| 1. |
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| 2. |
- Blissing, Annica
(författare)
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Thiopurine S-methyltransferase - characterization of variants and ligand binding
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects.Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function.Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (KD ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.
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| 3. |
- Costa Felicissimo, Viviane
(författare)
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Infrared - X-ray pump probe spectroscopy
- 2005
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The present thesis concerns theoretical studies of molecular interactions investigated by infrared and X-ray spectroscopic techniques, with emphasis on using the two technologies combined in pump probe experiments. Three main types of studies are addressed: the use of near-edge X-ray absorption fine structure spectra (NEXAFS) to manifest through-bond and through-space interactions; the role of hydrogen bonding on the formation of X-ray photoelectron spectra as evidenced by simulations of the water dimer; and the development of theory, with sample applications, for infrared X-ray pump probe spectroscopy - the main theme of the thesis. Ab initio calculations indicate that NEXAFS spectra give direct information about the through-bond and through-space interactions between vacant non-conjugated π* orbitals. It is found that the X-ray photoelectron spectrum of the water dimer differs strongly from the monomer spectrum in that two bands are observed, separated by the chemically shifted ionization potentials of the donor and the acceptor. The hydrogen bond is responsible for the anomalously strong broadening of these two bands. The studies show that X-ray core electron ionization of the water dimer driven by an infrared field is a proper technique to prove the proton transfered state contrary to conventional X-ray photoelectron spectroscopy. Our simulations of infrared X-ray pump-probe spectra were carried out using wave packet propagation techniques. The physical aspects of the proposed new X-ray spectroscopic method - phase sensitive Infrared - X-ray pump probe spectroscopy - are examined in detail in two sample applications - on the NO molecule and on the dynamics of proton transfer in core ionized water dimer. It is found that the phase of the infrared pump field strongly influences the trajectory of the nuclear wave packet on the ground state potential. This results in a phase dependence of the X-ray pump probe spectra. A proper choice of the delay time of the X-ray pulse allows to directly observe the X-ray transition in the proton transfered well of the core excited potential.
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| 4. |
- Drobin, Kimi
(författare)
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Antibody-based bead arrays for high-throughput protein profiling in human plasma and serum
- 2018
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Affinity-based proteomics utilizes affinity binders to detect target proteins in a large-scale manner. This thesis describes a high-throughput method, which enables the search for biomarker candidates in human plasma and serum. A highly multiplexed antibody-based suspension bead array is created by coupling antibodies generated in the Human Protein Atlas project to color-coded beads. The beads are combined for parallel analysis of up to 384 analytes in patient and control samples. This provides data to compare protein levels from the different groups.In paper I osteoporosis patients are compared to healthy individuals to find disease-linked proteins. An untargeted discovery screening was conducted using 4608 antibodies in 16 cases and 6 controls. This revealed 72 unique proteins, which appeared differentially abundant. A validation screening of 91 cases and 89 controls confirmed that the protein autocrine motility factor receptor (AMFR) is decreased in the osteoporosis patients.Paper II investigates the risk proteome of inflammatory bowel disease (IBD). Antibodies targeting 209 proteins corresponding to 163 IBD genetic risk loci were selected. To find proteins related to IBD or its subgroups, sera from 49 patients with Crohn’s disease, 51 with ulcerative colitis and 50 matched controls were analyzed. From these targeted assays, the known inflammation-related marker serum amyloid protein A (SAA) was shown to be elevated in the IBD cases. In addition, the protein laccase (multi-copper oxidoreductase) domain containing 1 (LACC1) was found to be decreased in the IBD subjects.In conclusion, assays using affinity-based bead arrays were developed and applied to screen human plasma and serum samples in two disease contexts. Untargeted and targeted screening strategies were applied to discover disease-associated proteins. Upon further validation, these potential biomarker candidates could be valuable in future disease studies.
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| 5. |
- Elfving, Anna
(författare)
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Transcriptional regulation of mouse ribonucleotide reductase
- 2011
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- All living organisms are made of cells and they store their hereditary information in the form of double stranded DNA. In all organisms DNA replication and repair is essential for cell division and cell survival. These processes require deoxyribonucleotides (dNTPs), the building blocks of DNA. Ribonucleotide reductase (RNR) is catalyzing the rate limiting step in the de novo synthesis of dNTPs. Active RNR is a heterodimeric protein complex. In S phase cells, the mouse RNR consists of the R1 and the R2 proteins. The R1/R2 RNR-complex supplies the cell with dNTPs required for DNA replication. Outside S-phase or in non-proliferating cells RNR is composed of R1 and p53R2 proteins. The R1/p53R2 RNR-complex supplies cells with dNTPs required for mitochondrial DNA replication and for DNA repair. An undisturbed dNTP regulation is important since unbalanced dNTP pools results in DNA mutations and cell death. Since unbalanced pools are harmful to the cell, RNR activity is regulated at many levels. The aim of this thesis is to study how the mouse RNR genes are regulated at a transcriptional level. We have focused on the promoter regions of all three mouse RNR genes. Primer extension experiments show that the transcription start of the TATA-less p53R2 promoter colocalizes with an earlier unidentified initiator element (Inr-element). This element is similar to the known Inr-element in the mouse R1 promoter. Furthermore, functional studies of the R1 promoter revealed a putative E2F binding element. This result suggests that the S phase specific transcription of the R1 gene is regulated by a similar mechanism as the R2 promoter which contains an E2F binding site. Finally we have established a method to partially purify the transcription factor(s) binding the upstream activating region in the mouse R2 promoter by phosphocellulose chromatography and affinity purification using oligonucleotides immobilized on magnetic beads. This method will allow us to further study the transcription factors responsible for activating expression of the R2 protein. This method has a potential to be utilized as a general method when purifying unknown transcription factors.
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| 6. |
- Larsson, Veronica J.
(författare)
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The roles of inner nuclear membrane proteins during interphase and mitosis
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The nuclear envelope (NE) consists of two concentric membranes, the outer nuclear membrane (ONM) and the inner nuclear membrane (INM). The LINC (linker of nucleoskeleton and cytoskeleton) complex spans both the ONM and the INM connecting the cytoskeleton to the nucleoskeleton and chromatin. Only a few of the known INM proteins have been functionally characterized and shown to have important roles in chromatin organisation. Defects in the genes coding for proteins in the INM and the nuclear lamina give rise to serious human diseases, called envelopathies.In 2009 (Buch et al. 2009) our group made two major discoveries. We showed for the first time, that an integral INM protein distributed along the microtubules of the mitotic spindle. This protein was therefore named Samp1, Spindle Associated Membrane Protein 1. The second discovery was that depletion of Samp1 caused detachment of the centrosome from the NE, suggesting that Samp1 is associated with the microtubule cytoskeleton both in interphase and mitosis.In this thesis we continued to investigate Samp1´s role during interphase. We also wanted to investigate the localisation of Samp1 in the mitotic spindle and possible function during mitosis. We show that the expression of Samp1 mutants and depletion of Samp1 affects the distribution and organisation of A-type lamins, the LINC complex protein Sun1 and the LINC complex associated protein emerin. Thus, in interphase Samp1 is functionally connected to the LINC complex and the A-type lamina network. The LINC complex can help explain how the centrosomes detach from the NE in Samp1 depleted cells. In mitotic cells, we found that depletion of Samp1 caused prolonged metaphase and aberrant mitotic phenotypes such as bi-nucleation, enlarged nuclei and micronuclei. We also showed that Samp1 interacts with RanGTPase and importin-β, which are key players in assembling the mitotic spindle. Samp1 also modulates the levels of importin-β and NuMA in the mitotic spindle, which could explain the mitotic phenotypes that we se in Samp1 depleted cells. Here we present evidence showing, for the first time, that an INM protein is present on kinetochore microtubules and have an essential role for correct chromosome segregation and spindle assembly.
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| 7. |
- Nordén, Jenny
(författare)
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Multifaceted adhesion properties of Helicobacter pylori in promotion of gastric disease
- 2011
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Helicobacter pylori is a Gram-negative spiral-shaped bacteria that resides in the gastric mucosa and adheres to the epithelial lining of the human stomach. H. pylori adhesion mediates inflammation of the epithelium, which can lead to more severe diseases such as chronic active gastritis and gastric carcinoma. In order to adhere tightly to the mucosal lining, H. pylori expresses adhesion proteins called adhesins. The best-charachterized adhesins are blood group antigen binding adhesin, BabA, and sialic acid binding adhesin, SabA. During chronic infection there is an adhesion/inflammation-associated balance between BabA- and SabA-mediated binding modes. BabA is the major mediator of binding when the stomach is in good health and the corresponding binding receptors are ABO blood group antigens such as the difucosylated Lewis b antigen that is expressed on MUC5AC mucins by the surface epithelium. However, in chronic inflammation of the gastric mucosa there are dynamic shifts in the glycosylation patterns; in inflamed gastric tissue, SabA is an important mediator of bacterial binding and adherence. The corresponding binding receptors for the SabA adhesin are complex sialylated antigens such as sLewis x (sLex) and sLewis a (sLea). In this thesis, I describe our findings that SabA also promotes H. pylori binding to red blood cells in the gastric mucosal small blood vessels. The minimal binding epitope on the erythrocyte surfaces is the sialylated NeuAcα2-3Gal-disaccharide and this bacterial adherence promotes hemagglutination, i.e. the rapid aggregation of the erythrocytes. Another important finding is that the binding properties of SabA are of polymorphic nature. In particular, clinical isolates demonstrate variant types of relative binding affinity for the series of sialyl-di Lewis x (sdiLex), sialyl-Lewis a (sLea) and sialyl-lactosamine (sLn). The relative binding to sialylated glycans is strain dependent and H. pylori strains J99 and SMI9 display different SabA-mediated binding modes for sialylated glycans. By introduction of the sabA gene from strain J99 into strain SMI9, the detailed binding mode of SMI9sabAJ99 was altered and preferably displayed the original binding mode of strain J99. Thus, the polymorphism of SabA-mediated binding is an inherent property of the adhesion protein itself, i.e. the polypeptide itself and is not encoded or influenced by the strain genome background. The individualized binding properties or polymorphism in binding modes provides SabA with the opportunity to adapt to individual hosts and to the inflammatory changes in glycosylation. SabA expression makes use of slipped-strand mispairing for H. pylori to swiftly attach and detach from the epithelium. Furthermore, the ability to fluently attach and detach from the epithelial surfaces is an important feature of H. pylori and its ability to evade the mucosal inflammatory responses, as well as from the rapid turnover and shedding of gastric mucosa. Here, I describe a novel blood group antigen binding outer membrane protein, FecA3. This is an adhesin candidate, which independently of BabA, binds the series of fucosylated blood group antigens. The similar binding modes of BabA and FecA3 for ABO blood group antigens is not fully understood but the expression of FecA3 is regulated by the nickel-responsive regulator NikR, which acts as a sensor regulated by the availability of free nickel ions. Ni2+increases when pH is lowered, thus regulation of FecA3 is suggested to be governed by local pH. Taken together, the high-affinity binding by BabA, and continuous biopanning for the fittest, in particular for the low-affinity binding of FecA3, might be contrasting but complementary properties and features of the different adhesins that are of importance during different stages of mucosal inflammation and disease development.
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| 8. |
- Norling, Karin, 1988
(författare)
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Studying the influence of the physicochemical properties of lipid nanoparticles for mucosal vaccine delivery
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lipid-based nanoparticles have attracted attention as promising pharmaceutical carriers. Reports of them having inherent adjuvant properties make them particularly interesting as vaccine vectors; however, the physicochemical profile of an ideal nanoparticle for mucosal vaccine delivery remains unknown. The aim of this thesis work is to contribute a better understanding of the connection between physicochemical properties of lipid nanoparticles used as vaccine carriers and the activation of the immune response at several different levels of complexity. As combined antigen and adjuvant, we used a novel fusion protein comprising the Cholera toxin A1 subunit, combined with either the M2e or Ealpha peptide and a dimer of the D subunit of Staphylococcus aureus protein A. This fusion protein was coupled to liposomes and lipodisks with systematically varied poly(ethylene glycol) (PEG) content, protein load, rigidity and size/shape. Firstly, a detailed characterization of the biological response in vitro and in vivo, in a mouse model, to two types of fusion protein-carrying lipid particles was performed. Compared with the free fusion protein, which is in itself already an effective vaccination compound, the result showed that the non-PEGylated liposomes more efficiently induce both cell- and antibody-mediated immune responses as well as protection against a lethal virus challenge than both free fusion protein and the PEGylated liposomes. Secondly, an in vitro study was performed, focusing on elucidating the effect of the physicochemical properties of the carrier particle on processing, in particular the antigen presentation in major histocompatibility complex class II (MHC II), by dendritic cells. Out of 6 different formulations, which varied with respect to PEGylation, fusion protein load, membrane rigidity, size and shape it was found that only the DSPC-based liposome formulation, the only liposome formulation in gel phase, was able to increase antigen presentation compared to free fusion protein. Additionally, this formulation lead to an increased amount of surface-bound MHC II, indicating that the liposomes themselves might have an immunostimulatory effect, making them a promising candidate for further evaluation as a vaccine carrier with inherent adjuvant properties.
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| 9. |
- Olsson, Thomas, 1977
(författare)
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Characterization of artificial and biological lipid vesicles using TIRF and SPR
- 2018
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Synthetic lipid vesicles serve as important mimics of cells and the natural membranes that they are enclosed by. As such they are frequently used as simplified models of the highly complex cell membrane to aid in-depth physicochemical and biological characterization of this essential biological structure. Lipid vesicles also fulfill fundamental biological functions, for example in intercellular signaling via extracellular vesicles and also as signal-substance containing intercellular secretory vesicles in the synapses of neurons and in secretory cells of the endocrine glands, which release their cargo to the extracellular space in response to external cues. Lipid-based nanoparticles are also of increasing importance as drug carriers, both for targeted release at specific tissues and for improved cellular uptake. There are today many techniques available to probe a multitude of lipid vesicle properties, including size, structure, content, molecular composition etc. In this thesis work, we have contributed improved means to quantify vesicle size using fluorescence microscopy by using total internal reflection fluorescence (TIRF) microscopy to correlate the measured distribution in fluorescence intensity of individual vesicles to their size, as measured by nanoparticle particle tracking analysis (NTA). A similar approach has been used before by others, but the formalism used that have won prevalence contains a mathematical error, which motivated the introduction of an improved expression for converting total vesicle intensity to vesicle size. We present the difference between the former and the latter formalism, as well as the possible negative impact of the former when used to draw conclusions for larger sized vesicles in a number of studies. One example when this type of analysis is crucial, is in studies were a certain vesicle property is correlated with vesicle size. One such example is studies of membrane protein function, which is often dependent of membrane curvature. In the second work, we used surface plasmon resonance (SPR) and amperometry as quantitative methods to investigate whether secretory dense core vesicles, isolated from bovine chromaffin cells from the medulla of adrenal glands, are able to maintain their high loading of catecholamine molecules after vesicle isolation and purification and how the vesicle catecholamine content is affected by vesicle exposure to osmotic stress. We found, as also previously reported by intracellular amperometry measurements in live cells, that dense core vesicles release part of the vesicle catecholamine content when exerted to a hyperosmotic shock, and also that this release occurs very rapidly in response to the applied osmotic stress. This work demonstrates the strength of using different complementary label-free measurements to account for the total number of catecholamine molecules in such vesicles and for monitoring molecule release from vesicle compartments in real-time. Further, by using knowledge gained about chromaffin vesicle size from TEM together with changes in refractive index as probed with SPR using suspensions based on ordinary and heavy water, we could estimate the hydration level of the dense protein core of the chromaffin vesicles.
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| 10. |
- Ponnandai Schanmugavel, Kumaravel, 1991
(författare)
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Development of a Yeast Model for Functional Analysis of Human Copper Transport Proteins
- 2018
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- ABSTRACT Copper (Cu) is an important trace element that plays a vital role in several biological processes. It mediates numerous biochemical functions to maintain cellular homeostasis making it an essential metal for sustaining life. Many proteins or enzymes that are involved in various biochemical pathways require copper as a cofactor (also a regulator). In human cells, the Cu uptake is mediated by high-affinity copper uptake protein (Ctr1), followed by cytoplasmic chaperone Atox1 that shuttles Cu from the plasma membrane to Wilson’s disease protein ATP7B (P-type ATPase), a membrane-bound protein located at the Golgi apparatus. ATP7B incorporates Cu to various Cu-dependent enzymes in the secretory pathway. The main biological role of ATP7B (or Wilson Disease Protein) is to maintain the copper balance inside the human cell. Genetic defects in ATP7B often leads to a nonfunctional protein where copper balance is impaired and this condition results in Wilson’s disease (WD). ATP7B is a large multi-domain membrane transport protein that shows typical characteristics of a P1b type ATPase. In contrast to its bacterial (CopA) or yeast (Ccc2) counterparts which have one or two metal binding domains (MBD) respectively, the human ATP7B has six cytosolic MBDs in the N-terminal region. The reason for the presence of these six MBDs in ATP7B is not completely understood, and neither is the ATP7B mediated copper release in the Golgi. In this thesis, the development of a novel yeast model system for investigating the functional role of ATP7B in copper transport is described. The system probes shuttling of copper via human Atox1 to ATP7B proteins when expressed in a yeast humanized model. Using this system, we investigated the roles of six metal binding domains (MBDs) in ATP7B (Paper 1) and examined the Cu release (paper II). The results address the importance of the yeast model for studying human Cu transport proteins, the role of MBDs in ATP7B mediated Cu transport, the role of Atox1 in shuttling Cu and the significance of the luminal loop in ATP7B for Cu release function. Overall, the yeast model system developed in this thesis has great future potential for studying human copper transport proteins, which are involved in genetic diseases such as WD. The designed system can be expanded by using system biology approaches, to gain further understanding on human copper transport as well as copper transport related disorders.
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