| 1. |
- Janols, Rebecka, 1981-
(författare)
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Evolving Systems – Engaged Users : Key Principles for Improving Region-wide Health IT Adoption
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many countries have formulated their eHealth visions and billions of dollars have been spent on supporting the eHealth development throughout the world. An important part of the development is the electronic patient record (EPR). To enable sharing and increase cooperation between care providers, most Swedish county councils have decided to use a region-wide EPR. The health professionals often experience numerous problems and consider the region-wide EPR to be too generic and require them to tailor their practices instead of the system evolving towards supporting their needs.The aim of the PhD research is to gain knowledge of adoption when deploying and using region-wide health IT systems. This is accomplished by studying, analysing and reflecting upon what region-wide health IT systems are and how professionals use them in their practice. In the research a grounded theory method has been used, which means that the empirical data, not theories and hypotheses, have driven the research process. The data-gathering methods have been interviews, observations, participating in meetings, questionnaires, seminars and conducting literature reviews.In order to be able to improve the adoption, a set of four key principles has been identified: (1) Evolving systems-Engaged users, (2) Treat IT deployment and usage as part of organisational development, (3) Identify, respect and support differences, and (4) Identify what must be customised and what can be centralised.These four principles challenge the traditional way of developing enterprise-wide IT and emphasise the importance that users must engage in the development, procurement and deployment process to identify their similar and unique needs and procedures. It is crucial that both the similarities and uniqueness are respected and supported. The similarities can be supported by a centralised, standardised solution, while uniqueness requires a customised solution. In order to accomplish that, the IT deployment and usage needs to be treated as an important part of the on-going organisational development, and the IT systems must evolve, i.e., be continuously developed in order to engage the users to participate.
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| 2. |
- Persson, Patrik
(författare)
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Aspects of Regulation of GFR and Tubular Function in the Diabetic Kidney : Roles of Adenosine, Nitric Oxide and Oxidative Stress
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Diabetic nephropathy is the main cause for initiation of renal replacement therapy and early symptoms in patients include increased glomerular filtration rate (GFR), decreased oxygen tension and albuminuria, followed by a progressive decline in GFR and loss of kidney function. Experimental models of diabetes display increased GFR, decreased tissue oxygenation and nitric oxide bioavailability. These findings are likely to be intertwined in a mechanistic pathway to kidney damage and this thesis investigated their roles in the development of diabetic nephropathy. In vivo, diabetes-induced oxidative stress stimulates renal tubular Na+ transport and in vitro, proximal tubular cells from diabetic rats display increased transport-dependent oxygen consumption, demonstrating mechanisms contributing to decreased kidney oxygenation. In control animals, endogenous adenosine reduces vascular resistance of the efferent arteriole via adenosine A2-receptors resulting in reduced filtration fraction. However, in diabetes, adenosine A2-signalling is dysfunctional resulting in increased GFR via increased filtration fraction. This is caused by reduced adenosine A2a receptor-mediated vasodilation of efferent arterioles. The lack of adenosine-signaling in diabetes is likely due to reduced local adenosine concentration since adenosine A2a receptor activation reduced GFR only in diabetic animals by efferent arteriolar vasodilation. Furthermore, sub-optimal insulin treatment also alleviates increased filtration pressure in diabetes. However, this does not affect GFR due to a simultaneously induction of renal-blood flow dependent regulation of GFR by increasing the filtration coefficient. In diabetes, there is decreased bioavailability of nitric oxide, resulting in alterations that may contribute to diabetes-induced hyperfiltration and decreased oxygenation. Interestingly, increased plasma concentration of l-arginine, the substrate for nitric oxide production, prevents the development of increased GFR and proteinuria, but not increased oxygen consumption leading to sustained intra-renal hypoxia in diabetes. This thesis concludes that antioxidant treatment directed towards the NADPH oxidase as well maneuvers to promote nitric oxide production is beneficial in diabetic kidneys but is targeting different pathways i.e. transport-dependent oxygen consumption in the proximal tubule by NADPH oxidase inhibition and intra-renal hemodynamics after increased plasma l-arginine. Also, the involvement and importance of efferent arteriolar resistance in the development of diabetes-induced hyperfiltration via reduced adenosine A2a signaling is highlighted.
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| 3. |
- Behra, Phani Rama Krishna
(författare)
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Comparative genomics of the genus Mycobacterium : Genome evolution, phylogeny and diversity
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The genus Mycobacterium includes more than 190 species, and many cause severe diseases such as tuberculosis and leprosy. According to the "World Health Organization", in year 2019 alone, 10 million people developed TB, and 1.4 million died. TB had been in decline in developed countries, but made its reappearance as an opportunistic pathogen targeting immuno-compromised AIDS victims. Also, non-tuberculosis mycobacteria (NTM) infections have emerged as a major infectious agent in recent times. NTM occupy diverse ecological niches and can be isolated from soil, tap water, and groundwater. This thesis has investigated the Mycobacterium species from a genomic perspective, focusing on the biology of virulence factors, mobile genetic elements, tRNAs, and non-coding RNAs and their evolutionary distribution and possible relationship with phenotypic diversity. As part of this study, we have sequenced 153 mycobacterial genomes, including type strains, environmental samples, isolates from hospital patients, infected fish, and outbreak samples in an animal facility at Uppsala University. We have provided a phylogenetic tree based on 387 (and 56) core genes covering most species (244 genomes) constituting the Mycobacterium genus. The core gene phylogeny resulted in 33 clades. Subsequently, we have covered different clade groups, such as, M. marinum, M. mucogenicum, M. chelonae and M. chlorophenolicum and investigated the NTM clade-specific genome diversity and evolution. Our examination of non-coding genes showed that the total number of tRNA genes per species varies between 42 and 90. Among the species with more than 50 tRNAs, additional tRNA genes are likely acquired through horizontal gene transfer (HGT), as supported by the presence of closely linked HNH endonuclease gene and GOLLD RNA. We have explored the presence of selenocysteine utility and the gene for selenoprotein "formate dehydrogenase" among 244 mycobacterial genomes. For the M. chlorophenolicum clade, we have explored genes with a role in the bioremediation process. Comparative genomics of M. marinum and M. chelonae clade groups suggest new clusters or subspecies. Mutational hotspots are relatively higher in M. marinum compared to that in M. tuberculosis and M. salmoniphilum. Relatively higher number of hotspots in M. marinum is likely related to its ability to occupy different ecological niches. Finally, the thesis uncovered IS elements, phage sequences, plasmids, tRNA, and ncRNA contributing to mycobacterial evolution.
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| 4. |
- Hedlund, Joel, 1978-
(författare)
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Bioinformatic protein family characterisation
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biological research is necessary; not only to further our understanding of the processes of life, but also to combat disease, hunger and environmental damage.Bioinformatics is the science of handling biological information. It entails integrating, structuring and analysing the ever-increasing amounts of available biological data. In practise it means using computers to analyse huge amounts of very complicated data taken from a field that is only partially understood, to see the hidden trends and connections, and to draw useful conclusions.My thesis work has mainly concerned the study of protein families, which are groups of evolutionarily related proteins. I have analysed known protein families and created predictive models for them, and developed algorithms for defining new protein families. My principal techniques have been sequence alignments and hidden Markov models (HMM). To aid my work, I have written a lot of software, including MSAView, a visualiser for multiple sequence alignments (MSA).In this thesis, the protein family of inorganic pyrophosphatases (H+-PPases) is studied, as well as the two protein superfamilies BRICHOS and MDR (medium-chain dehydrogenases/reductases). The H+-PPases are tightly membrane bound, proton pumping, dimeric enzymes with ~700-residue subunits and found in bacteria, plants and eukaryotic parasites, and which use pyrophosphate as an alternative to ATP. The BRICHOS superfamily is only present in higher eukaryotes, but encompasses at least 8 protein families with a wide range of functions and disease associations, such as respiratory distress syndrome, dementia and cancer. The sequences are typically ~200 residues with even shorter functional forms. Finally, MDR, is a large and complex protein superfamily; it currently has over 16000 members, it is present in all kingdoms of life, the pairwise sequence identity is typically around 25 %, the chain lengths vary as does the oligomericity, and the members are partaking in a multitude of biological processes. The member families include the classical liver alcohol dehydrogenase (ADH), quinone reductase, leukotriene B4 dehydrogenase, and many more forms. There are at least 25 human MDR genes excluding close homologues. There are HMMs available for detecting MDR superfamily membership, but none for the individual families.For the H+-PPase family, we characterised member sequences found using an HMM of a conserved 57-residue region thought to form part of the active site. This region was found to contain two highly conserved nonapeptides, mainly consisting of the four “very early” residues Gly, Ala, Val and Asp, compatible with an ancient origin of the family. The two patterns have charged amino acid residues at positions 1, 5 and 9, are apparent binding sites for the substrate and parts of the active site, and were shown to be so specific for these enzymes that they can be used for automated annotation of new sequences.For the BRICHOS superfamily, we were able to find three previously unknown member families; group A, which may be ancestral to the ITM2 families (integral membrane protein 2); group B, which is a close relative to the gastrokine families, and group C, which appears to be a truly novel, disjoint BRICHOS family. The C-terminal region of group C has nearly identical sequences in all species ranging from fish to man and is seemingly unique to this family, indicating critical functional or structural properties.For the MDR superfamily, we characterised and built stable HMMs for 17 member families using an empiric approach. From our experiences we were able to develop an algorithm for automated HMM refinement that uses relationships in data to produce stable and reliable classifiers, and we used it to produce HMMs for 86 distinct MDR families. We have made the program freely available and it can be readily applied to other protein families. We also developed a web site (http://mdr–enzymes.org) that makes our findings directly useful also for non-bioinformaticians.In our analyses of the 86 families, we found that MDR forms with 2 Zn2+ ions in general are dehydrogenases, while MDR forms with no Zn2+ in general are reductases. Furthermore, in Bacteria, MDRs without Zn2+ are more frequent than those with Zn2+, while the opposite is true for eukaryotic MDRs, indicating that Zn2+ has been recruited into the MDR superfamily after the initial life kingdom separations.Multiple sequence alignments (MSA) play a central part in most work on protein families, and are integral to many bioinformatic methods. With the ongoing explosive increase of available sequence data, the scales of bioinformatic projects are growing, and efficient and human-friendly data visualisation becomes increasingly challenging, but is still essential for making new interpretations and discovering unexpected properties of the data.Ideally, visualisation should be comprehensive and detailed, and never distract with irrelevant information. It needs to offer natural and responsive ways of exploring the data, as well as provide consistent views in order to facilitate comparisons between datasets. I therefore developed MSAView, which is a fast, modular, configurable and extensible package for analysing and visualising MSAs and sequence features. It has a graphical user interface and a powerful command line client, and can be imported as a package into any Python program. It has a plugin architecture and a user extendable preset library. It can integrate and display data from online sources and launch external viewers for showing additional details. It also includes two new conservation measures; alignment divergences, which indicate atypical residues or deletions, and sequence conformances, which highlight sequences that differ from their siblings at crucial positions.In conclusion, this thesis details my work in analysing two protein superfamilies and one protein family using bioinformatic methods; developing an algorithm for automated generation of stable and reliable HMMs, as well as a new conservation measure, and a software platform for working with aligned sequences.
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| 5. |
- Lysholm, Fredrik, 1981-
(författare)
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Bioinformatic methods for characterization of viral pathogens in metagenomic samples
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Virus infections impose a huge disease burden on humanity and new viruses are continuously found. As most studies of viral disease are limited to theinvestigation of known viruses, it is important to characterize all circulating viruses. Thus, a broad and unselective exploration of the virus flora would be the most productive development of modern virology. Fueled by the reduction in sequencing costs and the unbiased nature of shotgun sequencing, viral metagenomics has rapidly become the strategy of choice for this exploration.This thesis mainly focuses on improving key methods used in viral metagenomics as well as the complete viral characterization of two sets of samples using these methods. The major methods developed are an efficient automated analysis pipeline for metagenomics data and two novel, more accurate, alignment algorithms for 454 sequencing data. The automated pipeline facilitates rapid, complete and effortless analysis of metagenomics samples, which in turn enables detection of potential pathogens, for instance in patient samples. The two new alignment algorithms developed cover comparisons both against nucleotide and protein databases, while retaining the underlying 454 data representation. Furthermore, a simulator for 454 data was developed in order to evaluate these methods. This simulator is currently the fastest and most complete simulator of 454 data, which enables further development of algorithms and methods. Finally, we have successfully used these methods to fully characterize a multitude of samples, including samples collected from children suffering from severe lower respiratory tract infections as well as patients diagnosed with chronic fatigue syndrome, both of which presented in this thesis. In these studies, a complete viral characterization has revealed the presence of both expected and unexpected viral pathogens as well as many potential novel viruses.
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| 6. |
- Persson, Christel, 1968-
(författare)
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Sfärernas symfoni i förändring : lärande i miljö för hållbar utveckling med naturvetenskaplig utgångspunkt : en longitudinell studie i grundskolans tidigare årskurser
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This research deals with learning in science, including learning in environment for sustainable development. Learning in environment and sustainable development are obligatory perspectives in science as well as in other school subjects. The longitudinal study started in 2003, concerning 28 pupils nine years of age in a city in southern Sweden. Data collection has been caught in the pupils’ science lessons from year 2003 to 2006. In order to analyse the pupils’ development of concepts in science and in environment for sustainable development, I have videotaped a lot of sequences from the science lessons and followed up with questionnaires and questions in interviews. Stimulated recall is used to find the teacher’s intentions and reactions on the outcome of the lessons. The results are analysed according to the Earth System Science (ESS) model. It is a model, which describes the relations and interactions between the natural spheres: the atmosphere, hydrosphere, lithosphere as well as the biosphere, including man, and the technosphere/anthroposphere. The longitudinal approach resulted in important findings regarding the changes in the pupils’ answers over time. They develop complicated 'concept webs'. The concepts found among the pupils in this study are e.g. the hydrological cycle; life; soil; water in every day life; pollution; non-polluting busses as well as waste; collecting batteries; corrosion; greenhouse and the increasing greenhouse effect. Some concepts e.g. the hydrological cycle, life and soil can from the beginning be classified as concepts used in science, but also to describe what happens in the environment. Concepts as pollution; non-polluting busses; collecting batteries; corrosion; greenhouse and increasing greenhouse effect are used by the pupils to express relations and interactions in and between the natural spheres including man. The relation between man and nature is for the pupils an area of conflicts through the entire study when the pupils from a scientific approach will be aware of the impact on living ecosystems including themselves, today and in the future. The concepts are often connected to each other in a more or less complicated network, 'concepts webs'. The obtained results indicate that the Socratic dialogue is a possible and successful method to use for the development of pupils’ concepts in environmental questions and issues. Another finding in the study is how different methods, e.g. Play and learning, support environmental learning and learning for sustainable development during the science lessons. Play is important in integrated learning and gives opportunity to understand others’ perspectives, Theory of mind. The results indicate an integrated learning process by the pupils, implying in what way they express human impact on nature.
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| 7. |
- Widén, Pär
(författare)
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Bedömningsmakten : Berättelser om stat, lärare och elev, 1960‐1995
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Avhandlingen handlar om hur bedömningssystemet i bred bemärkelse presenterades i betygsdebatten i samband de politiska besluten om betygssystemet för gymnasiet mellan 1960 och 1995. Avhandlingen tar sin utgångspunkt i de berättelser som konstruerades under perioden. Syftet är att spåra och tolka förändringar i berättelserna om bedömning i anslutning till det politiska projektet att demokratisera och individualisera skolsystemet. Fokus är särskilt riktat mot de bedömningstekniker och innebörder för relationen mellan stat, lärare och elever som aktualiserades i samband med de två gymnasiereformerna. Härigenom synliggörs en viktig definitionskamp om statens förhållande till individen - lärare och elever - och hur makten i den nya enhetliga och demokratiska gymnasieskolan skulle förstås och organiseras. Avhandlingen pekar också på hur berättelser konkurrerar och hur en berättelse kan ges olika innebörder i förhållande till det politiska projektet att skapa en skola som tar till vara elevernas demokratiska möjligheter och lika villkor.
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| 8. |
- Zuo, Shusheng, 1968-
(författare)
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Quantitation, Purification and Reconstitution of the Red Blood Cell Glucose Transporter GLUT1
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein.The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization.In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.
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| 9. |
- Andersson, Michael R., 1974-
(författare)
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Functional aspects of inorganic phosphate transport
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Inorganic phosphate is an essential nutrient for all organisms. It is required for many cellular components as nucleic acids and phospholipids, and as energy-carrying compounds such as ATP. Thus, a regulated uptake of this pivotal nutrient is of outermost importance. Depending of the availability of phosphate in the surroundings the yeast Saccharomyces cerevisiae make use of two different systems for transporting phosphate into the interior of the cell: a low-affinity system that is active during surplus phosphate conditions and a high-affinity system that is active when the availability becomes limited. This thesis focuses on the high-affinity system, which is comprised of the Pho84 and Pho89 transporters. Of the two transporters, Pho84 is the predominant one, responsible for almost all phosphate uptake during low phosphate conditions, and the contribution of Pho89 is of minor importance. Hence Pho84 is by far the most well characterized phosphate transporter. Even though much is known about phosphate transporters in yeast little in known about how phosphate is transported. The work in this thesis aims to broaden the knowledge about the transport mechanism by the means of site-directed mutagenesis and functional characterization. Also the similarity of Pho84 to glucose sensors and the potential role of conserved residues in phosphate signaling are investigated. By the use of a high-affinity system deletion strain (∆Pho84 ∆Pho89), we also managed to investigate the functional importance of well conserved residues in Pho89. In summary: the work presented in this thesis has contributed to increase the knowledge about transport mechanisms in phosphate transporters.
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| 10. |
- Carlsson, Jonas, 1979-
(författare)
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Mutational effects on protein structure and function
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis several important proteins are investigated from a structural perspective. Some of the proteins are disease related while other have important but not completely characterised functions. The techniques used are general as demonstrated by applications on metabolic proteins (CYP21, CYP11B1, IAPP, ADH3), regulatory proteins (p53, GDNF) and a transporter protein (ANTR1).When the protein CYP21 (steroid 21-hydroxylase) is deficient it causes CAH (congenital adrenal hyperplasia). For this protein, there are about 60 known mutations with characterised clinical phenotypes. Using manual structural analysis we managed to explain the severity of all but one of the mutations. By observing the properties of these mutations we could perform good predictions on, at the time, not classified mutations.For the cancer suppressor protein p53, there are over thousand mutations with known activity. To be able to analyse such a large number of mutations we developed an automated method for evaluation of the mutation effect called PREDMUT. In this method we include twelve different prediction parameters including two energy parameters calculated using an energy minimization procedure. The method manages to differentiate severe mutations from non-severe mutations with 77% accuracy on all possible single base substitutions and with 88% on mutations found in breast cancer patients.The automated prediction was further applied to CYP11B1 (steroid 11-beta-hydroxylase), which in a similar way as CYP21 causes CAH when deficient. A generalized method applicable to any kind of globular protein was developed. The method was subsequently evaluated on nine additional proteins for which mutants were known with annotated disease phenotypes. This prediction achieved 84% accuracy on CYP11B1 and 81% accuracy in total on the evaluation proteins while leaving 8% as unclassified. By increasing the number of unclassified mutations the accuracy of the remaining mutations could be increased on the evaluation proteins and substantially increase the classification quality as measured by the Matthews correlation coefficient. Servers with predictions for all possible single based substitutions are provided for p53, CYP21 and CYP11B1.The amyloid formation of IAPP (islet amyloid polypeptide) is strongly connected to diabetes and has been studied using both molecular dynamics and Monte Carlo energy minimization. The effects of mutations on the amount and speed of amyloid formation were investigated using three approaches. Applying a consensus of the three methods on a number of interesting mutations, 94% of the mutations could be correctly classified as amyloid forming or not, evaluated with in vitro measurements.In the brain there are many proteins whose functions and interactions are largely unknown. GDNF (glial cell line-derived neurotrophic factor) and NCAM (neural cell adhesion molecule) are two such neuron connected proteins that are known to interact. The form of interaction was studied using protein--protein docking where a docking interface was found mediated by four oppositely charged residues in respective protein. This interface was subsequently confirmed by mutagenesis experiments. The NCAM dimer interface upon binding to the GDNF dimer was also mapped as well as an additional interacting protein, GFRα1, which was successfully added to the protein complex without any clashes.A large and well studied protein family is the alcohol dehydrogenase family, ADH. A class of this family is ADH3 (alcohol dehydrogenase class III) that has several known substrates and inhibitors. By using virtual screening we tried to characterize new ligands. As some ligands were already known we could incorporate this knowledge when the compound docking simulations were scored and thereby find two new substrates and two new inhibitors which were subsequently successfully tested in vitro.ANTR1 (anion transporter 1) is a membrane bound transporter important in the photosynthesis in plants. To be able to study the amino acid residues involved in inorganic phosphate transportation a homology model of the protein was created. Important residues were then mapped onto the structure using conservation analysis and we were in this way able to propose roles of amino acid residues involved in the transportation of inorganic phosphate. Key residues were subsequently mutated in vitro and a transportation process could be postulated.To conclude, we have used several molecular modelling techniques to find functional clues, interaction sites and new ligands. Furthermore, we have investigated the effect of muations on the function and structure of a multitude of disease related proteins.
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