An evaluation of the apxIVA based PCR-REA method for differentiation of Actinobacillus pleuropneumoniae

A restriction analysis of PCR (PCR-REA) amplified apxIVA gene has been suggested as an alternative method for serotyping of Actinobacillus pleuropneumoniae by Jaglic et al. [Jaglic, Z., Svastova, P., Rychlik, I., Nedbalcova, K., Kucerova, Z., Pavlik, I., Bartos, M., 2004. Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping. Vet. Microbiol. 103, 63-69]. The current study investigated whether this alternative method could distinguish between the reference strains of serovars 13-15 and the value of the method when applied to 47 field isolates representing serovars 1-3, 5, 7-9, 12 and 15 as well as non-typable isolates. The reference strains of serovars 13 and 14 had the same sized product after the apxIVA PCR, while the product for serovar 15 was of different size compared to all the other serovar reference strains. The CfoI digest profiles of the reference serovars 13 and 14 strains were different from each other and from all other serovars. The HpaII digest profiles of these two serovars were very similar to each other, but both were distinctively different from the other serovar profiles. The CfoI digest profile of serovar 15 strain was very similar to the serovars 3 and 12 strains except for two faint extra bands for serovar 15. The HpaII digest profiles of serovars 12 and 15 reference strains were identical. The PCR-REA method correctly recognized the serovar of 21 of 43 field isolates. It was concluded that the method was a useful additional tool to support, but could not replace, conventional serotyping.

Identification and immunogenicity of an immunodominant mimotope of Avibacterium paragallinarum from a phage display peptide library

Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

High resolution genetic and physical mapping of the I-3 region of tomato chromosome 7 reveals almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with duplications on Arabidopsis chromosomes 1, 2 and 3

The tomato I-3 gene introgressed from the Lycopersicon pennellii accession LA716 confers resistance to race 3 of the fusarium wilt pathogen Fusarium oxysporum f. sp. lycopersici. We have improved the high-resolution map of the I-3 region of tomato chromosome 7 with the development and mapping of 31 new PCR-based markers. Recombinants recovered from L. esculentum cv. M82 × IL7-2 F2 and (IL7-2 × IL7-4) × M82 TC1F2 mapping populations, together with recombinants recovered from a previous M82 × IL7-3 F2 mapping population, were used to position these markers. A significantly higher recombination frequency was observed in the (IL7-2 × IL7-4) × M82 TC1F2 mapping population based on a reconstituted L. pennellii chromosome 7 compared to the other two mapping populations based on smaller segments of L. pennellii chromosome 7. A BAC contig consisting of L. esculentum cv. Heinz 1706 BACs covering the I-3 region has also been established. The new high-resolution map places the I-3 gene within a 0.38 cM interval between the molecular markers RGA332 and bP23/gPT with an estimated physical size of 50-60 kb. The I-3 region was found to display almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with Arabidopsis thaliana chromosomes 1, 2 and 3. An S-receptor-like kinase gene family present in the I-3 region of tomato chromosome 7 was found to be present in the microsyntenous region of grape chromosome 12 but was absent altogether from the A. thaliana genome.

A PCR assay for gender assignment in dugong (Dugong dugon) and West Indian manatee (Trichechus manatus)

Gender assignment for some aquatic mammals in the field is difficult. Molecular sexing from tissue biopsies is possible as males are heterogametic. Here we describe a multiplex PCR assay that amplifies the male specific SRY gene and differentiates ZFX and ZFY gametologues in two sirenian species, dugong (Dugong dugon) and West Indian manatee (Trichechus manatus). The assay was validated with animals of known gender and proved accurate and robust to experimental failure.

A head-mounted display as a personal viewing device: Dimensions of subjective experiences

The use of head-mounted displays (HMDs) can produce both positive and negative experiences. In an effort increase positive experiences and avoid negative ones, researchers have identified a number of variables that may cause sickness and eyestrain, although the exact nature of the relationship to HMDs may vary, depending on the tasks and the environments. Other non-sickness-related aspects of HMDs, such as users opinions and future decisions associated with task enjoyment and interest, have attracted little attention in the research community. In this thesis, user experiences associated with the use of monocular and bi-ocular HMDs were studied. These include eyestrain and sickness caused by current HMDs, the advantages and disadvantages of adjustable HMDs, HMDs as accessories for small multimedia devices, and the impact of individual characteristics and evaluated experiences on reported outcomes and opinions. The results indicate that today s commercial HMDs do not induce serious sickness or eyestrain. Reported adverse symptoms have some influence on HMD-related opinions, but the nature of the impact depends on the tasks and the devices used. As an accessory to handheld devices and as a personal viewing device, HMDs may increase use duration and enable users to perform tasks not suitable for small screens. Well-designed and functional, adjustable HMDs, especially monocular HMDs, increase viewing comfort and usability, which in turn may have a positive effect on product-related satisfaction. The role of individual characteristics in understanding HMD-related experiences has not changed significantly. Explaining other HMD-related experiences, especially forward-looking interests, also requires understanding more stable individual traits and motivations.

Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR

The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek's disease virus serotype 1 (MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNA copies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assays were found to be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being less than 5% for Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and feather tips from experimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standard curves, and standard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quantification. These methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range of samples.

The development of an improved PCR-based marker system for Sw-5, an important TSWV resistance gene of tomato

Sw-5 is an important disease resistance gene of tomato, providing broad resistance to Tomato spotted wilt virus (TSWV). A cleaved amplified polymorphic sequence (CAPS) marker, closely linked to the gene, has been reported. Although the Sw-5 locus has been characterised, a gene-specific marker has not been developed. This paper presents a PCR-based marker-system that consists of the co-amplification of a dominant marker representing the Sw-5 gene sequence, and the modified CAPS marker as a positive control and indicator of genotype.

A multi-microprocessor architecture for solving partial differential equations

This paper presents the architecture of a fault-tolerant, special-purpose multi-microprocessor system for solving Partial Differential Equations (PDEs). The modular nature of the architecture allows the use of hundreds of Processing Elements (PEs) for high throughput. Its performance is evaluated by both analytical and simulation methods. The results indicate that the system can achieve high operation rates and is not sensitive to inter-processor communication delay.

Development of an immunomagnetic capture-reverse transcriptase-PCR assay for three pineapple ampeloviruses

A semi-automated, immunomagneticcapture-reverse transcription PCR(IMC-RT-PCR) assay for the detection of three pineapple-infecting ampeloviruses, Pineapple mealybug wilt-associated virus-1, -2 and -3, is described. The assay was equivalent in sensitivity but more rapid than conventional immunocapture RT-PCR. The assay can be used either as a one- or two-step RT-PCR and allows detection of the viruses separately or together in a triplex assay from fresh, frozen or freeze-dried pineapple leaf tissue. This IMC-RT-PCR assay could be used for high throughput screening of pineapple planting propagules and could easily be modified for the detection of other RNA viruses in a range of plant species, provided suitable antibodies are available.

Application of Haar functions for transmission line and transformer differential protection

The development of algorithms, based on Haar functions, for extracting the desired frequency components from transient power-system relaying signals is presented. The applications of these algorithms to impedance detection in transmission line protection and to harmonic restraint in transformer differential protection are discussed. For transmission line protection, three modes of application of the Haar algorithms are described: a full-cycle window algorithm, an approximate full-cycle window algorithm, and a half-cycle window algorithm. For power transformer differential protection, the combined second and fifth harmonic magnitude of the differential current is compared with that of fundamental to arrive at a trip decision. The proposed line protection algorithms are evaluated, under different fault conditions, using realistic relaying signals obtained from transient analysis conducted on a model 400 kV, 3-phase system. The transformer differential protection algorithms are also evaluated using a variety of simulated inrush and internal fault signals.

Kestävä kehitys globaalin ajan hyvinvointiyhteiskunnan haasteena

The objective of the dissertation was to determine the concept of sustainable development according to current understanding and to analyze the structuration of sustainable daily life and how it varies between different groups. The present dissertation is both a literature-based theoretical study and data-based empirical research. The theoretical framework of the study was a greated model of the Structuration of Sustainability in Everyday Life. The model is based on a synthesis of Giddens Theory of Structuration (1984), Spaargaren JA van Vliet's Theory of Consumption as Social Practices (2000) and principles of sustainable development. According to the model created, sustainable everyday life is generated in a context of internal and external factors compromising the interests of ecosystems, society and business. The literature used in the thesis included international and national statements on sustainable development and research into sustainability and the transition to sustainable societies. The data were collected at Helsinki Metropolia University of Applied Sciences. The discretionary sample consisted of students of social services (n = 210) and were collected using the semantic differential technique. The data were analyzed using quantitative and qualitative methods. The results showed that the value placed on ecological, economic and social sustainability increased with age. Activity in non governmental organizations was associated with the acceptance of sustainable development as a whole and especially with global responsibility. Women's everyday life promoted sustainability more than men´s. People living in Helsinki had more sustainable ways of living than those living in the surrounding municipalities because of greater recycling and the low importance given to ownership. Prefering vegetarian food turned out to be a real opportunity for a more sustainable way of living because there were few barriers identified. Contradictory human behavior occurred when advanced sustainable consumer were ready to risk their health. The importance of communality was high and it was considered an aspect of health. The most significant obstacles to sustainable development in daily life were high costs, lack of knowledge and busyness. Similar attitudes towards sustainable development translate into different people´s behavior, which indicates complexities of the behaviour change in the context of sustainable development. The role of non governmental organizations is significant in increasing global responsibility. Education presents an opportunity to increase sustainability, but there are challenges to face from system thinking and in understanding entities in a state of transition towards sustainable everyday life. The responsibility of policy makers is paramount because high costs create a barrier to a sustainable way of living. The implementation of the concept of sustainable development should be focused on the planetary ethics which cover humans, animals, plants and ecosystems. Keywords: Sustainable development, sustainable thinking, behaviour change  

Expression of functional GABAc receptors in the brain

γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the nervous system and acts via three distinct receptor classes: A, B, and C. GABAC receptors are ionotropic receptors comprising ρ subunits. In this work, we aimed to elucidate the expression of ρ subunits in the postnatal brain, the characteristics of ρ2 homo-oligomeric receptors, and the function of GABAC receptors in the hippocampus. In situ hybridization on rat brain slices showed ρ2 mRNA expression from the newborn in the superficial grey layer of the superior colliculus, from the first postnatal week in the hippocampal CA1 region and the pretectal nucleus of the optic tract, and in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three ρ subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, ρ2 mRNA expression clearly dominated over ρ1 and ρ3. GABAC receptor protein expression was confirmed in the adult hippocampus, superior colliculus, and dorsal lateral geniculate nucleus by immunohistochemistry. From the selective distribution of ρ subunits, GABAC receptors may be hypothesized to be specifically involved in aspects of visual image motion processing in the rat brain. Although previous data had indicated a much higher expression level for ρ2 subunit transcripts than for ρ1 or ρ3 in the brain, previous work done on Xenopus oocytes had suggested that rat ρ2 subunits do not form functional homo-oligomeric GABAC receptors but need ρ1 or ρ3 subunits to form hetero-oligomers. Our results demonstrated, for the first time, that HEK 293 cells transfected with ρ2 cDNA displayed currents in whole-cell patch-clamp recordings. Homomeric rat ρ2 receptors had a decreased sensitivity to, but a high affinity for picrotoxin and a marked sensitivity to the GABAC receptor agonist CACA. Our results suggest that ρ2 subunits may contribute to brain function, also in areas not expressing other ρ subunits. Using extracellular electrophysiological recordings, we aimed to study the effects of the GABAC receptor agonists and antagonists on responses of the hippocampal neurons to electrical stimulation. Activation of GABAC receptors with CACA suppressed postsynaptic excitability and the GABAC receptor antagonist TPMPA inhibited the effects of CACA. Next, we aimed to display the activation of the GABAC receptors by synaptically released GABA using intracellular recordings. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation were prolonged by TPMPA. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABAC receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABAC receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA. Taken together with the restricted expression pattern of GABAC receptors in the brain and their distinctive pharmacological and biophysical properties, our findings supporting extrasynaptic localization of these receptors raise interesting possibilities for novel pharmacological therapies in the treatment of, for example, epilepsy and sleep disorders.

Specific detection of the Old World screwworm fly, Chrysomya bezziana, in bulk fly trap catches using real-time PCR.

The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis-causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time-consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular-based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species-specific real-time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species-specific primers and an OWS-specific Taqman MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non-target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real-time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low.

The role of catechol-O-methyltransferase (COMT) and the effects of COMT inhibition in brain and in cardiovascular and renal pathophysiology

Catechol-O-methyltransferase (COMT) metabolizes catecholamines such as dopamine (DA), noradrenaline (NA) and adrenaline, which are vital neurotransmitters and hormones that play important roles in the regulation of physiological processes. COMT enzyme has a functional Val158Met polymorphism in humans, which affects the subjects COMT activity. Increasing evidence suggests that this functional polymorphism may play a role in the etiology of various diseases from schizophrenia to cancers. The aim of this project was to provide novel biochemical information on the physiological and especially pathophysiological roles of COMT enzyme as well as the effects of COMT inhibition in the brain and in the cardiovascular and renal system. To assess the roles of COMT and COMT inhibition in pathophysiology, we used four different study designs. The possible beneficial effects of COMT inhibition were studied in double-transgenic rats (dTGRs) harbouring human angiotensinogen and renin genes. Due to angiotensin II (Ang II) overexpression, these animals exhibit severe hypetension, cardiovascular and renal end-organ damage and mortality of approximately 25-40% at the age of 7-weeks. The dTGRs and their Sprague-Dawley controls tissue samples were assessed with light microscopy, immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography (HPLC) to evaluate the tissue damages and the possible protective effects pharmacological intervention with COMT inhibitors. In a second study, the consequence of genetic and pharmacological COMT blockade in blood pressure regulation during normal and high-sodium was elucidated using COMT-deficient mice. The blood pressure and the heart rate were measured using direct radiotelemetric blood pressure surveillance. In a third study, the effects of acute and subchronic COMT inhibition during combined levodopa (L-DOPA) + dopa decarboxylase inhibitor treatment in homocysteine formation was evaluated. Finally, we assessed the COMT enzyme expression, activity and cellular localization in the CNS during inflammation-induced neurodegeneration using Western blotting, HPLC and various enzymatic assays. The effects of pharmacological COMT inhibition on neurodegeneration were also studied. The COMT inhibitor entacapone protected against the Ang II-induced perivascular inflammation, renal damage and cardiovascular mortality in dTGRs. COMT inhibitors reduced the albuminuria by 85% and prevented the cardiovascular mortality completely. Entacapone treatment was shown to ameliorate oxidative stress and inflammation. Furthermore, we established that the genetic and pharmacological COMT enzyme blockade protects against the blood pressure-elevating effects of high sodium intake in mice. These effects were mediated via enhanced renal dopaminergic tone and suggest an important role of COMT enzyme, especially in salt-sensitive hypertension. Entacapone also ameliorated the L-DOPA-induced hyperhomocysteinemia in rats. This is important, since decreased homocysteine levels may decrease the risk of cardiovascular diseases in Parkinson´s disease (PD) patients using L-DOPA. The Lipopolysaccharide (LPS)-induced inflammation and subsequent delayed dopaminergic neurodegeneration were accompanied by up-regulation of COMT expression and activity in microglial cells as well as in perivascular cells. Interestingly, similar perivascular up-regulation of COMT expression in inflamed renal tissue was previously noted in dTGRs. These results suggest that inflammation reactions may up-regulate COMT expression. Furthermore, this increased glial and perivascular COMT activity in the central nervous system (CNS) may decrease the bioavailability of L-DOPA and be related to the motor fluctuation noted during L-DOPA therapy in PD patients.

Androgen receptor in transcriptional regulation and carcinogenesis

Androgens control a variety of developmental processes that create the male phenotype and are important for maintaining male fertility and normal functions of tissues and organs that are not directly involved in procreation. Androgen receptor (AR) that mediates the biological actions of androgens is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. Although AR was cloned over 15 years ago, the mechanisms by which it regulates gene expression are not well understood. A growing body of in vitro experimental evidence suggests that a complex network of proteins is involved in the androgen-dependent transcriptional regulation. However, the process of AR-dependent transcriptional regulation under physiological conditions is largely elusive. In the present study, a series of experiments were performed, including quantitative chromatin immunoprecipitation (ChIP) assays, to investigate AR-mediated transcription process using living prostate cancer cells. Our results show that the loading of AR and recruitment of coactivators and RNA polymerase II (Pol II) to both the promoter and enhancer of AR target genes are a transient and cyclic event that in addition to hyperacetylation, also involves dynamic changes in methylation, phosphorylation of core histone H3 in androgen-treated LNCaP cells. The dynamics of testosterone (T)-induced loading of AR onto the proximal promoters of the genes clearly differed from that loaded onto the distal enhancers. Significantly, more holo-AR was loaded onto the enhancers than the promoters, but the principal Pol II transcription complex was assembled on the promoters. By contrast, the pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the PSA promoter, but was unable to load onto the PSA enhancer and was incapable of recruiting Pol II, coactivators and following changes of covalent histone modifications. The partial antagonist cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading onto both the PSA promoter and enhancer at a comparable efficiency with androgen in LNCaP cells expressing mutant AR. However, CPA- and RU486-bound AR not only recruited Pol II and coactivator p300 and GRIP1 onto the promoter and enhancer, but also recruited the corepressor NCoR onto the promoter as efficiently as CDX. In addition, we demonstrate that both proteasome and protein kinases are implicated in AR-mediated transcription. Even though proteasome inhibitor MG132 and protein kinase inhibitor DRB (5, 6-Dichlorobenzimidazole riboside) can block ligand-dependent accumulation of PSA mRNA with same efficiency, their use results in different molecular profiles in terms of the formation of AR-mediated transcriptional complex. Collectively, these results indicate that transcriptional activation by AR is a complicated process, which includes transient loading of holo-AR and recruitment of Pol II and coregulators accompanied by a cascade of distinct covalent histone modifications; This process involves both the promoter and enhancer elements, as well as other general components of the cell machineries e.g. proteasome and protein kinase; The pure antiandrogen CDX and the partial antagonist CPA and RU486 exhibit clearly different profiles in terms of their ability to induce the formation of AR-dependent transcriptional complexes and the histone modifications associated with the target genes in human prostate cancer cells. Finally, by using quantitative RT-PCR to compare the expression of sixteen AR co-regulators in prostate cancer cell lines, xenografts, and clinical prostate cancer specimens we suggest that AR co-regulators protein inhibitor of activated STAT1 (PIAS1) and steroid receptor coactivator 1(SRC1) could be involved in the progression of prostate cancer.