Biochemistry and Cell Biology - Doctoral Theses
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Item Evaluation of biomarkers for the diagnosis, management and follow-up of women with gestational trophoblastic disease(University College Cork, 2024) Joyce, Caroline Martha; O'Donoghue, Keelin; McCarthy, Tommie V.; Coulter, John; Irish Research CouncilGestational Trophoblastic Disease (GTD) describes a spectrum of disorders arising from the abnormal proliferation of trophoblastic tissue. Human chorionic gonadotrophin (hCG) is an excellent biomarker for most forms of this disease as its concentration accurately reflects trophoblastic activity. Hydatidiform mole (HM) or molar pregnancy is the most common form of GTD which may be suspected on ultrasound but requires pathological confirmation for diagnosis. Prognosis is generally excellent however close surveillance with hCG monitoring is imperative to detect cases progressing to malignant disease, referred to as gestational trophoblastic neoplasia (GTN). Clinical management and treatment of women with GTN in specialist centres achieves cure rates approaching 100%, emphasising the importance of accurate hCG monitoring for early detection of disease persistence to ensure optimal outcome. This thesis aims to evaluate pathological, genetic, and biochemical biomarkers used in the diagnosis, monitoring, and follow-up of women with GTD. In this thesis, a scoping review was performed to generate evidence and identify gaps in the GTD knowledge base to inform areas for future research. Key findings were the need to standardise hCG immunoassays for effective GTD surveillance, to improve diagnostic reporting pathways and to establish more accurate incidence rates. In order to identify patient priorities and inform initiatives to enhance the quality of care, a service evaluation was performed with women enrolled in the National GTD Registry invited to participate. Feedback revealed a knowledge gap regarding GTD amongst healthcare professionals outside GTD centres. This study also revealed shortcomings in psychological support and bereavement counselling offered to women after a molar pregnancy. To enhance the diagnostic accuracy of HM reporting, an in-situ hybridisation ploidy assay was developed with a customised scoring system to aid partial hydatidiform mole (PHM) diagnosis. This innovative technique provides a reliable adjunct to morphological assessment for PHM classification. The accuracy of this ploidy technique was confirmed by evaluation with molecular short tandem repeat genotyping. A national pathology questionnaire was performed to gather information on GTD diagnostic rates. Data collected was cross-referenced with cases documented in the National GTD Registry. This revealed a concerning under-enrolment of women with GTD (42%) onto the National GTD Registry by their clinicians. An audit of HM diagnostic rates over a 3-year period was performed following the Implementation of the new ploidy technique to aid diagnosis . This revealed a higher local PHM incidence rate (1 in 296 births) than reported nationally in the pathology audit (1 in 484 births). An electronic questionnaire was distributed to all European laboratories offering hCG for GTD management to gather insights on 5 key areas: hCG methodology, quality parameters, reporting and operational procedures, and use of hCG for non-GTD testing. This revealed considerable inter- and intra-laboratory variability in practice, emphasising the need to promote standardisation and harmonisation of immunoassays for GTD-derived hCG. A review of five challenging cases managed at the Irish GTD centre was conducted to evaluate the role of serum hCG and molecular genotyping in guiding management decisions, treatment strategies, and risk stratification for women with molar pregnancy, GTN and GTD mimics. These complex cases confirmed the pivotal role of serum hCG and molecular genotyping in monitoring disease persistence, prognostic stratification, and clinical management Novel strategies to measure hCG were pursued to address the lack of centralised hCG testing for GTD in Ireland. A proof-of-concept study for remote capillary blood collection was performed to evaluate the clinical efficacy and user acceptability of this method of collection for hCG monitoring in early pregnancy. The study confirmed the equivalence of capillary and venous blood hCG testing, demonstrated complete clinical concordance, thereby offering an alternative to venepuncture for hCG measurement. To explore the accessibility of hCG testing in the community, a point-of-care testing (POCT) device was evaluated for its suitability to triage women with pregnancy of unknown location (PUL) in the early pregnancy unit. The Abbott iSTAT®1 POCT instrument was chosen for this purpose and was clinically concordant with central laboratory hCG results facilitating the use of clinical decision thresholds for PUL. The research presented in this thesis provides valuable clinical insights through a series of nine research studies and has broadened the knowledge base in GTD. The findings offer an opportunity to positively impact diagnostic practices. The knowledge gap regarding GTD amongst healthcare professionals revealed in the patient survey points to a need for targeted educational initiatives in this area. Widespread integration of the HER2 ploidy technique to support morphology and standardise HM reporting could improve diagnostic accuracy, addressing a suspected underdiagnosis of PHM nationally and possibly internationally. It also suggests the need to re-evaluate incidence rates following the introduction of advanced diagnostic techniques such as ploidy assessment and molecular genotyping. This thesis identified an under-registration of women with GTD in Ireland supporting the need for initiatives such as mandatory registration or advocacy by professional organisations to increase registration rates. The inter and intra-laboratory variation in hCG immunoassays revealed in the European laboratory survey merits renewed harmonisation efforts through pilot external quality assurance and sample exchange programmes. The use of novel methods to enhance the accessibility of hCG monitoring for GTD in the community, through capillary blood collection or use of point-of-care analysis, warrants further study. The positive patient feedback indicated a preference for this approach, suggesting better compliance with serial test monitoring, a priority for GTD management.Item The gut microbiome of the wild great tit (Parus major): drivers and fitness consequences(University College Cork, 2023) Somers, Shane Edmond; Quinn, John; Ross, R. Paul; Stanton, Catherine; Irish Research Council for Science, Engineering and Technology; European Research Council; Science Foundation IrelandThe gut microbiome plays a vital role in its host’s ecology. Clinical studies have shown gut microbes increase host health and fitness by providing digestive and immune functions, as well as aiding development. Natural variation in the microbiome is widely believed to affect host fitness in the wild but we are lacking experimental studies to test this. The microbiome varies with both host and environmental factors but most studies to date have focussed on individual factors and not adequately addressed the multiple overlapping and hierarchical drivers of microbiome variation working at environmental, host and microbial scales. This thesis investigates the role of the gut microbiota in host fitness, and how this is affected by and varies across contexts. Additionally, we address sources of variation in the gut microbiota at a host and environmental level, accounting for host ecology and drivers at different scales. We find that the host’s weight is correlated with microbiome diversity during development but that the direction of this relationship is context dependent. This shows that the microbiome interacts with the environment to determine host fitness and is important because it helps explain the contradictory findings linking diversity to weight. We also show that the interaction between the host, its microbiome and environment change with developmental stage. Specifically, we found that the microbiome of developed individuals is remarkably resilient to environmental perturbation, while developing individuals are much more sensitive, with important implications for future experiments. We developed a novel method for experimentally perturbing the microbiome that will allow microbiome researchers to begin testing hypotheses linking the microbiome to host ecology and evolution in natural settings. Finally, we show that welfare measures, such as environmental enrichment may interact with the gut microbiota to impact on host health and behaviour. In summary, I show that variation in the microbiome is linked to host ecology and that this variation is linked to host fitness.Item Interrogating annotated protein coding regions for hitherto undetected translation(University College Cork, 2023) Fedorova, Alla; Baranov, Pavel V.; Science Foundation IrelandRibosome profiling (Ribo-seq) is a technique that allows to capture ribosome protected fragments and sequence them. This powerful method enables discovery of not yet annotated proteoforms and translated open reading frames (ORFs), even ones that are hidden in annotated protein coding regions. Here we employed the Ribo-seq data together with comparative genomics analysis in order to discover non-AUG initiated proteoforms derived via alternative translation start sites that are in-frame with annotated starts. Production of such non-AUG proteoforms can be split into two scenarios. First, some nonAUG proteoforms are generated as alternative proteoforms in addition to annotated AUG- initiated ones. This phenomenon is called PANTs - Proteoforms with Alternative N-termini. The second scenario is when a non-AUG codon is used exclusively as the translation start for the generation of the main protein product from mRNA. In addition to discovery of non-AUG proteoforms, we rebuilt and upgraded an instance of the Galaxy platform for processing Ribo-seq data called RiboGalaxy. This update enabled prediction of novel translated ORFs from raw Ribo-seq reads by using only an internet browser with no need of local software. This update made working with Ribo-seq data more accessible to the scientific community. Chapter 1 is an introductory chapter which describes Proteoforms with Alternative N termini - PANTs. In particular, it covers different sources of PANTs, their functions and methods for their discovery. Chapter 2 covers the development of a pipeline for detection of non-AUG N-terminally extended proteoforms in the human genome which constitutes a phylogenetic approach and Ribo-seq-based approach. It also narrates the discovery of novel non AUG N-terminal extensions using the aforementioned pipeline and an attempt to describe the functionality of those non-AUG N-termini. Chapter 3 describes the phenomenon of exclusive non-AUG initiation when only non AUG initiated proteoform is generated from mRNA unlike Proteoforms with Alternative N-termini (PANTs) when both non-AUG and AUG proteoforms are generated from the same mRNA. Reported proteoforms were analysed and novel candidates predicted using Ribo-seq data. Chapter 4 reports the development of an update of RiboGalaxy - an interactive user friendly online platform for the processing Ribo-seq data which covers all the steps from preprocessing raw reads and quality control to transcriptomic and genomic alignments which then can be visualised and analysed in Trips-viz and GWIPS-viz - transcriptomic and genomic browsers for ribosome profiling data which altogether comprise the Riboseq.org resource. This platform enables preparing ribosome profiling data for subsequent detection of translated ORFs in Trips-viz. This update includes its backend moving to configuration manager (ansible), updating tools, their dependencies and reference indices and adding novel tools that allow to prepare files for easy upload to GWIPs-viz and Trips-viz.Item Exploring the camouflaged and non-coding genome: analysing difficult genomic regions in human disease genetics(University College Cork, 2023) Becerra Rodríguez , Maria de los Ángeles; Moore, Thomas F.; Baranov, Pavel V.; Science Foundation IrelandThe main objective of this thesis is to highlight the importance of investigating camouflaged regions, specifically segmental duplications, and non-coding regions, in the human genome. These regions, often overlooked due to their complexity, hold immense potential for uncovering novel insights into disease genetics. In pursuit of this objective, this thesis first focused on the study of camouflaged and non-coding regions in the context of schizophrenia. Schizophrenia is a complex psychiatric disorder specific to humans characterized by a combination of altered cognitive function, distorted perception, and disrupted social behaviour. Understanding the genetic underpinnings of schizophrenia is crucial for advancing our knowledge of its aetiology and developing more effective diagnostic and therapeutic approaches. Through comprehensive genomic analyses, novel insights were gained, which identified a novel duplication in a locus affecting a dopamine receptor implicated in neurotransmission. Additionally, small deletions in constrained non-coding regulatory regions were implicated in schizophrenia for the first time. Moreover, this thesis characterizes a long non-coding RNA (lncRNA) originating from the segmentally duplicated Pregnancy-Specific Glycoprotein locus. The lncRNA was expressed exclusively in oligodendrocytes, implicating it in the regulation of myelination processes in the brain. This lncRNA is human-specific, further emphasizing the biological relevance of camouflaged and non-coding regions in the context of human evolution. Throughout this thesis, a human-specific perspective was adopted, recognizing the unique genomic features that shape our species. By expanding our knowledge of difficult genomic regions, such as camouflaged and non-coding regions, this thesis aims to close the gap in the missing heritability problem and to gain a comprehensive understanding of the genetic architecture underlying complex disorders and uniquely human traits like schizophrenia.Item New photonic imaging systems for biomedical applications(University College Cork, 2023-06-07) Sen, Rajannya; Papkovsky, Dmitri B.; Andersson-Engels, StefanOxygen (O2) is one of the essential environmental parameters for living organisms, as it plays a vital role in their metabolism. Therefore, monitoring the distribution of O2 and its dynamics is essential for physiological studies. Assessment of tissue oxygenation by means of phosphorescent probes and optical microscopy has been increasingly used in biological and medical research. O2 imaging by PLIM is gaining popularity as it can provide an accurate, quantitative, and calibration-free mapping of O2 concentration in complex biological samples and is unaffected by external factors such as probe concentration, autofluorescence, photobleaching, optical alignment, excitation intensity, and scattering. Current PLIM platforms have limitations in image acquisition speed, sensitivity, penetration depth, accuracy, and general usability. TCSPC with raster scanning technique is frequently used in PLIM, but it is slow as it requires long pixel dwell times to measure long-lived phosphorescence. Other commonly used techniques include gated CCD and CMOS cameras, which lack single photon sensitivity and have a low frame rate. Moreover, the majority of the current PLIM systems use a microscopic format, while macroscopic systems are rarely available. We demonstrate a new macro-imager that overcomes many of these limitations. The imager is based on the new time-stamping Tpx3Cam optical camera, which combines a novel silicon optical sensor and a Timepix3 readout chip that has the capability to record the TOA and TOT at each pixel simultaneously. For TCSPC-PLIM applications, the Tpx3Cam imager was built by integrating the camera with an image intensifier that provided single photon sensitivity, an emission filter, and a macro lens using a Cricket adapter. Sample excitation was performed with an LED controlled by a pulse generator and synchronised with the camera. A custom-designed software was used to acquire the Tpx3Cam raw data in a binary format, and a C-language code was used to post-process the data. The resulting data matrix was then fitted with a bi-exponential function to determine the lifetime values. Planar phosphorescent O2 sensors and a resolution plate mask were used to characterize the imager. The performance and resolution of the imager were optimized with different image processing techniques, and the spatial and temporal resolution achieved were adequate for wide-field PLIM applications. Various commercial and non-commercial O2-sensitive phosphorescent materials were evaluated with the new imager. The lifetime signals were recorded for 20 s and the PLIM images generated by the imager showed good uniformity and lifetime contrast between the different materials with varying temperature and oxygenation conditions. The phosphorescence lifetime values were also consistent with those measured by alternative methods. Next, we demonstrated the application of the imager for mapping O2 concentration in live tissue samples stained with a PtBP-based NanO2-IR probe. The tissues were stained passively or by microinjections, and PLIM was performed under resting conditions and upon inhibition of respiration. The imager allowed accurate phosphorescence lifetime measurement of the probe and generated detailed O2 concentration maps of the tissue surface and even at depths of up to 0.5 mm inside the tissue. Lastly, we demonstrated the Tpx3Cam imager in model in vivo applications to map O2 concentration and tissue hypoxia in grafted tumours of laboratory animals. The probe delivery conditions in the animals were optimized to prevent adverse effects and enable long-term detection of the probe in the grafts. The live animals were injected with CT26 cells stained with the NanO2-IR probe to develop tumours with characteristic phosphorescence. The animals were monitored, and tumours were allowed to grow for 3, 7, 10 and 17 days. O2 imaging of live and euthanized animals at different time points produced phosphorescence lifetime values that reflected the hypoxic state of the tissue. Confocal PLIM microscopy was also performed on the excised tissues to demonstrate the distribution of the probe in the tumour. These results demonstrate that the new Tpx3Cam imager shows promising performance in PLIM mode with high spatial resolution. It can image objects up to several centimetres in size with flexible optical alignment (e.g., vertical or horizontal configuration). It can produce accurate lifetime values at a superior speed and sensitivity that can be converted to O2 concentration maps. The application of the imager ranges from the characterization of solid-state O2-sensors to O2 imaging of sizable biological samples, including respiring cells, live post-mortem animal tissues and organs and in vivo imaging of tissue hypoxia in animal models. Overall, the imager can be used for quantitative wide-field PLIM in physiological in vivo studies in a minimally invasive manner.