Quantification of human DNA in forensic samples is an important step during STR profiling because the STR genotyping is sensitive to the quantity of DNA used in the PCR reaction. This study focuses on the importance of quantification in the entire process of genetic analysis. Two real time PCR platforms (Roche LightCycler480 System and ABI 7900 RT PCR) were used to compare two commercial kits in terms of DNA quantification. It was found out that accuracy of absolute quantification values in commercial quantification kits is strongly dependent on the construction of calibration curve. Especially low template DNA samples were used to assess whether QuantifilerTM or Plexor® HY System can determinate a minimum quantification value (cut off value) below which STR profiles would consistently fail to be detected. The usage of Plexor® HY System enabled to determine the cut off quantification value more exactly probably due to different molecular background and chemistry used in this kit. Reliability and other issues connected with cut off value are discussed. In order to better understand the relationship between the quantity of DNA and the number of detectable loci series the dilution experiment with standard DNA007 was done. Quantitative and qualitative consequences of input DNA amount in evaluation of...

The crayfish plague pathogen, Aphanomyces astaci, is one of the most serious threats to European indigenous crayfish species, e.g., the noble crayfish (Astacus astacus). The only way to protect susceptible crayfish species from the disease is to prevent the dispersion of the pathogen to their populations. One of the most important sources of the crayfish plague pathogen in Central Europe is the spiny-cheek crayfish (Orconectes limosus), a species of North American origin, which can carry the parasite in its cuticle for years. Some literature sources claimed that the pathogen dispersion from the American vectors is restricted to periods of moulting or to the time before and after the crayfish death. However, experimental evidence for such hypotheses was lacking. The main aim of my thesis was to test these predictions, and the alternative scenario that the crayfish plague pathogen can be transmitted from the infected spiny-cheek crayfish also in other periods. For this purpose, experiments were set up to investigate A. astaci transmission from infected spiny-cheek crayfish to non-infected spiny-cheek or noble crayfish. As expected, the pathogen was transmitted to noble crayfish much more easily than to the uninfected American host. Nevertheless, we succeeded in the pathogen transmission also among spiny-cheek...

eIF3 and eIF4E are very important eukaryotic translation initiation factors. eIF3 is practically involved in every step of translation initiation, eIF4E is important mainly for its ability to bind the cap. Mammalian factor eIF3 consists of thirteen subunits, many subunits have a function apart from translation, such as in apoptosis and mitosis. It was proved that upregulated or downregulated expression of some subunits as well as upregulated expression of eIF4E is linked with different types of tumours and malignancies in human. In the first part of my work, I was examining the amount of transcripts of subunits eIF3a, b, d, e, f, g, h, i and j in cell lines which are used for study of acute lymphoblastic leukaemia. I tried to find if there is a difference in the amount of trancripts between lines or between lines and control line in these subunits. According to experiments and statistical analysis, I proved increased amount of mRNA for eIF3b subunit in control cell line NC-NC in comparison with other used leukaemic cell line except from line NALM6. Other differences were not statistically important. In the second part of my work, I was analysing 3' UTR region of transcripts of eIF4E1 and utilising of polyadenylation signals in this trancript. I used the leukeamic cell lines again. The experiments clearly...

Previous findings from this laboratory demonstrating changes in dopamine (DA) transporter and D2 receptors in the amygdaloid complex of subjects with major depression indicate that disruption of dopamine neurotransmission to the amygdala may contribute to behavioral symptoms associated with depression. Quantitative real-time RT-PCR was used to investigate the regional distribution of gene expression of DA receptors in the human amygdala. In addition, relative levels of mRNA of DA receptors in the basal amygdaloid nucleus were measured postmortem in subjects with major depression and normal control subjects. All five subtypes of DA receptor mRNA were detected in all amygdaloid subnuclei, although D1, D2, and D4 receptor mRNAs were more abundant than D3 and D5 mRNAs by an order of magnitude. The highest level of D1 mRNA was found in the central nucleus, whereas D2 mRNA was the most abundant in the basal nucleus. Levels of D4 mRNA were highest in the basal and central nuclei. In the basal nucleus, amounts of D4, but not D1 or D2, mRNAs were significantly higher in subjects with major depression as compared to control subjects. These findings demonstrate that the D1, D2 and D4 receptors are the major subtypes of DA receptors in the human amygdala. Elevated DA receptor gene expression in depressive subjects further implicates altered dopaminergic transmission in the amygdala in depression.

A common model for studying Chlamydia trachomatis and growing chlamydial stocks uses Lymphogranuloma venereum serovar L2 and non-polarized HeLa cells. However, recent publications indicate that the growth rate and progeny yields can vary considerably for a particular strain depending on the cell line/type used, and seem to be partially related to cell tropism. In the present study, the growth of invasive serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on collagen-coated microcarrier beads, as well as in HeLa cells grown in tissue culture flasks. Microscopy analysis revealed no difference in chlamydial attachment/entry patterns or in inclusion development throughout the developmental cycle between cell lines. Very comparable growth curves in both cell lines were also found using real-time PCR analysis, with increases in chlamydial DNA content of 400-500-fold between 2 and 36 h post-inoculation. Similar progeny yields with comparable infectivity were recovered from HEC-1B and HeLa cell bead cultures, and no difference in chlamydial growth was found in polarized vs. non-polarized HeLa cells. In conclusion, unlike other C. trachomatis strains such as urogenital serovar E, invasive serovar L2 grows equally well in physiologically different endometrial and endocervical environments, regardless of the host cell polarization state.

Mycoplasma gallisepticum (MG) can cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. Impacts of MG infections can include increased morbidity and mortality, decreased egg production, hatchability and feed efficiency. Biosecurity and bio-surveillance remain the primary means of deterrence as the current means of control are not wholly effective. Further applications towards the characterization of MG-related disease control requires proper understanding of the characteristics of MG infections with accurate and efficient quantification of in vivo MG populations. To this end, study was conducted to determine the in vivo MG populations in infected pullets and to determine impact of sampling schedule. The role of sampling in 3 distinct anatomical sites, and their associated MG populations were also compared. Results confirmed that sampling events did not affect MG populations and as a possible confounding factor, sampling frequency could be avoided for the future development of novel means of MG control.

Identification of genes specific to fiber development would improve the efforts in developing cotton plants with superior fiber quality. Through genetic introgression, 17 interspecific chromosome substitution lines (CS-B lines) of upland cotton in G. hirsutum (TM-1) have been developed and released recently. These substitution lines have TM1 as background and contain either whole chromosome or chromosome arms of G. barbadense (line 3-79) chromosomes. CS-B25 has chromosome 25 from G. barbadense substituted into TM-1 G. hirsutum was reported to show superior fiber properties. In this study, suppression subtraction hybridization (SSH) combined with Affymetrix cotton genome microarray arrays were used to identify differentially expressed genes in CS-B25. An SSH fiber cDNA library was constructed with differentially expressed genes identified in CS-B25. Microarray analysis showed that 23 genes were up-regulated and 9 down-regulated. Majority of these genes were involved in Ethylene signal pathway, Ubiquitin-proteasome pathway and cell wall synthesis.

De grampositiva bakterierna enterokocker tillhör normalfloran i tarmen hos människor samt djur. I detta arbete studeras vancomycin-variabla enterokocker (VVE) som är vanA positiva enterokocker, känsliga för vancomycin och kan utveckla resistens in vivo. Vancomycin-resistenta enterokocker (VRE) består av två arter bakterier Entrococcus faecalis och Enterococcus faecium. VVE består av Enterococcus faecium och är svåra att detektera med odlingsbaserad diagnostik och kan därför leda till att bakterien är underdiagnostiserad, samt att den tyst kan sprida sig inom sjukhusmiljön. VVE har en förmåga att kunna övergå från vancomycin-variabla till att vara vancomycin-resistenta fenotyper. Därför undersöks i denna studie vid vilken vancomycinkoncentration (5, 4, 3, 2 mg/L) som VVE kan anrikas i buljongerna inför amplifiering med hjälp av real-time polymerase chain reaction (real-time PCR). Resultatet visade att vancomycinkoncentrationen skulle behövas sänkas till 2 mg/L, dock kan bakgrundsfloran vara för hög vid denna koncentration av antibiotika. / The Gram-positive bacteria Enterococci, belong to the normal intestinal flora in humans and in animals. In the following study, vancomycin-variable enterococci (VVE) are investigated. VVE is a vanA-positive bacteria, meaning that they are sensitive to vancomycin and can develop a resistance in vivo. Vancomycin-resistant enterococci (VRE) consists of two different species of bacteria, Enterococcus faecalis and Enterococcus faecium. Furthermore, VVE consists of E. faecium and can be difficult to detect with a culture-based diagnostic andcan therefore lead to the bacterium being underdiagnosed. This in turn means that the bacteria can be quietly spread within the hospital. VVE has a unique ability, it can transition from vancomycin-variables to having vancomycin-resistant phenotypes. Therefore, this study investigates at what vancomycin concentration the VVE broths can be enriched in the broths before the qPCR. The results show that the vancomycin concentration is optimal at 2 mg/L. However, for the background flora this might be a too high concentration.

Francisella tularensis is the etiological agent of tularemia, a zoonotic disease with worldwide prevalence. F. tularensis is a highly pathogenic organism and has been designated as a potential biothreat agent. Currently there are four recognized subspecies of F. tularensis: tularensis (type A), holarctica (type B), mediasiatica, and novicida. In addition, genomic studies have further subdivided type A tularensis into two subclassifications, type A.I and type A.II. These two subclassifications differ in geographic distribution with type A.I appearing mainly in the Eastern United States and type A.II appearing mainly in the Western United States. Because of differences of virulence among the subspecies, it is important to be able to quickly identify each of the subspecies rapidly and accurately. This work describes the development of a multiplex real-time polymerase chain reaction (PCR) assay which was shown to be ~98% successful at identifying the known subspecies of F. tularensis. Furthermore, F. tularensis is thought be a genome in decay (losing genes) because of the relatively large number of pseudogenes present in its genome. We hypothesized that the observed frequency of gene loss/pseudogenes may be an artifact of evolution in response to a changing environment, and that genes involved in virulence should be under strong positive selection. Eleven arbitrarily chosen virulence genes were screened for positive selection along with 10 arbitrarily chosen housekeeping genes. Analyses of selection yielded one housekeeping gene and 7 virulence genes which showed significant evidence of positive selection. Our results suggest that while the loss of functional genes through disuse could be accelerated by negative selection, the genome decay in Francisella could also be the byproduct of adaptive evolution, as evidenced by several of its virulence genes which are undergoing strong, positive selection.

Objective: Reductions in sodium pump (SP) abundance can give rise to increases in contractile force in uterine and vascular smooth muscle as well as an increased secretion in secretory cells, including potentially those of the placenta. To determine whether the mouse might serve as a model for human pregnancy in terms of the SP and to determine whether changes in SP abundance anticipate or follow labor, we studied pregnant mice over the final trimester of their pregnancy. Study Design: C57Bl6 dams (n=46) were bred and studied during their pregnancy. Animals (n=4) were sacrificed at specific gestational time points. Other mice had labor induced with LPS on Gestational day 15 and were then studied at specific time points after induction. Specimens were studied for mRNA abundance as well as protein abundance using methods such as Real time RT-PCR and Western blot analysis. Data were analyzed by ANOVA with post hoc Duncan's pair-wise comparisons. Results: Levels of uterine SP α3 isoform mRNA were most abundant on day 14 near the beginning of the third trimester. There was a significant fall in SP &alpha3 mRNA abundance by day 18 with a slightly lower level on the day of birth but an increased SP α3 mRNA abundance by one day post partum. Contrary to the uterus, SP α3 mRNA levels in the placenta increased over the last trimester, from day 14 to the day of birth. Western blot analysis on the two tissues demonstrated a somewhat similar pattern. In the LPS studies of uterus and placenta, the SP α3 isoform protein abundance appeared to fall when compared to the 2 hour time point. Those animals which were injected with a vehicle control showed very little change in SP α3 abundance after injection. While protein levels were reduced, there was no significant reduction in mRNA for all specimens. Conclusion: Uterine SP α3 isoform protein expression fell late in mouse pregnancy but prior to labor and appeared to be mediated by reductions in its mRNA. These reductions paralleled changes observed in term pregnant women. Such reductions would increase the sensitivity of the uterus to agents causing contraction but may directly increase the force, duration and frequency of contractions. Placental SP α3 isoform protein expression had no significant change over the final trimester. However, unlike uterine protein, the placental protein may not be mediated by its mRNA. Reductions in SP α3 protein abundance were also seen in preterm labor produced by LPS induction. These changes may not be mediated by mRNA. Taken together, changes in the SP α3 isoform may represent a fundamental mechanism in the initiation and/ or progression of term labor and in preterm in mouse and potentially in human.