| 1. |
- Mottahedin, Amin
(författare)
-
Developing brain and systemic inflammation: a "Toll-like" link with consequences
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The developing brain is vulnerable to external insults, and perinatal brain injury (PBI) is a major cause of life-long neurological syndromes such as cerebral palsy. Currently, no pharmaceutical intervention is available. Hypoxia/ischemia (HI), infections and inflammation are implicated in the pathogenesis of PBI. However, the crosstalk between these etiologies is not fully understood. Toll-like receptors (TLR) 3 and TLR2 are responsible for sensing viral and bacterial infections and initiating the inflammatory response. The aim of this thesis was to investigate the effect of systemic inflammation induced by activation of these TLRs on neonatal HI brain injury. We demonstrate that intraperitoneal administration of TLR3 and TLR2 ligands (PolyI:C and P3C, respectively) prior to HI increases the brain injury in neonatal mice. PolyI:C and P3C induced neuroinflammation and altered microglial phenotype as assessed by RT-qPCR, multiplex cytokine assay or flow cytometry. PolyI:C also upregulated the pro-apoptotic gene, Fasl, expression and reduced activation of pro-survival signaling molecule Akt. On the other hand, P3C suppressed mitochondrial respiration, a major mechanism of cellular energy production. P3C, unlike other TLR agonists, induced marked infiltration of leukocytes to the cerebral spinal fluid and brain of neonatal mice and rats. Confocal microscopy, Cre recombinase-mediated gene targeting and in vitro cell transmigra-tion assay revealed the choroid plexus as a site of leukocyte entry. RNA sequencing of the choroid plexus followed by transcriptome cluster analysis and Ingenuity Pathway Analysis revealed potential mechanisms of leukocyte infiltration, including a specific chemotaxis signature and cytoskeleton-related pathways. Finally, we show that N-acetylcysteine treatment inhibits TLR2-mediated leukocyte trafficking in vivo and in vitro. To conclude, this thesis describe a TLR-mediated link between systemic inflammation and developing brain with detrimental consequences on HI brain injury, suggesting potential novel therapeutic strategies.
|
|
| 2. |
- Lai, Kuei-Hung
(författare)
-
Studies on anti-leukemic terpenoids from medicinal mushrooms and marine sponges with ChemGPS-NP-based targets investigation of lead compounds
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis investigates the anti-leukemic activity of terpenoids isolated from medicinal mushrooms and marine sponges, as well as their possible targets and mechanisms of action.In the first section, we focused on studying the triterpenoidal components of three triterpenoid-enriched medicinal mushrooms Antrodia cinnamomea, Ganoderma lucidum, and Poria cocos, which have been used in folk medicine for centuries and also developed into several contemporary marketed products. We isolated the major and characteristic triterpenoids from these mushrooms, together with six new lanostanoids (II-1–II-6). The anti-leukemic activity of the isolates was evaluated in vitro using MTT proliferative assay and seven of them exhibited potential anti-leukemic effect. The active lead compounds were further subjected to computational analyses utilizing the ChemGPS-NP tool. We established a database for the anti-leukemic relevant chemical space of triterpenoids isolated from these three medicinal mushrooms, which could be used as a reference database for further research on anti-leukemic triterpenoids. Our results indicated that the anti-leukemic effect of the active lead compounds was mediated not only through topoisomerases inhibition but also through inhibiting DNA polymerases.The second and third sections focused on isolation of anti-leukemic sesterterpenoids from sponges. The investigation of Carteriospongia sp. led to the isolation of two new scalarane-type sesterterpenoids (III-1 and III-2) and one known tetraprenyltoluquinol-related metabolite (III-3). All isolates exhibit an apoptotic mechanism of action against Molt 4 cells, found to be mediated through the disruption of the mitochondrial membrane potential (MMP) and inhibition of topoisomerase IIα expression. Detailed investigation of the apoptotic mechanism of action using molecular docking analysis revealed that compound III-1 might target Hsp90 protein. The apoptotic-inducing effect of III-3 was supported by in vivo experiment by suppressing the volume of xenograft tumor growth (47.58%) compared with the control.In the final section of this thesis we studied manoalide and its derivatives, sesterterpenoids isolated from the sponge Luffariella sp.. Manoalide has been studied as a potential anti-inflammatory agent for the last thirty years with more than 200 publications and 40 patents. However, the configurations at positions 24 and 25 were never revealed. In the current study, ten manoalide-type sesterterpenoids (IV-1–IV-10) were isolated from Luffariella sp. and their stereoisomers at positions 24 and 25 were identified and separated for the first time. The configuration at positions 24 and 25 showed to have a significant effect on the anti-leukemic activity of manoalide derivatives, with the 24R,25S-isomer exhibiting the most potent anti-leukemic activity. The apoptotic mechanism of action of compound IV-7 against Molt 4 cells was investigated, and the compound was found to trigger MMP disruption and intracellular reactive oxygen species (ROS) generation. Compound IV-7 also inhibited activity against both human topoisomerases, I and II. The in vivo experiment further supported the anti-leukemic effect of IV-7 with a 66.11% tumor volume suppression compared to the control.
|
|
| 3. |
- Bhandage, Amol K., 1988-
(författare)
-
Glutamate and GABA signalling components in the human brain and in immune cells
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glutamate and γ-aminobutyric acid (GABA) are the principal excitatory and inhibitory neurotransmitters in the central nervous system (CNS). They both can activate their ionotropic and metabotropic receptors. Glutamate activates ionotropic glutamate receptors (iGlu - AMPA, kainate and NMDA receptors) and GABA activates GABA-A receptors which are modulated by many types of drugs and substances including alcohol. Using real time quantitative polymerase chain reaction, I have shown that iGlu and/or GABA-A receptor subunits were expressed in the hippocampus dentate gyrus (HDG), orbitofrontal cortex (OFC), dorsolateral prefrontal cortex (DL-PFC), central amygdala (CeA), caudate and putamen of the human brain and their expression was altered by chronic excessive alcohol consumption. It indicates that excitatory and inhibitory neurotransmission may have been altered in the brain of human alcoholics. It is possible that changes in one type of neurotransmitter system may drive changes in another. These brain regions also play a role in brain reward system. Any changes in them may lead to changes in the normal brain functions.Apart from the CNS, glutamate and GABA are also present in the blood and can be synthesised by pancreatic islet cells and immune cells. They may act as immunomodulators of circulating immune cells and can affect immune function through glutamate and GABA receptors. I found that T cells from human, rat and mouse lymph nodes expressed the mRNAs and proteins for specific GABA-A receptor subunits. GABA-evoked transient and tonic currents recorded using the patch clamp technique demonstrate the functional GABA-A channel in T cells. Furthermore, the mRNAs for specific iGlu, GABA-A and GABA-B receptor subunits and chloride cotransporters were detected in peripheral blood mononuclear cells (PBMCs) from men, non-pregnant women, healthy and depressed pregnant women. The results indicate that the expression of iGlu, GABA-A and GABA-B receptors is related to gender, pregnancy and mental health and support the notion that glutamate and GABA receptors may modulate immune function. Intra- and interspecies variability exists in the expression and it is further influenced by physiological conditions.
|
|
| 4. |
|
|
| 5. |
- Lindberg, Frida A.
(författare)
-
The Biological Importance of the Amino Acid Transporter SLC38A10 : Characterization of a Knockout Mouse
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The biggest group of transporters, the solute carriers (SLCs), has more than 400 members, and about 30% of these are still orphan. In order to decipher their biological function and possible role in disease, there is a need for characterization of these. Around 25% of SLCs are estimated to have amino acids as substrates, including transporters belonging to the SLC38 family. The SLC38 members are sometimes referred to their alternative name: sodium-coupled neutral amino acid transporters (SNATs). One of these transporters, SNAT10 (or SLC38A10), has been characterized as a bidirectional transporter of glutamate, glutamine, alanine and aspartate, as well as having an efflux of serine, and is ubiquitously expressed in the body. However, its biological importance is not yet understood. The aim with this thesis was to characterize a mouse model deficient in SNAT10 protein in order to find the biological importance of this transporter. In paper I, this is done by using a series of behavioral tests, including the open field test, elevated plus maze, rotarod and Y-maze, among others. The SNAT10 knockout mouse was found to have an increased risk-taking behavior, but no motor or spatial working memory impairments. Furthermore, the knockout mouse was found to have a decreased body weight. In paper II, an additional behavioral characterization was performed by using the multivariate concentric square field™ (MCSF) test. The MCSF test is an arena with different zones associated to different behavioral traits, which generates a behavioral profile depending on where the mouse spends its time. The result from this test implies that the SNAT10 deficient mouse has a lower explorative behavior than its wild type littermates. In paper III, gene expression was studied in whole brain and some genes related to cell cycle regulation and p53 expression were found to be differentially expressed in the knockout brain. Additional gene expression was studied in kidney, liver, lung and muscle, but no changes were found. Plasma levels of histidine and threonine were altered in males, but no altered amino acid levels were found in knockout females, suggesting a possible sex-specific effect. These studies together imply that SNAT10 might be involved in processes related to risk-taking and explorative behavior in the open field and MCSF tests. SNAT10 deficiency also affected amino acid levels in plasma, indicating a disrupted amino acid homeostasis.
|
|
| 6. |
- Klawonn, Anna, 1985-
(författare)
-
Molecular Mechanisms of Reward and Aversion
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Various molecular pathways in the brain shape our understanding of good and bad, as well as our motivation to seek and avoid such stimuli. This work evolves around how systemic inflammation causes aversion; and why general unpleasant states such as sickness, stress, pain and nausea are encoded by our brain as undesirable; and contrary to these questions, how drugs of abuse can subjugate the motivational neurocircuitry of the brain. A common feature of these various disease states is involvement of the motivational neurocircuitry - from mesolimbic to striatonigral pathways. Having an intact motivational system is what helps us evade negative outcomes and approach natural positive reinforcers, which is essential for our survival. During disease-states the motivational neurocircuitry may be overthrown by the molecular mechanisms that originally were meant to aid us.In study I, to investigate how inflammation is perceived as aversive, we used a behavioral test based on Pavlovian place conditioning with the aversive inflammatory stimulus E. coli lipopolysaccharide (LPS). Using a combination of cell-type specific gene deletions, pharmacology, and chemogenetics, we uncovered that systemic inflammation triggered aversion by MyD88-dependent activation of the brain endothelium followed by COX1-mediated cerebral prostaglandin E2 (PGE2) synthesis. Moreover, we showed that inflammation-induced PGE2 targeted EP1 receptors on striatal dopamine D1 receptor–expressing neurons and that this signaling sequence induced aversion through GABA-mediated inhibition of dopaminergic cells. Finally, inflammation-induced aversion was not an indirect consequence of fever or anorexia but constituted an independent inflammatory symptom triggered by a unique molecular mechanism. Collectively, these findings demonstrate that PGE2-mediated modulation of the dopaminergic circuitry is a key mechanism underlying inflammation-induced aversion.In study II, we investigate the role of peripheral IFN-γ in LPS induced conditioned place aversion by employing a strategy based on global and cell-type specific gene deletions, combined with measures of gene-expression. LPS induced IFN-ɣ expression in the blood, and deletion of IFN-ɣ or its receptor prevented conditioned place aversion (CPA) to LPS. LPS increased the expression of chemokine Cxcl10 in the striatum of normal mice. This induction was absent in mice lacking IFN-ɣ receptors or Myd88 in blood brain barrier endothelial cells. Furthermore, inflammation-induced aversion was blocked in mice lacking Cxcl10 or its receptor Cxcr3. Finally, mice with a selective deletion of the IFN-ɣ receptor in brain endothelial cells did not develop inflammation-induced aversion. Collectively, these findings demonstrate that circulating IFN-ɣ binding to receptors on brain endothelial cells which induces Cxcl10, is a central link in the signaling chain eliciting inflammation-induced aversion.In study III, we explored the role of melanocortin 4 receptors (MC4Rs) in aversive processing using genetically modified mice in CPA to various stimuli. In normal mice, robust aversions were induced by systemic inflammation, nausea, pain and kappa opioid receptor-induced dysphoria. In sharp contrast, mice lacking MC4Rs displayed preference towards most of the aversive stimuli, but were indifferent to pain. The unusual flip from aversion to reward in mice lacking MC4Rs was dopamine-dependent and associated with a change from decreased to increased activity of the dopamine system. The responses to aversive stimuli were normalized when MC4Rs were re-expressed on dopamine D1 receptor-expressing cells or in the striatum of mice otherwise lacking MC4Rs. Furthermore, activation of arcuate nucleus proopiomelanocortin neurons projecting to the ventral striatum increased the activity of striatal neurons in a MC4R-dependent manner and elicited aversion. Our findings demonstrate that melanocortin signaling through striatal MC4Rs is critical for assigning negative motivational valence to harmful stimuli.The neurotransmitter acetylcholine has been implied in reward learning and drug addiction. However, the role of cholinergic receptor subtypes in such processes remains elusive. In study IV we investigated the function of muscarinic M4Rs on dopamine D1R expressing neurons and acetylcholinergic neurons, using transgenic mice in various reward-enforced behaviors and in a “waiting”-impulsivity test. Mice lacking M4-receptors from D1-receptor expressing neurons exhibited an escalated reward seeking phenotype towards cocaine and natural reward, in Pavlovian conditioning and an operant self-administration task, respectively. In addition, the M4-D1RCre mice showed impaired waiting impulsivity in the 5-choice-serial-reaction-time-task. On the contrary, mice without M4Rs in acetylcholinergic neurons were unable to learn positive reinforcement to natural reward and cocaine, in an operant runway paradigm and in Pavlovian conditioning. Immediate early gene expression mirrored the behavioral findings arising from M4R-D1R knockout, as cocaine induced cFos and FosB was significantly increased in the forebrain of M4-D1RCre mice, whereas it remained normal in the M4R-ChatCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality.
|
|
| 7. |
- Boknäs, Niklas, 1979-
(författare)
-
Studies on interfaces between primary and secondary hemostasis
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Our conceptual understanding of hemostasis is still heavily influenced by outdated experimental models wherein the hemostatic activity of platelets and coagulation factors are understood and studied in isolation. Although perhaps convenient for researchers and clinicians, this reductionist view is negated by an ever increasing body of evidence pointing towards an intimate relationship between the two phases of hemostasis, marked by strong interdependence. In this thesis, I have focused on factual and proposed interfaces between primary and secondary hemostasis, and on how these interfaces can be studied.In my first project, we zoomed in on the mechanisms behind the well-known phenomenon of thrombin-induced platelet activation, an important event linking secondary to primary hemostasis. In our study, we examined how thrombin makes use of certain domains for high-affinity binding to substrates, called exosite I and II, to activate platelets via PAR4. We show that thrombin-induced platelet activation via PAR4 is critically dependent on exosite II, and that blockage of exosite II with different substances virtually eliminates PAR4 activation. Apart from providing new insights into the mechanisms by which thrombin activates PAR4, these results expand our knowledge of the antithrombotic actions of various endogenous proteins such as members of the serpin superfamily, which inhibit interactions with exosite II. Additionally, we show that inhibition of exosite II could be a feasible pharmacological strategy for achieving selective blockade of PAR4.In my second project, we examined the controversial issue of whether platelets can initiate the coagulation cascade by means of contact activation, a hypothesis which, if true, could provide a direct link between primary and secondary hemostasis. In contrast to previous results, our findings falsify this hypothesis, and show that some of the erroneous conclusions drawn from earlier studies can be explained by inappropriate experimental models unsuitable for the study of plateletcoagulation interfaces.My third project comprised an assessment of the methodological difficulties encountered when trying to measure the ability of platelets to initiate secondary hemostasis by the release of microparticles expressing tissue factor. Our study shows that the functional assays available for this purpose are highly susceptible to error caused by artificial contact activation. These results could help to improve the methodology of future research and thus pave the way for new insights into the roles of tissue factor-bearing microparticles in the pathophysiology of various thrombotic disorders.From a personal perspective, my PhD project has been a fascinating scientific odyssey into the largely unexplored interfaces between primary and secondary hemostasis. Looking forward, my ambition is to continue our work exploring platelet-coagulation interactions and to translate these insights into clinically meaningful information, which may someday improve the treatment of patients with bleeding and/or thrombosis.
|
|
| 8. |
- Nowak, Christoph, 1986-
(författare)
-
Insulin Resistance : Causes, biomarkers and consequences
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The worldwide increasing number of persons affected by largely preventable diseases like diabetes demands better prevention and treatment. Insulin is required for effective utilisation of circulating nutrients. Impaired responsiveness to insulin (insulin resistance, IR) is a hallmark of type 2 diabetes and independently raises the risk of heart attack and stroke. The pathophysiology of IR is incompletely understood. High-throughput measurement of large numbers of circulating biomarkers may provide new insights beyond established risk factors.The aims of this thesis were to (i) use proteomics, metabolomics and genomics methods in large community samples to identify biomarkers of IR; (ii) assess biomarkers for risk prediction and insights into aetiology and consequences of IR; and (iii) use Mendelian randomisation analysis to assess causality.In Study I, analysis of 80 circulating proteins in 70-to-77-year-old Swedes identified cathepsin D as a biomarker for IR and highlighted a tentative causal effect of IR on raised plasma tissue plasminogen activator levels. In Study II, nontargeted fasting plasma metabolomics was used to discover 52 metabolites associated with glycaemic traits in non-diabetic 70-year-old men. Replication in independent samples of several thousand persons provided evidence for a causal effect of IR on reduced plasma oleic acid and palmitoleic acid levels. In Study III, nontargeted metabolomics in plasma samples obtained at three time points during an oral glucose challenge in 70-year-old men identified associations between a physiologic measure of IR and concentration changes in medium-chain acylcarnitines, monounsaturated fatty acids, bile acids and lysophosphatidylethanolamines. Study IV provided evidence in two large longitudinal cohorts for causal effects of type 2 diabetes and impaired insulin secretion on raised coronary artery disease risk.In conclusion, the Studies in this thesis provide new insights into the pathophysiology and adverse health consequences of IR and illustrate the value of combining traditional epidemiologic designs with recent molecular techniques and bioinformatics methods. The results provide limited evidence for the role of circulating proteins and small molecules in IR and require replication in separate studies and validation in experimental designs.
|
|
| 9. |
- Chebli, Jasmine
(författare)
-
Physiological roles of amyloid precursor protein in vivo - zebrafish as a model
- 2021
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyloid-beta precursor protein (APP) is an evolutionarily conserved transmembrane protein expressed in many different tissues. APP belongs to a gene family consisting of two other APP-like proteins (APLP1 and APLP2). APP has been shown to be involved in biological processes such as neurite outgrowth, neuronal migration, synapse formation and plasticity, and cell-cell interactions. APP also plays a central role in the development of Alzheimer's disease (AD). APP's physiological role has been difficult to understand and despite all research is not yet completely understood. The purpose of this thesis was to study the role of APP during early development with zebrafish as the main model system. We have focused on the zebrafish's Apps and have tried to understand their function with the help of genetic knockout models created using the CRISPR / Cas9 method. We report that appb mutants have weakened cell adhesions that give rise to changes in cell organization. We also report that the appb mutants are smaller but develop into fertile and healthy adult individuals. We also found defects in the formation of the trigeminal ganglia (TG) and that Appb seems to have a role in cell-cell interaction. The more widespread TG also consisted of fewer nerve cells, indicating that Appb promotes nerve cell formation. Furthermore, our studies demonstrate APP expression in cilia on sensory nerve cells and ependymal cells covering the brain chambers. The conserved expression of APP in ependymal cilia in mice and humans suggest an important and preserved function. Zebrafish with mutated App were found to have defects in the formation of both cilia and cerebral ventricles. To identify new signalling pathways through which Appb controls these functions, we studied protein changes in appb mutants using mass spectrometry. These studies highlight changes that both confirm known and suggest new regulations by appb, especially in neural development, cell adhesion and in gene regulation. Finally, we tried to answer the underlying mechanisms behind compensation within the App family. We found that mutations in the app genes activate expression of homologous genes via so-called transcriptional adaptation. In conclusion, the findings reported in this thesis showed that App is implicated already in early cellular adhesion and sensory neuronal differentiation processes and is located to several sensory cilia in vivo. The use of zebrafish as a model organism allowed us to gain valuable knowledge on the physiological roles of App.
|
|
| 10. |
- Rodriguez, Juan, 1983
(författare)
-
Targeting apoptosis-inducing factor as a novel therapeutic strategy for preventing perinatal brain injury
- 2020
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Perinatal complications such as asphyxia can cause brain injuries that are often associated with subsequent neurological deficits. The mechanisms of perinatal brain injury are not fully understood, but mitochondria play a prominent role, not only due to their central function in metabolism, but also because many proteins with apoptosisrelated functions are located in the mitochondrion. Among these proteins, CHCHD4 and apoptosis-inducing factor (AIF) have already been shown to make important contributions to neuronal cell death upon hypoxia-ischemia (HI), but a better understanding of the mechanisms behind these processes is required for the development of improved treatments. By inducing HI in 9-day-old mice, leading to moderate brain injury, we studied these mechanisms from multiple perspectives. First, we determined the effect of chchd4 haploinsufficiency, and we showed that neonatal mice with this genotype experienced less brain damage due to reduced translocation of AIF and Cytochrome c from the mitochondrion. Second, we characterized the role of a newly discovered AIF isoform (AIF2), which is only expressed in the brain and the functions of which are unknown. By using Aif2 knockout mice, we showed that under physiological conditions there is an increase in Aif1 expression (the ubiquitously expressed isoform) due to a compensatory effect of loss of Aif2 expression. As a result, these mice showed a higher degree of brain damage after HI and were more vulnerable to oxidative stress. Third, we used another transgenic mouse in which Aif was overexpressed by knocking in a proviral insertion of Aif, leading to an increased expression of Aif1 without affecting the expression of Aif2. This mouse also showed a higher degree of brain damage and higher levels of oxidative stress. Finally, we used a peptide designed to block the apoptotic function of AIF. The results in young mice showed that the neuroprotective effect of the peptide was greater in male mice than in female mice. In summary, this PhD project has opened new perspectives in the comprehension of the mechanisms by which CHCHD4 and AIF are crucial proteins for brain damage after HI, and it has showed that AIF is a promising therapeutic target for improving outcome after perinatal brain injury.
|
|
