| 1. |
- Gouveia, Leonor, 1990-
(författare)
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The role of PDGF-A in lung development, injury and repair
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The developmental processes that take place during embryogenesis depend on a great number of proteins that are important for cell-to-cell communication. Platelet-derived growth factors are known to be important for epithelial-mesenchymal interactions during development and organogenesis. However, many details are still lacking regarding organ-specific PDGF expression patterns and detailed cellular functions. This thesis aims to better describe the contribution of PDGF-A signaling to lung developmental and injury processes.To study the cell-specific expression patterns of PDGF-A we generated a reporter mouse that show LacZ expression in all PDGF-A positive cells. This mouse model was used to characterize PDGF-A expression in embryonic and adult mouse tissues (paper I).With the use of three different reporter mice, we described the cell type specific expression patterns of PDGF-A, PDGF-C and PDGFRα in mouse lungs, from embryonic day 10.5 (E10.5) when development is initiated, until adulthood (Postnatal day 60) when the lung is fully mature (paper II).A lung-specific Pdgfa knockout mouse was generated and the impact of the deletion was studied during lung development and adulthood. Mice lacking Pdgfa expression in the lung survived until adulthood but exhibited abnormal alveolar development. This phenotype was caused by the inability of myofibroblasts to assemble alpha smooth muscle actin ring around the forming alveoli (paper III).To investigate if PDGF-A is involved in the injury response mechanisms of the adult lung, we generated inducible lung-specific Pdgfa knockout mice. In homeostasis, adult Pdgfa deletion did not result in any apparent phenotype, whereas after hyperoxia-induced lung injury, preliminary data show that mutant mice exhibit substantially more alveolar damage and immune cell infiltration (paper IV).In conclusion, this thesis reports novel insights into the expression and role of PDGF-A and PDGFRα for the lung, both in development and adulthood.
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| 2. |
- Mihajlica, Nebojsa, 1983-
(författare)
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Pericyte Influence on Drug Delivery Across the Blood-Brain Barrier : Implications for Therapy of Neurodegenerative Diseases
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The blood-brain barrier (BBB) represents a complex interface between the brain parenchyma and systemic blood circulation, strictly controlling exchange of substances between the two sites. Pericytes are mural cells located on the abluminal membrane of the brain endothelium, involved in BBB formation and maintenance. Previous studies have implied that pericyte-deficiency causes alterations in BBB integrity for larger molecules, mainly by upregulated transcytosis pathways. The aim of the thesis was to examine the role of pericytes for small-molecular drug transport across the BBB, by providing a closer insight into different aspects of transport in a pericyte-deficient state. PDGF-B retention motif knockout mice were used as a well-established pericyte-deficient model. Small-molecular drugs, namely diazepam, digoxin, imatinib, levofloxacin, oxycodone and paliperidone were selected based on utilization of different BBB transport mechanisms. Surprisingly, the extent of BBB transport expressed as the unbound brain-to-unbound plasma partition coefficients indicated no difference between pericyte-deficient and control mice for all tested drugs. In addition, no difference was observed in the rate of BBB transport estimated by trans-cardiac in situ brain perfusion experiments. These results imply preserved BBB features in terms of tight junctions that limit para-cellular transport, as well as unaltered transporter functionality and expression. Thus, BBB aspects relevant for small-molecular drug transport seem to be maintained regardless of pericyte presence at the BBB. In addition, data from proteome and transcriptome analysis of the brain microvasculature fragments were in line with these findings, showing no difference in major transporter expressions at the BBB in pericyte-deficient mice. Finally, experiments with tyrosine kinase (TK) inhibitors suggested a potential relevance of the imatinib-like TK target profiles for the stabilization of compromised BBB integrity in pericyte-deficiency. In conclusion, the present thesis work provided comprehensive insight into pharmacokinetics of small-molecular drugs in a pericyte-deficient state. It represents an important initial platform for future extensive investigations of BBB transport in pericyte-deficiency, towards the ultimate goal of developing novel therapeutics for the treatment of different neurodegenerative diseases.
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| 3. |
- Castro, Marco
(författare)
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Cellular and molecular roles for CDC42 in angiogenesis
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Angiogenesis is the physiological process by which new blood vessels grow and critically depends on the interplay between the major vascular units: endothelial cells, pericytes and smooth muscle cells. Dysfunction and mispatterning of blood vessels are associated with the progression of many vascular complications, and therefore, understanding the causes of vascular dysmorphia is a central question in vascular biology. CDC42 is a small GTPase known to regulate a diverse array of cellular functions in endothelial cells, however, its contribution to vascular development in vivo remains incompletely understood. The overall aim of this thesis work is to investigate the role of CDC42 during angiogenesis in the central nervous system, using an inducible endothelial-specific Cdc42 knockout model.In Paper I, I investigate which CDC42-dependent functions operational in vivo are of relevance for angiogenic sprouting, and how they contribute to blood vessel morphogenesis. Analysis of distinct cellular behaviours shows that CDC42 is critically required for proper EC dispersion in the vasculature and that it regulates sprouting angiogenesis and endothelial axial polarity.In Paper II, I explore the in vivo consequences of Cdc42 deletion for vascular morphogenesis, leading to the appearance of capillary-venous malformations in the brain, resembling the human disease of cerebral cavernous malformations. I aimed to understand how this type of vascular malformations arise and was been able to identify the MEKK3-ERK5-KLF2/4 molecular signalling pathway and other cellular events as the trigger factors that may be responsible for these malformations.Paper III redirects focus to the physiological roles of another protein, GPR116, in modulating blood-brain barrier permeability and pathologic angiogenesis in the central nervous system.In summary, these findings reveal crucial roles of endothelial CDC42 during angiogenesis and further uncover its potential relevance in the molecular pathogenesis of cerebrovascular malformations.
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| 4. |
- Álvarez-Aznar, Alberto
(författare)
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Cdc42, orchestrator of vascular morphogenesis in the retina
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cdc42 is a small GTPase that controls many cellular functions related to cytoskeletal dynamics, such as migration, polarity, and proliferation. Despite what we know of Cdc42 in other cell types, not much research has been done on the vasculature. This thesis describes the consequences of Cdc42 deletion in two vascular cell types—endothelial and mural cells—during developmental angiogenesis.In paper I, we demonstrate through a combination of in vitro, in silico, and in vivo assays, that Cdc42-deficient endothelial cells migrate less and fail to distribute normally in areas of naturally occurring high proliferation during angiogenesis, causing vascular malformations with enlarged lumens. In addition, these cells present impaired filopodia formation, a disadvantage for the tip cell position, disturbed axial polarity and altered junctions.With an in vivo approach, in paper III we demonstrate that the deletion of Cdc42 in mural cells has consequences on the morphogenesis of the retinal vasculature. Cdc42-deficient mural cells proliferate less and cannot keep up with the nascent angiogenic vasculature, which results in a complete pericyte loss at the sprouting front. Furthermore, we describe that mural cells contribute to the remodeling of the vasculature, also after the initial phases of angiogenesis.The CreERT2 system is frequently used for conditional gene deletion and lineage tracing. Tamoxifen administration allows spatiotemporally controlled recombination of fluorescent reporters, and tracing of the labeled cells. However, in the course of our studies, we observed tamoxifen-independent recombination. In paper II, we describe this phenomenon in detail, using different combinations of CreERT2 and fluorescent reporter lines. We conclude that tamoxifen-independent recombination is a widespread occurrence, and that fluorescent reporter lines present varying levels of susceptibility to it.In summary, the work presented here sheds new light on the role of Cdc42 in the vasculature. Additionally, this thesis describes in detail an important feature of CreERT2 and reporter lines that should be taken into account when performing lineage-tracing experiments.
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| 5. |
- Erlandsson, Anna, 1973-
(författare)
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Neural Stem Cell Differentiation and Migration
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neural stem cells are the precursors of neurons, astrocytes and oligodendrocytes. During neural development, the division of stem cells takes place close to the lumen of the neural tube, after which they migrate to their final positions within the central nervous system (CNS). Soluble factors, including growth factors, regulate neural stem cell proliferation, survival, migration and differentiation towards specific cell lineages.This thesis describes the function of platelet-derived growth factor (PDGF) and stem cell factor (SCF) in neural stem cell regulation. PDGF was previously suggested to stimulate neuronal differentiation, but the mechanisms were not defined. This study shows that PDGF is a mitogen and a survival factor that expands a pool of immature cells from neural stem cells. The PDGF-treated cells can be stained by neuronal markers, but need further stimuli to continue their maturation. They can become either neurons or glia depending on the secondary instructive cues. Moreover, neural stem cells produce PDGF. Inhibition of this endogenous PDGF negatively affects the cell number in stem cell cultures. We find that SCF stimulates migration and supports the survival of neural stem cells, but that it has no effect on their proliferation or differentiation into neurons and glia. Intracellular signaling downstream from the receptors for PDGF and SCF includes activation of extracellular signal-regulated kinase (ERK). This investigation shows that active ERK is not needed for the differentiation of stem cells into neurons, at least not during early stages.Neural stem cells have a future potential in the treatment of CNS disorders. To be able to use neural stem cells clinically we need to understand how their proliferation, differentiation, survival and migration are controlled. The results presented in this thesis increase our knowledge of how neural stem cells are regulated by growth factors.
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| 6. |
- Kurtyka, Magdalena, et al.
(författare)
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The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier
- 2024
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Ingår i: European Journal of Cell Biology. - : Elsevier. - 0171-9335 .- 1618-1298. ; 103:2
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Tidskriftsartikel (refereegranskat)abstract
- Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood -brain barrier (BBB). Most molecules require either carrier- or receptor -mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium -enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBBenriched genes according to established selection criteria. As a result, we propose the high -affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.
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| 7. |
- Lindeberg, Jonas, 1971-
(författare)
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New Conditional Gene Targeting Methods : For Studying Neurotrophic Mechanisms in Selected Neuronal Populations
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Powerful techniques to manipulate the mouse genome have had a great impact on our understanding of biology. The major drawback with conventional transgenic methods is that the genetic alteration will be present in every cell of the animal, from conception and onwards. Many genes are normally expressed in several different organs and at different times and an effect observed in the transgenic mouse could therefore be derived from various tissues or be a developmental consequence. Furthermore, some mutations will result in a lethal phenotype and prevent investigations on the adult gene function. This is a particular problem when studying the brain since it is not yet fully developed at birth. Consequently, a conditional methodology is required, where precise chromosomal alterations can be achieved in restricted tissues at chosen times in the living animal. This issue has been successfully tackled during the last decade. Conditional gene targeting is today a fact.This thesis demonstrates that it is possible to use an internal ribosomal entry sequence for tissue-specific direction of conditional gene targeting tools. General cassettes were constructed and shown to work in cells. Moreover, a knock-in transgenic mouse expressing the Cre recombinase in catecholaminergic neurons was made. This strain will offer possibilities to study genetic mechanisms in dopamine and noradrenaline producing cells and to build mouse models for common human diseases involving such neurons.Furthermore, another transgenic mouse was created having the Cre recombinase under tight tetracycline-regulated control. This strain can mediate inducible genetic recombination in brain neurons and be completely silenced by feeding the mice the antibiotic doxycycline. Moreover, by altering the time-point of doxycycline administration, chromosomal manipulations can be achieved in different neuronal patterns. This transgene is a general tool that can be combined with any mouse having floxed genes and also with lines producing tetracycline transactivators in other locations.Finally, constructs aiming at conditional manipulation of the nerve growth factor gene and targeting of sensory neuronal populations are described.
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| 8. |
- Nahar, Khayrun
(författare)
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The role of PDGF-B in brain blood vessels
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The development of blood vessels is dependent on several molecular cues to form properly. A functional PDGF-B/PDGFR-b signaling is paramount for the investment of mural cells, that provide with support, to the developing vasculature. Mutations in PDGFB and PDGFRB are linked to PFBC, an age-dependent neurodegenerative condition manifested by vessel associated calcifications in the brain. The overall aim of the work presented in here was to investigate PFBC related calcifications and analyze the effects of impaired PDGF-B/PDGFR-b signaling on the formation of brain calcifications in different mouse models.In paper I, we functionally analyzed PFBC-related PDGFB and PDGFRB mutations in vitro. While all PDGFB mutations lead to abolished protein function, PDGFRB mutations have more diverse consequences. We also show that reduced Pdgfb and Pdgfrb levels are insufficient for the formation of brain calcifications in several mouse strains. Moreover, region-specific susceptibility factors seem to reside in PFBC pathogenesis that are distinct from pericyte coverage and BBB deficiency.In paper II, we described the molecular composition and cellular association of calcified nodules that develop in two mouse models of PFBC, Pdgfbret/ret and Slc20a2-/- mice. We show that the nodules are composed of pro- and anti-mineralization proteins and that they are in direct association with astrocytes and microgliaIn paper III, we analyzed the effects of EC-specific ablation of PDGF-B in adult brain vasculature. We report a substantial decrease of pericyte coverage and altered VSMC morphology and that this phenotype is inadequate to trigger the formation of calcifications or affect BBB integrity.The aim of paper IV was to molecularly define the adult mouse brain vasculature by taking advantage of the scRNAseq technique. Here, we describe a gradual change in expression profile along the arteriovenous axis: ECs present a continuum along the axis while mural cell expression profile is punctuated. In summary, this thesis present detailed description of calcifications formed in mouse models of PFBC and address the role of impaired PDGF-B/PDGFR-b signaling for the formation of nodules in mice. Furthermore, the scRNaseq analysis performed on healthy adult brain vasculature has paved the way for future analysis in mouse models of PFBC.
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| 9. |
- Paulsson, Johan F., 1976-
(författare)
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Proislet Amyloid Polypeptide (proIAPP) : Impaired Processing is an Important Factor in Early Amyloidogenesis in Type 2 Diabetes
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyloid is defined as extracellular protein aggregates with a characteristic fibrillar ultra-structure, Congo red affinity and a unique x-ray diffraction pattern. At present, 25 different human amyloid fibril proteins have been identified, and amyloid aggregation is associated with pathological manifestations such as Alzheimer’s disease, spongiform encephalopathy and type 2 diabetes. Amyloid aggregation triggers apoptosis by incorporation of early oligomers in cellular membranes, causing influx of ions. Amyloid is the only visible pathological islet alteration in subjects with type 2 diabetes, and islet amyloid polypeptide (IAPP) is the major islet amyloid fibril component. IAPP is produced by beta-cells and co-localized with insulin in the secretory granules. Both peptides are synthesised as pro-molecules and undergo proteolytic cleavage by the prohormone convertase 1/3 and 2. Although IAPP is the main amyloid constituent, both proIAPP and proIAPP processing intermediates have been identified in islet amyloid.The aim of this thesis was to study the role of impaired processing of human proIAPP in early islet amyloidogenesis. Five cell lines with individual processing properties were transfected with human proIAPP and expression, aggregation and viability were studied. Cells unable to process proIAPP into IAPP or to process proIAPP at the N-terminal processing site accumulated intracellular amyloid-like aggregates and underwent apoptosis. Further, proIAPP immunoreactivity was detected in intracellular amyloid-like aggregates in betacells from transgenic mice expressing human IAPP and in transplanted human beta-cells. ProIAPP was hypothesized to act as a nidus for further islet amyloid deposition, and to investigate this theory, amyloid-like fibrils produced from recombinant IAPP, proIAPP and insulin C-peptide/A-chain were injected in the tail vein of transgenic mice expressing the gene for human IAPP. Pancreata were recovered after 10 months and analysed for the presence of amyloid. Both IAPP and proIAPP fibrils but not des-31,32 proinsulin fibrils, caused an increase in affected islets and also an increase of the amyloid amount. This finding demonstrates a seeding capacity of proIAPP on IAPP fibrillogenesis. IAPP has been known for some time to trigger apoptosis in cultured cells, and a novel method for real time detection of apoptosis in beta-cells was developed. Aggregation of recombinant proIAPP and proIAPP processing intermediates were concluded to be inducers of apoptosis as potent as IAPP fibril formation.From the results of this study, a scenario for initial islet amyloidogenesis is proposed. Initial amyloid formation occurs intracellularly as a result of alterations in beta-cell processing capacity. When the host cell undergoes apoptosis intracellular proIAPP amyloid becomes extracellular and can act as seed for further islet amyloid deposition.
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