The antibiotic class β-lactams include drugs such as penicillins, cephalosporines, carbapenems and monobactams which mechanism of action is to inhibit cell-wall synthesis. Bacteria have developed several mechanisms to counter β-lactams. Bacteria can defend themselves from antibiotics by releasing enzymes that attack the antibiotic compound itself by hydrolysis, target alteration or redox reactions. Presence of antibiotics can also trigger a downregulation of genes coding for antibiotic binding proteins, as well as upregulation of proteins that serves as channel and pump proteins that ensure no accumulation of antibiotics occurs in the cytosol. The aim with the study was to investigate the presence of three plasmid-mediated genes (blaFOX, blaCIT(CMY-2) and blaMOX) coding for ampicillin resistance (pAmpC) in water isolates sampled from Helge River, Kristianstad. The detection of genes was done according to a previous optimized protocol for Real-Time PCR with SYBR™Green chemistry (duplex blaCIT(CMY-2)/blaMOX and singleplex blaFOX). The method proved not to be robust for multiplex PCR, only the singelplex for the gene blaFOX could produce valid results. 30 of 96 isolates were deemed as positive for the gene, whereas 27 of 79 were considered clinical relevant. Among the 27 isolates, 16 also harbored other genes for resistance (13 blaCTX-M, 2 blaOXA, 1 blaTEM and 1 blaSHV). One isolate carried on three resistancegenes (blaFOX, blaCTX-M och blaTEM). A majority of the positive isolates, 20 out of 27, were sampled near the pumpstation. The findings indicate that Helge river might be a reservoir for dissemination of antibiotic resistance genes.

Efter tappning av 450 mL helblod från frivilliga blodgivare separeras blodet till plasma, erytrocyter och trombocyter. Kontroller tas för att undersöka komponentframställningens resultat. I plasmaenheterna kontrolleras bland annat att antalet erytrocyter är under 6x109 per liter. Helblod innehåller normalt 4-6x1012 erytrocyter per liter. Massiv blödning är den främsta indikationen för plasmatransfusion. En transfusion är förknippad med risker såsom transfusion related acute lung injury (TRALI) och transfusion associated circulatory overload (TACO). Syftet med examensarbetet var att hitta en beslutsgräns för bestämning av erytrocytinnehållet i plasmakomponenter som framställs vid beredning av blodkomponenter inför transfusion. Gränsen skulle fastställas genom en jämförelse mellan teststickan Multistix 8 SG, Body fluid-programmet i hematologiinstrumentet Advia 2120 samt manuell räkning i Bürkers räknekammare. Analys skedde av 38 prover varav 18 prover fick tillsats av extra erytrocyter för att antingen överstiga kontrollgränsen eller finna övergången till stickans högsta nivå. Medelvärdet och medianen för de kvantitativa resultaten från Bürkers räknekammare för 20 godkända kontrollerna, fördelade efter teststickans kategorier, beräknades. Samtliga resultaten låg mellan 0,063x109/L och 2,08x109/L. Av de 38 prover som analyserades erhöll 37 ett resultat i form <10x109/L på Advia 2120. Utifrån de erhållna resultaten fastslogs gränsen vid vilken en komponentkontroll garanteras ett godkänt resultat till ≤2+ på teststickan. Vid resultat 3+ ska en konfirmerande kvantitativ analys utföras. Den slutsats som kunde dras var att teststickan Multistix 8 SG kan användas som en screeningmetod vid analys av erytrocytinnehållet i de tillverkade plasmakomponenterna. Slutsatsen blev även att det Adviaprogram som användes inte är lämpligt för analys av erytrocytinnehållet i plasma. / Following blood donation of 450 mL of whole blood from volunteer donors, the blood is separated into plasma, erythrocytes and thrombocytes. Controls are performed to investigate the separation performance. E.g. the plasma units are not allowed to contain more than 6x109 erythrocytes per liter. Whole blood does normally contain 4-6x1012 erythrocytes per liter. The primary indication for plasma transfusion is massive bleeding, a treatment mainly associated with risks such as transfusion related acute lung injury (TRALI) and transfusion associated circulatory overload (TACO). The aim of the thesis work was to find a decision limit for the determination of the erythrocyte content in plasma components produced prior to transfusion. The limit was to be determined by a comparison between the Multistix 8 SG test stick, Body fluid program in the Advia 2120 hematology instrument and manual count in the Bürker counting chamber. Analysis were performed on 38 samples, of which 18 samples were prepared by addition of extra erythrocytes to either exceed the control limit or find the transition point to the highest result level of the stick. The average and median of the quantitative results from the Bürker counting chamber for the 20 approved controls, broken down by the categories on the stick, were calculated. All results were between 0.063x109/L and 2.08x109/L. Of the 38 samples analyzed, 37 received a result <10x109/L on the Advia 2120. Based on these results, the decision limit at which a component control is guaranteed an approved result was determined to ≤2+ on the test stick. In the case of a 3+ result, a confirmatory quantitative analysis must be performed. The conclusion was that the test stick Multistix 8 SG could be used as a screening method for analyzing the erythrocyte content of the plasma components produced. The conclusion was also that the Adviaprogram used is not suitable for analysis of the erythrocyte content in plasma.

Slutsteget i immunförsvarets komplementaktivering innefattar en klyvning av komplementprotein C5, till C5a och C5b, vilket initierar bildandet av terminalt komplementkomplex (TCC) som i form av membran-attack-komplex (MAC) bildar cytotoxiska porer i bland annat gramnegativa bakterier. Bildandet av MAC kan blockeras av endogena regulatorer och TCC frisätts då som ett lösligt komplex, sC5b-9, i plasma. I examensarbetet studerades en variant av TCC, som i tidigare studier visats bildas oberoende av C3- och C5-konvertas när serum surgjorts till pH < 7,0 in vitro. Syftet med studien var att studera om detta atypiska TCC (aTCC) bildades hos grisar, som i en mekonium aspirationssyndrom (MAS)-modell, erhållit ett sänkt systemiskt pH in vivo. I syftet ingick också att etablera en ELISA-baserad metod för att analysera aTCC. I en sandwich ELISA användes monoklonal anti-C5a/C5a (desArg) (klon T13/9) som fångande antikropp och monoklonal anti-C9 (klon aE11) som detekterande antikropp för att analysera aTCC i plasmaprover från 18 MAS-grisar, samt i ett kontrollmaterial bestående av grisserum som surgjorts till pH 6,8 och 6,4 in vitro. Mängden aTCC i kontrollproverna ökade när pH sänktes men innehållet av aTCC i plasmaproverna minskade över MAS-studiens förlopp. När den relativa förändringen i aTCC relaterades till MAS-grisarnas slutliga pH kunde ett signifikant samband ses (p = 0,02) som visade att en större förändring i aTCC sammanföll med ett lägre slutligt pH. Nivåerna av aTCC var generellt sett högre i plasmaproverna jämfört med kontrollproverna vilket skulle kunnat bero på skillnader i plasma vs serum avseende aTCC eller att proverna kom från grisar med olika ålder och vikt. Avsaknad av grisspecifik standard och negativ kontroll samt lågt signal/brusförhållande bidrar till felkällor för analysen och denna kräver fortsatt optimering. / The late steps of complement activation involves a cleavage of complement protein C5, to C5a and C5b, which initiates the formation of terminal complement complex (TCC). The final complex is referred to as the membrane-attack-complex (MAC) which forms cytotoxic pores in, inter alia, gram-negative bacteria. The formation of MAC can be inhibited by endogenous regulators and the TCC is then released as a soluble complex, sC5b-9, in plasma. In the degree project, another type of TCC was studied, which in previous studies had shown to form independently of C3 and C5 convertases when serum was acidified to pH <7.0 in vitro. The purpose of the study was to investigate whether this atypical TCC (aTCC) was formed in piglets, which in a model of meconium aspiration syndrome (MAS), received a reduced systemic pH in vivo. The purpose was also to establish an ELISA for analyzing aTCC. Sandwich ELISA, with monoclonal anti-C5a / C5a (desArg) (clone T13/9) as a capture antibody and monoclonal anti-C9 (clone aE11) as a detection antibody, was used to analyse aTCC in plasma samples from 18 MAS piglets, and in control samples consisting of pig serum acidified to pH 6.8 and 6.4 in vitro. The amount of aTCC in the control samples increased when the pH was lowered, but the content of aTCC in the plasma samples decreased over the course of the MAS study. When the relative change in aTCC was related to the final pH of the MAS pigs, a significant relationship could be seen (p = 0.02) which showed that a major change in the aTCC coincided with a lower final pH. aTCC were generally higher in plasma samples compared with control samples, which could be due to differences in plasma vs serum for aTCC or that the samples came from pigs of different age and weight. Lack of pig-specific standard and negative control as well as low signal to noise ratio contribute to sources of error for the analysis and this requires continued optimization.

Marine bacteria play an important role in the marine nutrition cycles. About half of all sea-living bacteria can use light as an energy source, in addition to organic carbon compounds, which is important for survival as the seas becomes acidic and low in nutrition due to changing climate. The Flavobacteriaceae is a bacterial family that plays an important role in the degradation of complex organic compounds in natural environments. Dokdonia donghaensis MED134 belongs to the Flavobacteriaceae family and use both chemotrophy and phototrophy as a source of energy. For its phototrophic ability, the bacteria use proteorhodopsin, which is a membrane-bound proton pump. This allows the bacteria to survive and grow in nutrient-poor environments. The purpose of the study was to investigate with qPCR if there was a difference in gene expression for proteorhodopsin and isocitrate dehydrogenase in Dokdonia donghaensis MED134 depending on whether the bacteria had grown in constant light or darkness. Analysis with qPCR showed a significantly greater gene expression for proteorhodopsin in the bacteria grown in constant light for seven days, compared to those grown in constant light for three and four days and those grown in constant darkness. No significant difference could be demonstrated in gene expression for isocitrate dehydrogenase. This indicates that Dokdonia donghaensis MED134 in nutritional deficiency can use light as a source of energy for survival and further growth. By simulating climate change an adaptability could be seen through increased gene expression. Through this, further understanding of the role of bacteria in the marine ecosystem can be obtained and further research can be conducted as the marine climate issue becomes more relevant.

Herpes simplexvirus 1 och 2 orsakar godartade sjukdomar, men kan även orsaka mortalitet. Diagnostik av herpes simplexvirus 1 och 2 utförs numera framför allt med realtids- Polymerase chain reaction (PCR). I metoden amplifieras specifika deoxiribonukleinsyra(DNA)-sekvenser till miljontals kopior vilka sedan detekteras med fluorescein. I realtids-PCR sätts positiva och negativa kontroller. De positiva kontrollerna kan vara InHouse eller kommersiella. Tolkningen av resultatet inkluderar inspektion av kontrollerna. DNA utsätts för nedbrytningsprocesser av olika slag, och kan förvaras på olika sätt för att upprätthålla stabilitet. Syftet med studien var att jämföra laboratoriets InHouse-kontroller med två kommersiella kontroller, för att utvärdera vilken av dessa som var mest stabila över tid. Utvärderingen utfördes genom att analysera tre kontroller med realtids-PCR, efter att de hade förvarats i -20° C, i 5° C och i 20° C, och var spädda i TE-buffert eller i PCR-vatten. De kommersiella och InHouse-kontrollerna visade sig vara jämbördiga. Vidare studier som görs under längre tid, i större omfattning och där koncentrationerna är samma för varje kontroll, föreslås. / Herpes simplex viruses 1 and 2 which usually cause benign diseases but can even cause mortality. The diagnostics of herpes simplex virus 1 and 2 are performed with real-time Polymerase chain reaction (PCR). In the real-time PCR method, specific deoxyribonucleic acid (DNA) sequences are amplified into millions of copies which are then detected with fluorescein. Positive and negative controls are used in real-time PCR. The positive controls can be InHouse or commercial. The interpretation of the results includes inspection of the controls. DNA is subject to degradation processes of different kinds and can be stored in different ways to maintain stability. The purpose of the study was to compare the laboratory's InHouse controls with two commercial controls, to evaluate which of these were more stable over time. The evaluation was performed by analyzing the three controls with real-time PCR after they were stored in temperatures at -20° C, at 5° C and at 20° C, and were diluted in TE-buffer or in water. The commercial and InHouse controls proved to be equitable. Further studies carried out for a longer period of time, to a greater extent and where concentrations are the same for each control are suggested.

Forcerad exspiratorisk volym på en sekund (FEV1) och vitalkapacitet (VC) utgör grunden för spirometri som är ett diagnostiskt hjälpmedel vid lungsjukdomar. Spirometri utförs vanligtvis i sittande position, men kan utföras i stående position. Syftet med studien var att jämföra om det finns en signifikant skillnad för FEV1 och VC vid dynamisk spirometri mellan sittande och stående position hos studenter utan känd lungsjukdom. Datainsamlingen utfördes på Klinisk Fysiologi, Länssjukhuset Ryhov i Jönköping av legitimerad biomedicinsk analytiker. 13 frivilliga studenter i åldrarna 22-33 deltog i studien, fyra var män och nio var kvinnor. Genomsnittligt BMI var 21,9 kg/m2 . Manövrarna utfördes minst tre gånger i sittande och sedan stående position. Deltagare med längd över 175 cm fick stå på knä. Medianen för VC i sittande position var 4,5 liter respektive 4,4 liter i stående position. Medianen för FEV1 var 3,6 liter i samtliga kroppspositioner. Wilcoxon-rangsummetest påvisade ingen statistiskt signifikant skillnad för varken VC eller FEV1 mellan sittande och stående position. På grund av litet urval kan ingen generell slutsats dras av denna studie men kan utgöra underlag för fortsatta studier. Ytterligare studier med en större och mer spridd population krävs för att kunna dra generella slutsatser om kroppspositionens påverkan på FEV1 och VC.

The result of cryopreservation of semen is crucial for patients in need of fertility preservation.The cryopreservation method is not optimized since only 10 % the sperms are expected tosurvive the treatment. The sperms are exposed to many risk factors such as oxidative stress,osmotic chock and ice crystallization. To minimize the risks, use of cryoprotectants is needed.The use of cryoprotectants helps the cell to dehydrate as penetrating cryoprotectants cancreate space between ice crystals and cell membrane.Two studies were performed. In study one, two different freezing medias (SpermFreezesolution and Cryoprotect II effect on sperm motility after freezing in were compared). Studytwo investigate whether the motility were best preserved if semen froze with all the contentsof the ejaculate or if the sample should be concentrated, with removal of seminal plasma,epithelium cells and dead sperm cells by gradient centrifugation.The project was performed according to recommended instructions for each freezing media.In total, 55 samples were collected for the first study and 23 samples were collected for thesecond study. The sperm motility was measured both before freezing and after thawing.The Cryoprotect II medium preserved the cells better than the SpermFreeze medium(p=0,006). Using SpermFreeze, higher rate of motility was obtained when centrifugation wereperformed before freezing (p=0,033), while this was not observed when using Cryoprotect II(p=0,055). In conclusion Nidacon preserved sperm more effectively than Vitrolife freezemedium. Vitrolife’s freezing medium preserved the samples better if centrifugation wereperformed before freezing. Nidacons freezing medium gave the same result for the samplesno matter centrifugation were performed before or after freezing.

CYP2D6, a member of the cytochrome P450 enzyme system, has a central role in drug metabolism, it metabolizes 25 % of clinically used drugs. The gene that codes for the enzyme displays a high degree of polymorphism, which effects enzyme functions to various degrees. Aside from smaller mutations like SNPs, alleles may also feature duplications or deletion of the whole gene. Due to the clinical relevance of these mutations, a simple and precise method for genotyping is needed. In this study, a method based on pyrosequencing for copy number analysis was evaluated, wherein the copy number was determined by relative quantification to a reference gene CYP2D8P. During evaluation of the method, several adjustments were tried for optimization, including adjustments of annealing temperature and primer concentration. The results showed a difficulty in distinguishing between copy numbers using the method, as well as a high coefficient of variation. Therefore, further optimization is required before the method could be implemented into clinical practice.

Malignant lymphomas are a group of cancerous diseases that develop from lymphocytes and primarily affect lymph nodes. Being the sixth most common cancer type in Sweden, lymphoma is a societal problem that needs to be tackled by improving care and treatment of patients. This study was designed to examine the blood cell composition in lymphoma patients and well as determine whether the use of cryopreserved cells affected the analysis outcome. An evaluation of the methods used was also performed. Frozen peripheral blood from lymphoma patients as well as fresh and frozen blood from healthy controls was used. The cells of interest were monocytes, granulocytes, Treg, NKT, iNKT, B and T cells plus the dendritic cell activation protein CCR7. Three immunophenotyping methods were used. Method one was used in staining surface cell markers while the other two were for both surface and intracellular staining using two distinctive kits. The results showed no significant difference in immune cell composition between patients and blood donors. Limited patient samples and the lack of female blood donors could explain the unexpected result. A substantial difference in Treg cells was observed in fresh and frozen tested samples as well as T cell outcomes in method one compared to the other two methods. There were fewer Treg cells in frozen samples, which probably was due to cryopreservation while the lack of fixation in method one led to the loss of CD4+ T cells. Overall, the methods used were adequate but definitely require some improvements.

Escherichia coli are bacteria found in bowels of warm blooded animals. Most subspecies are harmless and part of the normal gut flora. However, E. coli have the ability to exchange genetic material with other bacteria, and some E. coli have acquired genes coding for virulence factors. VTEC, E. coli with the ability to produce verotoxin are commonly found in cattle, but certain types can cause severe disease in humans, known as enterohaemorrhagic E. coli, EHEC.     In this study, isolates of E. coli and other bacteria in the family Enterobacteriaceae from calves were subtyped and clustered using MALDI-TOF. Ten strains were selected for experimental competition analysis against E. coli MG1655.     The aim of the study was to identify strains of bacteria with the potential to outcompete VTEC in the cattle host and decrease the risk of human infections.     Three of the bacterial strains were able to outcompete the laboratory strain, and in future studies these strains can be analysed when competing against VTEC. The rest of the strains were outcompeted. Four known strains of VTEC were analysed competing the laboratory strain, showing weak ability to compete. Finally, a highly pathogenic strain of VTEC was analysed against Escherichia coli Nissle 1917, known for its ability to outcompete many strains of bacteria. Nissle could not outcompete the tested VTEC strain under the tested conditions.     In conclusion the majority of the bacterial strains isolated from calves were identified as E. coli and three of the isolates showed good ability to compete against the laboratory strain.