Litteraturstudier har genomförts med syftet att utveckla minst en ny detektionsmetod som skulle kunna ersätta den metod som företaget Cavidi använder sig av idag. Cavidi hade specificerat krav som metodförslaget skulle uppfylla. Dessa krav var att metoden skulle vara snabb, lättanvänd, billig, ha hög känslighet och kunna förvaras i rumstemperatur. Två lovande metoder som baseras på två olika principer valdes ut. Den ena metoden bygger på att enkelsträngat DNA med en specifik nukleinsyrakomposition syntetiseras. Denna sekvens har egenskapen att spontant bilda en sekundärstruktur som kan bilda komplex med en fluorofor. Detta ökar dess fluorescens. Ökningen detekteras med fluorescensspektroskopi. Den andra metoden baseras på ett optomagnetiskt fenomen vilket innebär att ett magnetiskt fält påverkas av polariserat ljus. Metoden går ut på att virioner först renas fram från blodplasma och att de fäster på jonbytarkulor under rådande buffertförhållanden. Magnetiska nanopartiklar tillsätts som binder till jonbytarkulornas lediga ytor. Om många virioner har bundit till jonbytarkulorna finns det en större mängd fria nanopartiklar i lösningen. Antalet fria nanopartiklar i lösningen är proportionellt mot mängden HIV i provet och kan då detekteras med en fotodetektor. Fördelarna med dessa metoder är att processen blir billigare, snabbare och har en hög känslighet. Metoderna är lättanvända och använder färre komponenter jämfört med Cavidis nuvarande metod. Därmed blir Cavidis produkter billigare och tillgängliga för fler människor.

In the human body, the main function of the healthy thyroid gland is the regulation of the metabolism and hormone production. Included in the thyroid are organized structured and uniformly shaped follicles ranging from 50-500 μm in diameter. Pathologies lead to morphological changes of these follicles, affecting the density and size, but can also lead to an absence. In this study optical coherence tomography (OCT) was used to examine pathological thyroid tissue by extracting structural information of the follicles from image segmentation. However, OCT images usually include a high amount of speckle noise which affects the segmentation outcome. Due to that, the OCT images need to be improved. The aim of this thesis was to investigate the appropriate filtering methods to enhance the images and thus improve the segmentation outcome. The images of pathological thyroid tissues with a size of 0:5-1 cm where scanned by a spectral domain OCT system (Telesto II, Thorlabs GmbH, Germany) using a center wavelength of 1300nm. The obtained 2D and 3D images were saved as .oct file as well as implemented and visualized in a MATLAB graphical user interface (GUI) for further processing. For image improvement, four filtering enhancement methods were applied to the 2D images such as the enhanced resolution imaging (ERI), adaptive Wiener filter, discrete wavelet transform (DWT) and multi-frame wavelet transform (WT). The processed images were further converted to grayscale and binary images for intensity-based segmentation. The output of all methods were compared and evaluated using signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), enhanced number of looks (ENL), edge profiles and outcome of the segmented images. It was demonstrated that the complex DWT (cDWT) with a higher threshold and the multi-frame WT using the haar wavelet showed enhanced results over the other filtering methods. The computed SNR could be increased up to 52% and the ENL value up to 4802%, applying the multi-frame WT, while the CNR could be increased up to 106% for cDWT. The lowest obtained gradient was equal to an intensity decrease of -61% and -68% for multi-frame WT and cDWT, respectively. The filtering method could increase the smoothness of the image while the edge sharpness could be kept. The segmentation could detect both small and large follicles. ERI did not show any improvement in the segmentation but could enhance the structural detail of the image. Larger neighbourhoods of the adaptive Wiener filter showed a highly blurred image and led to merged follicles in the image segmentation. The wavelet filters DWT and multi-frame WT gave most satisfying results since high and low frequencies were divided into subbands, where individual information on vertical, horizontal and diagonal edges was stored. Applied cDWT had an even higher amount of subbands, so that more information on signal and speckle noise could be specified. Due to this fact, it was possible to achieve a decreased noise level while edge sharpness where maintained. Using a multi-frame image an increased SNR was obtained, as the intensity information stayed constant over the individual frames while the noise information changed. Wavelet based filtering showed higher improved results in comparison to the adaptive Wiener filter or the ERI in the 2D domain. By applying filtering methods in higher dimensions such as 3D or even 4D, better results in noise reduction are expected. Improved settings for the individual filtering methods as well as enhancement in segmentation are part of the future work.

In the last decades, the prevalence of obesity increased dramatically worldwide. According to the WHO, 2.1 billion people (30% of the population) around the world are obese or overweight. Effective ways to decrease food intake are needed. Evidence from emerging studies indicates an association between feeding behavior and the IL-6 expression in the central nervous system (CNS). The study aimed to investigate the effect of IL-6 modulation on feeding behavior and learning and memory process in the ventral hippocampus (vHPC). To implement this aim two groups of rats were used, the first group was exposed to the reduction of the IL-6 expression (knockdown), whereas the other one to microinjections of exogenous IL-6 (EX IL-6). Both experimental groups were subjected to a set of behavioral and molecular tests specific for investigating memory process, emotional/affective behavior and food intakes like novel object recognition, Morris water maze, and social interaction test. The results for IL-6 knockdown (KD) showed improvement in the short term memory, but did not affect the food intake. On the other hand, EX IL-6 caused an increase in the locomotor’s activity and the food intake during the 24 hours, but at the same time caused impairment in the spatial and learning memory. Taken together, these results provide new insight on the role of IL-6 outside of inflammation highlighting its ability to modulate hippocampus-dependent mnemonic process, and affective and feeding behaviors in the vHPC, however several questions still remain not addressed and the study require further investigation.

No description available.

No description available.

In recent years non-vitamin K dependent oral anticoagulants (NOACs) have started to replace warfarin for treatment and prevention of deep venous thrombosis (DVT), pulmonary embolism (PE) and stroke in patients with and without atrial fibrillation. There is a need for a simple and rapid method to detect the presence of these drugs in patient plasma. To meet these new demands, MediRox is developing a screening assay based on a novel prothrombin time (PT) method for rapid detection of NOACs in plasma. The assay is semi-quantitative and by dividing the International Normalised Index (INR) from a NOAC sensitive PT method with the INR from a NOAC insensitive PT method, NOAC containing samples be detected while plasma from normal donors and with warfarin are excluded.   The purpose of this project is to develop prototypes of assay quality controls for detection of NOACs in plasma.   The results show that the method used for the NOAC control prototypes is applicable and the PT ratio is comparable to patient samples for the low, medium and high concentrations of NOAC. The effect of lyophilisation indicates that the PT ratios for the NOAC control prototypes were nearly unaffected by the lyophilisation. The in-use stability at room temperature (20-25oC) for all NOAC control prototypes were at least 24 hours.   The methodology for production needs to be further optimised to increase the commutability to patient samples with very high concentrations of NOAC. The data indicates that the effect of lyophilisation is minimal and the stability of the NOAC control prototypes are satisfying, which is promising for future product development of NOAC controls.

Viral infections are constantly increasing and impose a large threat to the public health. Alphaviruses are responsible for several animal and human diseases and have a large medical importance with few treatments available today. Alphaviruses are small, spherical single stranded RNA viruses, and are most often transmitted by mosquito vectors. Alphaviruses contains a domain of nonstructural proteins that compose the replication machinery. The domain is crucial for viral replication to occur and is therefore an interesting target for antiviral therapy. With the focus on Chikungunya and Semliki Forest Virus this work investigates the events in the cells on molecular level during infections. To do this a panel of Camelid derived single domain antibodies are developed to target the nonstructural proteins of Chikungunya and Semliki Forest Virus. Binding of the produced nanobodies to the viral proteins was investigated by biochemical methods including immunoprecipitations, western blot, and ELISA. Cell lines that express nsP-specific nanobodies in the cytosol were employed for infection- and plaque assays with Semliki Forest Virus in order to determine the antiviral potential of the new nanobodies. Three of the nanobodies proved to bind two different nonstructural proteins of the viruses, providing opportunities for further investigations and a possible use of these nanobodies to identify viral vulnerabilities that could be exploited for antiviral intervention.

Surface plasmon resonance (SPR) is a label-free, versatile and highly sensitive method for studying molecular interactions in real time. It is widely used by industry and academia alike in fields ranging from Alzheimer’s disease research to detection of heavy metals. In this review, studies published during the last 10 years using Biacore or other SPR instruments were compiled and compared. Trends were also identified in the field. Amine coupling was found to be the most common ligand strategy for proteins, and most SPR research related to the field of medicine. Furthermore, three main purposes of an SPR experiment were identified: To determine the affinity between a pair of molecules, kinetics between a pair of molecules or to detect a certain molecule in a solution. The results presented are often related to these three purposes, and are most often presented and evaluated in terms of kinetic, affinity and sensitivity constants. SPR can be used for studying a broad range of molecular interactions, and an overview was obtained by dividing up the field into different parts based on molecular interactions and SPR methods. The study of molecular interactions using SPR was divided into protein-protein interactions (PPIs), antibody-antigen, protein-biomolecule interactions, interactions between proteins and small molecules, and non-conventional SPR methods. Non-conventional SPR methods include localized surface plasmon resonance (LSPR) and SPR imaging (SPRi), which are both based on the same optical sensing principles as SPR.

Advancements in the field of modified messenger RNA (mRNA) has led to new ventures in the pharmaceutical industry. However, new drug products demand new analytical methods to ensure the efficacy and purity of the drug. Capillary gel electrophoresis (CGE) with UV detection shows great potential for separation of mRNA samples due to the equal mass-to-charge ratio of mRNA and the flexible parameters of the CGE methods. This thesis investigates the optimal parameters of the capillary electrophoresis method, sample treatment procedure and sieving medium composition for enhanced shortmers separation of mRNA by CGE analysis. An RNA ladder with 100-1000 nucleotides and EPO mRNA with 900 nucleotides were used as model compounds. The effect of capillary dimensions and separation temperature on the resolution of the RNA peaks was established through comparative experiments. Sample treatment processes were evaluated to achieve an optimal conformation of the mRNA for CGE analysis. By heating the mRNA sample for 15 min at 80°C all multimers were seemingly eradicated. Moreover, it was found that addition of 4 M of urea to mRNA sample before heating resulted in improved peak shape. A sieving medium consisting of a mix of the two polymers polyvinylpyrrolidine (PVP) and hydroxyethyl cellulose (HEC) proved to have beneficial qualities for separation. The addition of sucrose as viscosity modifier in the sieving medium surprisingly further enhanced the resolution. Moreover, during the project a heavy wash was established which drastically improved repeatability of the analyses through more efficient regeneration of the capillary. ISSN:

Antibodies are a class of proteins that are made by the immune system to recognize harmful organisms and molecules. Their exceptional capability of specifically recognizing molecules has been investigated for over a century and information thereof has been utilized for a variety of applications including vaccine and generation of therapeutic antibodies. Occasionally, instead of protecting the host against pathogens, antibodies can recognize constituents of the host and thereby cause an autoimmune reaction that eventually can lead to a disease. Therefore, it is of great interest to understand what the antibodies bind to and their specificities.   The last decades of technical development and availability of protein and peptide microarrays have enabled large-scale profiling of antibodies and precise determination of their specificities through epitope mapping. In this thesis the aim was to use affinity proteomics tools to profile antibodies, determine their specificities, and discover potential associations of autoantigens to disease by analyzing blood-derived samples with microarray-based methods.   In Paper I, 57 serum samples from patients with the suggested autoimmune disease narcolepsy, were analyzed on planar antigen microarrays with 10,846 human protein fragments. Verification on an independent sample collection consisting of serum samples from 176 individuals, revealed METTL22 and NT5C1A as two potential autoantigens. In Paper II, antibodies from 53 plasma samples from patients with first-episode psychosis, a condition suggested to have a partial autoimmune component, were analyzed on planar antigen microarrays with 2,304 human protein fragments. After a follow-up study of the patients, antibodies toward an antigen representing the three proteins, PAGE2, PAGE2B, PAGE5, was found associated to an increased risk of developing schizophrenia. In Paper III, serum and plasma samples from patients with the autoimmune diseases multiple sclerosis and narcolepsy, were epitope mapped on high-density peptide microarrays with approximately 2.2 million peptides. Technical and biological verification, by using other microarray technology and analyzing  samples from 448 patients, revealed one peptide for multiple sclerosis and narcolepsy, representing the proteins MAP3K7 and NRXN1, with higher antibody reactivity towards in each group, respectively. In Paper IV, purified polyclonal antibodies raised against a surface antigen found on malaria-infected erythrocytes, were profiled on the peptide microarrays representing all proteins found on malaria-infected erythrocytes derived from Plasmodium falciparum. Then, different Plasmodium falciparum strains were analyzed by immunofluorescence microscopy and western blots, using the epitope mapped antibodies. The performance of the immunoassays were compared to the identified epitopes, and validated by RNA sequencing.   In conclusion, these investigations describe multiplex methods to identify and characterize antibodies, their disease association and epitopes. Follow-up studies are needed to determine their potential use and clinical value. / <p>QC 20170905</p>