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Determining the phosphorylation of low-abundance peptides in small amount of clinical samples is challenging. In this approach, in-house construction of IMAC columns plus esterification of the peptides are the key features that improve sensitivity. Phosphorylation events within cancer cells often become dysregulated, leading to oncogenic signaling and abnormal cell growth. Phosphopeptides derived from aberrantly phosphorylated proteins that are presented on tumors and not on normal tissues by human leukocyte antigen (HLA) class I molecules are promising candidates for future cancer immunotherapies, because they are tumor specific and have been shown to elicit cytotoxic T cell responses. Robust phosphopeptide enrichments that are suitable for low input amounts must be developed to characterize HLA-associated phosphopeptides from clinical samples that are limited by material availability. We present two complementary mass spectrometry–compatible, iron(III)-immobilized metal affinity chromatography (IMAC) methods that use either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) in-house-fabricated columns. We developed these protocols to enrich for subfemtomole-level phosphopeptides from cell line and human tissue samples containing picograms of starting material, which is an order of magnitude less material than what is commonly used. In addition, we added a peptide esterification step to increase phosphopeptide specificity from these low-input samples. To date, hundreds of phosphopeptides displayed on melanoma, ovarian cancer, leukemia and colorectal cancer have been identified using these highly selective phosphopeptide enrichment protocols in combination with a program called 'CAD Neutral Loss Finder' that identifies all spectra containing the characteristic neutral loss of phosphoric acid from phosphorylated serine and threonine residues. This methodology enables the identification of HLA-associated phosphopeptides presented by human tissue samples containing as little as nanograms of peptide material in 2 d.

There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Cytotoxic T cell infiltration is well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) target and kill tumor cells. However, the specific antigens that drive these CD8+ T cell responses have not been well characterized. Recently, phosphopeptides have emerged as strong candidates for tumor-specific antigens, as dysregulated signaling in cancer leads to increased and aberrant protein phosphorylation. Here, we identify 120 HLA-I phosphopeptides from primary CRC tumors, CRC liver metastases and CRC cell lines using mass spectrometry and assess the tumor-resident immunity against these posttranslationally modified tumor antigens. Several CRC tumor-specific phosphopeptides were presented by multiple patients’ tumors in our cohort (21% to 40%), and many have previously been identified on other malignancies (58% of HLA-A*02 CRC phosphopeptides). These shared antigens derived from mitogenic signaling pathways, including p53, Wnt and MAPK, and are therefore markers of malignancy. The identification of public tumor antigens will allow for the development of broadly applicable targeted therapeutics. Through analysis of TIL cytokine responses to these phosphopeptides, we have established that they are already playing a key role in tumor-resident immunity. Multifunctional CD8+ TILs from primary and metastatic tumors recognized the HLA-I phosphopeptides presented by their originating tumor. Furthermore, TILs taken from other CRC patients’ tumors targeted two of these phosphopeptides. In another cohort of CRC patients, the same HLA-I phosphopeptides induced higher peripheral T cell responses than they did in healthy donors, suggesting that these immune responses are specifically activated in CRC patients. Collectively, these results establish HLA-I phosphopeptides as targets of the tumor-resident immunity in CRC, and highlight their potential as candidates for future immunotherapeutic strategies.

ObjectiveTo understand parental perspectives regarding universal newborn screening (UNS) for congenital cytomegalovirus (cCMV) in Canada.DesignA qualitative, patient-led study using the Patient and Community Engagement Research approach consisting of online focus groups and in-depth individual interviews to understand parental preferences regarding UNS for cCMV. Data were analysed iteratively using inductive thematic analysis and narrative story analysis.SettingCanada-wide study conducted via video conference from October to December 2023.Patients12 participants from five Canadian provinces who self-identified as 18 years of age or older and as having parental lived experience with cytomegalovirus (CMV) or cCMV participated in the study.ResultsWe identified three themes: (1) attitudes about UNS for cCMV, including participants’ unanimous support for UNS and confirmation that parental anxiety is not a deterrent for screening, (2) cCMV diagnosis, including the importance of coupling cCMV diagnosis with access to treatment and medical support and (3) awareness of cCMV, where participants shared their frustration about the lack of public and pregnant people’s awareness of cCMV.ConclusionsParental anxiety is not a deterrent for UNS for cCMV. Children with cCMV and their families deserve every opportunity to attain their best possible outcomes. UNS offers children with cCMV access to early intervention if they need it, and also helps to raise awareness and education to prevent future CMV infections.

Background Although omic-based discovery approaches can provide powerful tools for biomarker identification, several reservations have been raised regarding the clinical applicability of gene expression studies, such as their prohibitive cost. However, the limited availability of antibodies is a key barrier to the development of a lower cost alternative, namely a discrete collection of immunohistochemistry (IHC)-based biomarkers. The aim of this study was to use a systematic approach to generate and screen affinity-purified, mono-specific antibodies targeting progression-related biomarkers, with a view towards developing a clinically applicable IHC-based prognostic biomarker panel for breast cancer. Methods We examined both in-house and publicly available breast cancer DNA microarray datasets relating to invasion and metastasis, thus identifying a cohort of candidate progression-associated biomarkers. Of these, 18 antibodies were released for extended analysis. Validated antibodies were screened against a tissue microarray (TMA) constructed from a cohort of consecutive breast cancer cases (n = 512) to test the immunohistochemical surrogate signature. Results Antibody screening revealed 3 candidate prognostic markers: the cell cycle regulator, Anillin (ANLN); the mitogen-activated protein kinase, PDZ-Binding Kinase (PBK); and the estrogen response gene, PDZ-Domain Containing 1 (PDZK1). Increased expression of ANLN and PBK was associated with poor prognosis, whilst increased expression of PDZK1 was associated with good prognosis. A 3-marker signature comprised of high PBK, high ANLN and low PDZK1 expression was associated with decreased recurrence-free survival ( p  < 0.001) and breast cancer-specific survival (BCSS) ( p  < 0.001). This novel signature was associated with high tumour grade (p < 0.001), positive nodal status (p = 0.029), ER-negativity (p = 0.006), Her2-positivity (p = 0.036) and high Ki67 status (p < 0.001). However, multivariate Cox regression demonstrated that the signature was not a significant predictor of BCSS (HR = 6.38; 95% CI = 0.79-51.26, p  = 0.082). Conclusions We have developed a comprehensive biomarker pathway that extends from discovery through to validation on a TMA platform. This proof-of-concept study has resulted in the identification of a novel 3-protein prognostic panel. Additional biochemical markers, interrogated using this high-throughput platform, may further augment the prognostic accuracy of this panel to a point that may allow implementation into routine clinical practice.

This thesis is the first full-length study to exclusively examine theatrical entertainment in the Royal Navy and the first study to put forward historical and contextual evidence of a 20thcentury shipboard theatrical repertoire. This thesis documents and analyses shipboard performances created for and by serving personnel on the upper decks, the aircraft hangars, and the messes of naval vessels at sea. This thesis draws upon written evidence from diaries and memoirs in formal archive holdings, interviews with naval personnel, and visual materials including photographs and commission books to provide a detailed historical, contextual analysis of a limited number of case studies in order to illustrate a broader nexus of theatrical practice within the Royal Navy. Chapter One reveals theatrical entertainment to be an important aspect of the navy's unofficial heritage and pinpoints how theatrical productions and the activities associated with them including 'spinning dits' and sketch writing help to forge an exclusive naval identity among shipboard companies. Chapter Two investigates the shipboard entertainment produced by the Special Service Squadron (SSS) during the World Cruise (1923 -1924) and the production of children's parties during the latter half of the 20thcentury. This chapter highlights the strategic value of entertainment at sea in enabling displays of soft power that counter hard power displays in gunboat diplomacy. Chapter Three examines the motivations for and functions of Crossing the Line and SODS opera; two theatrical forms that both endorse and subvert naval hierarchies. The final chapter examines theatrical entertainments produced during the First World War on SS Gourko and by R.F Scott's company during the British National Antarctic Expedition (1901-1904) and considers the value of theatre-making as a survival strategy. By adopting a case study approach, this thesis aims to not only consider the functions different theatrical forms in different sites, times, and situations serve for performers and their audiences but to open up new meanings of the theatrical in each context and to reveal what these practices tell us about naval shipboard communities more broadly.

Graphite bricks make up a significant part of the core of an Advanced Gas-cooled Reactor (AGR). The graphite moderates the neutrons vital to the continuation of the fission chain reaction and provides support and stability for the entire core. During operation, the graphite can be oxidised due to the extreme conditions inside the core and so undergo weight loss. Differential shrinkage caused by neutron interaction throughout the brick can also cause radial cracking to occur. The effects of the oxidation, weight loss and cracking reduce the ability of the graphite to function as a moderator. The effects also have the potential of reducing the structural integrity of the brick, causing movement and structural instability of the entire core. It is, therefore, vital to monitor the condition of the graphite bricks and to understand how the changes in the graphite's properties and structure may affect the safe operation of the reactor. This report firstly looks briefly at the effect of irradiation on the graphite brick; the mechanisms leading to weight loss and cracking. The report then considers various methods which can be used to inspect the deterioration of graphite blocks within the cores of AGRs deriving quantitative and qualitative information on density and crack profiling. These methods will be considered for use both on small samples trepanned from the core and in-situ blocks within the reactor core, requiring non-destructive techniques. The inspection methods considered in this report are: Electrical Impedance Tomography (EIT); Four point probes; Eddy Current Tomography; and Electromagnetic Inductance Tomography (EMT). There are two main contributions of this thesis. First, the development an EIT methodology using outward facing probes, which were best suited to the geometry of the graphite bricks within the AGR. Proof of principle was established using both modelling and laboratory testing. The second contribution is the development of commercial grade EMT equipment, which can be used on-site to determine the conductivity of trepanned samples. The method was successfully demonstrated in the laboratory; however, further development will be required for use on-site, due to the sampling speed required.

Immunotherapies signify a major development in the fight against increasing cancer morbidity and mortality. However, they have been limited by a lack of tumour-specific targets. MHC class I associated phosphopeptides represent a novel class of potentially tumour-specific targets, since dysregulation of signalling in cancers leads to aberrant phosphorylation. Using an autologous model of cancer, in healthy individuals, it was shown that a significant proportion of all anti-tumour cytotoxic memory T cell responses target phosphopeptides. In colorectal cancer (CRC), there is an established association between memory CD8+ T cell infiltration and survival. CRC and oesophageal adenocarcinoma tumours and cell lines were used to identify 134 tumour-associated phosphopeptides. Approximately 65% of these derive from well-defined cancer pathways and are thus markers of malignancy. Multifunctional tumour-infiltrating lymphocytes were present in primary and metastatic tumours that recognised these phosphopeptides. Furthermore, healthy donors have pre-existing memory T cell responses to many CRC-associated phosphopeptides. Phosphopeptide-specific T cells were readily expanded ex vivo and killed CRC cell lines. Therefore, MHC class I associated phosphopeptides are ideal immunotherapeutic targets, as immunity must spare healthy tissue. Immunity to tumour-associated phosphopeptides represents a biological strategy for distinguishing tumour from healthy tissue. These phosphopeptides are potential sHLA-associated cancer biomarkers and immunotherapeutic targets.