COMPARISON OF PRIMERS FOR RAPD-PCR FROM ENVIRONMENTAL ISOLATES OF CRYPTOCOCCUS NEOFORMANS, CRYPTOCOCCUS ALBIDUS AND CRYPTOCOCCUS LAURENTII COMPLEX

Various organisms have been characterized by molecular methods, including fungi of the genus Cryptococcus. The purposes of this study were: to determine the discriminatory potential of the RAPD (Random Amplified Polymorphic DNA) primers, the pattern of similarity of the Cryptococcus species, and discuss their useful application in epidemiological studies. We analyzed 10 isolates of each specie/group: C. albidus, C. laurentii complex, C. neoformans var. grubii, all from environmental source, and two ATCC strains, C. neoformans var. grubii ATCC 90112, and C. neoformans var. neoformans ATCC 28957 by RAPD-PCR using the primers CAV1, CAV2, ZAP19, ZAP20, OPB11 and SEQ6. The primers showed a good discriminatory power, revealing important differences between them and between species; the SEQ6 primer discriminated a larger number of isolates of three species. Isolates of C. laurentii showed greater genetic diversity than other species revealed by all six primers. Isolates of C. neoformans were more homogeneous. Only the primer CAV2 showed no amplification of DNA bands for C. albidus. It was concluded that the use of limited number of carefully selected primers allowed the discrimination of different isolates, and some primers (e. g., CAV2 for C. albidus) may not to be applied to some species.

Comparison of application times for ice packs used to relieve perineal pain after normal birth: a randomised clinical trial

Aims and objectives. To compare the effect of an ice pack applied for 10, 15 and 20 minutes to relieve perineal pain after birth. Background. Perineal pain after vaginal birth, with or without vaginal trauma, is one of the most common morbidities reported for postnatal women. Cryotherapy has been used in postpartum period to relieve perineal pain and investigated in several studies. However, cryotherapy treatment protocols in perineal care vary widely regarding temperature, frequency and duration of the application. Design. A controlled trial, randomised for two groups and with a third group as a historical control. Method. The intervention was carried out in a maternity hospital in Sao Paulo, Brazil. The study population consisted of three groups of 38 women who used an ice pack on the perineum, in a single application: group A-10 minutes; group B-15 minutes; group C-20 minutes (historical control from another clinical trial). Participants perineal pain magnitude was evaluated through a numerical scale (010), at four different points: before the cryotherapy; immediately after and at 20 and 40 minutes after cryotherapy. Results. After application of the ice pack, there was no statistical difference when comparing the perineal pain among groups in the second, third and fourth evaluations. Most of the postnatal women reported pain relief, with 72.8% reporting a decrease in pain >50%; 21.9% reported a decrease between 3050%. All postnatal women subjected to cryotherapy were favourable to the procedure. Conclusion. There is no difference in pain scores following ice pack application in three different times (10, 15 and 20 minutes) in women who report moderate or intense perineal pain after normal delivery. Relevance for clinical practice. Ice treatment is safe, and application times of 10 or 15 minutes are as beneficial as an application time of 20 minutes to relieve perineal pain.

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Comparison of two laboratory-developed PCR methods for the diagnosis of Pulmonary Tuberculosis in Brazilian patients with and without HIV infection

Abstract Background Direct smear examination with Ziehl-Neelsen (ZN) staining for the diagnosis of pulmonary tuberculosis (PTB) is cheap and easy to use, but its low sensitivity is a major drawback, particularly in HIV seropositive patients. As such, new tools for laboratory diagnosis are urgently needed to improve the case detection rate, especially in regions with a high prevalence of TB and HIV. Objective To evaluate the performance of two in house PCR (Polymerase Chain Reaction): PCR dot-blot methodology (PCR dot-blot) and PCR agarose gel electrophoresis (PCR-AG) for the diagnosis of Pulmonary Tuberculosis (PTB) in HIV seropositive and HIV seronegative patients. Methods A prospective study was conducted (from May 2003 to May 2004) in a TB/HIV reference hospital. Sputum specimens from 277 PTB suspects were tested by Acid Fast Bacilli (AFB) smear, Culture and in house PCR assays (PCR dot-blot and PCR-AG) and their performances evaluated. Positive cultures combined with the definition of clinical pulmonary TB were employed as the gold standard. Results The overall prevalence of PTB was 46% (128/277); in HIV+, prevalence was 54.0% (40/74). The sensitivity and specificity of PCR dot-blot were 74% (CI 95%; 66.1%-81.2%) and 85% (CI 95%; 78.8%-90.3%); and of PCR-AG were 43% (CI 95%; 34.5%-51.6%) and 76% (CI 95%; 69.2%-82.8%), respectively. For HIV seropositive and HIV seronegative samples, sensitivities of PCR dot-blot (72% vs 75%; p = 0.46) and PCR-AG (42% vs 43%; p = 0.54) were similar. Among HIV seronegative patients and PTB suspects, ROC analysis presented the following values for the AFB smear (0.837), Culture (0.926), PCR dot-blot (0.801) and PCR-AG (0.599). In HIV seropositive patients, these area values were (0.713), (0.900), (0.789) and (0.595), respectively. Conclusion Results of this study demonstrate that the in house PCR dot blot may be an improvement for ruling out PTB diagnosis in PTB suspects assisted at hospitals with a high prevalence of TB/HIV.

Estimation of lung vital capacity before and after coronary artery bypass grafting surgery: a comparison of incentive spirometer and ventilometry

Abstract Background Measurement of vital capacity (VC) by spirometry is the most widely used technique for lung function evaluation, however, this form of assessment is costly and further investigation of other reliable methods at lower cost is necessary. Objective: To analyze the correlation between direct vital capacity measured with ventilometer and with incentive inspirometer in patients in pre and post cardiac surgery. Methodology Cross-sectional comparative study with patients undergoing cardiac surgery. Respiratory parameters were evaluated through the measurement of VC performed by ventilometer and inspirometer. To analyze data normality the Kolmogorov-Smirnov test was applied, for correlation the Pearson correlation coefficient was used and for comparison of variables in pre and post operative period Student's t test was adopted. We established a level of ignificance of 5%. Data was presented as an average, standard deviation and relative frequency when needed. The significance level was set at 5%. Results We studied 52 patients undergoing cardiac surgery, 20 patients in preoperative with VC-ventilometer: 32.95 ± 11.4 ml/kg and VC-inspirometer: 28.9 ± 11 ml/Kg, r = 0.7 p < 0.001. In the post operatory, 32 patients were evaluated with VC-ventilometer: 28.27 ± 12.48 ml/kg and VC-inspirometer: 26.98 ± 11 ml/Kg, r = 0.95 p < 0.001. Presenting a very high correlation between the evaluation forms studied. Conclusion There was a high correlation between DVC measures with ventilometer and incentive spirometer in pre and post CABG surgery. Despite this, arises the necessity of further studies to evaluate the repercussion of this method in lowering costs at hospitals.

A comparison for a multiscale study of structural lineaments in southern Brazil: LANDSAT-7 ETM+ and shaded relief images from SRTM3-DEM

This paper presents a comparison of descriptive statistics obtained for brittle structural lineaments extracted manually from LANDSAT images and shaded relief images from SRTM 3 DEM at 1:100, 000 and 1:500, 000 scales. The selected area is located in the southern of Brazil and comprises Precambrian rocks and stratigraphic units of the Paraná Basin. The application of this methodology shows that the visual interpretation depends on the kind of remote sensing image. The resulting descriptive statistics obtained for lineaments extracted from the images do not follow the same pattern according to the scale adopted. The main direction obtained for Proterozoic rocks using both image types at a 1:500, 000 scale are close to NS±10, whereas at a 1:100, 000 scale N45E was obtained for shaded relief images from SRTM 3 DEM and N10W for LANDSAT images. The Paleozoic sediments yielded the best results for the different images and scales (N50W). On the other hand, the Mesozoic igneous rocks showed greatest differences, the shaded relief images from SRTM 3 DEM images highlighting NE structures and the LANDSAT images highlighting NW structures. The accumulated frequency demonstrated high similarity between products for each image type no matter the scale, indicating that they can be used in multiscale studies. Conversely, major differences were found when comparing data obtained using shaded relief images from SRTM 3 DEM and Landsat images at a 1:100, 000 scale.

Accuracy, precision and robustness of different methods to obtain samples from silages in fermentation studies

The objective of this study was to evaluate accuracy, precision and robustness of two methods to obtain silage samples, in comparison with extraction of liquor by manual screw-press. Wet brewery residue alone or combined with soybean hulls and citrus pulp were ensiled in laboratory silos. Liquor was extracted by a manual screw-press and a 2-mL aliquot was fixed with 0.4 mL formic acid. Two 10-g silage samples from each silo were diluted in 20 mL deionized water or 17% formic acid solution (alternative methods). Aliquots obtained by the three methods were used to determine the silage contents of fermentation end-products. The accuracy of the alternative methods was evaluated by comparing mean bias of estimates obtained by manual screw-press and by alternative methods, whereas precision was assessed by the root mean square prediction error and the residual error. Robustness was determined by studying the interaction between bias and chemical components, pH, in vitro dry matter digestibility (IVDMD) and buffer capacity. The 17% formic acid method was more accurate for estimating acetic, butyric and lactic acids, although it resulted in low overestimates of propionic acid and underestimates of ethanol. The deionized water method overestimated acetic and propionic acids and slightly underestimated ethanol. The 17% formic acid method was more precise than deionized water for estimating all organic acids and ethanol. The robustness of each method with respect to variation in the silage chemical composition, IVDMD and pH is dependent on the fermentation end-product at evaluation. The robustness of the alternative methods seems to be critical at the determination of lactic acid and ethanol contents.

Comparison of primers for RAPD-PCR from environmental isolates of Cryptococcus neoformans, Cryptococcus albidus and Cryptococcus laurentii complex

Various organisms have been characterized by molecular methods, including fungi of the genus Cryptococcus. The purposes of this study were: to determine the discriminatory potential of the RAPD (Random Amplified Polymorphic DNA) primers, the pattern of similarity of the Cryptococcus species, and discuss their useful application in epidemiological studies. We analyzed 10 isolates of each specie/group: C. albidus, C. laurentii complex, C. neoformans var. grubii, all from environmental source, and two ATCC strains, C. neoformans var. grubii ATCC 90112, and C. neoformans var. neoformans ATCC 28957 by RAPD-PCR using the primers CAV1, CAV2, ZAP19, ZAP20, OPB11 and SEQ6. The primers showed a good discriminatory power, revealing important differences between them and between species; the SEQ6 primer discriminated a larger number of isolates of three species. Isolates of C. laurentii showed greater genetic diversity than other species revealed by all six primers. Isolates of C. neoformans were more homogeneous. Only the primer CAV2 showed no amplification of DNA bands for C. albidus. It was concluded that the use of limited number of carefully selected primers allowed the discrimination of different isolates, and some primers (e.g., CAV2 for C. albidus) may not to be applied to some species.

Comparison of child self-reports and parent proxy-reports on quality of life of children with attention deficit hyperactivity disorder

Abstract Background Attention deficit hyperactivity disorder (ADHD) is a neurobiological condition that affects 3%–7% of the pediatric population and significantly compromises the quality of life (QoL) of these individuals. The aim of the current study was to compare child self-reports and parent proxy reports on the QoL of children with ADHD. Methods Forty-five children with ADHD, combined type, aged 8–12 years without comorbidities, were compared with 43 typically developing children. PedsQL™ 4.0 (Pediatric QoL Inventory™) Generic Core Scales (physical, emotional, social, and school functioning) were completed by families and children self-reporting their health-related QoL. Results Children with ADHD reported themselves significantly lowered their PedsQL™ scores on all dimensions in comparison to typically developing children. Statistically significant differences were observed in social functioning (p = 0.010), school functioning (p <0.001), psychosocial health (p <0.001), and total score (p = 0.002). The physical functioning and emotional functioning dimensions did not differ significantly between groups, with p = 0.841 and p = 0.070, respectively. Parents of children with ADHD also reported lower PedsQL™ scores, with statistically significant differences in all dimensions. The relationship between child self-reports and parent proxy reports indicated that there is greater agreement among children with ADHD, except for the school functioning. Conclusions This suggests that children with the disorder and their parents have a perception of the functional limitations the disorder brings. It is therefore important to undertake studies to verify the QoL in children with ADHD that aim to provide and measure the scope of the well-being of these children.

Zooplankton biomass estimation from digitized images: a comparison between subtropical and Antarctic organisms

The measurement of mesozooplankton biomass in the ocean requires the use of analytical procedures that destroy the samples. Alternatively, the development of methods to estimate biomass from optical systems and appropriate conversion factors could be a compromise between the accuracy of analytical methods and the need to preserve the samples for further taxonomic studies. The conversion of the body area recorded by an optical counter or a camera, by converting the digitized area of an organism into individual biomass, was suggested as a suitable method to estimate total biomass. In this study, crustacean mesozooplankton from subtropical waters were analyzed, and individual dry weight and body area were compared. The obtained relationships agreed with other measurements of biomass obtained from a previous study in Antarctic waters. Gelatinous mesozooplankton from subtropical and Antarctic waters were also sampled and processed for body area and biomass. As expected, differences between crustacean and gelatinous plankton were highly significant. Transparent gelatinous organisms have a lower dry weight per unit area. Therefore, to estimate biomass from digitized images, pattern recognition discerning, at least, between crustaceans and gelatinous forms is required.

Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Studies on the interaction of surfactants and neutral cyclodextrins by capillary electrophoresis. Application to chiral analysis

In the present study, mixed systems composed of SDS in the presence of neutral cyclodextrins were considered. Firstly, the effect of the CDs on the CMC of the surfactant was evaluated by CE experiments. Furthermore, a new CE approach based on electric current measurement was developed for the estimation of the stoichiometry as well as of the binding constants of SDS-CDs complexes. The results of these investigations were compared to those obtained with a different technique, electronic paramagnetic resonance (EPR). The obtained results suggested that methylated CDs, in particular (2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD), strongly affect the micellization of SDS in comparison to the other studied CDs. This effect also paralleled the chiral CD-MEKC performance, as indicated by the enantioresolution of (+/-)-Catechin, which was firstly selected as a model compound representative of important chiral phytomarkers. Then a CD-MEKC system, composed of sodium dodecyl sulfate as surfactant (90 mM) and hydroxypropyl-beta-cyclodextrin (25 mM) as chiral selector, under acidic conditions (25 mM borate – phosphate buffer, pH 2.5) was applied to study the thermal epimerisation of epi-structured catechins, (-)-Epicatechin and (-)-Epigallocatechin, to non epi-structured (-)-Catechin and (-)-Gallocatechin. The latter compounds, being non-native molecules, were for the first time regarded as useful phytomarkers of tea sample degradation. The proposed method was applied to the analysis of more than twenty tea samples of different geographical origins (China, Japan, Ceylon), having undergone different storage conditions and manufacturing processes.

Foetal heart rate recording: analysis and comparison of different methodologies

Monitoring foetal health is a very important task in clinical practice to appropriately plan pregnancy management and delivery. In the third trimester of pregnancy, ultrasound cardiotocography is the most employed diagnostic technique: foetal heart rate and uterine contractions signals are simultaneously recorded and analysed in order to ascertain foetal health. Because ultrasound cardiotocography interpretation still lacks of complete reliability, new parameters and methods of interpretation, or alternative methodologies, are necessary to further support physicians’ decisions. To this aim, in this thesis, foetal phonocardiography and electrocardiography are considered as different techniques. Further, variability of foetal heart rate is thoroughly studied. Frequency components and their modifications can be analysed by applying a time-frequency approach, for a distinct understanding of the spectral components and their change over time related to foetal reactions to internal and external stimuli (such as uterine contractions). Such modifications of the power spectrum can be a sign of autonomic nervous system reactions and therefore represent additional, objective information about foetal reactivity and health. However, some limits of ultrasonic cardiotocography still remain, such as in long-term foetal surveillance, which is often recommendable mainly in risky pregnancies. In these cases, the fully non-invasive acoustic recording, foetal phonocardiography, through maternal abdomen, represents a valuable alternative to the ultrasonic cardiotocography. Unfortunately, the so recorded foetal heart sound signal is heavily loaded by noise, thus the determination of the foetal heart rate raises serious signal processing issues. A new algorithm for foetal heart rate estimation from foetal phonocardiographic recordings is presented in this thesis. Different filtering and enhancement techniques, to enhance the first foetal heart sounds, were applied, so that different signal processing techniques were implemented, evaluated and compared, by identifying the strategy characterized on average by the best results. In particular, phonocardiographic signals were recorded simultaneously to ultrasonic cardiotocographic signals in order to compare the two foetal heart rate series (the one estimated by the developed algorithm and the other provided by cardiotocographic device). The algorithm performances were tested on phonocardiographic signals recorded on pregnant women, showing reliable foetal heart rate signals, very close to the ultrasound cardiotocographic recordings, considered as reference. The algorithm was also tested by using a foetal phonocardiographic recording simulator developed and presented in this research thesis. The target was to provide a software for simulating recordings relative to different foetal conditions and recordings situations and to use it as a test tool for comparing and assessing different foetal heart rate extraction algorithms. Since there are few studies about foetal heart sounds time characteristics and frequency content and the available literature is poor and not rigorous in this area, a data collection pilot study was also conducted with the purpose of specifically characterising both foetal and maternal heart sounds. Finally, in this thesis, the use of foetal phonocardiographic and electrocardiographic methodology and their combination, are presented in order to detect foetal heart rate and other functioning anomalies. The developed methodologies, suitable for longer-term assessment, were able to detect heart beat events correctly, such as first and second heart sounds and QRS waves. The detection of such events provides reliable measures of foetal heart rate, potentially information about measurement of the systolic time intervals and foetus circulatory impedance.

Isotope geochemical and geochronologic studies of basement units in the Central Western Carpathians (Slovakia)

In dieser Arbeit werden geochronologische und isotopen-geochemische Daten zur Entwicklung der Zentralen Westlichen Karpathen präsentiert. Die Karpathen bilden die östliche Fortsetzung der Alpen und können in drei Alpine Grundgebirgsdecken unterteilt werden, von denen zwei, die Veporische und die Gemerische, bearbeitet wurden. In der Veporischen Einheit wurden polymetamorphe Grundgebirgseinheiten untersucht, um deren genaue Altersstellung zu definieren und sie isotopengeochemisch zu klassifizieren. Dagegen wurde in der der Gemerischen Einheit, welche die Veporische Einheit überlagert, ein spezialisierter S-Typ Granit im Detail untersucht, um die petrogenetischen Prozesse, die zur magmatischen Entwicklung dieses Granits geführt haben, zu identifizieren. U-Pb Datierungen an Zirkonen der Veporischen Grundgebirgseinheiten zeigen für die gesamte Veporische Einheit ordovizische Entsehungsalter an (440-470 Ma). Diese Datierungen revidieren publizierte kambrische Entstehungsalter dieses Grundgebirges. Die Isotopensignatur (epsilon Nd und 87Sr/86Sr) der ordovizischen Grundgebirgseinheiten, bestehend aus stark überprägten Amphiboliten und Gneissen, ist von der Signatur der sich im Norden anschliessenden Tatrischen Einheit gut unterscheidbar. Die Bleiisotopenzusammensetzung dieser Gesteine ist stark krustal geprägt und überschneidet sich mit der der Tatrischen Einheit. Zusammen mit den T-DM Altern sind diese Einheiten vergleichbar mit prävariskischen Einheiten der Alpen. Somit kann das ordovizische Grundgebirge zu den peri-Gondwana Terranen gezählt werden, die an einem aktiven Kontinentalrand im Norden von Gondwana gebildet wurden. In den Gesteinen der Veporischen Einheit wurde im Weiteren eine starke metamorphe überprägung und intensiver felsischer Magmatismus karbonischen Alters erkannt (320-350 Ma). Dieses Ereignis ist zeitgleich mit dem Magmatismus, welcher hauptsächlich in der sich im Norden anschliessenden Tatrischen Einheit beobachtet wird. Dieser gehört der variskischen Orogenese an. Intensive alpine Deformation und Metamorphose konnte in der südlichen Veporischen Einheit anhand der Einzelzirkondatierungen und der Isotopendaten der ordovizischen Einheiten nachgewiesen werden. Am Dlha Dolina Granit in der Gemerischen Einheit können starke Fraktionierungs- und Auto-Metasomatose-Effekte beobachtet werden. Durch die magmatische Fraktionierung wird eine Anreicherung der SEE erzeugt, wogegen die Metasomatose die SEE stark verarmt. Es kommt sogar zur Ausbildung eines Tetraden Effektes im SEE Muster, welche den starken Einfluss von Fluiden während der spät-magmatischen Phase belegt. Gesamtgesteins Pb-Pb Daten beschränken das minimale Intrusionsalter dieses Granites auf 240 Ma. Dieses Alter ist in guter übereinstimmung mit den Sr-Isotopendaten der magmatisch dominierten Gesteine, wohingegen die stark metasomatisch geprägten Gesteine ein zu radiogenes 87Sr/86Sri aufweisen. Während dieser Arbeit wurde intensiv mit der Blei-Isotopenzusammensetzung von Gesamtgesteinsproben gearbeitet. Um die Auswertung dieser Daten optimieren zu können wurde ein Computerscript für das GPL Programm Octave erstellt. Die Hauptaufgabe dieses Scripts besteht darin, Regressionen für geochronologische Anwendungen gemäss York (1969) zu berechnen. Ausserdem können mu und kappa-Werte für diese Regressionen berechnet und eine Hauptkomponentenanalyse, welche hilfreich für den Vergleich von zwei Datensätzen ist, durchgeführt werden. Am Ende der vorliegenden Arbeit wird die analytische Methode für einen Mikrowellen beschleunigten Säureaufschluss von granitoidem Material zur Bestimmung der Sr- und Nd-Isotopenzusammensetzung und der Elementkonzentrationen vorgestellt. Diese kombinierte Methode nutzt ein TIMS für die Sr und Nd Isotopenmessungen und eine Einzelkollektor-ICPMS zur Bestimmung der SEE, Rb und Sr Konzentrationen, welche mithilfe von relativen Sensitivitätsfaktoren gegenüber einem internen Standard quantifiziert werden. Diese Methode wird durch Messungen von internationalen Referenzmaterialien bewertet. Die Ergebnisse zeigen eine Reproduzierbarkeit von <10% für die Elementkonzentrationen und von <5% für Elementverhältnisse.

Surface plasmon fluorescence spectroscopy and microscopy studies for biomolecular interaction studies

ABSTARCT Biotechnology has enabled the modification of agricultural materials in a very precise way. Crops have been modified through the insertion of new traits or the inhibition of existing gene functions, named Genetically Modified Organism (GMO), and resulted in improved tolerance of herbicide and/or increased resistance against pests, viruses and fungi. Commercial cultivation of GMO started in 1996 and increased rapidly in 2003 according to a recently released report by the International Service for the Acquisition of Agri-Biotech Applications (ISAAA), depicted continuing consumer resistance in Europe and other part of the world. Upon these developments, the European Union regulations mandated labeling of GMOs containing food and as a consequence, the labeling of GMO containing product in the case of exceeding the1% threshold of alien DNA is required. The aim of the study is to be able to detect and quantify the GMO from the mixture of natural food components. The surface plasmon resonance (SPR) technique combined with fluorescence was used for this purpose. During the presented studies, two key issues are addressed and tried to solve; what is the best strategy to design and built an interfacial architecture of a probe oligonucletide layer either on a two dimensional surface or on an array platform; and what is the best detection method allowing for a sensitive monitoring of the hybridisation events. The study includes two parts: first part includes characterization of different PNAs on a 2D planar surface by defining affinity constants using the very well established optical method “Surface Plasmon Fluorescence Spectroscopy”(SPFS) and on the array platform by “Surface Plasmon Fluorescence Microscopy” (SPFM), and at the end comparison of the sensitivity of these two techniques. The second part is composed of detection of alien DNA in food components by using DNA and PNA catcher probes on the array platform in real-time by SPFM.