Bakgrund: Endoplasmatiskt retikulum upprätthåller proteinhomeostasen genom syntes och degradering av proteiner. Tre signalvägar reglerar processen via ”unfolded protein response” på olika sätt. Inositol-requiring enzyme 1 alpha är en signalväg aktiverad av en störd proteinhomeostas. Stressen uppstår om cellen utsätts för kemiska substanser t.ex. i samband med djurförsök vid framtagning av läkemedel. Den aktiverade signalvägens respons leder bl.a. till splicing av X-box-binding Protein 1 mRNA. Syfte: Projektets syfte är att minska behovet av djurförsök genom att möjliggöra ökad implementering av 3R-principen. Frågeställningen är om den fluorescerande plasmiden pEGFP-XBP1∆dDBD-STOP-tagRFPt som binder till X-box binding protein 1 mRNA kan användas för detektion av celler vars endoplasmatiska retikulum är stressat. Metod: Plasmid pEGFP-XBP1∆dDBD-STOP-tagRFPt renades ur DH5-Alpha Escherichia coli med JETstar 2.0 Plasmid Purification Midiprep Kit. Humana epiteliala njurceller 293 transfekterades med plasmiden varpå de doserades med tunicamycin. Kontrollen utgjordes av dimetylsulfoxid. Mikroskopering skedde i fluorescensmikroskopet Zeiss Axiovert 200. Mjukvaran AxioVision Ver. 4.8.1.0. Carl Zeiss Imaging Solutions framställde cellerna på dataskärm. Resultat: Hypotestestets signifians bestämdes till p < 0,05. Resultatet antyder att antalet ER- stressade celler ökar med ökande inkubationstid (p = 0,001 – 0,966). En mindre antydan finns också mot en ökande koncentration av tunicamycin (p = 0,096 – 0,690). Slutsats: En rimlig slutsats kan inte dras då antalet utförda analyser är för få. / Background: Endoplasmic reticulum maintains protein homeostasis by protein synthesis and degradation. Three signal paths regulates the process by ”unfolded protein response” in different ways. Inositol-requiring enzyme 1 alpha is one path activated by a disturbed protein homeostasis. Stress starts if the cell is exposed to chemical substances as in connection with scientific animal experiments when pharmaceuticals are developed. The response of the activated signal path leads e.g. to splicing of X-box-binding protein 1 mRNA. Object: The object of the project is to reduce the need of scientific animal experiments by enabling increased implementation of the 3R principle. The question is if the fluorescent plasmid pEGFP-XBP1∆dDBD-STOP-tagRFPt binding to X-box binding protein 1 mRNA can be used for detection of cells whose endoplasmatic reticulum is stressed. Method: Plasmid pEGFP-XBP1 ∆ dDBD-STOP-tagRFPt was purified from DH5-Alpha Escherichia coli with JETstar 2.0 Plasmid Purification Midiprep Kit. Human epithelial kidney cells 293 were transfected with the plasmid whereupon they were dosed with tunicamycin. The control was made off dimethylsulfoxide. Microscopy was done in the fluorescence microscope Zeiss Axiovert 200. The software AxioVision Ver. 4.8.1.0. Carl Zeiss Imaging Solutions imaged cells on the computer screen. Result: The significans of the hypothesis test was set to p < 0,05. The result suggests that the number of ER-stressed cells increase with increasing incubation time (p = 0,001 – 0,966). A smaller hint is also pointing toward increasing concentrations of tunicamycin (p = 0,096 – 0,690). Conclusion: A reasonable conclusion cannot be made because of too few tests.

Leber’s hereditary optic neuropathy (LHON) is a genetic disease that causes the patients to become blind, first in one eye and then the other, around the ages of 10-75 years. The disease is caused by mutations in the mitochondrial DNA, which disturbs the respiratory chain leading to the deterioration of the retinal ganglion cells. This study’s aim is to optimize a multiplex amplification-refractory mutation system PCR for detection of three primary mutations causing LHON. This was done through a series of PCRs, including PCR aimed at the ß-globin gene, conventional simplex PCR and a simplex ARMS-PCR aimed at the three primary mutations causing LHON. This study was, however, terminated prematurely due the Covid-19 outbreak and the optimization of the ARMS-PCR could therefore not be done. This study’s aim was adapted to the new circumstances to instead provide guidance on how to perform the optimization using the results from the PCRs that were done before the termination. The results found that for the ARMS-PCR 2 mM of magnesium would suffice as a start point overall and the need to solve the problems with the two 14484 plasmids was evident. The ARMS-PCR is one of many methods that can be used to the detect single nucleotide polymorphism, but its availability and robustness makes this a method worth optimizing. To continue with the optimization of the ARMS-PCR several factors would have to be tested, including annealing temperature, primer concentrations and magnesium concentration.

The pig production in Sweden is divided into conventional and organic production, with the organic production divided into EU organic and KRAV production. Pigs are divided into different age categories: weaned pigs, growing pigs, fattening pigs, gilts/sows before farrowing and dry sows. Roundworm, nodular worm, whipworm, coccidia, red stomach worm, threadworm and lungworm are common parasites in pigs affecting growth, feed conversion and economy. Organic pigs are more exposed to parasites because of outdoor stay and deworming could help if used with care. The aim of this project was to investigate parasites in pig herds with organic production. The investigation would increase the knowledge of parasites in different age categories in currently used production systems and contribute to good recommendations about parasite control and treatment. A modified McMaster technique was used to identify and quantify parasite eggs with microscope. Larval culturing and microscopy were used to distinguish eggs from nodular worm and red stomach worm. Roundworm, nodular worm, whipworm and coccidia were found in all age categories. Threadworm was only identified in dry sows and gilts/sows before farrowing. Nodular worm and coccidia showed highest quantity of positive samples at herd and sample level followed by roundworm and whipworm. Roundworm and nodular worm had highest quantity of eggs per gram faeces followed by whipworm and threadworm. The results corresponded to previous studies in parasite occurrence despite new conditions. More samples, herds and environmental factors should be investigated in conjunction with parasite occurrence to get broader knowledge and to give good recommendations in parasite control and treatment.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the field of bacterial diagnostics and is also used for routine analysis in smaller clinical laboratories. For identification of moulds, pre-analytical steps are more complicated and time consuming than for bacteria, and the choice of reference library has a big impact on the utility of MALDI-TOF MS. The aim of this study was to investigate if MALDI-TOF MS is applicable for identification of moulds belonging to the genus Aspergillus at the hospital laboratory in Gävle. Therefore, strains belonging to the genus Aspergillus and Penicillium, were analysed with MALDI-TOF MS after 2, 4 and 7 days of incubation. Two different extraction protocols were used and compared. Mass spectra were compared to reference spectra in two different databases: MSI-2 and RUO Compass library/BDAL (Bruker). Of the strains included, 97 % were correctly identified to species complex level with MSI-2. Only 25 % were identified to species level with RUO Compass library/BDAL (Bruker). However, totally 56 % were correctly identified to species complex level if a lower score value limit than recommended for identification, were applied. Significantly raised score values were observed with one of the protein extraction protocols used. Although, in most cases, the strains were considered identified to species complex level with either method. This pilot study conveys the feasibility of MALDI-TOF MS for identification of Aspergillus species in a clinical laboratory. While there are still issues to address, applying MALDI-TOF MS has the potential to allow for quicker and more precise identification, also in this specific clinical setting.

Hudens normalflora består av lågpatogena bakterier där stafylokockerna är de vanligaste förekommande bakterierna. Studier visar att antimikrobiella medel kan förändra hudbakteriepopulationer och att dessa förändringar kan leda till kritiska konsekvenser för hudens försvar. Propylenglykol är en substans som klassas som antimikrobiell och bakteriedödande. Propylenglykol har ett brett användningsområde och används ofta som hjälpmedel i en mängd olika läkemedel. Den finns bland annat i kosmetika såsom hudprodukter där den fungerar som fuktbindande och som konserveringsmedel. Vanliga koncentrationer av propylenglykol som fuktighetsbevarande ämne i topikaler är cirka 15 % och i kosmetika finns propylenglykol i koncentrationerna <0,1 % - >50 %. Det saknas i nuläget forskning om propylenglykols effekt på hudens bakterieflora. Syftet med studien var att med olika koncentrationer av propylenglykol bestämma MIC (minsta hämmande koncentration) och MBC (minsta baktericida koncentration) på vanliga stafylokocker (S. epidermidis, S. aureus, S. hominis och S. capitis) som ingår i hudens normalflora. För att bestämma MIC och MBC användes buljongspädningsmetoden där propylenglykol späddes ut i olika koncentrationer med buljong i en mikrotiterplatta. Sedan tillsattes valda testbakterier och OD600 mättes i 24 timmar. Resultatet visade att MIC och MBC för propylenglykol var 12,5 % respektive 25 % på vanliga stafylokocker som finns på huden. Vid koncentrationer av propylenglykol på 12,5 % hämmades synlig bakterietillväxt av S. epidermidis, S. aureus, S. hominis och S. capitis och vid 25 % uppstod en baktericid effekt på bakterierna. Mer forskning behövs dock för att få reda på hur hudens bakterier påverkas av propylenglykol och konsekvenserna av det. / The normal flora of the human skin is consisting of low pathogen bacteria, where the staphylococci are the most common bacteria. Studies show that antimicrobial substances can alter populations of skin bacteria and that these alterations can lead to critical consequences for the resistance of the skin. Propylene glycol is a substance that is classified as antimicrobial and bactericidal and the substance has a wide area of use and is frequently used as a supportive substance in various pharmaceuticals. Propylene glycol can be found in cosmetics and skincare products where it functions as moisture-binding and preservative. Normal concentrations of propylene glycol as moisture-binding substance in topicals is approximately 15 % and in cosmetics the concentration of propylene glycol is <0,1 % - >50 %. As of today, there are few scientific studies regarding the effects of propylene glycol to the bacterial flora of the human skin. The purpose of this study was to determine MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) for normal staphylococci (S. epidermidis, S. aureus, S. hominis och S. capitis) included in the normal flora of the skin using various concentrations of propylene glycol. To be able to determine MIC and MBC the broth dilution method was used, where propylene glycol was diluted in various concentrations with broth in a microtiter plate. Hereafter, selected test bacteria were added and OD600 was measured during 24 hours. The results implicated that MIC and MBC for propylene glycol were 12,5 % and 25 % for common staphylococci located on the skin. At concentrations of propylene glycol of 12,5 %, visible bacterial growth of S. epidermidis, S. aureus, S. hominis and S. capitis was inhibited and at 25 % a bactericidal effect occurred on the bacteria. It shall be noted that further research is needed to find out how the skin's bacteria are affected by propylene glycol and its consequences.

Helicobacter pylori-infektion är en av de ledande orsakerna till utvecklingen av maligniteter i ventrikeln. Tillämpning av pålitliga analytiska metoder är därför väsentlig för en korrekt diagnostik och behandling av infektionen. Syftet med studien var att ge en översikt av icke-invasiva metoder som tillämpas för påvisning av H. pylori och utvärdera vilken metod som är bäst lämpad, med avseende på metodens prestandaegenskaper och det kliniska tillståndet hos patienten. En systematisk litteraturöversikt utfördes, genom sökning efter vetenskapliga artiklar med inklusions- och exklusionskriterier i databaserna PubMed och CINAHL. Utvalda artiklar kvalitetsgranskades och 20 studier inkluderades i resultatet. Sammanställt hade fecesantigentester en sensitivitet och specificitet på 92,64% respektive 91,47%, antikroppstester hade 97,20% respektive 81,59%, urea utandningstester hade 91,40% respektive 91,70% och polymeraskedjereaktionen hade 75,45% respektive 98,30%. Därutöver hade kliniska tillstånd såsom atrofisk gastrit, intestinal metaplasi och gastrointestinal blödning en negativ påverkan på metodernas diagnostiska tillförlitlighet. Studien konstaterade att beträffande metodens prestanda är fecesantigentester mest lämpliga för påvisning av H. pylori- infektion. Vid allvarligare kliniska åkommor bör minst två icke-invasiva diagnostiska metoder tillämpas för att säkerställa pålitliga resultat. / Helicobacter pylori infection is one of the leading causes of ventricular pathologies. Reliable analytic methods are therefore crucial for the correct diagnosis and treatment of the infection. The aim of this study was to provide an overview of non-invasive diagnostic methods used for the detection of H. pylori and to evaluate which method is most suitable, considering its performance and the clinical condition of the patient. A systematic literature review was conducted, searching peer-reviewed research articles with inclusion and exclusion criteria on the databases PubMed and CINAHL. An assessment of the selected articles quality resulted in the inclusion of 20 articles. Overall, stool antigen tests had a sensitivity and specificity of 92,64% and 91,47% respectively, antibody tests 97,20% and 81,59% respectively, urea breath tests 91,40% and 91,70% respectively, and the polymerase chain reaction 75,45% and 98,30% respectively. Furthermore, conditions such as atrophic gastritis, intestinal metaplasia and gastrointestinal bleeding had a negative impact on the diagnostic accuracy of the methods. This study concluded that, regarding the methods performance, stool antigen tests are more suitable for detecting a H. pylori infection. With the mentioned clinical conditions, at least two non- invasive diagnostic methods should be used to ensure reliable results.

Ekokardiologiska undersökningar erbjuder en lätt, snabb och icke-invasiv metod för diagnostik av vänster förmaksvolym (LAV). I dagsläget är hjärt-magnetresonanstomografi (CMR) referensmetod för mätningen. Dock är CMR både tid- och kostnadskrävande, vilket leder till att tvådimensionell ekokardiografi (2DE) istället används. Den relativt nya metoden tredimensionell ekokardiografi (3DE) erbjuder en intressant valmöjlighet vid bedömning av förmaksvolym. Det är studiens syfte att ställa 2DE mot 3DE, gällande modaliteternas korrelation av vänster förmaksvolym, med CMR som referensmetod. Studien undersöker även förhållandet för modaliteternas intra- och inter-bedömar variation. Studien har använt databaserna Pubmed och Medline där systematiska litteratursökningar genomförts under april månad år 2020. Flera inklusions och exklusions kriterier har använts, studien har endast använt artiklar publicerade från 2010 och framåt. 3DE uppvisar en högre korrelation mot CMR vid volymbestämning av LAV. Resultatet visade även att 3DE har en lägre variation mellan både intra- och inter-bedömare än 2DE. 3DE har både en starkare korrelation mot CMR och en lägre bedömarvariation än 2DE, dock lider denna modalitet av begränsningar i dagsläget. Begränsningarna är: tid, personalens erfarenhet, brist på referensvärden samt kostnad för klinikerna. Författarna rekommenderar användning av 3DE för säkrare bedömning av LAV. Fortsatt kunskap som ger adekvata referensvärden och standardiserade mjukvarusystem behövs. / Echocardiological examinations offer an easy, quick, and non-invasive method for diagnosing the left atrium volume (LAV). Cardiac magnetic resonance imaging (CMR) is currently the golden-standard method. However, CMR is time-consuming and costly. Instead two-dimensional echocardiography (2DE) modality is used. The relatively new three-dimensional echocardiography (3DE) method offers an interesting choice when assessing LAV. It’s the purpose of this study to plot 2DE against 3DE regarding LAV correlation and with CMR as reference. The study will examine the relationship between modalities intra- and interobserver variation. Pubmed and Medline databases were used, and systematic literature searches were carried out during April 2020. Inclusion and exclusion criterias have been adopted; the study has only used articles published from 2010 onwards. 3DE shows a higher correlation with CMR in assessment of LAV. The result also showed that 3DE has a lower variation between both intra- and interobserver. 3DE has a stronger correlation to CMR and a lower variation than 2DE, however, 3DE suffers from limitations. These limitations are time, staff experience, lack of reference values and cost to clinics. The authors recommend 3DE for more accurate LAV assessment. Further research for more adequate reference-values and software systems is needed.

Nanoparticles have been used in industry and in medicine due to their properties which give them beneficial uses. This usage of the nanoparticles has risen the question about how harmful they are to the human body, the connection between the exposure to nanoparticles, and many diseases that occur in the body. Methods This study focused on the effect of nanoparticles in a whole human blood loop model. The blood was incubated with Silica, Titanium dioxide and Palladium particles in heparinized loops without any anticoagulants added. The blood’s cell count was analyzed with a cell counter and then complement, and contact system’s markers were analyzed with ELISA to detect a presence of activations in the systems. Experiments one to five were an optimization of test settings. Results An activation of the contact system was initiated in the loops containing the aggregated titanium dioxide nanoparticles. A high platelets consumption up to 73.8 % was observed as well as two visible clots. On top of that, blood smears showed micro-clots in the blood incubated with the aggregated nanoparticles. Conclusion Nanoparticles initiated an activation in the contact system in the aggregated form in comparison with the dispersed form. Further and deeper studies should be executed to observe the importance of the single or the aggregated form in the actual effect on the immune system.

Background: The level of activity in an individual can be the difference between health and illness. Physical inactivity can cause diseases such as osteoporosis and type-2 diabetes. It has been reported that children live an increasingly inactive life, with less than the recommended a total of 60 minutes daily for children and adolescents of 6-17 years of age. Objective: The objective was to compare two activity diaries and how the results correspond to measurements by an activity meter. Material and methods: This study included 12 children who each carried an activity meter for four days to measure Total Energy Expenditure. In parallel, they filled in two different activity diaries. In the diaries two different calculation methods were used, with a Physical Activity Ratio value or a Metabolic Equivalent of Task value which then was inserted into equations to calculate Total Energy Expenditure. Anthropometric measurements were obtained by use of a stadiometer, a caliper and a bioimpedance scale. Results: The results from the Physical Activity Ratio diary indicated a better match with the results from the activity meter. Conclusions: Between the two diaries significant difference in how the activities were estimated were found, where an overestimation could be seen in the diary that used the Metabolic Equivalent of Task. Differences could also be seen between the activity meter and both diaries, also here the difference were bigger with the Metabolic Equivalent of Task diary. The Physical Activity Ratio diary was better matched with the activity meter.

Under senare år har en särskilt hög resistensutveckling observerats hos gramnegativa bakterier inom familjen Enterobacteriaceae. Den främsta resistensmekanismen utgör produktion av så kallade "extended-spectrum β-lactamases" (ESBL) och särskilt oroväckande är karbapenemaser (ESBLCARBA) som har förmåga att bryta ner ett flertal olika grupper av β-laktamantibiotika. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) har utforskats som metod för snabb detektion av karbapenemasaktivitet genom analys av nedbrytning av antibiotika. Syftet med denna studie var att utvärdera om MALDI-TOF MS kan användas som metod för att detektera enzymatisk nedbrytning av Ceftolozan/Tazobaktam samt att undersöka vilka enzymer som uppvisar nedbrytning av antibiotikan. Sju karbapenemasproducerande isolat och en β-laktamasnegativ kontrollstam användes i studien. Isolaten inkuberades 120 min respektive 270 min med antibiotika (1mg/ml) i en buffertlösning (0,08% ammoniumbikarbonat, pH 8). Efter centrifugering analyserades supernatanten med MALDI-TOF MS. Nedbrytning av Ceftolozan detekterades hos samtliga karbapenemasproducerande stammar, utom hos E. coli med NDM-1 produktion. Nedbrytningstoppar av Tazobaktam detekterades emellertid enbart hos stammar med OXA-48 och NDM-7 produktion. Tydligast nedbrytning sågs efter 120 min. För tydligare visualisering av nedbrytningstoppar bör metoden dock optimeras med avseende på matrix, buffert och antibiotikakoncentration. / In recent years, an alarming increase of antibiotic resistance has been observed in Gram-negative bacteria, classified in the family Enterobacteriaceae. The main resistance mechanism is the production of extended-spectrum β-lactamases (ESBL). Particularly worrisome is the production of carbapenemases (ESBLCARBA) due to their ability to hydrolyze a broad range of β-lactams. Recently, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has been investigated as a method for rapid detection of carbapenemase activity through observation of antibiotic degradation. The aim of this study was to investigate whether MALDI-TOF MS can be used as a method to detect degradation of Ceftolozane/Tazobactam as well as to examine which enzymes that possess the ability to hydrolyze the antibiotic. A total of seven carbapenemase-producing strains were used in the study. The experiment also included a β-lactamase-negative isolate as a negative control. The strains were incubated with antibiotic (1mg/ml) in a buffered solution (0,08% ammonium bicarbonate, pH 8) for 120 min and 270 min. The supernatant, after centrifugation, was analyzed by MALDI-TOF MS. All the carbapenemase-producing strains demonstrated hydrolysis of Ceftolozane, except for NDM-1 producing E. coli. However, mass peaks corresponding to the degradation of Tazobactam were only detected in strains producing OXA-48 and NDM-7. The degradation of Ceftolozane/Tazobactam was most apparent after 120 minutes. However, to better enable detection of mass peaks, further optimization is needed in regard to appropriate matrix, buffer and antibiotic concentration.