| 1. |
- 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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| 2. |
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| 3. |
- Mastinu, Enzo, 1987
(författare)
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Embedded Controller for Artificial Limbs
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Promising developments are currently ongoing worldwide in the field of neuroprosthetics and artificial limb control. It is now possible to chronically connect a robotic limb to bone, nerves and muscles of a human being, and use the signals sourced from these connections to enable movements in the artificial limb. It is also possible to surgically redirect a nerve, deprived from its original target muscle due to amputation, to a new target in order to restore the original motor functionality. Intelligent signal processing algorithms can now utilize the bioelectric signals gathered from remaining muscles on the stump to decode the motor intention of the amputee, providing an intuitive control interface. Unfortunately for patients, clinical implementations still lag behind the advancements of research, and the conventional solutions for amputees remained basically unchanged since decades. More efforts are therefore needed from researchers to close the gap between scientific publications and hospital practices.The ultimate focus of this thesis is set on the intuitive control of a prosthetic upper limb. It was developed an embedded system capable of prosthetic control via the processing of bioelectric signals and pattern recognition algorithms. It includes a neurostimulator to provide direct neural feedback modulated by sensory information from artificial sensors. The system was designed towards clinical implementation and its functionality was proven by amputee subjects in daily life. It also constitutes a research platform to monitor prosthesis usage and training, machine learning based control algorithms, and neural stimulation paradigms.
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| 4. |
- Tyagi, Nisha
(författare)
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Live Biotherapeutics : importance of formulation and lyophilization parametersand an example of a clinical application
- 2023
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In recent years, probiotics have expanded from their traditional classification as “health promoting food” to the development of live biotherapeutic products (LBP). Traditional probiotics are marketed as food/dietary supplements while LBPs are drug products intended for treatment or prevention of diseases. This type of products offers several advantages over traditional drugs, but also entail potential challenges with development, manufacturing, and demonstration of clinical safety. To obtain a sufficient quality, LBPs are typically produced by cultivation in a bioreactor, followed by formulation and lyophilization.In the first part of the project, the impact of lyophilization parameters on physicochemical and biological properties of Limosilactobacillus reuteri R2LC was evaluated. Using sucrose as a lyoprotectant gave a better freeze-drying survival, vitality and storage stability than using trehalose. A high concentration (20%) of sucrose sometimes resulted in a collapsed structure and 15% gave the overall best properties of the lyophilized bacteria. Interestingly, vitality was positively affected by using a higher concentration (1010 cfu/ml) of bacteria. Another observation was that introducing an annealing step in the process was positive when using sucrose as lyoprotectant, but no effect was seen when using trehalose.The second part of the project describes evaluation of the genetically modified L. reuteri R2LC expressing the human chemokine CXCL12 (ILP100-Topical) in a phase 1 trial on wound healing. The product was safe and well-tolerated. In addition, it gave a larger proportion of healed wounds (76 %) on Day 32 when compared to saline/placebo (59 %) (p=0.020) and the time of wound healing was reduced by 6 days on average and by 10 days at highest dose. Also, ILP100-Topical increased the density of CXCL12+ cells in the wounds and local wound blood perfusion.
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| 5. |
- Stepulane, Annija, 1994
(författare)
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Antibacterial elastomeric materials for biomedical applications
- 2022
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- An ageing population in combination with scientific and clinical advancements have led to a steady increase in the use of medical devices. Elastomeric biomaterials – materials displaying rubber-like mechanics – have found widespread applicability in the production of both short- and long-term medical devices. Despite the prevalence of such devices, issues with medical device-associated infections remain. By surface colonization of bacteria, systemic infection can arise resulting in patient suffering and increased burden on the healthcare system. Consequentially, development of antibacterial elastomers capable of withstanding bacterial surface colonization has been proposed as an effective strategy for prevention and mitigation of medical device-associated infections. In this thesis, two alternative strategies to develop antibacterial polydimethylsiloxane (PDMS) elastomers have been proposed and evaluated. In the first strategy, PDMS surface modification with multifunctional hydrogel microparticle coating has been developed. Using a coating of antimicrobial peptide (AMP) RRPRPRPRPWWWW-NH2 functionalized hydrogel particles, high antibacterial activity was reported against Staphylococcus epidermidis and Staphylococcus aureus. As an additional functionality, the ability of the coating to encapsulate and release therapeutic substances was investigated, resulting in a sustained delivery of polar, amphiphilic, and nonpolar drugs. In the second strategy, modification of bulk PDMS by synthesis of drug-eluting PDMShydrogel blends was proposed. PDMS and triblock copolymer (diacrylated Pluronic F127, DA-F127) hydrogel blends were prepared with varying ternary PDMS–DA-F127–H2O compositions. The test compositions chosen resulted in stable elastomers with tailorable mechanics and ordered self-assembled nanostructure. The variation in composition offered potential for sustained delivery of polar and nonpolar drugs, demonstrating potential for production of drug-eluting and antibacterial elastomeric devices with tailorable mechanics.
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| 6. |
- Löfhede, Johan, 1978
(författare)
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Classification of Burst and Suppression in the Neonatal EEG
- 2007
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The brain requires a continuous supply of oxygen and even a short period of reduced oxygen supply risks severe and lifelong consequences for the affected individual. The delivery is a vulnerable period for a baby who may experience for example hypoxia (lack of oxygen) that can damage the brain. Babies who experience problems are placed in an intensive care unit where their vital signs are monitored, but there is no reliable way to monitor the brain directly. Monitoring the brain would provide valuable information about the processes going on in it and could influence the treatment and help to improve the quality of neonatal care. The scope of this project is to develop methods that eventually can be put together to form a monitoring system for the brain that can function as decision-support for the physician in charge of treating the patient.The specific technical problem that is the topic of this thesis is detection of burst and suppression in the electroencephalogram (EEG) signal. The thesis starts with a brief description of the brain, with a focus on where the EEG originates, what types of activity can be found in this signal and what they mean. The data that have been available for the project are described, followed by the signal processing methods that have been used for pre-processing, and the feature functions that can be used for extracting certain types of characteristics from the data are defined. The next section describes classification methodology and how it can be used for making decisions based on combinations of several features extracted from a signal. The classification methods Fisher’s Linear Discriminant, Neural Networks and Support Vector Machines are described and are finally compared with respect to their ability to discriminate between burst and suppression. An experiment with different combinations of features in the classification has also been carried out. The results show similar results for the three methods but it can be seen that the SVM is the best method with respect to handling multiple features.
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| 7. |
- 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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| 8. |
- 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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| 9. |
- 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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| 10. |
- Petrou, Georgia, 1991-
(författare)
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Investigating mucin interactions with diverse surfaces for biomedical applications
- 2019
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mucous membranes are covered with mucus, a viscoelastic hydrogel that plays an essential role in their protection from shear and pathogens. The viscoelasticity of mucus is owing to mucins, a group of densely glycosylated proteins. Mucins can interact with a wide range of surfaces; thus, there is big interest in exploring and manipulating such interactions for biomedical applications. This thesis presents investigations of mucin interactions with hydrophobic surfaces in order to identify the key features of mucin lubricity, as well as describes the development of materials that are optimized to interact with mucins. In Paper I we investigated the domains which make mucins outstanding boundary lubricants. The results showed that the hydrophobic terminal domains of mucins play a crucial role in the adsorption and lubrication on hydrophobic surfaces. Specifically, protease digestion of porcine gastric mucins and salivary mucins resulted in the cleavage of these domains and the loss of lubricity and surface adsorption. However, a “rescue” strategy was successfully carried out by grafting hydrophobic phenyl groups to the digested mucins and enhancing their lubricity. This strategy also enhanced the lubricity of polymers which are otherwise bad lubricants. In Paper II we developed mucoadhesive materials based on genetically engineered partial spider silk proteins. The partial spider silk protein 4RepCT was successfully functionalized with six lysines (pLys-4RepCT), or the Human Galectin-3 Carbohydrate Recognition Domain (hGal3-4RepCT). These strategies were aiming to either non-specific electrostatic interactions between the positive lysines and the negative mucins, or specific binding between the hGal3 and the mucin glycans. Coatings, fibers, meshes and foams were prepared from the new silk proteins, and the adsorption of porcine gastric mucins and bovine submaxillary mucins was measured, demonstrating enhanced adsorption. The work presented demonstrates how mucin-material interactions can provide us with valuable information for the development of new biomaterials. Specifically, mucin-based and mucin-inspired lubricants could provide desired lubrication to a wide range of surfaces, while our new silk based materials could be valuable tools for the development of mucosal dressings.
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