Syftet med arbetet var att försöka utveckla en metod för att isolera lipoprotein(a), ett lipoprotein som korrelerar i hög grad till kardiovaskulär sjukdom, från plasma. Det har varit problematiskt att på ett enkelt sätt separera lipoprotein(a) från LDL på grund av dess strukturella likheter. Som inspiration användes en artikel där separationen utfördes med anjonbyteskromatografi-HPLC (high performance liquid chromatography). Målet var att applicera metoden på ett FPLC-system (fast protein liquid chromatography) med en gelfiltreringskolonn och en anjonbyteskolonn där det kaotropiska saltet natriumperklorat användes som elueringsmedel under anjonbyteskromatografin. Resultaten kontrollerades med DLS (dynamic light scattering), SDS-PAGE och Western blot med antikroppar riktade mot lipoprotein(a). Resultaten visade på goda möjligheter till en bra separation vid fortsatta studier.

Background: Neuronal plasticity and regeneration in cancer are understudied aspects of cancer research. Studies have shown that neurogenesis and axonogenesis are associated with cancer progression and metastatic potential. Purpose: The purpose of this project was to optimize an immunofluorescence stain to observe nerve development and regeneration in cancer tissue, with the use of antibodies against neurofilament light chain (Nf-L), growth associated protein 43 (gap-43), and doublecortin X (DCX). Material and method: Staining optimization included evaluations of antigen retrieval, tissue permeabilization, antibody dilution, and duration of primary antibody incubation. The analyses were tested on colorectal- and lung cancer tissues. Results: The detection of Nf-L was not successful in any combination of the analyses or on ether tissue. The staining Gap-43 showed the best results using antigen retrieval with pepsin in HCl and primary antibody dilution 1:500 combined with incubation overnight at 4 °C. Staining for DCX needs more evaluation due to non-specific binding in lung cancer tissue. The stain showed the best results with antigen retrieval performed with pepsin in HCl, primary antibody dilution 1:250 combined with 1 hour incubation at room temperature of the primary antibody. Permeabilization has to some degree shown good results in combination with antigen retrieval with pepsin in HCl. Conclusion: A good protocol was established for Gap-43 detection, but the procedures for Nf-L and DCX detections need to be optimized.

Activated Partial Thromboplastin Time (APTT) is a screening method used to detect defects in the intrinsic pathway. Different preanalytical variables such as centrifugation and thawing can affect the given test results from APTT analysis. The purpose of the study was to evaluate how the results for APTT were affected when frozen citrate plasma was thawed in a heating cabinet or a water bath, and how the results for APTT were affected if the samples were single or double centrifuged before freezing. A total of 40 fresh samples were collected for each study purpose and analyzed. To study how thawing conditions affected frozen samples, aliquoted plasma was frozen at –20°C for a week and then thawed in either a water bath or in a heating cabinet. To study how single and double centrifugation affected the test results, one of the two aliquots per sample was centrifuged again while the other remained single centrifuged. The samples were then stored frozen for one week and afterwards thawed. The results showed statistically significant differences between fresh samples and frozen samples thawed in a heath incubator or water bath. Regarding centrifugation conditions, both single and double centrifugation yielded statistically significant differences in comparison to fresh plasma. All the evaluated sample treatments showed statistically significant differences in comparison to fresh samples. However, in all cases the mean percentage change was ≤10%, which indicates no clinical significance. In conclusion APTT-samples seem to be sensitive to preanalytical variables and further studies are needed to evaluate their effects.

Due to recent declines regarding pollinators, The Swedish Civil Contingencies Agency launched a project investigating possible biological threats towards wild bumblebees. This led to an extensive collection of bumblebees from areas in southern Sweden during the summer of 2018. The purpose of this project was to investigate the prevalence of ten bumblebee pathogens and see if there were any difference in prevalence in areas with and areas without commercial bumblebees. This study also aimed to investigate if the prevalence of the pathogens tended to fluctuate during the season. Of the collected bumblebees (Bombus spp) the ones from the late summer season were analyzed in this project, and data from the already analyzed samples from the early summer season was used. To screen the bumblebees for the pathogens a laboratory process was followed with dissections – homogenization – DNA and RNA extractions – conversion of RNA to cDNA and lastly real-time quantitative PCR. The results showed significant differences between areas with and without commercial bumblebees for some pathogens, but the results were only consistent between early and late season for Apicystis bombi and Black queen cell virus. Apicystis bombi had a higher prevalence (2,0 and 3,0 for late and early season) in areas with commercial bumblebees. Black queen cell virus had a higher prevalence (1,3 and 1,6 for late and early season) in areas without commercial bumblebees. For the seasonal fluctuation a significant difference between early and late season could be seen for the pathogens – Crithidia bombi, Apicystis bombi, Locustacarus buchneri and Sphaerularia bombi.

A prototype of a haptic and virtual reality simulator has been developed for simulation of the bone milling and material removal process occurring in several operations, e.g. temporal bone surgery or dental milling. The milling phase of an operation is difficult, safety critical and very time consuming. Reduction of operation time by only a few percent would in the long run save society large expenses. In order to reduce operation time and to provide surgeons with an invaluable practicing environment, this licentiate thesis discusses the introduction of a simulator system to be used in both surgeon curriculum and in close connection to the actual operations. The virtual reality and haptic feedback topics still constitute a young and unexplored area. It has only been active for about 10-15 years for medical applications. High risk training on real patients and the change from open surgery to endoscopic procedures have enforced the introduction of haptic and virtual reality simulators for training of surgeons. Increased computer power and the similarity to the successful aviation simulators also motivate to start using simulators for training of surgical skills. The research focus has been twofold: 1) To develop a well working VR-system for realistic graphical representation of the skull itself including the changes resulting from milling, and 2) to find an efficient algorithm for haptic feedback to mimic the milling procedure using the volumetric Computer Tomography (CT) data of the skull. The developed haptic algorithm has been verified and tested in the simulator. The visualization of the milling process is rendered at a graphical frame rate of 30 Hz and the haptic rendering loop is updated at 1000 Hz. Test results show that the real-time demands are fulfilled. The visual and haptic implementations have been the two major steps to reach the over all goal with this research project. A survey study is also included where the use of VR and haptic simulators in the surgical curriculum is investigated. The study starts with a historical perspective of the VR and haptic topics and is built up by answering different questions related to this topic and the implementation of simulators at the medical centres. The questions are of general concern for those developing surgical VR and haptic simulators. Suggested future work includes modelling, development and validation of the haptic forces occurring in the milling process and, based on this, implementation in the simulator system. Also, further development of the simulator should be done in close cooperation with surgeons in order to get appropriate feedback for further improvements of the functionality and performance of the simulator. / QC 20101112

Background: Urinary tract infection (UTI) is a common bacterial infection that is usually diagnosed by symptoms such as dysuria and frequency, and the golden standard is to take a urine culture to identify bacteria that may cause UTI. This method was founded with the idea that normal urine is sterile, but this is now being questioned because of growing evidence of a urinary microbiota thus giving a new approach to methods for UTI diagnosis. Aim: To identify and re-evaluate findings of bacteria from urine cultures in the ongoing paradigm shift of a potential urinary microbiome, and dysbiosis as a cause for UTI. Materials and Methods: This study used MALDI-TOF MS to identify approximately 250 bacteria isolates that had been cultured by Expanded Quantitative Urine Culture (EQUC) from 162 women with symptoms of cystitis. EQUC had allowed the bacteria to grow in both CO2 and anaerobic conditions, which differs from standard techniques.   Results and Conclusion: Escherichia coli and Enterococcus faecalis dominated the results of most frequently identified bacteria. However, other bacteria were commonly present within the same culture which is traditionally considered as contamination but may now indicate a urinary flora. Anaerobic bacteria – such as Porphyromonas sp. – were also identified, but their connection to UTI is unclear. Lactobacillus sp. – which are associated with a healthy flora in women – were found in urine cultures and often in smaller quantities which could suggest dysbiosis. More research on Lactobacillus sp. and their correlation with UTI is suggested for a more accurate indication of urinary dysbiosis in women.

Treponema pallidum (T. pallidum) is the bacteria that causes syphilis, which is a sexually transmitted disease that might give the carrier a multisystemic infection. A combination of serological tests is needed to put a diagnosis of T. pallidum infection, because the bacteria cannot be cultured in vitro. Serological tests categorize intro nontreponemal test and treponemal test, which both are needed for a correct diagnosis. The principle of a nontreponemal test is the measure of antibodies directed towards lipoid antigens, whereas the principle of a treponemal test is the measure of antibodies directed towards specific T. pallidum antigens. One kind of a treponemal test that are used are treponemal antibody test which is a manual agglutination test. At Sundsvall County hospital TP-PA was used for detection of antibodies, this kit is no longer available for purchase which means that a new kit must be validated to take its place. The purpose of this study was to evaluate the possible replacing kit, TP-HA. To validate TP-HA, ten positive serum samples were analyzed and compared with the two kits. A positive kit-control were also used to confirm the results of this study. The results showed that TP-PA was more sensitive than TP-HA according to a Wilxocon ranking test (p<0.05). Because TP-PA has to be replaced due to stricter in vitro diagnostic regulation (IVD-R laws), TP-HA will be taking its place even though it differs in sensitivity. This might effect tests with a lower titre, they might give a negative result even though the test has a low concentration of antibodies which needs to be further evaluated.

Background: Cystic fibrosis (CF) is a hereditary disorder caused by genetic mutations, which affect the chloride ion channels, leading to disrupted salt balance in different organs. A lack of properly functioning chloride ion channels can lead to formation of thick mucus, which hinders organ function, especially in the lungs where repeated inflammation occurs. Early diagnosis is critical to prevent further deterioration of the patient's condition. Current method of analysis of CF diagnostics uses conductivity meters to measure sweat electrolytes. However, current guidelines suggest using a chloridometer to directly measure chloride concentration, is the most reliable marker of cystic fibrosis. The aim of this project was to conduct a comprehensive evaluation of the new instrument's safety, reliability, validity, and conformity of the reference range to international chloride meter guidelines. Additional aims were to investigate the effect of storage conditions on sweat chloride concentration levels and examine the effect of increased salt intake on sweat test results. The study recruited healthy participants and took samples of their sweat by inducing sweat gland secretion. The chloride ion concentration was determined using a coulometric method. The results of the study found that the new method was reliable and matched international protocols. It also revealed that an increased salt consumption can impact chloride concentration in sweat, but not to an extent that it can affect medical decisions. Additionally, the study demonstrated that sweat samples can be frozen for up to two weeks without affecting the outcome of the chloride determination.

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Sleep is very important for health and academic performance. The amount of sleep is variable and depends among other things, on the age of the individual. Today there are many varieties of motion sensors that can be used in healthcare and clinical research for sleep registration, such as Actigraph GT9X Link and Actiwatch 2. The purpose of this study was to compare these two sensors, Actigraph and Actiwatch, for sleep registration by analyzing different sleep variables. The more precise sensor of the two will be used in the Energymetabolic laboratory in the case of survey or research. The method involved participants wearing both sensors for two nights and one day and trying to live as normal as possible. A sleep diary was used to complete the registrations, with related questions about sleep. There were 17 healthy participants between the ages 21 and 31. The results showed that the registrations from both sensors have varying spread and deviations. Overall the results were similar between the sensors and the sleep diary, and it was shown that there is no significant difference between the sensors. However, Actigraph deviated on most occasions from the diary. The Energymetabolic laboratory have access to many Actigraph GT9X Link sensors. This means that an evaluation has to be made to decide whether the cost of replacing the Actigraph GT9X Link sensors with Actiwatch 2 is worth it for the small improvement in precision.