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  • Boivie, Patrik, 1976- (författare)
  • Cerebrovascular accidents associated with aortic manipulation during cardiac surgery
  • 2005
  • Doktorsavhandling (övrigt vetenskapligt)abstract
    • Background: Despite the successful development in cardiac surgery, cerebrovascular accidents (CVA) remain a devastating complication. Aortic atherosclerosis has been identified as a major risk factor for CVA. The present thesis addresses this question in relation to aortic manipulation during cardiac surgery, being divided into a clinical (I-II) and an experimental part (III-V).Material and methods: Consecutive cardiac surgery cases (n=2641) were analyzed. Patients with CVA were extracted from a database designed to monitor clinical symptoms. Patient records were used to confirm clinical data and diagnosis. Subdivision was made into three groups: control subjects, immediate, and delayed CVA, being analyzed for neurological symptoms (I). Patients with CVA who also had been investigated with computer tomography (CT) (n=77) were further evaluated in terms of hemispheric and vascular distribution of lesions. The CT-findings were compared with CVA symptoms (II). An aortic perfusion model was developed using cadaver aorta onto which multiple cross-clamp manipulations were applied (III). Washout samples of perfusate were analyzed by computerized image processing and with subdivision into different particle spectra. The model was further developed with the introduction of intraluminal manipulation from cannula and intra-aortic filter (IV). A technique for macro-anatomic mapping of plaque distribution of cadaver thoracic aorta was developed (V). Variation in plaque density was analyzed in different anatomical segments, monitored by digital image analysis. Hazards associated with surgical manipulation were studied by superimposing cannulation and cross-clamp sites onto the aortic maps in a blinded fashion.Results: The incidence of immediate and delayed CVA was 3.0% and 0.9%, respectively. Aortic quality was a strongly associated with immediate but not delayed CVA. Left-sided symptoms of immediate CVA were significantly more frequent than of the contra-lateral side. Positive signs on CT were seen in 66% of the CVA patients. Right-hemispheric lesions were more frequent compared with the contra-lateral side and the middle-cerebral artery territory dominated. Aortic cross-clamping produced a substantial output of particulate matter. Manipulation by intra-aortic filter produced a significant washout of embolic particles that escaped the filter, although some particles were captured. Cannulation was an additional source of embolic material. In terms of plaque distribution was the anterior wall of the ascending part and arch of the aorta more affected than its posterior side. However, observing a plaque in the anterior wall of this aortic segment predicted to 83% a concomitant plaque in the posterior wall. Increased age correlated positively with plaque density. The theoretical chance of interfering with a plaque during cannulation and/or clamp positioning was 45.8%.Conclusions: Both CT scans and clinical symptoms confirmed that CVA after cardiac surgery had a right-hemispheric predominance. The perfusion model resulted in a profound output of material during cross-clamp maneuvers. The intra-aortic filter successfully collected particles but also generated embolic debris on its own. Aortic cannulation was an additional source of embolic debris. Plaques were frequently found in the cadaveric aorta, and there was a high risk of plaque interference during surgical manipulation. As expected, plaque density was age-dependent.
  • Chilkova, Olga, 1976- (författare)
  • Functional and structural properties of eukaryotic DNA polymerase epsilon
  • 2006
  • Doktorsavhandling (övrigt vetenskapligt)abstract
    • In eukaryotes there are three DNA polymerases which are essential for the replication of chromosomal DNA: DNA polymerase alpha (Pol alpha), DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon). In vitro studies of viral DNA replication showed that Pol alpha and Pol delta are sufficient for DNA replication on both leading and lagging DNA strands, thus leaving the function of Pol epsilon unknown. The low abundance and the reported protease sensitivity of Pol epsilon were holding back biochemical studies of the enzyme. The aim of this study was to characterize the structural and functional properties of eukaryotic Pol epsilon.We first developed a protocol for over-expression and purification of Pol epsilon from the yeast Saccharomyces cerevisiae. Pol epsilon consists of four subunits: Pol2 (catalytic subunit), Dpb2, Dpb3 and Dpb4. This four-subunit complex was purified to homogeneity by conventional chromatography and the subunit stoichiometry of purified Pol epsilon was estimated from colloidal coomassie-stained gels to be 1:1:1:1. The quaternary structure was determined by sedimentation velocity and gel filtration experiments. Molecular mass (371 kDa) was calculated from the experimentally determined Stokes radius (74.5 Å) and sedimentation coefficient (11.9 S) and was in good agreement with a theoretical molecular mass calculated for a heterotetramer (379 kDa). Analytical sedimentation equilibrium ultracentrifugation experiments supported the proposed heterotetrameric structure of Pol epsilon.By cryo-electron microscopy and single-particle image analysis we determined the structure of Saccharomyces cerevisiae Pol epsilon to 20-Å resolution. The four-subunit complex was found to consist of a globular domain, comprising the Pol2 subunit, flexibly connected to an elongated domain, including Dpb2, Dpb3 and Dpb4 subunits. We found that Pol epsilon requires a minimal length of 40 base pairs of primer-template duplex to be processive. This length corresponds to the dimensions of the elongated domain.To characterize the fidelity by which Pol epsilon synthesizes DNA, we purified wild type and exonuclease-deficient Pol epsilon. Wild type Pol epsilon synthesizes DNA with a very high accuracy. Analysis of the exonuclease-deficient Pol epsilon showed that Pol epsilon proofreads more than 90% of the errors made by its polymerase activity. Exonuclease-deficient Pol epsilon was shown to have a specific spectrum of errors not seen in other DNA polymerases: a high proportion of transversions resulting from T-dTTP, T-dCTP and C-dTTP mispairs. This unique error specificity and amino acid sequence alignment suggest that the structure of the polymerase active site of Pol epsilon differs from those of other members of B family DNA polymerases.With recombinant proteins and circular single-stranded DNA templates, we partially reconstituted DNA replication in vitro, in which we challenged Pol epsilon and Pol delta in side-by-side comparisons regarding functional assays for polymerase activity and processivity, as well as physical interactions with nucleic acids and PCNA. We found that Pol epsilon activity and “on-DNA” PCNA interactions are dependent on RPA-coated template DNA. By the surface plasmon resonance technique, we showed that Pol epsilon has a high affinity for DNA and low affinity for immobilized PCNA. By contrast, Pol delta was found to have low affinity for DNA and high affinity for PCNA. We suggest that a possible function of RPA is to regulate down the DNA synthesis through Pol epsilon, and that the mechanism by which Pol epsilon and Pol delta load onto the template is different due to different properties of the interaction with DNA and PCNA.
  • Werner, Thomas, 1971- (författare)
  • Peptidoglycan recognition proteins in Drosophila melanogaster
  • 2004
  • Doktorsavhandling (övrigt vetenskapligt)abstract
    • The fruit fly Drosophila melanogaster is an excellent model organism to study the innate immune response, because insects and mammals share conserved features regarding the recognition and destruction of microorganisms and Drosophila is easily accessible to genetic manipulation. In my present study, I identified a new family of pattern recognition molecules for bacterial peptidoglycan in Drosophila, the Peptidoglycan Recognition Proteins (PGRP). This family of proteins is widespread in the animal kingdom, for instance there are 4 PGRP genes in humans with unknown function. So far, all tested PGRPs (from insects and mammals) have been shown to bind peptidoglycan. In Drosophila, we found and characterized 13 PGRP genes, which fall into two classes: Short PGRPs and Long PGRPs. To the short group belong PGRP-SA, SB1, SB2, SC1A, SC1B, SC2, and SD with short transcripts and predicted extracellular proteins. The long members are PGRP-LA, LB, LC, LD, LE, and LF with long transcripts and predicted intracellular and membrane spanning proteins. Transcripts from the 13 different PGRP genes are present in immune competent organs, and the majority are inducible by infection. The transcriptional regulation of the inducible PGRP genes occurs either via the imd/Relish or in some cases Toll/Dif pathway. My RNAi experiments in mbn-2 cells revealed that the peptidoglycan recognition protein PGRP-LC is a major activator of the imd/Relish pathway. In PGRP-LC deficient mbn-2 cells, Relish signalling is almost entirely blocked. However, the complex PGRP-LC gene generates three alternative splice forms, each of them carrying one of three possible PGRP domains, LCx, LCy, and LCa. I found that in the tissue culture system PGRP-LCa plays a specific role in the recognition of Gram-negative bacteria, while PGRP-LCx is crucial for the recognition of Gram-positive and Gram-negative bacteria, and peptidoglycan. Targeted mutagenesis of the PGRP-LCa isoform in vivo shows that the situation is more complicated than in the cell culture experiments. In conclusion, PGRPs constitute a highly diversified family of proteins, including key players of the innate immune response.
  • Antonsson, Åsa, 1972- (författare)
  • Regulation of NF-κB by Calmodulin
  • 2003
  • Doktorsavhandling (övrigt vetenskapligt)abstract
    • Cells experience numerous external signals which they must respond to. Such signals arriving at the cell surface are transduced via various signal transduction pathways and often ultimately result in regulation of transcription. NF-κB is a family of transcription factors involved in the regulation of genes important for processes such as immune and inflammatory responses, cell growth, development and cell survival. NF-κB proteins are normally kept inactive in the cytoplasm due to masking of their nuclear localisation signal (NLS) by inhibitory IκB proteins. A large number of stimuli lead to the activation of IκB-kinase (IKK). Active IKK phosphorylates IκB and thereby labels it for ubiquitination and, subsequently, degradation by the proteasome. Liberated NF-κB enters the nucleus, where it takes part in the regulation of its target genes.Calmodulin (CaM) is a ubiquitous Ca2+-binding protein which is considered to be the predominant intracellular Ca2+ sensor. CaM plays a major role in the Ca2+-dependent regulation of a wide variety of cellular processes, including transcription. CaM regulates transcription both indirectly through CaM-dependent kinases and phosphatases and directly through interaction with transcription factors.CaM was found to bind directly and in a Ca2+-dependent fashion to the two NF-κB family members c-Rel and RelA. The CaM-NF-κB interactions were strongly enhanced by NF-κB activating stimuli and this enhancement was blocked by the addition of IκB, suggesting that c-Rel and RelA can bind CaM after their signal-induced release from IκB. Compared to wild-type c-Rel, CaM binding-deficient mutants were shown to exhibit an increased nuclear accumulation and transcriptional activity on Ca2+-regulated cytokine promoters. The results suggest that CaM can inhibit transport of c-Rel, but not of RelA, to the nucleus and thereby differentially regulate the activation of NF-κB proteins following cell stimulation. CaM was also found to affect NF-κB activity indirectly through the action of a CaM-dependent kinase (CaMK). Studies of the events leading to IκBα phosphorylation revealed that CaM and CaMKII inhibitors blocked phorbol ester induced activation of IKK. Furthermore, CaM and CaMKII inhibitors also blocked T cell receptor/CD3 induced IκBα degradation, and expression of an inhibitor-resistant derivative of the γ isoform of CaMKII caused the inhibitors lose their effect on phorbol ester induced IκBα degradation. Finally, expression of a constitutively active CaMKII resulted in the activation of NF-κB. These results identify CaMKII as a mediator of IKK activation, specifically in response to T cell receptor/CD3 and phorbol ester stimulation.In conclusion, this thesis describes the identification of CaM as a dual regulator of NF-κB proteins, acting both directly and indirectly to affect the activity of this family of transcription factors.
  • Jonsson, Bertil, 1956- (författare)
  • Interaction between humans and car seats studies of occupant seat adjustment, posture, position, and real world neck injuries in rear-end impacts
  • 2008
  • Doktorsavhandling (övrigt vetenskapligt)abstract
    • Background: The latest generation of rear-end whiplash protection systems, as found in the WHIPS Volvo and SAHR Saab, have reduced injury rates by almost 50% in comparison with the previous generation of seat/head restraint systems. Occupant behaviour, such as seated posture and seat adjustment settings, may affect the injury risk. Method: Five studies were conducted. Studie I was an injury outcome study based on insurance data. Studies II-IV investigated seat adjustment, occupant backset, and cervical retraction for drivers and occupants in different postures and positions in the car, during stationary and driving conditions. Study V compared the occupant data from studies II and III with a vehicle testing tool, the BioRID dummy, using the protocols of the ISO, RCAR, and the RCAR-IIWPG.Results: Female drivers and passengers had a threefold increased risk for medically-impairing neck injury in rear-end impacts, compared to males. Driver position had a double risk compared with front passenger seat position. Female drivers adjusted the driver seat differently to male drivers; they sat higher and closer to the steering wheel and with more upright back support. The volunteers also adjusted their seat differently to the ISO, RCAR, and RCAR-IIWPG protocol settings; both sexes sat further away from the steering wheel, and seat back angle was more upright then in the protocols. In stationary cars, backset was highest in the rear seat position and lowest in the front passenger seat position. Males had a larger backset than females. Cervical retraction decreased and backset increased for both sexes when posture changed from self-selected posture to a slouched posture. The BioRID II dummy was found to represent 96th percentile female in stature, and a 69th percentile female in weight in the volunteer group.Conclusions: Risks in car rear-end impacts differ by sex and seated position. This thesis indicates the need for a 50th percentile female BioRID dummy and re-evaluation of the ISO, RCAR, and RCAR-IIWPG protocols, and further development of new safety systems to protect occupants in rear-end impacts.
  • Li, ShuShun, 1963- (författare)
  • Thrombospondin 1, an autocrine regulator in T cell adhesion and migration
  • 2005
  • Doktorsavhandling (övrigt vetenskapligt)abstract
    • Lymphocytes, the principal cells of the immune system, perform the immune function throughout the body by their unique capacity to circulate in blood stream and lymphatic vessels and migrate in lymphoid and non-lymphoid tissues. The mechanisms regulating lymphocyte adhesion and migration, interactions with cells and components within the extracellular matrix are not fully understood. The aim of this work has been to elucidate molecular mechanisms governing T lymphocyte adhesion and migration by endogenous molecules. The studies presented in this thesis have shown that thrombospondin-1 (TSP-1) is expressed in T lymphocytes with a high turnover, manifested by variable cell surface expression, and is regulated by SDF-1a, adhesion to fibronectin and collagen type IV. The TSP-1 binding site of calreticulin (CRT), spanning amino acid 19-32, was shown to be a major triggering factor for T cell migration within a three-dimensional collagen type 1 matrix. The chemokine SDF-1a stimulated migration via a calreticulin-TSP-1 pathway. Endogenous calreticulin binding to the N-terminal domain of endogenous TSP-1 elicited a motogenic signal to the T cells through the C-terminal domain of TSP-1 and its cell surface receptor integrin-associated protein (IAP, CD47). Inhibition experiments of ligand binding of CD91 by receptor associated protein (RAP) and small interfering RNA technology indicated that CD91 is an important factor in TSP-1-mediated T cell adhesion and migration. These results unveil an autocrine mechanism of CRT-TSP-1-CD47-CD91 interaction for the control of T cell motility and migration within 3D extracellular matrix substrata. The data demonstrated that T cell adhesion and migration are sequential events governed by a series of interacting cell surface molecules comprising a CRT-TSP-1-CD47-CD91 pathway where endogenous TSP-1 functions as the hub. Ligation of the CD3/T cell antigen receptor complex determines T cell adhesion through this mechanism. CRT interaction with the N-terminal domain of TSP-1 elicits cytoplasmic spreading, and augments adhesion, while a counter-adhesive motogenic pathway, triggering interaction of the C-terminal domain of TSP-1, induces migration. CD91-dependent internalization of TSP-1 is a crucial event of this motogenic pathway. In conclusion, the studies provide a novel mechanism governing T cell adhesion and migration within extracellular matrix substrata.
  • Loh, Edmund, 1981- (författare)
  • RNA-mediated virulence gene regulation in the human pathogen Listeria monocytogenes
  • 2010
  • Doktorsavhandling (övrigt vetenskapligt)abstract
    • The Gram-positive human pathogen Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. The majority of its virulence genes are encoded on a 9-kb pathogenicity island and are controlled by the transcriptional activator PrfA. Expression of these genes is maximal at 37°C and minimal at 30°C in a mechanism involving an RNA thermosensor. This thesis brings up different aspects of RNA-mediated regulation, including regulatory RNA structures within coding mRNA controlling expression to 5-untranslated RNA (5´-UTR) that controls downstream genes (cis-acting) as well as small non-coding RNAs (ncRNAs) that bind other target RNA (trans-acting). We investigated the importance of the coding region of the prfA-mRNA for its expression. Various lengths of prfA-mRNA were fused with reporter genes. Our finding suggested that the first 20 codons of prfA-mRNA were essential for efficient translation in Listeria monocytogenes. Translation of the shorter constructs was shown to be reduced. The expression level showed an inverse correlation with the RNA secondary structure stability in the beginning of the coding region. Riboswitches have previously been known to control expression of their downstream mRNA in a cis-acting manner. A trans-acting S-adenosylmethionine-binding riboswitch termed SreA was identified in Listeria monocytogenes. It was found to control the expression of the virulence regulator PrfA, by binding to the prfA-UTR and thereby affecting its translation. We examined the RNA locus encoding different virulence factors in Listeria monocytogenes. Several of them were preceded by 5´-UTRs of various lengths. We speculate that these 5´-UTRs could control expression of the downstream mRNA, provided they are of sufficient length. These findings prompted us to examine where and when Listeria monocytogenes switches on gene expression. Tiling array was used to compare RNAs isolated from wild-type and mutant bacteria grown at different growth conditions. Antisense RNAs covering parts of or whole open-reading frames as well as 29 new ncRNAs were identified. Several novel riboswitches possibly functioning as upstream terminators were also found. My thesis work compiles together a variety of novel RNA-mediated gene regulatory entities. A first coordinated transcriptional map of Listeria monocytogenes has been set up. My work has also revealed that the expression of the virulence regulator PrfA is controlled at several levels, indicating the importance of both the 5´-UTR and the coding RNA for regulated expression.
  • Dahl, Lina, 1981- (författare)
  • Stem cell function and organ development analysis of Lhx2 function in hematopoietic stem cells and eye development
  • 2010
  • Doktorsavhandling (övrigt vetenskapligt)abstract
    • When a multicellular organism suffers damages to tissues/organs it heals itself by either substituting the lost cellular matrix by scar formation or by regenerating the lost tissue. Regeneration likely occurs by a recapitulation of the developmental process that formed the organ. Many processes regulating organ development are based on epithelial-mesenchymal interactions and a strict control of organ specific stem/progenitor cells. Elucidation of the molecular basis of these processes is therefore vital in order to develop novel therapies in regenerative medicine. The LIM homebox gene Lhx2 is interesting in this context since Lhx2 has been shown to be important for the formation of several organs by regulating epithelial-mesenchymal interactions and progenitor cell function. Targeted inactivation of Lhx2 leads to a lethal anemia due to malformed liver and severe neural abnormalities such as hypoplasia of the forebrain and anophtalmia. Thus, elucidation of the mechanisms of the function of Lhx2 in different organ systems would give important insights into the molecular mechanisms regulating epithelial-mesenchymal interactions and stem/progenitor cell function. To elucidate the function of Lhx2 in the hematopoietic system Lhx2 was initially expressed in hematopoietic progenitor cells derived from ES cells differentiated in vitro using retroviral vectors. This approach led to the generation of hematopoietic stem cell (HSC)-like cell lines suggesting that Lhx2 could impact HSC function. However neither the specificity nor the efficiency of the Lhx2-induced phenotype could be determined using this approach. To be able to elucidate the function of Lhx2 in the hematopoietic system, an ES cell line with inducible Lhx2 expression was generated. Lhx2 expression induces self-renewal of a distinct hematopoietic progenitor cell from which HSC-like cell lines were established. Down-regulation of Lhx2 in these HSC-like cell lines leads to a rapid loss of stem cell character, providing a good model to study the molecular function of Lhx2 in hematopoietic stem/progenitor cells. A global gene expression analysis was performed comparing the Lhx2+ stem cell population to the Lhx2- differentiated progeny. This approach identified genes putatively linked to self-renewal/differentiation of HSCs. A considerable proportion of the genes showed an overlapping gene expression pattern with Lhx2 expression in tissue of non-hematopoietic origin suggesting that Lhx2 function in stem/progenitor cells partly overlap with Lhx2 function during organ development. In order to define other Lhx2-dependent progenitor cell populations and to generate a tool to analyze the function of Lhx2 in organ development a new transgenic mouse model was generated. By using a specific part of the Lhx2 promoter to drive expression of Cre recombinase in vivo (Lhx2-Cre mice) we have been able to define the first eye committed progenitor cells in the forebrain. By using the Lhx2-Cre mice it will be possible to distinguish the function of genes during eye development from their function in the patterning of the forebrain e.g. the eye field transcription factors. Conditional inactivation of Lhx2 in these eye specific progenitor cells causes an immediate developmental arrest. The transgene is also active in Lhx2-/- embryonic forebrain, but re-expression of Lhx2 in Lhx2-/- progenitor cells only promote formation of retinal pigment epithelium cells. Analysis of genes expressed by the Lhx2+ stem cell population allowed us to define novel genes putatively linked to Lhx2 function in eye development. Thus, we have defined the progenitor cells in the forebrain committed to eye development and the expansion and patterning of these progenitors are dependent on Lhx2. Although commitment to eye development is Lhx2-independent, Lhx2 might be important for the acquisition of the oligopotent fate of these progenitor cells.
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