Coming dissertations at MedFak
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Vestibular schwannoma : Clinical, Epidemiological and Biochemical perspectives
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-523760
Vestibular schwannoma (VS) is a slow growing benign tumour originating in the Schwann cells surrounding the vestibulocochlear nerve. Over recent decades, the incidence rate for VS has steadily increased, with greater numbers of patients with smaller tumours being diagnosed. Today, it is estimated that around 1 in 500 people will suffer from VS in their lifetime. The most common symptom of VS is unilateral hearing loss, tinnitus or dizziness. The growth rate of the tumour is unpredictable and not related to degree of symptoms. The overall aim of this thesis was to provide new knowledge that could be used to improve routines for treatment and clinical guidelines for future patients with sporadic VS.
A local clinical quality database was used to identify patients with VS treated at Uppsala University hospital. The information in the database of patients with VS was used to analyze postoperative complications after translabyrinthine surgery, hearing outcomes after hearing preservation middle cranial fossa surgery, both postoperative and after more than 10 years of follow up, and the risk of enduing a fall-related injury. The proteome of the human endolymphatic sac endolymph in six patients with VS was described.
13% of the translabyrinthine operated patients (93 of 700) suffered from one or more complications postoperatively. Increasing age and tumour size were both risk factors for postoperative facial nerve dysfunction. Greater tumour size increased the risk for intracranial hemorrhage. 60 out of 84 patients with VS operated on through middle fossa surgery had preserved hearing after surgery. After 10 years, the hearing had deteriorated symmetrically in the tumour ear and the contralateral ear. There was no increased risk for fall-related injuries among patient with VS compared to VS-free controls. Studying subgroups, an increased risk of fall-related injury was displayed among middle-aged patients before being diagnosed with VS and postoperatively in patients treated with middle fossa surgery. A total of 1,211 proteins were detected in the ES endolymph, of which 110 were unique for the endolymph. To further improve the knowledge regarding patients with VS, a joint national guideline program would be desirable.
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Tertiary lymphoid structures in glioblastoma : Discovery, Characterization and Therapeutic Induction
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-523782
Glioblastoma (GBM) is an incurable brain cancer with a median survival of less than two years from diagnosis. The tumor microenvironment plays a major role in GBM progression through sustaining immunosuppression and poor lymphocytic infiltration. Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that form in inflamed tissues and are associated with positive prognosis in numerous cancers outside the central nervous system. Prior to this work, TLS had not been reported or studied in GBM. In this thesis, we aimed to characterize TLS in glioma patients, and to investigate immunotherapeutic approaches that could reprogram the GBM microenvironment to induce these structures, promote anti-tumor responses and prolong survival.
In Paper I, we discovered the presence of TLS in low grade and high grade glioma tissues, and found that they correlated with increased T cell infiltration inside the tumor. Moreover, we demonstrated that agonistic CD40 therapy (αCD40) induced the formation of TLS with a follicle-like organization in murine glioma models. αCD40 also promoted a population of CD11b+ regulatory B cells, which inhibited T cell activation. These cells were not present within the TLS, indicating that TLS formation and the induction of CD11b+ B cells were likely two independent processes.
In Paper II, we employed murine glioma models to study the therapeutic effect of cytokines involved in lymphoid tissue development, and selected LIGHT as the most promising candidate. To therapeutically deliver LIGHT to the tumor microenvironment, we engineered an AAV vector targeted to the brain endothelial cells to express LIGHT (AAV-LIGHT). AAV-LIGHT promoted the formation of TLS and functional high endothelial venules. Moreover, AAV-LIGHT strengthened effector and memory CD8+ T cell responses, and boosted a population of TCF1+CD8+ stem-like T cells. This was associated with a prolonged survival, indicating that AAV-LIGHT is a promising therapeutic candidate for the treatment of GBM.
In Paper III, we coupled advanced spatial transcriptomics of human GBM tissue and time point experiments in murine glioma models to investigate the stages of TLS assembly. We found that TLS formation is a step-wise process, where each stage is characterized by specific cell components and pathways. Understanding the steps underlying TLS assembly will be critical to develop efficient TLS-inducing immunotherapies.
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Pitfalls in β-cell ion imaging with fluorescent indicators and their use for real-time detection of somatostatin secretion
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-523019
Fluorescent ion indicators have become indispensable tools in cell physiology. Dye indicators can easily be loaded into most types of cells, while genetically encoded indicators are advantageous in allowing specific cellular or subcellular targeting. Comparing responses of dye and protein-based indicators may provide useful insights into indicator properties and cellular processes. Here, a few genetically encoded Ca2+ indicators and fluorescent Ca2+ dyes were compared in insulin-secreting β-cells. Recordings of depolarization-triggered changes of the cytoplasmic Ca2+ concentration ([Ca2+]i) beneath the plasma membrane with total internal reflection fluorescence microscopy demonstrated distinct [Ca2+]i spikes with protein-based indicators, while the dyes mainly reported stable [Ca2+]i elevations. The spikes reflected Ca2+ release from the endoplasmic reticulum, triggered by autocrine purinergic receptor activation from exocytotic release of ATP. The indicator-dependent differences were unrelated to Ca2+ binding affinity and buffering and probably reflected slower Ca2+ dissociation kinetics of the protein indicators. In glucose-stimulated mouse islets, the dye Fura-2 reported the characteristic [Ca2+]i response with an initial lowing followed by rapid increase, which was abolished by hyperpolarization with the K+-channel opener diazoxide. The simultaneously present genetically encoded indicator R-GECO1 failed to detect the lowering and reported a spurious [Ca2+]i elevation also in the presence of diazoxide, responses that could be ascribed to pH sensitivity of the indicator. Recordings with fluorescent H+ indicators demonstrated that glucose increases cytoplasmic pH in β-cells. Elevations of [Ca2+]i counteracted the alkalinization and [Ca2+]i oscillations in glucose-stimulated islets were associated with anti-phasic oscillations of [Ca2+]i and pH. A [Ca2+]i imaging-based reporter cell assay for real-time detection of the islet hormone somatostatin was generated by transfecting HeLa cells with somatostatin receptor 2, the G-protein Gα15 and R-GECO1. The reporter cells detected somatostatin secretion from islets imaged in the same view-field as dose-dependent [Ca2+]i elevations. Mouse and human islets released somatostatin in response to high K+, glucose, and the hormones GLP-1 and ghrelin. In glucose-stimulated mouse islets, bursts of somatostatin release were synchronized with islet [Ca2+]i oscillations. Analyses of islets from human donors indicated that type 2 diabetes is associated with hypersecretion of somatostatin. In conclusion, this thesis highlights potential pitfalls with fluorescent ion indicators in β-cell signalling studies and provides new insights into β-cell regulation of [Ca2+]i and pH. Moreover, it introduces an assay for real-time detection of somatostatin secretion from islets that holds promise for studies of the role of this hormone under normal conditions and in diabetes.