| 1. |
- Fälth Savitski, Maria, 1979-
(författare)
-
Improved Neuropeptide Identification : Bioinformatics and Mass Spectrometry
- 2008
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bioinformatic methods were developed for improved identification of endogenous peptides using mass spectrometry. As a framework for these methods, a database for endogenous peptides, SwePep, was created. It was designed for storing information about endogenous peptides including tandem mass spectra. SwePep can be used for identification and validation of endogenous peptides by comparing experimentally derived masses of peptides and their fragments with information in the database. To improve automatic peptide identification of neuropeptides, targeted sequence collections that better mimic the peptidomic sample was derived from the SwePep database. Three sequence collections were created: SwePep precursors, SwePep peptides, and SwePep predicted. The searches for neuropeptides performed against these three sequence collections were compared with searches performed against the entire mouse proteome, and it was observed that three times as many peptides were identified with the targeted SwePep sequence collections. Applying the targeted SwePep sequence collections to identification of previously uncharacterized peptides yielded 27 novel potentially bioactive neuropeptides.Two fragmentations studies were performed using high mass accuracy tandem mass spectra of tryptic peptides. For this purpose, two databases were created: SwedCAD and SwedECD for CID and ECD tandem mass spectra, respectively. In the first study, fragmentation pattern of peptides with missed cleaved sites was studied using SwedCAD. It was observed that peptides with two arginines positioned next to each other have the same ability to immobilize two protons as peptides with two distant arginines. In the second study, SwedECD was used for studying small neutral losses from the reduced species in ECD fragmentation. The neutral losses were characterized with regard to their specificity and sensitivity to function as reporter ions for revealing the presence of specific amino acids in the peptide sequence. The results from these two studies can be used to improve identification of both tryptic and endogenous peptides.In summary, a collection of methods was developed that greatly improved the sensitivity of mass spectrometry peptide identification.
|
|
| 2. |
- Källback, Patrik
(författare)
-
Development and Application of Software Tools for Mass Spectrometry Imaging
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mass spectrometry imaging (MSI) has been extensively used to produce qualitative maps of distributions of proteins, peptides, lipids, neurotransmitters, small molecule pharmaceuticals and their metabolites directly in biological tissue sections. Moreover, during the last 10 years, there has been growing demand to quantify target compounds in tissue sections of various organs. This thesis focuses on development and application of a novel instrument- and manufacturer-independent MSI software suite, msIQuant, in the open access format imzML, which has been developed specifically for quantitative analysis of MSI data. The functionality of msIQuant facilitates automatic generation of calibration curves from series of standards that can be used to determine concentrations of specific analytes. In addition, it provides many tools for image visualization, including modules enabling multiple interpolation, low intensity transparency display, and image fusion and sharpening. Moreover, algorithms and advanced data management modules in msIQuant facilitate management of the large datasets generated following rapid recent increases in the mass and spatial resolutions of MSI instruments, by using spectra transposition and data entropy reduction (at four selectable levels: coarse, medium, fine or superfine) before lossless compression of the data. As described in the thesis, implementation of msIQuant has been exemplified in both quantitative (relative or absolute) and qualitative analyses of distributions of neurotransmitters, endogenous substances and pharmaceutical drugs in brain tissue sections. Our laboratory have developed a molecular-specific approach for the simultaneous imaging and quantitation of multiple neurotransmitters, precursors, and metabolites, such as tyrosine, tryptamine, tyramine, phenethylamine, dopamine, 3-methoxytyramine, serotonin, gamma-aminobutyric acid (GABA), and acetylcholine, in histological tissue sections at high spatial resolution by matrix-assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) MSI. Chemical derivatization by charge-tagging primary amines of analytes significantly increased the sensitivity, enabling mapping of neurotransmitters that were not previously detectable by MSI. The two MSI approaches have been used to directly measure changes in neurotransmitter levels in specific brain structures in animal disease models, which facilitates understanding of biochemical mechanisms of drug treatments. In summary, msIQuant software has proven potency (particularly in combination with the reported derivatization technique) for both qualitative and quantitative analyses. Further developments will enable its implementation in multiple operating system platforms and use for statistical analysis.
|
|
| 3. |
- Sköld, Karl, 1974-
(författare)
-
Neuropeptidomics – Methods and Applications
- 2006
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The sequencing of genomes has caused a growing demand for functional analysis of gene products. This research field named proteomics is derived from the term proteome, which by analogy to genome is defined as all proteins expressed by a cell or a tissue. Proteomics is however methodologically restricted to the analysis of proteins with higher molecular weights. The development of a technology which includes peptides with low molecular weight and small proteins is needed, since peptides play a central role in many biological processes. To study endogenous peptides and hormones, the peptidome, an improved method comprising rapid deactivation in combination with nano-flow liquid chromatography (LC) and mass spectrometry (MS) was developed. The method has been used to investigate endogenous peptides in brains of mouse and rat. Several novel peptides have been discovered together with known neuropeptides. To elucidate the post mortem time influence on peptides and proteins, a time course study was performed using peptidomics and proteomics technologies. Already after three minutes a substantial amount of protein fragments emerged in the peptidomics study and some endogenous peptides were drastically reduced with increasing post mortem time. Of about 1500 proteins investigated, 53 were found to be significantly changed at 10 minutes post mortem as compared to control. Moreover, using western blot the level of MAPK phosphorylation was shown to decrease by 95% in the 10 minutes post mortem sample. A database, SwePep (a repository of endogenous peptides, hormones and small proteins), was constructed to facilitate identification using MS. The database also contains additional information concerning the peptides such as physical properties. A method for analysis of LC-MS data, including scanning for, and further profiling of, biologically significant peptides was developed. We show that peptides present in different amounts in groups of samples can be automatically detected.The peptidome approach was used to investigate levels of peptides in two animal models of Parkinson’s disease. PEP-19, was found to be significantly decreased in the striatum of MPTP lesioned parkinsonian mice. The localization and expression was further investigated by imaging MALDI MS and by in situ hybridization. The brain peptidome of reserpine treated mice was investigated and displayed a number of significantly altered peptides. This thesis demonstrates that the peptidomics approach allows for the study of complex biochemical processes.
|
|
| 4. |
- Svensson, Marcus, 1972-
(författare)
-
Neuropeptidomics – Expanding Proteomics Downwards
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biological function is mainly carried out by a dynamic population of proteins which may be used as markers for disease diagnosis, prognosis, and as a guide for effective treatment. In analogy to genomics, the study of proteins is called proteomics and it is generally performed by two-dimensional gel electrophoresis and mass spectrometric methods. However, gel based proteomics is methodologically restricted from the low mass region which includes important endogenous peptides. Furthermore, the study of endogenous peptides, peptidomics, is compromised by protein fragments produced post mortem during conventional sample handling. In this thesis nanoflow liquid chromatography and mass spectrometry have been used together with improved methods for sample preparation to semi-quantitatively monitor peptides in brain tissue. The proteolysis of proteins and rise of fragments in the low mass region was studied in a time-course study up to ten minutes, where a potential marker for sample quality was found. When rapidly denatured brain tissue was analyzed, the methods enabled detection of hundreds of peptides and identifications of several endogenous peptides not previously described in the literature. The identification process of endogenous peptides has been improved by creating small targeted sequence collections from existing databases. In applications of the MPTP model for Parkinson’s disease the protein and peptide expressions were compared to controls. Several proteins were significantly changed belonging to groups of mitochondrial, cytoskeletal, and vesicle associated proteins. In the peptidomic study, the levels of the small protein PEP-19 was found to be significantly decreased in the striatum of MPTP administered animals. Using imaging mass spectrometry the spatial distribution of PEP-19 was found to be predominant in the striatum and the levels were concordantly decreased in the parkinsonian tissue as verified by immunoblotting.
|
|
| 5. |
- Boman, Andrea, 1978-
(författare)
-
Lysosomal network proteins as biomarkers and therapeutic targets in neurodegenerative disease
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The pre-symptomatic stage of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) occurs several decades before the clinical onset. Changes in the lysosomal network, i.e. the autophagosomal, endosomal and lysosomal vesicular system, are among the first alterations observed. There are currently no treatments to slow or cure neurodegenerative diseases, and there is a great need for discovery of treatment targets in cellular pathways where pathology pre-dates the neuronal death. It is also crucial to be able to diagnose neurodegenerative diseases earlier, both to enable early intervention treatment and aid in selecting clinical trial populations before the patient has widespread pathology.This thesis aims at investigating the potential of lysosomal network proteins as biomarkers and therapeutic targets in neurodegenerative disease.A targeted search for lysosomal network proteins was performed in cerebrospinal fluid (CSF) from AD patients, and seven proteins: early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), lysozyme, microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were elevated. The levels of EEA1, LAMP-1, LAMP-2, LC3, lysozyme and Rab3 were also measured in CSF from parkinsonian syndrome patients: PD, clinically diagnosed 4-repeat tauopathy, pathologically confirmed corticobasal degeneration (CBD) and pathologically confirmed progressive supranuclear palsy (PSP) patients. LAMP-1 and LAMP-2 were decreased in PD. LC3 and lysozyme levels were increased in 4-repeat tauopathy patients. EEA1 was decreased and lysozyme increased in PSP, and LAMP-1, LAMP-2, LC3 and lysozyme were increased in CBD. The lysosomal network proteins had different CSF protein profiles in all the parkinsonian syndromes, as well as in AD. It should be emphasized that only a select few of the lysosomal network proteins were observed to be changed, rather than a general change in lysosomal network proteins, which implicates the involvement of these seven proteins in specific pathological processes. The most interesting candidates, LAMP-2 and lysozyme, were selected for further study for their involvement in the pathology of AD.Lysozyme was found to co-localise with Aβ plaques in AD patients and overexpression prolonged survival and improved the activity in a Drosophila model of AD. Lysozyme was found to alter the aggregation pathway of Aβ1-42, to counteract the formation of toxic Aβ species and to protect from Aβ1-42 induced cell toxicity. Aβ1-42 in turn was found to increase the expression of lysozyme in both neuronal and glial cells. These data suggest that lysozyme levels rise in AD as a compensatory response which is protective against Aβ associated toxicity.LAMP-2 mRNA and protein were found increased in brain areas relevant for AD pathology and various cellular models showed complex involvement of LAMP-2 in Aβ related pathology, with extensive crosstalk between LAMP-2 and Aβ. Exposure to oligomeric Aβ1-42 caused an upregulation of LAMP-2 and in turn, overexpression of LAMP-2 caused a reduction in secreted levels of Aβ1-42, as well as changing the generation pattern of Aβ and affecting clearance and secretion of Aβ1-42. These data indicate that the increased levels of LAMP-2 in AD could be an attempt to regulate Aβ generation and secretion.In summary, this thesis reports that utilising lysosomal network proteins as biomarkers and novel therapeutic targets for neurodegenerative diseases holds great promise.
|
|
| 6. |
- Domellöf, Magdalena Eriksson, 1977-
(författare)
-
Cognitive and motor dysfunction in the early phase of Parkinson's disease
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disease. The diagnosis is based on a combination of the motor signs: tremor, bradykinesia, rigidity and postural abnormalities. Mild Cognitive Impairment (MCI) is common early in the disease and a large proportion of patients with PD develop dementia (PDD). Associations between motor symptoms and cognitive decline have been suggested but the results are inconclusive due to differences in the selection of participants and variables tested. Large population based studies with comprehensive neuropsychological investigation in newly diagnosed cases with PD followed prospectively are rare. The aim of this thesis was to improve characterization and understanding of cognition in PD, and to explore the relationship to motor impairment in the early phase of PD.Methods: All new patients with suspected idiopathic parkinsonism in the catchment area (142 ooo inhabitants) were examined during a period of five years and four months. Among other investigations, a comprehensive neuropsychological evaluation was carried out in 119 of 148 patients with PD together with 30 age matched healthy controls. Assessments were repeated after one three and five years.Results: Patients performed worse than healthy controls in a majority of neuropsychological tests. MCI at the time of diagnosis were found in 36% according to recently published MCI criteria. Thirty % were cognitively impaired using another definition. One fourth of the patients developed PDD within five years after diagnosis and 25 % of those with MCI at baseline reversed back to normal cognition. Age and MCI were significant predictors of dementia. Education was an independent predictor for severe cognitive dysfunction at diagnosis but did not predict PDD. Patients with MCI converting to PDD had worse performance on visuospatial function, semantic fluency, episodic memory, mental flexibility and conceptual thinking. There were no differences in cognitive performance between patients with predominant Postural and Gait Disturbances (PIGD) and the tremor dominant subtype at the baseline investigation and belonging to the PIGD subgroup at baseline did not predict PDD. Dementia converters declined more rapidly than non-converters in posture/gait function. Associations between bradykinesia and measures of executive functions and working memory were found, and between posture and gait disturbances and visuospatial function. Some of these associations were persistent after one year. Patients receiving the dopamine agonist pramipexole performed significantly worse on a measure of verbal fluency at the one year follow up.Conclusions: The differences in proportions of cognitively impaired in the different studies emphasize the value of joint criteria for PD-MCI. Even when using such criteria, a substantial proportion of patients revert back to normal function. The increase in motor disability in patients with PDD could have several different causes that need to be further investigated. Associated motor and cognitive dysfunctions could reflect common pathophysiological processes in partly shared networks. Both dopaminergic and non-dopaminergic motor and cognitive functions seems to be involved in PDD which suggests that pharmacological treatment in PD needs to go beyond the scope of dopaminergic deficiency in search for new therapies that would also be effective for non-motor symptoms.
|
|
| 7. |
- Holmqvist, Tomas, 1974-
(författare)
-
Signaling via Orexin Receptors : A Pharmacological Study
- 2004
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The orexin receptors are a pair of newly discovered G-protein coupled receptors which are activated by the neuropeptides orexins and play a role in sleep/vigilance, apetite/metabolism and neuroendocrine regulation. On a cellular level receptor activation results in, to name but a few effects, elevation of intracellular calcium and depolarisation. All cellular effects display an uncommon dependence of extracellular Ca2+, which has been shown to be due to influx of extracellular Ca2+ as a primary response.Here we provide evidence for a high specificity of orexin receptors for orexin peptides over other neuropeptides, despite previous reports of the opposite. Other neuropeptides could neither displace orexin-A from orexin receptors, nor affect functional responses induced by orexin peptides via orexin receptors. In an effort to assess the determinants of orexin-A binding to orexin receptors orexin-A was truncated/mutated and tested for functional responses. It was found that alterations in the orexin-A sequence had more prominent effects on the activation of OX1 than on OX2 receptors.When the signaling of orexin receptors was investigated in neuron-like cells it was found that they couple to Ca2+-metabolism and PLC activation in a manner similar to that in non-neuronal cells. Investigations of OX1 receptor regulation of adenylyl cyclases showed orexin receptors to have a dual effect on the production of cAMP. A high-affinity inhibitory coupling and a low-affinity stimulatory coupling. The stimulatory coupling was determined to consist of two components, a low potency GS-coupling and a high-potency PKC coupling.In conclusion we have shown that orexin receptors are preferentially activated by orexin peptides and the receptors couple to Ca2+-metabolism in a similar way in different contexts. Orexin receptors couple to both the phospholipase C and the adenylyl cyclase pathway and to some extent these pathways converge in the production of cAMP.
|
|
| 8. |
- Patra, Kalicharan
(författare)
-
Modulation of Neuronal Functions : the Role of SLC10A4
- 2014
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mental health of a person depends on the correct functioning of the brain. The brain and the spinal cord contain many types of cells, of which one important type are called the neurons. Neurons are special in the way they connect to each other to form large networks. The chemicals called transmitters are packed at the nerve endings into tiny packets called vesicles and when a signal arrives these vesicles fuse immediately to the attached cell surface and release their contents. The role of the synaptic vesicular transporter proteins is to ensure proper packing of transmitter molecules that can be released upon stimulation. Vesicular packing is an important process. The carrier proteins involved in packing work in coordination to determine the amount and type of transmitters to be packed. Missing a carrier protein from the vesicles might lead to improper packing and inaccurate signaliing. These signaling molecules are known for their implications in many psychiatric and neurological disorders like Alzheimer’s disease, Parkinson’s disease, Schizophrenia, and attention deficit to name just a few. How a vesicular transporter can affect the modulatory functions of aminergic neurons is the subject of this thesis. This thesis reports on the effects of the loss of a vesicular orphan transporter. Study I demonstrates the localization of this protein to monoaminergic and cholinergic terminals. It reports the effect of the loss of Slc10A4 on vesicular dopamine uptake, synaptic clearance of dopamine and hypersensitivity of animals to dopamine related psychostimulants. Study I also provides evidence for ATP as a possible ligand for SLC10A4 protein. Study II provides data on the clinical relevance of Slc10A4 in playing a protective role against vulnerability to epilepsy. It reports that loss of Slc10A4 renders the animals hypersensitive to cholinergic drugs. Study III provides a closer look at individual cholinergic synapses at neuromuscular junctions in mice lacking Slc10A4. The structural and electrophysiological properties of the NMJ are found compromised because of the loss of this vesicular protein. Taken together, this thesis presents a SV protein’s perspective of viewing at modulation of synaptic transmission.
|
|
| 9. |
- Svenningsson, Per
(författare)
-
Striatal adenosine A2A receptors
- 1998
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The nucleoside adenosine exerts a modulatory action in the central nervous system by activating G-protein coupled receptors. Four such receptors are cloned and pharmacologically characterized: A1, A2A, A2B and A3. Caffeine, the most widely consumed psychostimulant, acts as an adenosine receptor antagonist predominantly on the first two. Adenosine A1 receptors influence neuronal excitability and neurotransmitter release in an inhibitory manner and are expressed in most brain areas. On the other hand, adenosine A2A receptors, the subject of the present thesis, are predominantly expressed in striatum, the major area of the basal ganglia. Striatum processes sensorimotor, associative and limbic information that it receives in a topographically organized way from cerebral cortex, thalamic subnuclei and the midbrain. Striatal efferents go to substantia nigra and the internal part of globus pallidus (the entopeduncular nucleus in rodents) either directly (the striatonigral pathway), or indirectly, via the external part of globus pallidus and the subthalamic nucleus (the striatopallidal pathway). By combining radioactive and non-radioactive in situ hybridization and highly sensitive riboprobes, A2A receptor mRNA was found, in primates as well as rodents, to be extensively co-expressed with dopamine D2 receptor mRNA in striatopallidal neurons, but not in cholinergic interneurons. A2A receptor mRNA was largely segregated from dopamine D1 receptor mRNA. The phosphoprotein DARPP-32 is highly enriched in striatal projection neurons and plays an important role in integrating intracellular responses exerted by the major striatal neurotransmitters, neuromodulators and neuropeptides. In striatal slices, simultaneous stimulation of A2A and D1 receptors was found to exert an additive effect on phosphorylation, and thereby activation, of DARPP-32, suggesting that A2A receptors regulate the activity of DARRP-32 in the striatopallidal pathway and that D1 receptors do so in the striatonigral. The present study also shows that the biphasic effect of caffeine on locomotion is mirrored by a biphasic regulation of immediate early genes in striatum. Blockade of adenosine A2A receptors appears crucial for the stimulatory action of caffeine. Our data suggest that endogenous adenosine, acting via A2A receptors, has a facilatatory effect on the expression of immediate early genes in the striatopallidal pathway that does not require intact dopaminergic neurotransmission. A2A receptors are, therefore, likely to be important for coordinating neurotransmission through the basal ganglia in a parallel manner. However, there are also important network interactions between adenosine A2A receptors and dopamine D1 receptors on immediate early gene expression in striatum and globus pallidus, partly resembling those between D1 and D2 receptors. In conclusion, these studies strongly suggest that endogenous adenosine, acting via A2A receptors, is of considerable importance for regulating neurotransmission in the striatopallidal pathway. The stimulation by caffeine in doses resembling those achieved following normal human use appears largely due to modulation of this pathway. These results further imply that adenosine A2A receptors may be a possible target for future development of drugs for treatment of Parkinson's disease, affective disorders and schizophrenia.
|
|
| 10. |
- Viereckel, Thomas, 1987-
(författare)
-
United in Diversity : A Physiological and Molecular Characterization of Subpopulations in the Basal Ganglia Circuitry
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Basal Ganglia consist of a number of different nuclei that form a diverse circuitry of GABAergic, dopaminergic and glutamatergic neurons. This complex network is further organized in subcircuits that govern limbic and motor functions in humans and other vertebrates. Due to the interconnection of the individual structures, dysfunction in one area or cell population can affect the entire network, leading to synaptic and molecular alterations in the circuitry as a whole. The studies in this doctoral thesis aimed at characterizing restricted subpopulations of neurons in the Basal Ganglia circuitry and their importance in the wider function of the network. To this end, we identified subpopulations of neurons in the subthalamic nucleus (STN), substantia nigra (SN) and ventral tegmental area (VTA), characterized their molecular profile and investigated their physiological role in the circuitry.Within the mouse STN, reduction of glutamatergic neurotransmission in a subpopulation expressing Paired-like homeodomain transcription factor 2 (Pitx2) led to structural alterations in the nucleus as well as biochemical alterations of the dopaminergic system in the Nucleus accumbens (NAc) and changes in reward-related behavior. In the ventral midbrain, we identified and characterized novel marker genes selective to the VTA or SN. Of these, transient receptor potential cation channel subfamily V member 1 (TrpV1) marks a population of mainly glutamatergic neurons in the VTA which project to the NAc, while gastrin releasing peptide (Grp) is expressed in a population of dopaminergic neurons neuroprotected in Parkinson's disease. Furthermore, we discovered that disruption of glutamatergic co-release of dopaminergic neurons expressing dopamine transporter (DAT), diminishes fast EPSCs and glutamate release but does not affect the acquisition of reward-related behavioral tasks. To selectively quantify glutamate release from specific subpopulations, we devised a technique combining glutamate-amperometry and optogenetics. This was used to measure glutamate released from Pitx2-expressing synaptic terminals in the Globus pallidus as well as DAT- or TrpV1-expressing terminals in the NAc.In summary, this doctoral thesis has furthered understanding of the function and importance of specific subpopulations within the Basal Ganglia circuitry and provides a novel means to investigate glutamate in the intact rodent brain within clearly defined, restricted cell populations.
|
|
