Coming dissertations at MedFak
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Advanced molecular tools for diagnostic analyses of RNA and antibodies in situ and in solution
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-522118
Advanced molecular diagnostics uses in vitro biological assays to detect nucleic acids or proteins even in low concentrations across samples, allowing for the identification of biomarkers, monitoring the course of the disease over time, and selection of appropriate therapy. In this thesis, I focus on development and early applications of several molecular tools of expected value in research, and eventually also clinically.
In papers I and II, proximity extension assay (PEA) was for the first time modified to measure specific antibody responses, rather than protein levels as in the standard PEA. We call the method AbPEA and the technique was used to sensitively measure antibody responses to the spike protein or the nucleocapsid of SARS-CoV-2. We demonstrated that AbPEA has high specificity, sensitivity, and broad dynamic range, along with multiplexing potential, offering performance similar to that of other methods for antibody measurements. We demonstrated utilization of blood and saliva samples in paper I and paper II, respectively, which further establish that our approach has great potential for large-scale screening and biobanking.
In paper III, we aimed to investigate how the protein composition of extracellular vesicles (EVs) differed among blood samples collected from healthy individual or ones with either mild or severe COVID-19. Proximity barcoding assay was applied to obtain a comprehensive overview of the protein composition of large numbers of individual EVs, demonstrating interesting differences.
In paper IV, we enhanced padlock-RCA-based RNA genotyping in situ by using another newly developed technology for highly selective detection of DNA or RNA sequence variants, referred to as super RCA (sRCA). Our analysis showed that this approach can improve the selectivity for sequence variants during in situ detection of mutant or wild-type transcripts, and the signals representing superRCA reaction products are prominent and easily distinguished from any background.
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Traumatic brain injury in elderly patients
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-521958
The increase of elderly traumatic brain injury (TBI) patients constitutes a considerable challenge. The aim was therefore to specifically study elderly TBI patients with respect to patient characteristics, neurointensive care (NIC) and outcome, and to identify age specific features, which may be important for selection of patients and optimization of NIC in the elderly. Data from the Uppsala TBI-registry and collected physiological monitoring data from the NIC unit were analysed.
Between 1996–1997 and 2008–2009, patients ≥60 years had doubled from 16% to 30%. Despite the increase of elderly an overall favorable outcome was maintained at around 75% between the two periods and the elderly showed favorable outcome in slightly more than 50%.
Analysis of characteristics and outcome between 2008–2010 showed that fall accidents and acute subdural hematoma were more common in the elderly ≥65 years. Admission status and NIC treatment did not differ depending on age, except that a larger proportion of the elderly had surgery. Elderly ≥65 years showed a favorable outcome in 51% compared to 72% in the young.
Studies of patients ≥60 years treated 2008–2014 showed that high age, multiple injuries, low Glasgow coma motor score on admission and the use of mechanical ventilation were negative prognostic factors.
Elderly had different secondary insult patterns with a higher percentage of good monitoring time (%GMT) with high cerebral perfusion pressure (CPP), high mean arteria blood pressure (MAP) and high systolic blood pressure (SBP) and less %GMT with high intracranial pressure (ICP), low CPP and low MAP. On the contrary to the young, high %GMT with SBP>180 was associated with favorable outcome in the elderly, indicating that blood pressure probably should be treated differently in the elderly.
Elderly had worse pressure autoregulation (higher values of PRx) and spent longer time with higher PRx. Elderly also had higher optimal CPP and spent lower %GMT with CPP close to optimal CPP. High PRx correlated with mortality in elderly but pressure autoregulation influenced outcome less in the elderly.
Overall, the results show that elderly TBI patients differ in many aspects and more studies are warranted to increase knowledge and optimize NIC.
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Cell-autonomous and paracrine mechanisms underlying Pik3ca-driven vascular malformations
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-521712
Vascular malformation is a benign overgrowth of blood or lymphatic vessels leading to life-threatening consequences for affected patients. Activating mutations in the TIE2 receptor cause the majority of venous malformations (VMs), while somatic activating mutations in PIK3CA, leading to the overactivation of the PI3K-AKT pathway, cause both VMs and lymphatic malformations (LMs). Although molecular inhibitors targeting the PI3K-AKT-mTOR pathway, such as rapamycin, have shown beneficial effects, they are not curative. This thesis aimed to explore the endothelial cell-autonomous and paracrine mechanisms underlying Pik3ca-driven pathological vascular growth to identify a rationale for improved and curative therapies for vascular malformations.
In Paper I, we reported that one of the most common causative mutations, PIK3CAH1047R, gives rise to two distinct LM subtypes known as macrocystic and microcystic LM in humans. Using a transgenic mouse model with temporally controlled LEC-specific activation of Pik3caH1047R, we found that the growth of microcystic LM is dependent on both the upstream pro-lymphangiogenic VEGF-C-VEGFR3 and the downstream AKT-mTOR signalling. Combination treatment targeting both signalling pathways led to effective inhibition of lesion growth in mice, suggesting a novel therapeutic approach for LM patients. In Paper II, we explored further the endothelial cell-autonomous and paracrine mechanisms underlying microcystic LM growth in mice. Using single-cell RNA sequencing, we identified a new immune-interacting subtype of dermal lymphatic capillary endothelial cells, termed iLECs. We showed that in Pik3ca mutant mice, iLECs produce factors that recruit pro-lymphangiogenic VEGF-C-producing macrophages. Macrophage depletion, inhibition of their recruitment, and anti-inflammatory COX-2 treatment resulted in decreased lymphatic growth, indicating a critical role of paracrine signalling between iLECs and immune cells in the pathogenesis of microcystic LM. In Paper III, we described distinct lymphatic vessel responses to oncogenic PI3K activation in different organs. We observed that while lymphatic vessels in the skin form microcystic LM through vessel sprouting, in certain other organs, they form large cysts reminiscent of macrocystic LM. Finally, we used mice with a BEC-specific activation of Pik3caH1047R to compare disease mechanisms in VM to those in LM in Paper II and to focus further on the former in Paper IV.