasises the role of transporters in the dye-based staining of the intact bacteria.

Autoimmune disease is characterised by the development of abnormal immune responses directed against antigenic components of the host i.e. self-antigen. Multiple sclerosis (MS) is a chronic autoimmune, inflammatory disease directed against an unknown antigen in the central nervous system. This leads to focal demyelinating lesions and axonal damage. MS is caused by a complex interplay between multiple genetic and environmental risk factors and its incidence is increasing in both developed and developing countries worldwide. Current disease-modifying therapies non-specifically suppress components of the inflam- mation in MS. They are effective but with significant adverse events. There is an unmet need for safe targeted immunotherapies and for treatments that actively promote remyelination. This thesis examines two potential novel therapeutic strategies in autoimmunity and MS. The first approach arose from the observation, by previous research at GSK, that there is a population of CD4+ lymphocytes with apparent constitutive pSTAT1 expression independent of any stimulus and refractory to JAK-1 inhibition. This pSTAT1hi population had been found to be enriched in patients with systemic lupus erythematosus. The starting hypothesis of this thesis was that these cells might be pathogenic in autoimmune disease. However, following deep FACS phenotyping of this lymphocyte subset, the constitutive pSTAT1hi population was demonstrated to be an artefact of non-specific antibody binding, resulting in termination of this project. The remainder of the thesis focuses on the immunological effects of bexarotene. This retinoid X receptor agonist is currently being tested in a phase II clinical trial for its ability to promote remyelination [“CCMR One” trial]. Here, we focused on the ability of bexarotene to modulate the human Th17/iTreg axis in vitro, owing to literature reporting that RXR ligands augment RAR agonists’ effect on murine Th17/iTreg cells. We show that bexarotene promotes the differentiation of human Foxp3+ iTregs from naive CD4+ T cells independent of RAR engagement, whilst concomitantly inhibiting the differentiation of Th17 cells and their secretion of IL-17a. Bexarotene was not found to alter the methylation status of the Treg-specific demethylated region, with iTregs remaining fully methylated within this study. Next, I examined the peripheral blood of patients receiving six months of oral bexarotene treatment within the CCMR One trial. There was no change in serum IL-17a, the frequency of CD25hi CD127 Foxp3+ Tregs, nor in the secretion of IL-17a from effector T cells. Mirroring the data in vitro, no change was observed in the TSDR methylation status of Tregs isolated from these patients. Taken together, these results demonstrate the ability for RXR-specific ligands to modulate the Th17/iTreg axis in favour of iTreg differentiation in vitro, and thus their potential in the treatment of those autoimmune diseases which are driven by a failure of self-tolerance and imbalance of Tregs and inflammatory Teffectors.

The loss of efficiency and performance of bioprocesses on scale-up is well known, but not fully understood. This work addresses this problem, by studying the effect of some fermentation gradients (pH, glucose and oxygen) at a larger scale in a bench-scale two compartment reactor (PFR + STR) using the cadaverine-producing recombinant bacterium, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT. The initial scale down strategy increased the magnitude of these gradients by only increasing the mean cell residence time in the plug flow reactor (τ_PFR). The cell growth and product related rate constants were compared as the τ_PFR was increased; differences were significant in some cases, but only up to 2 min residence time. For example, losses in cadaverine productivity when compared to the control fed-batch fermentation on average for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 42 % and 46 % respectively. This indicated that the increasing the τ_PFR alone does not necessarily increase the magnitude of fermentation gradients. The new scale-down strategy developed here, increased the magnitude of fermentation gradients by not only increasing the τ_PFR, but also considering the mean frequency at which the bacterial cells entered the PFR section (f_m). The f_m was kept constant by reducing the broth volume in the STR. Hence, the bacterial cells also spent shorter times in the well mixed STR, as the τ_PFR was increased (hypothesised as giving the bacterial cells less time to recover the non-ideal PFR section of the SDR). On adoption of this strategy cadaverine productivity decreases for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 32 % and 53 % respectively. Thus, highlighting that loss in performance is most likely to occur as the magnitude of heterogeneity within the fermentation environment increases. However, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT did show some resilience in its biomass productivity. It was only marginally affected in the harshest of conditions simulated here.

Abnormal activation of brain microglial cells is widely implicated in the pathogenesis of schizophrenia. The disrupted balance of microglia phenotypes has been hypothesized to influence the clinical course of the disease and affect symptom severity. Previously, the pathophysiology of microglial activation was considered to be intrinsic to the central nervous system. We hypothesised that due to their perivascular localization, microglia can also be activated by factors present in circulating blood. We applied a high-content functional screening platform, to characterize alterations in microglial intracellular signalling cascades induced by schizophrenia patient serum relative to control serum. Using automated sample preparation, fluorescent cellular barcoding and flow cytometry, the applied platform is capable of detecting multiple parallel cell signalling responses in microglia. First, we exposed a human microglia cell line to serum isolated from first-onset drug-naïve schizophrenia patients (n=60) and healthy controls (n=79). We were able to show that peripheral blood serum obtained from schizophrenia patients induced differential microglial cell signalling network responses in vitro. We subsequently assessed whether antipsychotic drug-treatment can normalise the abnormal microglial signalling responses previously identified by exposing microglia cells to serum from antipsychotic treated schizophrenia patients (n=15) and controls (n=17). In addition, in order to assess microglia activation in vivo, we obtained positron emission tomography (PET) imaging data from collaborators, who used a radiotracer to assess potential altered microglia activation in patients suffering from schizophrenia. Finally, as a proof of concept study, we attempted to validate these findings by assessing the effect of serum collected from first-onset drug-naïve schizophrenia patients (n=9), controls (n=12) as well as serum isolated from the same patients subjected to six weeks of clinical treatment with the antipsychotic olanzapine (n=9). This study aimed to identify normalisation of previously detected differences in microglia signalling pathways based on successful in vivo treatment. We demonstrate that peripheral blood serum isolated from schizophrenia patients, independent of their treatment status, is sufficient to trigger microglial cell signalling network responses in vitro, which are indicative of altered STAT3 signalling. We further explored the composition of the serum for differential expression of analytes, previously associated with neuropsychiatric disorders, and the utility of the detected microglial cellular phenotype as a target for novel drug discovery.

Rheumatoid arthritis (RA) is a chronic, heterogeneous, autoimmune disease that causes inflammation of synovial joints leading to pain, stiffness and swelling. If left untreated, RA results in irreversible joint destruction and long term disability. Initial treatment with glucocorticoids and other immunosuppressive agents suppresses inflammation. However, many of these drugs are not well-tolerated due to extensive side effects or are simply ineffective. The discovery of tumour necrosis factor-α (TNF) as a key mediator of inflammation in RA led to the development of monoclonal anti-TNF antibody therapy. Since then, other biologic drugs targeting immune pathways have been developed for RA, including interleukin-6 (IL-6) blockade, B cell depletion, and T cell co-stimulation blockade. Not all patients will respond to their first biologic drug and currently there is no way to predict which patient will respond to each different class of drug. Generally, 3-6 months are required to determine clinical efficacy, during which time joint inflammation proceeds. Therefore, discovering biomarkers to predict treatment response is a research priority. Biologic drugs target immune pathways. As single cell technology advances and has increasing capacity to identify subtle changes in many cell subsets, I hypothesise that studying the blood immune cell landscape will define cellular biomarker profiles relevant to each individual patient's disease. In the first chapter I developed an optimal protocol that allowed the collection and processing of PBMCs from distant locations. The second chapter utilises this protocol to examine innate and adaptive immune cell populations by 17-channel flow cytometry from 100 RA patients prior to commencing a biologic drug. The final chapter develops and utilises a 37-channel mass cytometry protocol that is superior at deeply immunophenotyping immune cells. Collectively this thesis identifies a number of candidate cellular biomarkers which are associated with biologic drug treatment response. In particular, patients with a low number of CD28+ Tregs, or aberrant inflammatory cytokine production upon stimulation of T cells, had reduced response to anti-TNFs. As the complexity of single cell technologies increases it is more likely that a meaningful correlation will be revealed to inform treatment response.

Tuberculosis is one of the most prevalent infectious diseases globally due to the successful survival mechanisms displayed by Mycobacterium tuberculosis (Mtb). Mtb primarily infects alveolar macrophages (AMs) and is able to live intracellularly for extended periods of time due to a number of virulence factors which inhibit the antibacterial mechanisms of the AMs. This aspect of the Mtb life cycle means TB treatments suffer from poor bioavailability and efficacy. Additionally, the rise in resistant strains of Mtb means the use of higher doses and the use of alternative second and third line drugs which increase the risk of systemic toxicity. Drug encapsulation is a novel approach that can provide more favourable drug pharmacokinetics and pharmacodynamics. The aim of this project was to develop a liposomal drug delivery system to target Mtb infected alveolar macrophages. The system involved the encapsulation of two drugs; the antibiotic gatifloxacin (GFLX) and Mtb virulence factor inhibitor CV7. The hypothesis was that the two different antibacterial mechanisms would work in synergy and increase the efficacy of the treatment. AM targeting and receptor-mediated endocytic uptake was encouraged by the presence of a ligand attached to the surface of the liposome. Furthermore a pH-sensitive release mechanism was to be incorporated into the liposome to encourage the release of the encapsulated drugs in the vicinity of the intracellular bacteria. The intention was to produce a drug delivery system to enable a TB therapy regime of fewer, lower doses to increase compliance and reduce systemic toxicity by increasing efficacy through improved bioavailability. GFLX was successfully encapsulated using a weak base active loading method. To establish encapsulation efficiency, a homogeneous fluorescence assay able to quantify intra- and extra-liposomal gatifloxacin simultaneously was developed. pH-sensitive release of the payload could be achieved using a pH-sensitive peptide with a novel design based on chimeric structure, namely P3. CV7 was successfully encapsulated using a weak acid active loading method. CV7 liposomes were able to be functionalised by the incorporation of a mannose ligand on the surface of the liposome. An inhibition assay using the target enzyme of CV7, MptpB, was optimised to assess efficacy of liposomally encapsulated and released CV7. Flow cytometry and confocal microscopy studies confirmed that the liposomal formulations were internalised by the target macrophage cell line, J774a.1. Mannose liposomes conveyed superior uptake kinetics. Further confocal microscopy showed that after internalisation the liposomes entered the endolysosomal pathway and colocalised with BCG. A BCG-macrophage infection model was used to determine the intracellular efficacy of the liposomal formulations. Encapsulated CV7 displayed increased efficacy over free CV7, while encapsulation in functionalised liposomes showed better efficacy still. The encapsulation of GFLX did not increase the efficacy of GFLX and synergy between the two drugs was not achieved. In conclusion, the liposomal encapsulation of CV7 increased uptake of the drug by the target cell line and facilitated colocalisation of the drug with the target pathogen thereby increasing efficacy. Such a formulation could potentially increase bioavailability and efficacy in vivo for a more tolerable TB therapy.

The development of a malaria vaccine is necessary for disease eradication. Successful vaccine candidates to date have targeted the asymptomatic, pre-erythrocytic stage of the disease, however even the most efficacious vaccines are only partially protective. Research undertaken in our laboratory has demonstrated that one such regimen, using an 8 week prime-boost viral vector approach of ChAd63 ME-TRAP and MVA ME-TRAP, induces sterile efficacy in 21% of vaccinees, with a key role identified for TRAP-specific CD8+ T cells. The work described in this thesis explores the most immunogenic regimen by which to administer these two pre-erythrocytic malaria vaccines. A shortening of the prime-boost interval from 8 to 4 weeks, and the addition of an extra ChAd63 ME-TRAP priming vaccination, both demonstrated improved T cell immunogenicity over the standard 8 week regimen. Further to this, novel assays were developed to aid the evaluation of vaccine-induced immune responses. Adaptations of the existing methodology for ELISpot analysis and to whole blood flow cytometry techniques, enabled more detailed analyses of paediatric vaccine-induced T cell responses in The Gambia. This work also permitted the comparison of vaccine immunogenicity in this paediatric population, with malaria-naïve and malaria-exposed adult vaccinees. The results suggest that vaccine-induced T cell responses in infants of 8 weeks and older are comparable to that of adults. A second approach involved the development of a novel functional assay. This assay quantitatively measured the in vitro inhibition of intrahepatic Plasmodium parasite development using T cells from ChAd63.MVA ME-TRAP vaccinated volunteers. The assay demonstrated the ability of CD8+ T cells to inhibit parasite development in a TRAP-specific manner, and provides a platform with which to further explore pre-erythrocytic immune responses.

Abstract: Across all domains of life, the surface of all cells is coated in a dense array ofcarbohydrates appended to proteins and lipids, collectively referred to as glycans. The interactionbetween glycans and glycan-binding proteins, also known as lectins, are at the interface of nearlyevery biological interaction at the cellular level. Unlike other biological macromolecules (nucleicacids and proteins), glycan biosynthesis is not template driven and glycans can be branchedpolymers, resulting in heterogenous structures that are difficult to predict, and which are verystructurally complicated. Glycans terminating with the monosaccharide sialic acid are capable ofengaging with the Siglec lectin family. Siglecs facilitate many cellular functions, however, all Siglecfunctions are initiated by engaging with a glycan terminating with sialic acid. In humans, Siglecsare a family of fifteen cell surface receptors, which are generally found on immune cells. Theimmunomodulatory properties of Siglecs motivates a better understanding of their physiologicaland pathophysiological roles. Therefore, the first step in understanding the biology of a Siglecstarts with understanding the ligands of that Siglec. Nevertheless, the complexity and diversity ofglycans coupled with multiple Siglecs often being expressed by a single cell makes deconvolutingSiglec-glycan interactions challenging. Through the development and optimization of approachesthat enable the systematic description of Siglec ligands a better understanding of the roles ofSiglecs in health and disease can be established. Insights gained by describing Siglec ligandscan then be leveraged to investigate the function of the Siglec and potentially apply thesediscoveries to develop novel therapeutics towards the treatment of many pathologies such ascancer, bacterial/viral infection, autoimmune disease, neurodegenerative disease and manyothers.iiiIn Chapter 2, improvements with respect to versatility, activity, yield, and storage of apreviously established soluble version of a Siglec referred to as a Siglec-Fc-chimera were made.The versatility of this construct was demonstrated by its use in many different approaches fordescribing Siglec-ligands including a cell-based glycan array, bead assay, ELISA, and massspectrometry-based assay. Using these different approaches many insights were made withrespect to Siglec ligands. Two examples of new insights are: the relationship between cis andtrans binding of Siglecs on primary human immune cells and the glycan ligands of CD33. It isnoteworthy that the tools and approaches developed in this Chapter were extensively applied inthe later Chapters.In Chapter 3, a novel liposomal nanoparticle formulation was developed for optimalengagement of Siglecs by glycolipids. Using this formulation, the human and murine Siglec familywas screened against a panel of glycolipids. During this interrogation, it was revealed thatglycolipids are ligands for Siglec-6. Additionally, the glycolipid binding profiles between humanand murine Siglecs were compared and the appropriateness of murine models to study humansSiglec-glycolipid interactions was assessed.Building on the work from Chapter 3, the ability of Siglec-6 to bind glycolipids wassystematically dissected using a panel of synthetic glycolipids referred to as neoglycolipidscoupled with mutagenesis studies. These studies revealed that a solvent exposed tryptophanresidue is critical for Siglec-6 engagement of sialosides when presented from a liposome.Additionally, Siglec-6-liposome binding was probed on genuine human cells and tissuesdemonstrating that glycolipid bearing liposomes can target Siglec-6 in physiologically relevantcontexts. A possible biological role was ascribed to Siglec-6 when it was found that Siglec-6 canfacilitate the internalization of natural nanoparticles, known as extracellular vesicles, in aglycolipid-dependant manner. In summary, the work described above demonstrates howsystematic description of Siglec ligands leads to the development of probes which can be appliedto learn more about the biological roles Siglecs play in health and disease.

Botryococcus braunii is a microscopic, colonial green alga that may be found in fresh and brackish waters throughout the globe. B. braunii is unique in that it constitutively synthesises and secretes copious amounts of various long-chain (C23-C40) hydrocarbons, generically termed “botryococcenes”. Botryococcanes, the hydrogenated forms of botryococcenes, comprise 1% of the fossil hydrocarbons found in petroleum deposits and in oil-shales. Microfossils identified as Botryococcus by optical and scanning electron microscopy are also abundant in these strata, but the actual identity and precise relationship between these microfossils and extant Botryococcus species is not known. In this investigation, the relationship between living Botryococcus algae and microfossils identified as Botryococcus using traditional palaeontological analysis and light-microscopy was investigated by analysis of ancient DNA (aDNA). The material used was identified in sediments from Boswell Lake (British Columbia, Canada), a Holocene lake that had remained undisturbed since the glacial retreat. New flow-cytometry methods were developed to rapidly purify enough of the relevant microfossils, from which aDNA was extracted and sequenced. Pollen grains were purified using the same flow-cytometry method and from the same horizons as the Botryococcus microfossils and used to age the sedimentary horizons by 14C radiocarbon dating. Samples of the purified microfossils were imaged by scanning electron microscopy for comparison with published images of fossils identified as Botryococcus from kerogens. In addition, metaDNA from the relevant horizons was extracted and sequenced by NGS, and a chemical analysis for botryococcene derivatives performed using two-dimensional gas chromatography (2D-GC). The genomic analyses show that the sub-fossils identified in Boswell Lake are likely to be representatives of B. braunii, race B. The geochemical analysis identified hydrocarbons that migrate as botryococcenes on 2D-GC in the strata whence the sub-fossils were purified. The SEM images indicate that the microfossils purified from Boswell Lake have similar morphologies to those found in kerogens. Taken together, these data strongly support the proposition that petroleum and kerogen deposits are unusually rich in B. braunii and that these algae have a lineage potentially dating 500 million years. The metagenomic analysis enabled similar conclusions to be reached regarding the presence of B. braunii within the sediment, without the need for targeted microfossil purification. While this analysis was less precise due to the under-representation of algal genomes in the public sequence databases, the metagenomics approach employed was particularly well suited to the temporal analysis of prokaryotic microcosms within Lake Boswell, the succession of which could be associated with periods of climatic variation. The analytical methods described herein are generally applicable to understanding microbial systems over geological periods, and may be used to generate important insights into the cause and effect relationships between microbial populations and environmental perturbation.

A collection of twenty three selectively mono-protected di- and triamines, masked with the Boc, Fmoc or Ddiv protecting groups, were synthesised via continuous flow synthesis in a self-assembled meso-scale PTFE flow reactor. The continuous flow strategy offered direct access to the mono-protected compounds in good yields, especially in the case of the Fmoc carbamates which circumvented the use of another sacrificial protecting group. Two of the mono-Boc-protected carbamates were used as starting materials to generate N-alkylglycine monomers; synthesised via tandem mono-alkylation and Fmoc carbamation, linked by an in-line scavenging protocol using a silica-based trisamine scavenger resin. The final step of the monomer synthesis employed catalytic transfer hydrogenolysis using 20% Pd(OH)2/C and 1,4- cyclohexadiene. The three-step flow procedure gave access to two monomers, with one of them being a novel N-alkylglycine unit bearing a triethylene glycol bridge. The monomers were used as building blocks to assemble new oligo-N-alkylglycines (peptoids) via microwave-assisted solid phase synthesis. Three different types of peptoids were synthesised: (i) oligo-N-(6-aminohexyl)glycines (“standard” peptoids), (ii) oligo-N-{2-[2-(2-aminoethoxy)ethoxy]ethyl}glycines (“triethylene glycol” [TEG] peptoids) and (iii) hetero-oligomers of alternating “standard” and “TEG” monomers (“hybrid” peptoids). The peptoids were evaluated for their cellular permeability and cytotoxicity with HeLa, HEK-293 and CHO cells. All the peptoids were shown to be non-cytotoxic at 10 μM based on cell proliferation assays. In general, it was found that the cellular uptake of the hybrid peptoids outperformed their standard and TEG analogues. Flow cytometry and confocal microscopy results revealed that the hybrid nonamer had the highest cellular uptake efficiency of all the peptoids synthesised. At a concentration of 1 μM, it outperformed the second best molecular transporter (standard nonamer) by a factor of seven.

Polymyalgia rheumatica (PMR) is a chronic inflammatory condition which affects the elderly, causing aching and stiffness of the neck, shoulders and pelvis, as well as more systemic manifestations such as fever, malaise and fatigue. PMR shares symptoms with rheumatoid arthritis (RA) and both are associated with significantly elevated circulating concentrations of interleukin-6 (IL-6), which is thought to play a key role in the pathogenesis of these diseases. In RA, IL-6 is derived from synovial cells in the joints. In PMR, however, the source of IL-6 is unknown. PMR patients do not exhibit the same joint involvement as in RA but they do have elevated circulating IL-6 concentrations, thus, it was hypothesised that the source of IL-6 in PMR may be one of the circulating peripheral blood mononuclear cell (PBMC) types, which are all capable of producing IL-6. Blood samples were taken from untreated PMR patients, RA patients with active disease and healthy controls (HC) of similar age and gender. To account for known circadian variations in circulating IL-6, samples were taken at a standard time. IL-6 was quantified in plasma and serum using cytometric bead array (CBA) and enzyme-linked immunosorbent assay. The biological activity of the IL-6 was tested for the first time in PMR using a B cell proliferation assay. Using immunostaining and flow cytometry, constitutive intracellular IL-6 was measured in CD3+, CD14+, CD19+, CD123+ and CD11c+ PBMCs. Intracellular IL-6 was also determined following PBMC stimulation in vitro, to determine potential differences in cell responsiveness. Concentrations of secreted IL-6 in the culture supernatants of resting and stimulated PBMC were determined by CBA in parallel cultures. Other cytokines were also quantified in order to examine PMR and RA pathologies more broadly. Finally, the results of the cytokine assays were compared with patient reported severity of fatigue and the four different components of fatigue (emotional, living, physical and cognitive). Circulating IL-6 was significantly elevated above HC in both serum and plasma of PMR and RA patients, and this IL-6 was found to be biologically active. PBMC in all subjects constitutively produced low levels of intracellular IL-6 and very low concentrations of secreted IL-6 in parallel cultures. Overall, responses to in vitro stimulation were variable but no significant differences were observed between PMR, RA and HC samples. Secreted IL-6, in contrast, increased dramatically following stimulation of all cultures, suggesting intracellular staining may not reflect the secretory capability of these cells, but also confirming that there were no differences between PBMC responses of PMR, RA and HC groups. A significant correlation was observed between circulating IL-6 concentrations in PMR and RA patients, and physical fatigue, living fatigue and total fatigue. Broader cytokine analysis demonstrated that IL-6 alone was significantly elevated in PMR patients. Taken together, circulatory IL-6 in active PMR is found to be elevated in the absence of other inflammatory cytokines. It is biologically active and correlates strongly with physical and living aspects of fatigue. Circulating PBMCs are not the source of this elevated IL-6 in PMR patients, suggesting that neutrophils, vascular endothelium or muscle tissue may instead be involved.

Cell separation based on antibody-targeted magnetic beads has been widely used in a number of applications in immunology, microbiology, oncology and more recently, in the isolation of circulating tumour cells (CTCs) in cancer patients. Although other cell separation techniques such as size based cell filtration and Fluorescence Activated Cell Sorting have also been in popular use, immunomagnetic cell isolation possesses the advantages of high throughput, good specificity and reduced cell stress. However, certain fundamental features of the cell-bead interface are still unknown. In this study, some of the key features of the cell-bead synapse were investigated in an effort to improve the efficiency of immunomagnetic cell isolation and reduce its dependence on high expressing cell surface markers. A clinically relevant antibody fragment (Fab) against tyrosine kinase receptor HER2 was applied to study the immunomagnetic isolation of HER2 expressing cancer cells. First, the minimum number of target proteins required on a cell for it to be isolated was determined. Second, the importance of the primary antibody affinity was investigated, using a series of Fab mutants with known kinetics and it was shown that despite starting with sub-nanomolar affinity, improving Fab affinity increased cell isolation. Third, the influence of the connection between the primary antibody and the bead was studied by comparing Fab bridged to the magnetic bead via a secondary antibody, Protein L or streptavidin; the high affinity biotin-streptavidin linkage increased isolation sensitivity by an order of magnitude. Fourth, the effect of manipulating cytoskeletal polymerization and cell membrane fluidity using small molecules was tested; cholesterol depletion decreased isolation and cholesterol loading increased cell isolation. The insights from these observations were then applied to isolate a panel of cell lines expressing a wide range of surface HER2. While the standard approach isolated less than 10% of low HER2 expressing cancer cells from spiked rabbit and human blood, our enhanced approach with the optimized cholesterol level, antibody affinity and antibody-bead linkage could specifically isolate more than 80% of such cells. The final part of this work focussed on developing an antibody clamp that could physically restrict the antigen within its binding site on the Fab and prevent antigen dissociation, using the HER2-Fab complex and the anti-myc peptide antibody 9E10. Work from this thesis provides useful insights into the molecular and cellular parameters guiding immunomagnetic cell isolation and can be used to extend the range of target receptors and biomarkers for tumour cell isolation and other types of cell separation, thereby enhancing the power and capacity of this approach.

Experimental studies into the use of cold atmospheric plasmas for inactivating foodborne pathogens are presented in this thesis. Eliminating the possibility that treatment delivered by a plasma to a population or assemblage of micro-organisms is unevenly distributed is an essential pre-requisite to attempting to interpret inactivation kinetics with a view to elucidating mechanisms of inactivation. A filtration method of depositing cells evenly on the surface of a membrane without cell stacking was developed and used throughout the work described here. Two atmospheric plasma systems were evaluated and each brought about microbial inactivation in a distinct way. A pulsed radio frequency plasma jet operated at 3.47 MHz caused gross morphological changes to L. innocua whereas a low frequency air mesh plasma system operated at a frequency of 24 kHz led to the inactivation of these bacteria without inducing observable structural changes. Changing the operating parameters of the plasma jet system had a significant effect on the composition of the reactive plasma species generated as revealed by changes to the mode of inactivation of bacteria. In addition to inactivating bacteria, the pulsed plasma jet was shown to be highly effective in degrading and removing amyloid aggregates from the surface of mica coupons. Amyloids have widely been used as a non-infectious model for prions, and the results obtained here show potential for the application of gas plasma technology for removing prions from abiotic surfaces in medical and other applications. It has widely been assumed that bacterial envelopes are the principal sites at which reactive plasma species bring about damage to cells. However, changing the composition of the bacterial membranes of E. coli and Listeria innocua by cultivating them at widely different temperatures to induce changes proved not to result in enhanced inactivation. Flow cytometry was also used to provide additional insights into possible mechanisms of inactivation. The following fluorescent dyes were used either singly or in combination; SYTO 13, DiBAC4(3), cFDA and PI. The results obtained with the dyes DiBAC4(3) and PI showed that Gram positive bacteria became depolarised prior to the bacterial membrane becoming compromised, possibly suggesting that the inactivating plasma species are affecting membrane proteins responsible for maintaining the bacterial charge. Differences between the fluorescent dye staining of Gram negative and Gram positive species were obtained using SYTO13 and PI demonstrating that the different membrane structures affect their interaction with the plasma. In additional studies, the air mesh plasma was used to treat multi-drug resistant strains of Methicillin resistant Staphylococcus aureus (MRSA) in an attempt to reverse antibiotic resistance. MRSA PM 64 was shown to reverse its antibiotic resistance to Oxacillin, Kanamycin and Trimethoprim. Culturing the bacteria in a nutrient limited media led to increased resistance towards plasma treatment and maintenance of their high levels of antibiotic resistance.

The cancer stem cell hypothesis has recently re-emerged as a compelling paradigm for the development and progression of neoplastic disease. The hypothesis proposes that a specific subset of “cancer stem cells” (CSC), believed to share many features with normal stem cells, is exclusively responsible for maintaining tumour growth and driving progression. If the CSC hypothesis applies, it may require re-evaluation of the clinical approach to neoplasia. Spontaneous cancer in the domestic dog represents a significant welfare problem, with dogs developing many tumours strongly reminiscent of those affecting humans. This study sought to investigate whether cells with characteristics of CSC are identifiable in canine cancer. Assays to identify, isolate and characterise CSC were adapted to the canine system, and cancer cell lines and spontaneous tumours of diverse origin evaluated for the presence of candidate populations. Whilst analysis of surface expression patterns did not identify specific subpopulations within canine cancer cell lines, these were detectable in cells derived directly from primary tumours. Assays for stem cellassociated drug resistance mechanisms could also be used to identify subsets of putative canine CSC. Formation of “tumourspheres” by canine cancer cell lines was found to be highly density-dependent, so a potentially unreliable method of isolating CSC. Expression of the cell surface glycoprotein CD44 was associated with cellular proliferation status, although it may not represent a stable canine CSC marker. The NFκB survival pathway, associated with apoptosis resistance of some putative CSC, was constitutively active in canine cancer cell lines; suppression using specific inhibitors could reduce cell viability, indicating that this may represent a rational therapeutic target. Overall, these studies demonstrated that CSC assays may be adapted to the canine model system, although they require rigorous interrogation to distinguish apparent CSC attributes from basic biological properties. Cell lines have provided a stable background upon which to optimise assays, but appear less likely to demonstrate discrete CSC subpopulations. Putative CSC subsets may be more readily identifiable within heterogeneous primary tumour cells. The application of some of these adapted assays within a clinical setting may enable further characterisation of individual patients’ tumours, and inform therapeutic regimes for improved treatment outcomes.

Micro-irradiation systems are used to analyse the effect of ionizing radiation at the cellular and tissue level, targeting individual cells within a population with a controlled low dose. Cell survival experiments using micro-irradiation systems are limited by factors including: 1) the radiation attenuation and optical properties of the chosen cell dish substrate, 2) the registration of the cell dish before and after irradiation or between multiple imaging modalities and 3) the analysis of the cell or colony growth after irradiation. In this thesis, a set of tools have been developed to improve micro-irradiation experiments and to increase the accuracy of information provided by the cell survival data. The first contribution, the substrate cell dish evaluation, provides a set of characteristics defining the substrates used for micro-irradiation experiments based on minimal energy loss and optical clarity using unstained cell imaging. The second contribution was the development of a novel and low cost fiducial marking device for micro-irradiation experiments using an 808 nm laser and providing marks suitable for imaging with multiple modalities. The minimum focused spot diameter was calculated as 22.9 urn and the device was used to create fiducial marks with diameters ranging from 20 urn to 130 urn. The third contribution, the development of a cell counting methodology for use with a lens-free imaging device, has been shown to accurately count thousands of cells suitable for immediate analysis. Approximately 1000 cell colonies, containing 17 729 cells on 11 cell dishes were used for testing and training for automatic cell counting. Validation of the cell counting method showed that 76% and 89% of the cell colonies were counted within a ± 20% and ± 30% error of the ground truth, respectively. Further development of the fiducial marking device, by modifying the choice of laser and making it suitable for multiple types of cell dish substrates, would increase the applications of the device. Development of the cell counting methodology for different cells line, and for cells grown on multiple types of substrates, would make the system suitable for analysis of a wide variety of cell survival studies. The cell counting methodology, applied to the CyMap lens-free imaging device, has the potential to be an extremely useful and cost effective tool for cell survival studies.

Recently, laser light sources of different regimes have emerged as an essential tool in the biophotonics research area. Classic applications include, for example: manipulating single cells and their subcellular organelles, sorting cells in microfluidic channels and the cytoplasmic delivery of both genetic and non-genetic matter of varying sizes into mammalian cells. In this thesis several new findings specifically in the optical cell sorting as well as in the photo-transfection study fields are presented. In my optical cell sorting and guiding investigations, a new technique for enhancing the dielectric contrast of mammalian cells, which is a result of cells naturally engulfing polymer microspheres from their environment, is introduced. I explore how these intracellular dielectric tags influence the scattering and gradient forces upon these cells from an externally applied optical field. I show that intracellular polymer microspheres can serve as highly directional optical scatterers and that the scattering force can enable sorting through axial guiding onto laminin coated glass coverslips upon which the selected cells adhere. Following this, I report on transient photo-transfection of mammalian cells including neuroblastomas (rat/mouse and human), embryonic kidney, Chinese hamster ovary as well as pluripotent stem cells using a tightly focused titanium sapphire femtosecond pulsed laser beam spot. These investigations permitted advanced biological studies in femtosecond laser transfection: firstly, the influence of cell passage number on the transfection efficiency; secondly, the possibility to enhance the transfection efficiency via whole culture treatments of cells thereby, synchronizing them at the mitotic (M phase) as well as the synthesis phases (S phase) of the cell cycle; thirdly, this methodology can activate the up-regulation of the protective heat shock protein 70 (hsp70). Finally, I show that this novel technology can also be used to transfect mouse embryonic stem (mES) cell colonies and the ability of differentiating these cells into the extraembryonic endoderm.

Considerable variation exists both within and between species in nuclear DNA content. Despite there being no obvious functional role for much of this DNA, many studies have reported phenotypic correlations with genome size at various taxonomic levels. This suggests that DNA plays a functional role beyond the traditionally understood mechanisms. One such example of a phenotypic correlation with DNA content is present in the genus Silene, where a negative correlation between DNA content and flower size exists within and between species. This relationship is consistent with the direction of sexual dimorphism in DNA content (caused by heteromorphic sex-chromosomes) and flower size in the most studied species in the genus: S. latifolia. This thesis takes a comparative approach between two closely related species in the genus (S. latifolia and S. marizii), which differ markedly in their nuclear DNA content, in order to investigate the nature and phenotypic impacts of variation in DNA content. A phenotypic survey from a number of S. marizii populations reveals that the pattern of DNA content variation in this species is very different to that in S. latifolia. In particular, phenotypic correlations with DNA content appear be much weaker, whilst sexual dimorphism in DNA content, when present, appears to occur in either direction. A survey of interspecific hybrids suggests that this may be due to an enlarged S. marizii X-chromosome and that DNA content in hybrids may be biased with regard to their parents. Repetitive elements may be significant constituents of plant genomes. A study of Ty1-copia class retrotransposons in the two species reveals that they are present as a large and highly heterogeneous population. Phylogenetic analysis of these elements suggests a substantial degree of genetic isolation between the two species. Finally, an assessment of the flow-cytometric method, used to estimate DNA content, reveals substantial error associated with the method, but only limited evidence for stoichiometric effects.

The aim of this work was to determine whether the number of FcεRI positive dendritic cells and the levels of expression and occupancy of this receptor by IgE were increase in atopic asthmatics compared to normal subjects. A method has been successfully developed to identify peripheral blood dendritic cells by flow cytometry as cells staining brightly for HLA-DR but negative for a cocktail of lineage specific antibodies (CD3, CD14, CD19, CD16 and CD56). PBMCs were purified from 35 subjects who had been characterised as to their atopic and asthmatic status. Three-colour flow cytometry was used to determine the levels of expression of FcεRI and CD23 by blood dendritic cells, and their ability to bind IgE in vitro. Statistical analysis revealed that the total expression of FcεRI on the surface of dendritic cells from normal and asthmatic subjects was not significantly different. However, dendritic cells from asthmatics had higher levels of IgE bound to the cell surface in vivo, and could further bind more IgE in vitro than dendritic cells from normal subjects. Dendritic cells were not found to express significant levels of CD23. The subunit composition of the receptor was resolved by RT-PCR on purified dendritic cells, and showed that in the peripheral blood, mRNA transcripts were present for the FcεRI-α, -β and -γ subunits. In conclusion, total levels of FcεRI expression by dendritic cells in the blood from normal and asthmatic subjects were not different, in marked contrast to receptor expression by dendritic cells in the lung. It is possible therefore that the local environment of the asthmatic lung leads to this upregulation of surface FcεRI. It does appear that the function of the receptor, and in particular its capacity to bind IgE, may be different in health and disease. An improved understanding of the expression of FcεRI by dendritic cells in health and disease may open new avenues for therapy to combat the suffering caused by asthma.

The effects of altered membrane fatty acid composition on the toxic interactions of heavy metals with Saccharomyces cerevisiae were examined. Saccharomyces cerevisiae was enriched with the polyunsaturated fatty acids (PUFAs) linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Incorporation of the exogenous PUF As resulted in them comprising greater than 65% and 40% of the total fatty acids in whole-cell and plasma membrane lipids, and nuclear membrane lipids, respectively. Incorporation of the exogenous PUF As had no discernible adverse effects on cell division. However, inhibition of cell division in the presence of Cd(N03)2 was accentuated by growth in the presence of the di-unsaturated fatty acid linoleate. Furthermore, susceptibility to both Cd2+ - and Cu2+ -induced plasma membrane permeabilisation and whole cell toxicity was markedly accentuated in PUF A-enriched cells, and increased with the degree of fatty acid unsaturation. The increased sensitivity ofPUFA-enriched cells to membrane permeabilisation and whole-cell toxicity was correlated with increased levels of lipid peroxidation in these cells. Cu2+ - and Cd2+_ induced lipid peroxidation was rapid and associated with a decline in plasma membrane lipid order, detected by fluorescence depolarization measurements. Levels of the lipid peroxidation products thiobarbituric acid-reactive substances (TBARS) and conjugated dienes were markedly higher in PUF A-enriched cells, compared with unsupplemented cells, following exposure to cadmium or copper. Thus, lipid peroxidation was demonstrated as a major means of heavy metal toxicity in a microorganism for the first time. In addition, the effects ofPUFA-enrichment on the interactions of heavy metals with cellular nucleic acids were examined. Exposure ofPUFA-enriched cells to the redox-active metals chromium and copper resulted in the uncoupling of DNA synthesis from cell division, leading to sequential S phases. For example, DNA levels of up to 8C were evident in 18:3-enriched cells after only 4.5 h exposure to 100 JJ.M Cu(N03h. Using flow cytometry, the heterogeneity in susceptibility to copper toxicity of exponential phase S. cerevisiae was also examined. Susceptibility towards copper toxicity was demonstrated to be cell cycle stage-dependent, whereby G2/M phase cells were found to be the most susceptible towards copper toxicity. Staining with the oxidantsensitive probe 2',7' -dichlorodihydrofluorescein diacetate (H2DCFDA) revealed that the greater copper sensitivity of G2/M phase cells correlated with elevated endogenous levels of reactive oxygen species in these cells.

This Thesis investigates the possibility of an extrathymic pathway of T cell development in the amphibian, Xenopus laevis. Initial studies examined the ontogenetic development of T cell surface antigen expression on both splenocytes and thymocytes in larvae at 7 days through to 6-8 month old adults, using the technique of dual-colour flow cytometry and employing a panel of T and B cell specific monoclonal antibodies. Flow cytometric analysis of splenocytes from thymectomized (Tx) Xenopus was then addressed. This revealed that early (5-7 day) larval thymectomy resulted in the ablation of T cell surface antigen expression, as defined by the monoclonal antibodies 2B1 (anti- CD5), AM22 and F17 (both anti-CD8), XT-1 (anti-XTLA-1) and D4.3 (putative anti-aβ T cell receptor). Lack of these markers was still evident in 8 month old Tx frogs, confirming the effectiveness of the operation. In vitro studies showed that no T cell marker expression could be induced on the surface of splenocytes from Tx animals following stimulation with concanavalin A (ConA), phytohaemagglutinin (PHA) or the potent mitogenic agent, phorbol myristate acetate (PMA). Studies were also carried out to investigate whether in vitro stimulation induced apoptosis. Flow cytometric studies revealed that CD5(^dull) expression could be induced on splenocytes from control Xenopus following stimulation with PMA. The nature of this induced CD5(^dull) expression was investigated further in order to determine why this phenomenon was only seen in control animals. These experiments involving mixing of T and B cell populations, revealed that CD5(^dull) expression was being induced upon the surface of Xenopus B cells, and that this PMA-induced expression required the presence of T cells, and was blocked by a protein kinase C (PKC) inhibitor. Finally, an additional search for extrathymic T cells involved examining the intestine and liver of both control and Tx Xenopus, these tissues being sites of extrathymic T cell development in higher vertebrates. The intestine of control Xenopus was shown to contain T lymphocytes with a surface phenotype distinct to that found in spleen or liver. Studies in Tx Xenopus showed that although expression of some T cell markers was ablated in liver and gut, CD5(^dull) and CD8(^dull) (as determined by the mAb AM22) lymphocytes persisted in these organs. However, proliferative studies showed that these 'T-like' cells were unable to respond to mitogenic stimulation with ConA, suggesting that they are not functional T cells.

Veterans returning from deployment in the Middle East have displayed chronic asthmatic-like symptoms after exposure to burn pits and sandstorms. These soldiers also encounter stressful environments during deployment, introducing another factor that could alter their immune response. In this thesis, we have investigated the impact of acute stress on lung inflammation in murine models, focusing on the role of innate lymphoid cells (ILCs) during exposure to the allergen Alternaria alternata (Alt) and burn pit constituents (BPCs) which include dioxins, hydrocarbons, and particulate matter. Our previous studies have shown that combinatory exposure to Alt and BPCs induces a mixed type 2 and non-type 2 inflammatory response, characterized by increased neutrophilia and a shift from ILC2 to ILC1-like populations. Single-cell RNA sequencing (scRNA-seq) of lung samples showed upregulation of neutrophilia-associated genes and stress-related genes in ILCs from Alt+BPC-challenged mice, indicating that stress exacerbates inflammatory responses. Here, we present a novel model to reflect an acute stress event followed by inhalation of BPCs or Alt focusing on analysis of lung granulocytes and innate lymphoid cells. Results indicated a significant decrease in different ILC subpopulations of stressed mice after either allergen or BPC exposure. This decline reflects reduced neutrophilia in non-type 2 inflammation and trending decreases in eosinophilia in type 2 inflammation. This study concludes that acute stress alters the immune response to lung inflammation. These findings suggest that stress, combined with BPC exposure, might contribute to the respiratory issues observed in exposed military personnel.

Understanding nanoparticle-cell interactions at single-nanoparticle and single-cell resolutions is crucial to improving the design of next-generation nanoparticles for safer, more effective, and more efficient applications in nanomedicine. This study partly focuses on recent advances in the continuous high-throughput analysis of nanoparticle-cell interactions at the single-cell level. We highlight and discuss the current trends in continual flow high-throughput methods for analyzing single cells centered around flow cytometry techniques. This study further discusses the challenges and opportunities with current flow cytometry approaches and provides proposed directions for innovation in the high-throughput analysis of nanoparticle-cell interactions. With the information obtained from the investigation of the single-cell analysis methods, it was clear than such analysis methods at the single-cell level are necessary to understand cell behavior and nanoparticle-cell interactions. However, current methods, such as inductively coupled plasma mass spectrometry and microscopy, have some disadvantages, such as being labor-intensive and can affect nano-bio interactions. Therefore, we used flow cytometry (FCM) as a label-free technique to enhance our knowledge of nanoparticle-cell interactions. To understand how 100 nm gold nanoparticles (AuNPs) affect cellular behavior, we incubated the NPs with RAW 264.7 cells to examine the change in granularity. Upon confirming this change, we wanted to investigate how using different nanoparticles and cell types can impact nano-bio interactions. Our results show that larger nanoparticles increase the side scattering (SSC) readings, hence the complexity of cells. Next, we performed a kinetics analysis experiment to understand how the uptake of NPs influences cells over a period of 24 hrs. We found that NP uptake increases with time but reaches a plateau at higher NP concentrations towards the end of the investigation period. Additionally, we investigated if the endocytosis pathway for Heparosan (HEP)-coated 100 nm AuNPs can be determined. Despite using inhibitors from different pathways, we have not been able to confirm which pathway HEP-coated 100 nm AuNPs get uptaken by.

Hematopoietic stem and progenitor cells (HSPCs) form a complicated system that must fulfill the needs of immune, platelet, and red blood cell development throughout life. It is thought that hematopoietic stem cells (HSCs) become active and generate multilineage progenitors which differentiate to make bipotent followed by lineage committed progenitors. This process of differentiation depends on a complex interplay of intrinsic and extrinsic cellular factors including cytokines and growth factors, epigenetic regulation, signaling pathways, and gene regulatory networks. Cell states were previously identified using small sets of surface markers and functional assays. These approaches were limited in their ability to fully characterize the molecular and functional heterogeneity within the hematopoietic system, as they relied on bulk cell populations rather than individual cells. The advent of single-cell multi-omics technologies has revolutionized our ability to characterize cell states, allowing for the simultaneous capture of multiple molecular features at the single-cell level. These approaches have recently become higher throughput with advancements in microfluidics and sequence barcoding techniques. At the same time, methods in flow cytometry, such as new fluorescent dyes, multi-laser systems, increased detection channels, and spectral unmixing algorithms have made it possible to measure more features at a single-cell level. With the advancement of measuring molecular features at the single cell level, analysis pipelines continue to optimize in throughput and efficiency.In this dissertation, I present my work focusing on the identification and isolation of hematopoietic progenitor cells. Singe-cell omics techniques have made it possible to identify rare and previously unknown cell types. Through the application of high throughput analysis pipelines, we can reconstruct the global structure of hematopoiesis and begin to associate cell states with different stages of developmental trajectories. We built an interactive viewer for the human cell atlas bone marrow tissue project, spanning 100,000 hematopoietic cells across 8 donors. Next, we built a software package for the construction of high dimensional flow cytometry datasets using Infinity Flow. Next, we used cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to generate a comprehensive cell atlas of murine hematopoietic stem and progenitor cells in the bone marrow. Finally, we integrated CITE-seq with Infinity Flow to streamline the development of fluorescence-activated cell sorting (FACS) panels for the isolation of transcriptionally discrete cell populations. These findings have important implications for understanding disease states, as identifying rare cell types that are dysregulated in disease can provide insights into underlying mechanisms and potential therapeutic targets.

Abstract: To combat microbial invaders, immune cells elicit a range of potent antimicrobial effector responses coordinated by complex intracellular signalling events. These events are initiated by the engagement of cell surface-expressed proteins, termed immunoregulatory receptors, that bind to and recognize foreign bodies. The transduction of extracellular events across the cell membrane by immune receptors allows for fine-tuning of immunological responses against microbes and for the resolution of tissue injury. Leukocyte immune-type receptors (LITRs) are a polymorphic and polygenic group of immunoregulatory proteins originally discovered in channel catfish (Ictalurus punctatus; IpLITRs) that share structural and phylogenetic similarities with mammalian members of the immunoglobulin superfamily (IgSF). IpLITRs contain both putative stimulatory and inhibitory receptor-types that regulate innate immune cellular functions through their cytoplasmic tail (CYT) regions, inducing both classical and unique intracellular signalling networks. Previous characterization of IpLITR-types has allowed our lab to examine how immune receptors associate with adaptor signalling molecules and form homo- and heterodimers, inhibit cellular cytotoxicity through classical and non-classical inhibitory components, induce differing modes of target capture and engulfment through an inhibitory receptor-type, as well as cross-inhibiting the phagocytic response using novel CYT signalling network mechanisms. Our lab has identified new LITR-types within the zebrafish genome (Danio rerio; DrLITRs) that have been shown to be ubiquitously expressed during embryogenic development and zebrafish adulthood. While some of these DrLITR-types are indicative of classical stimulatory or inhibitory receptors, DrLITR 1.2 was discovered to contain both activating and inhibitory motifs (i.e., immunoreceptor tyrosine-based activation motif; ITAM and immunoreceptor tyrosine-based inhibitory motif; ITIM) within the same receptor CYT. This arrangement is unusual as these motifs typically exist on separate stimulatory (i.e., ITAM-containing) or inhibitory (i.e., ITIM-containing) immunoregulatory receptors that co-engage to fine-tune cellular signalling and effector responses. My overall objective in this thesis was to examine the role both an ITAM and an ITIM play in controlling DrLITR-mediated signalling. My research aims were, i) to create DrLITR 1.2 as well as motif dysfunctional receptor constructs to generate DrLITR-expressing AD-293 cells, ii) to use these newly created construct-expressing cells to examine the phagocytic capacity of DrLITR 1.2 using a flow cytometric phagocytosis assay, and iii) to examine the novel ITIM-dependent augmentation of DrLITR 1.2-mediated phagocytosis. My results show that engagement of DrLITR 1.2-expressing cells with phagocytic targets resulted in a robust phagocytic response dependent on the presence of a functional ITAM within the receptor CYT. In addition, I also show that the ITIM motif surprisingly enhances the overall phagocytic response of the receptor while simultaneously decreasing the receptor’s ability to bind to targets. Utilizing confocal fluorescence microscopy, I show that the ITIM-associated inhibitory signalling molecule SHP-2 is localized to the bead-cell interface during the phagocytic engulfment of targets relying on the presence of a functional ITIM within the receptor CYT. Similarly, the ITAM-associated stimulatory signalling molecule Syk was also shown to be recruited to the phagocytic synapse and was dependent on a functional ITAM. Using pharmacological inhibition profiling, I also show that DrLITR 1.2 uses signalling molecules indicative of other ITAM-containing receptors and that the ITIM may play a role in protecting DrLITR 1.2 from crosstalk inhibition. Overall, the data presented in this thesis provides the first functional characterization of teleost immune receptors containing both an ITAM as well as an ITIM within the same receptor CYT. This thesis uncovers new information of how immunoreceptor tyrosine-based motifs fine-tune immunoregulatory receptor-mediated signalling responses.

Cancer refers to a group of diseases containing more than 200 different subtypes. Cancer is a heterogeneous disease by nature, meaning that there are differences among tumors of the same type in different patients, and there are differences among cancer cells within a single tumor of one patient. Since cancer is not a single disease, nor does it have a single cause, it proves to be incredibly hard to diagnose and treat. The ability to study cellular markers, cell and tissue spatial arrangement, and gene function are all integral parts of cancer diagnostic and treatment efforts. Here, I first present a review of current techniques for quantitative tissue imaging at cellular resolution. I broadly divide current imaging techniques into three categories: fluorescence-based, mass spectrometry-based, and sequencing-based. In this work, I primarily concentrate on fluorescence-based methods, with the focus being on our recently developed theory Multiplexing using Spectral Imaging and Combinatorics (MuSIC). The basis for MuSIC is to create combinations of fluorescent molecules (whether it be small molecule fluorophores or fluorescent proteins) to create unique spectral signatures. I then present a protocol for labeling antibodies with combinations of small molecule fluorophores, which I refer to as MuSIC probes. I use fluorescent oligonucleotides (oligos) to arrange the fluorophores at specified distances and orientations from one another in order to produce complex fluorescence spectra when the probe is excited. This labeling protocol is demonstrated using a 3-probe experimental setup, bound to Protein A beads, and analyzed via spectral flow cytometry. When translating this method to staining human cells, our staining intensity was not comparable to that of a conventional antibody labeling kit. Therefore, next I present an improved method to label antibodies with MuSIC probes with increased signal intensity. I re-arrange the oligo-fluorophore arrangement of the MuSIC probe to emit an increased fluorescent signal. Then I validate this approach by comparing the staining intensity of MuSIC probe-labeled antibodies to a conventional antibody labeling kit using human peripheral blood mononuclear cells. Lastly, I present simulation theories for the multiplexing capabilities of MuSIC probes for various biological and diagnostic applications. First, I present a theory for high-throughput genetic interaction screening using MuSIC probes generated from 18 currently available fluorescent proteins. Simulation studies based on constraints of current spectral flow cytometry equipment suggest our ability to perform genetic interaction screens at the human genome-scale. Finally, I adapt this simulation protocol to generate MuSIC probes from 30 currently available small-molecule fluorophores. Using the same constraints as before, I predict that I can perform cell-type profiling of 200+ analytes. I hope that the work presented here provides a foundation for the use of combination probes for various biological and disease applications and ultimately help to better diagnose and treat different types of cancer.

Aptamers are single stranded DNA or RNA sequences that bind with high affinity to a target molecule. The discovery of sequences that can bind to a target of interest starts with a mixture of random sequences which are refined through repeated incubation with the target followed by amplification of binding sequences in a process known as systematic evolution of ligands by exponential enrichment (SELEX). Aptamers produced by multiple SELEX rounds can assist bioanalytical scientific research through detecting molecules of interest in biosensors and have the potential to improve drug delivery to specific cells in humans. However, developing aptamers capable of useful application is difficult due to multiple unique challenges. Quantifying the ability for a pool of aptamers produced by SELEX to bind to a target has no single perfect method. In the field of aptamer research, scientists employ a variety of binding affinity tests which contain specific advantages and disadvantages. Reported values for binding affinity vary between different methods. When a pool does show evidence of most sequences binding, identifying the best sequences in a mixture of more than thousands of unique base pair arrangements provides a separate challenge. Finally, even after finding a sequence with evidence of strong binding to a desired target, there remains uncertainty if the aptamer will bind to the target as well in a real-world application as it does in a carefully controlled lab environment. This study examines aptamers at three distinct stages of development for separate capillary electrophoresis SELEX (CE-SELEX) experiments. For an aptamer already selected against transferrin receptor 1 (TfR1) with high affinity, a combination of flow cytometry and confocal microscopy provided evidence the aptamer did not significantly internalize into cancerous liver cells used to model human drug delivery. A separate aptamer pool, previously developed over 4 rounds of selection against low-density lipoprotein receptor (LDLR), is analyzed with preexisting bioinformatic tools as well as custom Python code on next generation sequencing (NGS) data, revealing a primer dimer sequence from polymerase chain reaction (PCR) which impacted the selection of aptamer length candidates but remained at a lower frequency than one possible aptamer candidate. After performing 4 rounds of CE-SELEX against mouse leptin, testing the binding affinity of rounds 1-3 with CE showed weak evidence of binding in early rounds which was lost in later rounds after correcting for an earlier false positive result. Documenting CE problems and optimization, even for an unsuccessful aptamer selection, may help improve future CE research, while detailing the experimental set up, discovery, and correction of the false positive result is important to help prevent future misleading conclusions.

CD4+ T cells play a pivotal role in generating and regulating effective immune responses against invading pathogens by orchestrating intracellular calcium (Ca2+) signaling through the synthesis of Ca2+ signaling secondary messengers such as nicotinic acid adenine dinucleotide phosphate (NAADP), cyclic adenosine 5-diphosphate ribose (cADPR), and inositol 1,4,5 triphosphate (IP3). These signaling events significantly impact downstream immune responses. In contrast, in the tumor microenvironment, upregulation of Ca2+ signals are associated with cancer cell proliferation, malignancy, and immune response suppression. NAADP being the most potent secondary messenger, its signaling pathway remains elusive and controversial. NAADP is believed to bind to accessory proteins such as Jupiter microtubule-associated homolog 2 (JPT2) and an Sm-like proteinLsm 12. These are thought to influence the poorly characterized 'two-pore channels' (TPCs) in the endo-lysosomal compartments of cells. Notably, previous studies using PF-543, a sphingosine kinase-1 (SphK-1) inhibitor, effectively diminished NAADP-mediated Ca2+ signals in sea urchin eggs, and U2OS cells, warranting further investigation of the involvement of accessory proteins, TPCs, and SphK-1 in this pathway. To shed light on JPT2's role in influencing T cell function, we conducted experiments to investigate its impact on T cell activation events. Our findings indicate that the knockdown of JPT2 by siRNA did not affect early T cell activation events, such as the phosphorylation of tyrosine kinases LCK and ZAP-70, but instead influenced Ca2+ release and later events, such as the phosphorylation of the critical transcription factor NF- κB, a process which is well-known to be Ca2+ dependent and responsible for T cell proliferation and differentiation. Additionally, our investigation into a library of PF-543 analogs on murine T cells and sea urchin eggs revealed a correlation between SphK-1 inhibition andCa2+ release. Our results suggest that JPT2 in mouse cells may be post-translationally modified.Overall, our research, advances the understanding of the complex NAADP-mediated Ca2+ signaling pathway, sheds light on the involvement of JPT2 and SphK-1 in regulating T cell function, and opens promising avenues for the development of targeted therapeutic strategies to modulate Ca2+ signaling in both immune responses and cancer treatment.

IntroductionScientific literature regarding the characterization of lymphocyte subpopulations of the cecal appendix is sparse, with few precedents limited to immunohistochemical techniques.MethodsWe conducted a prospective pilot study to characterize lymphocyte subpopulations of the cecal appendix in children. Participants were divided into three groups: (1) patients without histological acute appendiceal inflammation, (2) patients with histological uncomplicated acute appendicitis, and (3) patients with histological complicated acute appendicitis (gangrenous, perforated). A fresh sample of the base of the appendix was taken from all patients and a flow cytometric study was performed. Quantitative variables were compared using Kruskal-Wallis test and Mann-Whitney U test.ResultsThis study included 57 patients divided into Group 1 (n = 5), Group 2 (n = 37), and Group 3 (n = 15). Median values (IQR) of the percentage of B-lymphocytes were 67.8 [66.8-68.1] in group 1, 61.15 [53.74-66.4] in group 2, and 52.1 [33-62.02] in group 3 (p = 0.02). Median values (IQR) of the percentage of NK-lymphocytes were 0.26 [0.2-0.3] in group 1, 0.55 [0.37-0.66] in group 2, and 0.84 [0.35-1.45] in group 3 (p = 0.008). Median values (IQR) of the percentage of T-lymphocytes were 31.9 [31.7-33.1] in group 1, 37.68 [32.15-45.69] in group 2, and 46.9 [37.03-67] in group 3 (p = 0.02). Pair comparisons of groups 2 and 3 also showed significant differences in the percentage of B lymphocytes (p = 0.03) and NK-lymphocytes (p = 0.02).ConclusionsSignificant differences in lymphocyte subpopulations were identified according to the histologic grade of the cecal appendix. More specifically, a lower percentage of B-lymphocytes and a higher percentage of T- and NK-lymphocytes were observed in cases of acute appendicitis. These findings must be confirmed and their etiopathogenic, diagnostic, and prognostic implications elucidated in future studies with larger sample sizes.

BACKGROUND: Distinguishing patients with axial spondyloarthritis (axial SpA) from patients with other causes of chronic back pain remains a challenge. The lack of reliable biomarkers contributes to the diagnostic delay in axial SpA. Recently, macrophage migration inhibitory factor (MIF) has been proposed as a candidate diagnostic and prognostic biomarker. MIF is a proinflammatory cytokine that was shown to be upregulated in several autoimmune diseases, including axial SpA. The putative role of CD8+ T cells in the disease process suggests further that serum markers of cytotoxicity might have value as serological biomarkers in axial SpA, and that subpopulations of cytotoxic lymphocytes might deserve attention as candidate cellular biomarkers. OBJECTIVE: The goal of this study was to compare serum levels of MIF and other candidate serum proteins in patients with axial SpA and controls, and to develop a flow cytometry panel to analyze cytotoxic lymphocyte cell subpopulations in these cohorts, including KIR+CD8+ T cells, Granzyme B+ CD8+ T cells, MAIT cells, and InEx cells. METHODS: Study subjects were recruited from the Brigham and Women’s Hospital Orthopedic and Arthritis Center. Four cohorts were compared: healthy controls (HC), patients with chronic low back pain (cLBP), axial SpA patients not on a biologic (axSpA/-), and axial SpA patients treated with a TNF inhibitor (axSpA/TNFi). Study subjects were matched for age, sex, and race, when possible. Serum was evaluated using the LEGENDplex Human CD8/NK panel (BioLegend) for thirteen markers including IL-17A, IL-6, TNF, granzyme B, and perforin. CRP and MIF were evaluated by DuoSet ELISA (R&D Systems). A high-dimensional flow cytometry panel was designed to evaluate 14 cell populations of interest. RESULTS: The severity of back pain in the cLBP controls and axSpA/- patients was comparable (BASDAI Q2 mean 5.0 +/- 1.9 vs. 5.0 +/- 3.0). axSpA/- patients had higher back pain, BASDAI and ASDAS scores than axSpA/TNFi patients consistent with higher disease activity in the biologic naïve group. Serum CRP values were significantly higher in axSpA/- patients compared with HC, cLBP controls, and axSpA/TNFi patients (P= 0.01, P=0.0029, P=0.004 respectively). Serum MIF levels were not statistically different between all four groups (P= 0.8069). Additionally, there were no statistically significant differences between the groups for any of the markers included in the LEGENDplex Human CD8/NK panel. A 32-color staining panel was developed to evaluate cytotoxic cell populations. CONCLUSION: In contrast to a previous study, we did not find differences in serum MIF levels between axial SpA patients and controls. Of the evaluated serum biomarkers, only CRP values correlated with active axial SpA. We have developed a promising flow cytometry panel that will help analyze subpopulations of cytotoxic cells. This ultimately could shed light on a candidate cellular biomarker. Our results underscore the need for more research into diagnostic biomarkers in axial SpA.

Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related death in the United States. PDAC is highly metastatic with the liver being the most common site of metastasis. Notably, even after surgical resection of early stage of PDAC tumors, recurrence often occurs in the liver instead of the primary site that was surgically treated, suggesting the existence of micrometastasis in the liver of early-stage diseases. There is currently no approach to treat or prevent liver metastasis besides chemotherapy that is highly toxic and lacks efficacy. Therefore, new therapeutic targets need to be investigated to make a difference in patients’ outcomes. My thesis research project is to investigate small ubiquitin-like modification (SUMOylation) as a target to prevent and treat PDAC liver metastasis. SUMOylation is a reversible post-translational modification involving the attachment of a SUMO protein covalently to a target protein. Previous studies have shown that SUMOylation is required for KRas and c-Myc-dependent oncogenesis that are main drivers of PDAC progression. In addition, SUMOylation has been established as a target for activation of anti-tumor immunity. My findings suggest that SUMOylation inhibition can block PDAC liver metastasis. The mechanisms could be through both immune-mediated and tumor-specific effects.

Regulatory mechanisms of the immune system are essential for human health by maintaining homeostasis and preventing disease states such as excessive inflammation and autoimmunity. These vital functions are coordinated by a wide range of cells from multiple lineages that employ unique and inventive techniques to suppress immune overactivation. Here, we describe four instances of novel regulatory mechanisms, which all in their own capacities are rooted in host and microbial glycans. In one case, we report that FoxP3+ regulatory T cells (Tregs) respond to a dual cytokine signal discovered downstream of the microbial factor polysaccharide A (PSA) by eliciting robust immunosuppression through the production of IL-10 and the selective proliferation of IL-10-expressing Tregs. In another study, we find that the suppressive T cell phenotype downstream of PSA that has been the focus of decades of work is preceded by the ability of PSA to polarize an anti-inflammatory macrophage subtype. Another study examines the cellular compensation that occurs during genetic ablation of IL-10 in FoxP3+ Tregs, where type I regulatory T cells (Tr1) in the colon upregulate their production of IL-10 to prevent the spontaneous development of disease. In the final study, we describe a new role for CD22 on macrophages that transduces tissue sialylation status and drives the polarization of anti-inflammatory macrophages that are critical for shaping immune tone over time. Using the modalities described in this work, multiple mouse models of autoimmune and inflammatory diseases, including asthma, multiple sclerosis, and inflammatory bowel disease were therapeutically prevented or reversed, demonstrating the astounding power of glycans and the immune cells that respond to them.

Transplantation requires that an organ be removed from the circulation of thedonor and then reintroduced to the circulation of the recipient. Organs from deceaseddonors are often removed from the donor in one hospital and then transported on ice toanother hospital for the transplant procedure. Consequently, transplanted organs aresubjected to varying periods of ischemia before reperfusion in the recipient. This resultsin ischemia reperfusion injury (IRI) to the organ. For transplanted kidneys, IRI isfrequently severe enough to cause delayed function requiring dialysis and even totalfailure of the graft. Identifying mechanisms that mediate this injury remains a crucial goalof transplantation.It is known that IRI causes a surge of apoptosis. During normal homeostaticturnover of cells, apoptotic bodies are cleared without inducing inflammation or immuneresponses by C1q, the initial component of the complement system. In patients with therare genetic deficiency of C1q, apoptotic bodies are not effectively cleared and immuneresponses develop to autoantigens in the apoptotic bodies. We examined the role of C1qin modulating the early immune response to IRI in renal transplants by investigating theabsence of C1q on early graft injury in an MHC mismatched murine transplant model.We found that A/J kidney grafts transplanted into C1q KO mice exhibited high levels ofurinary injury marker, neutrophil gelatinase associated lipocalin (Ngal) within 1 and 2days posttransplant and decreased survival compared to WT mice. Immunohistologydemonstrated increased platelet aggregates in the renal medulla typical of unresolvedischemia induced injury in grafts undergoing early rejection 5 days after transplantation.Sources of C1q were identified during IRI in renal transplants by isolating infiltrating andresident Ly6C+ cell populations from the graft. Resident Ly6C+ cells in the donor kidneywere found to express high levels of C1q even 6 days after transplantation. Localavailability of C1q could decrease inflammatory responses occurring within the graft andextend graft survival.

Background: Extracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. However, in order to perform validation studies and screenings of patient samples, it is still necessary to develop general techniques permitting rapid handling of small amounts of biological samples from large numbers of donors. Results: Here we describe a method that, using just a few microliters of patient’s plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV ζ-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs. Conclusions: This optimized high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new ones.

Viruses in wastewater present public-health challenges as well as public-health opportunities. I consider both herein. I begin with a systematic literature review of nearly 300 studies, published from 2000 to 2018, that document applications of flow cytometry (FCM) to ensure microbial water quality and hence facilitate safe and effective water treatment, distribution, and reuse. I find that while there is a large body of evidence supporting widespread adoption of FCM as a routine method for microbial water-quality assessment, key knowledge gaps impede the technique from realizing its full potential. One of these gaps is robust protocols for FCM-based analysis of waterborne viruses. In this dissertation, I hypothesize that a fractional factorial experimental design is a better alternative to the “pipeline” strategy commonly followed for FCM protocol optimization. I then demonstrate my approach, using a fractional factorial experimental design to optimize staining of the bacteriophage T4 prior to FCM analysis. My results yield a specific protocol for reliably identifying and quantifying T4 bacteriophage through FCM.I also explain why manual gating of FCM data using a series of two-dimensional plots—the typical approach to FCM data analysis—is problematic, especially with respect to applications of FCM to facilitate advanced water treatment and reuse. I suggest that algorithmic clustering approaches could expedite and improve FCM data analysis, and could even help position FCM as a technique for real-time microbial water-quality monitoring. I test this theory by generating FCM data from two solutions: (i) a mixed-target solution containing a variety of environmentally relevant viral surrogates, and (ii) an environmental-spike solution comprising T4 bacteriophage in a wastewater matrix. I first analyze these data through manual gating, and then compare results to results obtained through algorithmic clustering: specifically, by coupling the OPTICS ordering algorithm with either manual or automated identification of clusters from the OPTICS-ordered data. I demonstrate that OPTICS-assisted clustering can in some cases work as well or better than manual gating of FCM data—and is certainly far faster and less labor-intensive. OPTICS-assisted clustering can also point to features in FCM data that are difficult to detect through manual gating alone. However, I also find that more needs to be done to position OPTICS as a reliable tool for automated, objective analysis of FCM data from environmental samples, especially data generated from challenging biological targets like viruses in challenging matrices like wastewater.I explore wastewater-borne viruses as a public-health opportunity through the lens of the COVID-19 pandemic. Wastewater-based epidemiology (WBE) quickly became recognized as a useful complement to clinical testing following the pandemic’s onset. However, little is known about sub-community relationships between wastewater and clinical data. I present a novel framework for probabilistically aligning wastewater and clinical data with high spatial granularity. I use this framework to uncover clear sub-regional (i.e., sub-city) and building/neighborhood-scale correlations between wastewater and clinical data collected through the Healthy Davis Together (HDT) pandemic-response initiative in Davis, CA. In addition, I hypothesize that multiple imputation (using an expectation maximization-Markov chain Monte Carlo (MCMC) approach) of non-detects in wastewater qPCR data is less likely to bias results than more commonly used non-detect handling methods (e.g., censoring or single imputation). I use the HDT data to test this hypothesis. I find that while results obtained using different non-detect handling methods are similar, they are not the same—indicating the importance of specifying non-detect handling method in WBE studies. I also find that the EM-MCMC method yields somewhat better agreement between clinical and wastewater data than do the other non-detect handling methods examined. Refinements to the algorithm, tuning parameters, and variable groupings used in this dissertation could further recommend the EM-MCMC method for wastewater-data analysis in the future.I conclude the dissertation with a discussion of lessons learned from my experience helping launch, grow, and manage the HDT WBE program. Conducting WBE requires significant investments of time, money, labor, and expertise. Given that much information gleaned from wastewater is not directly actionable, and/or duplicates information from other sources, it is prudent to consider whether these investments are worth it. I present seven recommendations for end users seeking to incorporate WBE into COVID-19 response: (1) avoid redundancy between clinical testing and WBE; (2) emphasize statistical thinking, data analysis, and data management; (3) define action thresholds; (4) monitor fewer sites more frequently; (5) build on existing infrastructure and programs for wastewater collection and analysis; (6) be prepared to adapt as the pandemic evolves; and (7) keep an eye on the future, including by proactively searching for emerging variants of concern.

Large-scale immune monitoring is becoming routinely used in clinical trials to identify determinants of treatment responsiveness, particularly to immunotherapies. Flow cytometry remains one of the most versatile and high throughput approaches for single-cell analysis; however, manual interpretation of multidimensional data poses a challenge when attempting to capture full cellular diversity and provide reproducible results. We present FlowCT, a semi-automated workspace empowered to analyze large data sets. It includes pre-processing, normalization, multiple dimensionality reduction techniques, automated clustering, and predictive modeling tools. As a proof of concept, we used FlowCT to compare the T-cell compartment in bone marrow (BM) with peripheral blood (PB) from patients with smoldering multiple myeloma (SMM), identify minimally invasive immune biomarkers of progression from smoldering to active MM, define prognostic T-cell subsets in the BM of patients with active MM after treatment intensification, and assess the longitudinal effect of maintenance therapy in BM T cells. A total of 354 samples were analyzed and immune signatures predictive of malignant transformation were identified in 150 patients with SMM (hazard ratio [HR], 1.7; P < .001). We also determined progression-free survival (HR, 4.09; P < .0001) and overall survival (HR, 3.12; P = .047) in 100 patients with active MM. New data also emerged about stem cell memory T cells, the concordance between immune profiles in BM and PB, and the immunomodulatory effect of maintenance therapy. FlowCT is a new open-source computational approach that can be readily implemented by research laboratories to perform quality control, analyze high-dimensional data, unveil cellular diversity, and objectively identify biomarkers in large immune monitoring studies. These trials were registered at www.clinicaltrials.gov as #NCT01916252 and #NCT02406144.

Simple Summary Flow cytometry allows detailed characterization of large numbers of cells and plays an important role in the diagnosis of acute myeloid leukemia. To facilitate analysis of flowcytometric data, reference databases of normal bone marrow samples and samples from acute myeloid leukemia patients, together with new software tools, are required. We here report on the building of a large database of acute myeloid leukemia patients (n = 1142) and 22 normal samples. We report on the quality assessment procedure used and its validation, discuss potential pitfalls, and provide possible solutions for avoiding such flaws in the construction of other databases. Our data show that obtaining and collecting reproducible flow cytometric data over time and across centers is feasible, but also that strict quality assessment remains crucial, even when standardized protocols for staining and instrument settings are being used in a multicenter setting. Flowcytometric analysis allows for detailed identification and characterization of large numbers of cells in blood, bone marrow, and other body fluids and tissue samples and therefore contributes to the diagnostics of hematological malignancies. Novel data analysis tools allow for multidimensional analysis and comparison of patient samples with reference databases of normal, reactive, and/or leukemia/lymphoma patient samples. Building such reference databases requires strict quality assessment (QA) procedures. Here, we compiled a datase

Cryptophytes (class Cryptophyceae) are bi-flagellated eukaryotic protists with mixed nutritional modes and a cosmopolitan distribution in marine and freshwater environments. Here we present a survey of cryptophyte abundance at the Scripps Institution of Oceanography Pier and in San Diego Bay. From 2011 to 2017, cryptophytes were consistently present off the pier with significant biotic and abiotic interactions but no observable seasonality. Weekly to bi-weekly high-throughput sequencing in 2016 revealed 16 unique cryptophyte sequences, with clade 4 and particularly 2 species dominant, one of which appears to be phylogenetically novel. One high cell abundance bloom in 2016 appears to be monospecific in origin and the second may be driven by grazing on an influx of an atypical Synechococcus clade. In the San Diego Bay, tidal forces appear to cyclically concentrate cryptophyte abundance at choke points in its bathymetry. A clade 4 sequence not dominant off the pier in 2016 is dominant in the back bay.