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Coming dissertations at MedFak

  • CRISPR/Cas9-based therapies and the role of astrocytes in Alzheimer’s disease and Parkinson’s disease Author: Evangelos Konstantinidis Link: Publication date: 2022-09-08 10:42

    Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative disorders. Whereas the AD brain features plaques of amyloid-beta (Aβ) and neurofibrillary tangles of tau, the PD brain is characterized by Lewy bodies and Lewy neurites containing α-synuclein (αSyn). Rare familial disease forms have illustrated a central involvement of these proteins in the respective pathogeneses. Mutations in the genes for the presenilins (PSEN1, PSEN2) result in AD by an increased generation of the more aggregation prone Aβ42 peptide, whereas mutations in the αSyn gene (SNCA) cause PD by affecting aggregation of αSyn.

    This thesis has investigated the gene editing tool CRISPR/Cas9 as a potential treatment strategy against AD and PD. When targeting PSEN1 M146L in patient fibroblasts, the increased Aβ42/Aβ40 ratio was partially restored and the treatment typically normalized the mutation-induced conformation of presenilin 1. Moreover, the treatment did not cause any major off-target effects across the genome. For SNCA, both the wild-type form and the A53T mutant were targeted. Lentivirus-mediated delivery of CRISPR/Cas9 to patient fibroblasts and HEK293T cells led to a targeting efficiency of up to 87%. However, treatment of A53T mutant patient fibroblasts only resulted in low and inconsistent targeting efficiencies.

    During the course of AD, progressive cellular dysfunction and degeneration cause widespread neuronal death. Apart from neurons, also glial cells are affected by the disease process. Astrocytes, the most abundant glial cell type, play a key role in maintaining brain homeostasis. However, in a neurodegenerative environment, astrocytes enter a reactive and inflammatory state that can potentially harm nearby neurons.

    To further investigate the role of astrocytes in AD, we generated a co-culture system of human induced pluripotent stem cell-derived neurons and astrocytes. We observed a differential effect of direct and remote astrocytic control on neuronal viability and functionality. Physical astrocytic contact combined with the presence of Aβ resulted in increased phagocytosis and clearance of dead cells as well as a reduced neuronal activity. However, indirect contact via conditioned media from control astrocytes improved the viability of neurons, whereas addition of Aβ led to hyperactivity. Analyses of long-term astrocytic cultures revealed a persistent reactive state accompanied by a limited Aβ degradation capacity and severe cellular stress.

    Overall, this thesis has explored novel gene therapeutic strategies for AD and PD as well as contributed with knowledge regarding the role of astrocytes in AD progression.

  • Evolutionary trajectories of Klebsiella pneumoniae : From experimental biofilm evolution to a hospital outbreak Author: Greta Zaborskytė Link: Publication date: 2022-09-01 10:29

    Bacterial evolution is closely intertwined with our lives. As their hosts, we shape how bacteria evolve by imposing numerous selective pressures during the time bacteria spend in our bodies. As a result, they adapt in various ways to colonize us or infect us better. In this thesis, I present studies aimed to expand the knowledge on the pathoadaptive changes in Klebsiella pneumoniae, which is a bacterial pathogen of critical importance worldwide. 

    In Paper I, we present a new 3D-printed device for growing and studying surface-attached bacterial biofilms. The special aim was to increase the ease of use and versatility, and we have used this biofilm device to screen for biofilm capacity, perform experimental evolution and fundamental biofilm analysis in subsequent studies.

    In Paper II, we study within-host evolution by analyzing 110 isolates originating from the same multidrug-resistant K. pneumoniae clone that caused an outbreak at Uppsala University Hospital between 2005 and 2010. We whole-genome sequenced these isolates and phenotypically characterized them to show that the clone has undergone extensive changes in individual patients, leading to increased biofilm formation capacity, attenuation of systemic virulence, and altered colonization potential.

    In Paper III, we exploit an experimental evolution approach to decipher evolutionary trajectories towards increased biofilm formation. We show how fast this trait can be acquired in different K. pneumoniae strains by a strong convergent evolution, mostly targeting genes involved in capsule, fimbriae, and c-di-GMP-related regulatory pathways. Importantly, this genetic parallelism extends beyond in vitro observations as we find an extensive overlap with clinical outbreak isolates that carry signatures from within-host evolution.

    The experimental evolution experiments revealed interesting genetic changes not only in the known structures or pathways but also in completely novel factors. In Paper IV, we explore a previously uncharacterized T6SS effector that is involved in biofilm formation in K. pneumoniae and strongly enhances this phenotype upon acquiring a single and specific point mutation. We demonstrate that the toxin acts as a DNase and that this mutation results in changes at multiple levels, including protein stability, toxicity, and transcriptional profiles, which collectively lead to the formation of biofilms.

  • Insulin promotes GABA signalling modulation in both the mouse hippocampus and human CD4+ T cells Author: Hayma Hammoud Link: Publication date: 2022-08-30 09:05

    Gamma-aminobutyric acid (GABA) is the most common inhibitory neurotransmitter in the adult mammalian brain, where it mediates several biological functions. Rapid inhibition is predominantly mediated by the activation of GABA-A receptors that are ubiquitously expressed across the central nervous system in a cell-, circuit-, or region-specific manner. This work contains four studies where GABA signalling and the modulatory effects of insulin are examined.

    In paper I, we used the patch-clamp technique to record synaptic and extrasynaptic GABA-A receptor-activated tonic currents from the granule cells of the dentate gyrus (DG) and CA3 pyramidal neurons along the dorsoventral axis of the mouse hippocampus. The results suggested cell type-specific variations in the inhibitory tone along the longitudinal hippocampal axis. In paper II, we analyzed both cell types in the mouse hippocampus and aimed to determine the effects of insulin on GABA signalling along the dorsoventral axis in the wild-type and Alzheimer’s disease animal model, tg-APPSwe mice. Physiological concentration of insulin modulated GABAergic synaptic and extrasynaptic tonic currents based on neuronal subtype and position along the axis in young wild-type mice. Furthermore, insulin normalized GABA-activated currents in aged tg-APPSwe mice to levels similar to those recorded in wild-type mice.

    In addition to the brain, GABA is present in blood and acts as a signalling molecule in immune cells. We show in paper III that GABA modulated cytokine release and the proliferation of activated CD4+ T cells isolated from normal individuals. These effects were differentially regulated by extracellular glucose concentration; the effects decreased as extracellular glucose concentration increased. GABA also modulated the expression of several genes and metabolism-related proteins in activated CD4+ T cells. Furthermore, insulin treatment increased the expression of the rho2 subunit of GABA-A receptors in activated CD4+ T cells and further enhanced the GABA effects. In paper IV, we observed that GABA inhibited the proliferation and altered cytokine release in a glucose concentration-dependent manner in activated CD4+ T cells isolated from type I diabetic patients. The findings of papers III and IV indicated a subset of samples called “non-responders”, wherein GABA did not affect the proliferation of activated CD4+ T cells but increased the release of a number of cytokines.

    The findings in my thesis increase the understanding of the modulatory effects of insulin on GABA signalling when GABA functions as a neurotransmitter and as an immunoregulatory molecule.