<p>Aromatase is the enzyme that catalysis the conversion of androgens into estrogens. It´s a member of P450 cytochrome family and is encoded by the CYP19-gene. The enzyme aromatase has an important role in regulating physiological and behavioral sexual mechanisms. This includes for instance activation, motivation and maintenance of the reproductive behaviors. The sexual behavior is affected by a complex series of events that requires the connection of endogenous hormonal and neurochemical changes with social interactions, especially between the opposite sexes. The aim of the present study was to examine how social interactions effect the aromatase expression and activity in the guppy brain. Guppy males were introduced into four different social conditions: Isolated, all male conditions, heterospecific (with zebrafish females) and conspecific female guppies. The focal males were kept under these conditions for two respectively four days. The sexual behavior, of each of the focal males was recorded daily during 10 minutes. The males with the guppy females showed, in contrast to the males in the other groups, a high frequency of reproductive behaviors. The brains of the focal males were collected and the brain aromatase activity was measured using tritiated water assay. I have also tried to analyze the gene-expression of aromatase with RT-PCR. However I was unable to analyze the results with the RT-PCR, because of possible primer-dimerization. Due to the limited time schedule, we were not able to solve the problem. ANOVA performed on the aromatase activity, revealed no significant difference between the different treatment groups. The variance was highest in the zebrafish category and lowest in the isolated males. There was no significant correlation between the mean number of reproductive behaviors and the aromatase activity in males that were together with guppy females. The results do not support the hypothesis that social interactions can affect the brain aromatase activity in guppy males.</p>

<p>Chlamydia and gonorrhea are major causes of sexually transmitted infections (STI) in adolescents worldwide. The infections are caused by <em>Chlamydia trachomatis</em> or <em>Neisseria gonorrhoeae, </em>bacteria with clinical manifestations such as urethritis, prostatitis and epididymitis among men, and urethritis, cervicitis and upper genital tract infection (i.e. pelvic inflammatory disease) among women. However, in many cases of genital tract infection, the etiology remains uncertain. In light of this, <em>Mycoplasma genitalium</em> was somewhat accidentally isolated in 1980 after prolonged incubation of urogenital specimens from men with non-gonococcal urethritis. Following the initial isolation in 1980, repeated attempts have been made to recover the extremely fastidious organism from clinical samples by culture techniques, but isolates have been rare and difficult to obtain. With the development of PCR methods in the early 1990s, detection of <em>M. genitalium</em> infection became more feasible.</p><p>The aim in paper <strong>I</strong> was to compare three different PCR assays (conventional and real-time 16S rRNA gene PCR as well as real-time <em>Mycoplasma genitalium</em> adhesin protein (MgPa) gene PCR) for detection of <em>M. genitalium</em>. The study also determined the prevalence of <em>M. genitalium</em>. Clinical specimens collected from STI attendees, 381 men and 298 women, were used to determine the prevalence of <em>M. genitalium</em> and 213 of these specimens were used in the PCR comparative study. The prevalence of <em>M. genitalium</em> infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %) respectively. In the PCR comparative study, <em>M. genitalium </em>DNA were detected in 61/76 (80.3 %) of true-positive specimen by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Hence, real-time MgPa gene PCR is well suited for clinical diagnosis of <em>M. genitalium</em> in urogenital specimens from men and women.</p><p>The aim in paper <strong>II</strong> was to determine whether a patients’ endocervical swab specimen can be transported in first void urine (FVU) as combined specimens in detection of <em>Mycoplasma genitalium </em>by real-time PCR. The study also compared two different DNA extraction methods (manual Chelex DNA extraction and automated BioRobot M48 DNA extraction) for observation of possible PCR inhibition. Clinical specimens collected from 329 women attending a STI clinic were used in the study. A total of 100 endocervical swab specimens transported in FVU was used in the PCR inhibition analysis. <em>M. genitalium</em> was detected in 25/329 (7.6 %) women. Endocervical swab specimens transported in FVU demonstrate higher sensitivity compared to both FVU alone and specimens transported in 2-SP medium detecting 24/25 (96 %), 22/25 (88 %) and 17/25 (68 %) of <em>M. genitalium</em> positive women, respectively. Automated BioRobot M48 DNA extraction was shown to be superior to manual Chelex extraction leaving no PCR inhibition and slightly higher DNA yield and/or better sensitivity. The results from these two studies are important knowledge in establishing the future diagnostic level of this STI in our county and also nationally.</p>

<p> </p><p>Genotyping can be used to link genetic variation among individuals to certain diseases or conditions. Some known disorders and states that are dependent on single nucleotide polymorphism (SNPs) are lactose intolerance, venous thrombosis, hereditary hemochromatosis and the difference in sensibility among people to metabolise drugs.</p><p>In this project a new kit, TaqManÒ Sample-to-SNP KitÔ <strong>for extraction of DNA and preparation of the extract for genotyping with real-time PCR and allelic discrimination, was evaluated. QIAamp® DNA Blood Biorobot® MDx Kit was used as the reference method.</strong></p><p>The purpose of the comparison was to find a method that makes DNA extraction from blood samples cheaper and faster, but with the same reliability as the reference procedure.</p><p>The results of the evaluation showed a complete agreement of the genotype results between the methods tested, which means that the new method was as reliable as the reference method. The costs of reagents and material would be reduced with 52% if the new method is adopted, that alone would result in a cost reduction of 144 000SEK a year with a sample volume of 650 samples/month. The time for DNA extraction would also be reduced with the new procedure.</p><p> </p>

<p>Reliable methods to analyze food for the presence of almond are important – not only for those allergic to almond, but also for monitoring the compliance with labelling regulations (EG directive 2003/89). Until now the Swedish National Food Administration has used methods like rocket immunoelectrophoresis and real-time PCR to detect almond in food. These methods are, however, not sensitive enough for protecting the most sensitive individuals. Therefore, the performance of a commercial ELISA kit was tested with regard to specificity/cross reactivity and limit of detection for almond both in solution and in different matrixes.</p><p>The limit of quantitation was at least 3,1 ppm (mg/kg) in solution and similar concentrations were measured in bisquits and chocolate. The ELISA method was about 100-fold more sensitive than rocket immunoelectrophoresis and PCR.</p><p>The specificity of the test kit was evaluated against a number of different nuts and seeds. No important cross reactivity was found. The antibodies against almond used in the kit can not differentiate between almond and apricot kernel. For such purposes the PCR method could be used.</p>

<p>Invasive Candida infection is an increasing cause of morbidity and mortality in the immunocompromised patient. Molecular diagnosis based on genomic amplification methods, such as real time PCR, has been reported as an alternative to conventional culture for early detection of invasive candidiasis. The template DNA extraction step has been the major limitation in most reported nucleic acid based assays, due to problems in breaking fungal cell walls and incomplete purification in PCR inhibitor substances.</p><p>The aim of this study was to compare enzymatic cell wall disruption using recombinant lyticase with mechanical disruption using glass beads. The QIAamp tissue kit was compared with two automated DNA extraction robots, the BioRobot M48 and NucliSens easyMAG, to determine their sensitivity, reliability and duration for DNA release of C. albicans. Mechanical cell wall disruption shortened and facilitated the extraction procedure, but the quantity of released DNA was significantly lower than when enzymatic cell wall disruption was used. Use of robots did not significantly shorten the DNA extraction time, compared with manual DNA extraction. However the NucliSens easyMAG resulted in a higher yield of target DNA compared to the BioRobot M48 and the manual QIAamp tissue kit.</p> / <p>Invasiva svampinfektioner är ett stort problem hos patienter med dåligt immunförsvar. Förekomst av invasiva svampinfektioner har ökat under senare år och medför hög dödlighet. En svampinfektion som inte snabbt diagnostiseras och behandlas kan bli livshotande om patientens kondition är dålig. Candida albicans är den vanligaste orsaken till invasiva svampinfektioner. Med traditionell svampidentifiering kan det ta dagar till veckor att isolera och artbestämma svampen. En snabbare metod att detektera Candida är att använda sig av molekylärbiologiska metoder som påvisar svampens arvsmassa, DNA. Svampar har en cellvägg som är svår att bryta ner och därför är DNA extraktionssteget ett av de mest rapporterade problemen vid DNA svampdiagnostik.</p><p>Syftet med denna studie var att jämföra enzymatisk och mekanisk cellväggsnedbrytning av C. albicans med hjälp av enzymet lyticase respektive glaskulor. Vi jämförde också en manuell metod med två automatiska robotar för att bestämma deras känslighet, tillförlitlighet och tidsåtgång för DNA-extraktion från C. albicans. De slutsatser som nåtts är att den enzymatiska cellväggsnedbrytningen var känsligare men betydligt mer tidskrävande än den mekaniska cellväggsnedbrytningen. Denna studie visade även att en av de automatiska systemen extraherade signifikant mer DNA än den manuella metoden.</p>

Bovine Spongiform Encephalopathy (BSE) belongs to a group of neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) which affect many species. From 1986 more than 184,000 cattle in the UK have been confirmed to be infected with this disease, and in Canada total losses to the economy reached $6 billion. This study examines the gene expression in three major innate immunity components: complement system, toll-like receptors, interleukins, and selected proteins of their signaling pathways. Quantitative real time polymerase chain reaction analyses were performed on caudal medulla samples to identify differentially expressed genes between non-exposed and orally challenged animals. In general, immune genes were down-regulated in comparison to non-challenged animals during first 12 months of disease with a tendency to be up-regulated at terminal stage of BSE. The results from this study provide a basis for further research on the mechanisms modifying immune responses and altering progression of the disease. / Animal Science

Genotyping can be used to link genetic variation among individuals to certain diseases or conditions. Some known disorders and states that are dependent on single nucleotide polymorphism (SNPs) are lactose intolerance, venous thrombosis, hereditary hemochromatosis and the difference in sensibility among people to metabolise drugs. In this project a new kit, TaqManÒ Sample-to-SNP KitÔ for extraction of DNA and preparation of the extract for genotyping with real-time PCR and allelic discrimination, was evaluated. QIAamp® DNA Blood Biorobot® MDx Kit was used as the reference method. The purpose of the comparison was to find a method that makes DNA extraction from blood samples cheaper and faster, but with the same reliability as the reference procedure. The results of the evaluation showed a complete agreement of the genotype results between the methods tested, which means that the new method was as reliable as the reference method. The costs of reagents and material would be reduced with 52% if the new method is adopted, that alone would result in a cost reduction of 144 000SEK a year with a sample volume of 650 samples/month. The time for DNA extraction would also be reduced with the new procedure.

Aromatase is the enzyme that catalysis the conversion of androgens into estrogens. It´s a member of P450 cytochrome family and is encoded by the CYP19-gene. The enzyme aromatase has an important role in regulating physiological and behavioral sexual mechanisms. This includes for instance activation, motivation and maintenance of the reproductive behaviors. The sexual behavior is affected by a complex series of events that requires the connection of endogenous hormonal and neurochemical changes with social interactions, especially between the opposite sexes. The aim of the present study was to examine how social interactions effect the aromatase expression and activity in the guppy brain. Guppy males were introduced into four different social conditions: Isolated, all male conditions, heterospecific (with zebrafish females) and conspecific female guppies. The focal males were kept under these conditions for two respectively four days. The sexual behavior, of each of the focal males was recorded daily during 10 minutes. The males with the guppy females showed, in contrast to the males in the other groups, a high frequency of reproductive behaviors. The brains of the focal males were collected and the brain aromatase activity was measured using tritiated water assay. I have also tried to analyze the gene-expression of aromatase with RT-PCR. However I was unable to analyze the results with the RT-PCR, because of possible primer-dimerization. Due to the limited time schedule, we were not able to solve the problem. ANOVA performed on the aromatase activity, revealed no significant difference between the different treatment groups. The variance was highest in the zebrafish category and lowest in the isolated males. There was no significant correlation between the mean number of reproductive behaviors and the aromatase activity in males that were together with guppy females. The results do not support the hypothesis that social interactions can affect the brain aromatase activity in guppy males.

PCR is a rapid, reproducible method for nucleic acid detection. However, this technology displays significant deficiencies when applied in clinical microbiology. This work’s aim was to improve current diagnostics and provide sensitive and quantitative real-time PCRs. Paper I describes the development of a sensitive and specific quantitative real-time PCR for the detection of Streptococcus pneumoniae, based on the Spn9802 DNA fragment. Applied to nasopharyngeal aspirates from 166 pneumonia patients, Spn9802 PCR had a sensitivity of 94% and a specificity of 98%. In Paper II the performance of a ply gene PCR for identification of pneumococcal lower respiratory tract infection (LRTI) was evaluated on bronchoalveloar lavage fluids. At the detection limit 103 genome copies/mL, 89% sensitivity but only 43% specificity was achieved. Paper III shows that S. pneumoniae DNA is detectable in plasma from acutely febrile patients. Sensitivities were low (26-42%) for detection of pneumococcal pneumonia, for bacteraemic pneumococcal pneumonia they were 60-70%. Paper IV describes evaluation of four PCR targets for Haemophilus influenzae detection. A real-time PCR based on the P6 gene was developed and applied to 166 CAP patients, using cut-off of 104 genome copies/mL the assay had a sensitivity of 97% and a specificity of 96%. In paper V, the two real-time PCRs presented in papers I and IV were combined with a PCR for detection of Neisseriae meningitidis. The analytical sensitivity of this multiplex real-time PCR was not affected by using a mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae) in single tubes. Applied to 156 LRTI patients, this PCR had sensitivities over 90% for S. pneumoniae and H. influenzae, and specificities of 89% and 96%, respectively. In conclusion, real-time PCR assays are useful for the diagnosis of S. pneumoniae and H. influenzae. They enable detection after antibiotic installation, and quantification increases the etiological specificity of pneumonia.

Reliable methods to analyze food for the presence of almond are important – not only for those allergic to almond, but also for monitoring the compliance with labelling regulations (EG directive 2003/89). Until now the Swedish National Food Administration has used methods like rocket immunoelectrophoresis and real-time PCR to detect almond in food. These methods are, however, not sensitive enough for protecting the most sensitive individuals. Therefore, the performance of a commercial ELISA kit was tested with regard to specificity/cross reactivity and limit of detection for almond both in solution and in different matrixes. The limit of quantitation was at least 3,1 ppm (mg/kg) in solution and similar concentrations were measured in bisquits and chocolate. The ELISA method was about 100-fold more sensitive than rocket immunoelectrophoresis and PCR. The specificity of the test kit was evaluated against a number of different nuts and seeds. No important cross reactivity was found. The antibodies against almond used in the kit can not differentiate between almond and apricot kernel. For such purposes the PCR method could be used.