Formal development and quantitative verification of dependable systems

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Experimental poisoning by Senecio brasiliensis in calves: quantitative and semi-quantitative study on changes in the hepatic extracellular matrix and sinusoidal cells

Extracellular matrix plays an important role in chronic hepatic lesions and has been studied in experimental intoxication models. However in cattle, studies on chronic disease have focused on the hepatocellular damage and extracellular matrix (ECM) changes are usually overlooked. There are no specific studies on the hepatic ECM in either normal or chronically damaged bovine liver. Thus an experimental model of hepatic toxicity model using Senecio brasiliensis poisoned calves was designed. Senecio brasiliensis contains pyrrolizidine alkaloids which cause either acute or chronic progressive dose dependent liver damage. Five calves were orally fed with 0.38g of dry leaves of S. brasiliensis/kg/day for 24 days. Liver needle biopsy specimens were obtained every 15 days for 60 days. Clinical signs of digestive complications appeared at 3rd week. One calf died on 45th day and four were evaluated up to 60th day. Biopsy samples were processed for routine light microscopy, immuno-histochemistry and transmission electron microscopy. From 30th day on progressive liver damage characterized by hepatocellular ballooning, necrosis, apoptosis and megalocytosis, centrilobular, pericellular and portal fibrosis were seen by light microscopy. Quantitative and semi-quantitative measurements of hepatic ECM components were performed before and after the onset of lesions. Morphometric analysis of total collagen and elastic fiber system was conducted. Total collagen and I and III collagen types progressively increased in throughout the liver of affected calves. Changes in location, amount and disposition of the elastic fiber system were also observed. Then numbers of Kupffer cells were significantly increased at 30th day and total numbers of sinusoidal cells were significantly increased at 45th and 60th days. Liver damage was progressive and irreversible even after the exposure to the plant was discontinued. Severe fibrotic lesions occurred mainly in portal tracts, followed by veno-occlusive and pericellular fibrosis. Collagen types I and III s were present in every normal and damaged liver, with predominance of type I. In affected calves the increase of total collagen and elastic fibers system paralleled the number of total sinusoidal cells.

Ultrasound call detection in capybara

The vocal repertoire of some animal species has been considered a non-invasive tool to predict distress reactivity. In rats ultrasound emissions were reported as distress indicator. Capybaras[ vocal repertoire was reported recently and seems to have ultrasound calls, but this has not yet been confirmed. Thus, in order to check if a poor state of welfare was linked to ultrasound calls in the capybara vocal repertoire, the aim of this study was to track the presence of ultrasound emissions in 11 animals under three conditions: 1) unrestrained; 2) intermediately restrained, and 3) highly restrained. The ultrasound track identified frequencies in the range of 31.8±3.5 kHz in adults and 33.2±8.5 kHz in juveniles. These ultrasound frequencies occurred only when animals were highly restrained, physically restrained or injured during handling. We concluded that these calls with ultrasound components are related to pain and restraint because they did not occur when animals were free of restraint. Thus we suggest that this vocalization may be used as an additional tool to assess capybaras[ welfare.

The Effects of Anesthetic Induced Loss of Consciousness on Quantitative Electroen Cephalogram, and Bispectral and Spectral Entropy Indices. Studies on Healthy Male

Changes in the electroencephalography (EEG) signal have been used to study the effects of anesthetic agents on the brain function. Several commercial EEG based anesthesia depth monitors have been developed to measure the level of the hypnotic component of anesthesia. Specific anesthetic related changes can be seen in the EEG, but still it remains difficult to determine whether the subject is consciousness or not during anesthesia. EEG reactivity to external stimuli may be seen in unconsciousness subjects, in anesthesia or even in coma. Changes in regional cerebral blood flow, which can be measured with positron emission tomography (PET), can be used as a surrogate for changes in neuronal activity. The aim of this study was to investigate the effects of dexmedetomidine, propofol, sevoflurane and xenon on the EEG and the behavior of two commercial anesthesia depth monitors, Bispectral Index (BIS) and Entropy. Slowly escalating drug concentrations were used with dexmedetomidine, propofol and sevoflurane. EEG reactivity at clinically determined similar level of consciousness was studied and the performance of BIS and Entropy in differentiating consciousness form unconsciousness was evaluated. Changes in brain activity during emergence from dexmedetomidine and propofol induced unconsciousness were studied using PET imaging. Additionally, the effects of normobaric hyperoxia, induced during denitrogenation prior to xenon anesthesia induction, on the EEG were studied. Dexmedetomidine and propofol caused increases in the low frequency, high amplitude (delta 0.5-4 Hz and theta 4.1-8 Hz) EEG activity during stepwise increased drug concentrations from the awake state to unconsciousness. With sevoflurane, an increase in delta activity was also seen, and an increase in alpha- slow beta (8.1-15 Hz) band power was seen in both propofol and sevoflurane. EEG reactivity to a verbal command in the unconsciousness state was best retained with propofol, and almost disappeared with sevoflurane. The ability of BIS and Entropy to differentiate consciousness from unconsciousness was poor. At the emergence from dexmedetomidine and propofol induced unconsciousness, activation was detected in deep brain structures, but not within the cortex. In xenon anesthesia, EEG band powers increased in delta, theta and alpha (8-12Hz) frequencies. In steady state xenon anesthesia, BIS and Entropy indices were low and these monitors seemed to work well in xenon anesthesia. Normobaric hyperoxia alone did not cause changes in the EEG. All of these results are based on studies in healthy volunteers and their application to clinical practice should be considered carefully.

Abdominal and pelvic ultrasound study of the maned wolf (Chrysocyon brachyurus)

The aim of the present study was the ultrasound characterization of the abdominal and pelvic regions of five maned wolves kept in captivity at the Triage Center of Wild Animals of the Federal University of Viçosa (Centro de Triagem de Animais Silvestres, Universidade Federal de Viçosa). This characterization included descriptions of ultrasonographic aspects and measurements of various structures using B-mode ultrasound. Biometric data were collected to assess the existence of significant linear correlations between these measurements and the measurements obtained by ultrasound. Additionally, hematological and serum biochemistry evaluations of the animals were performed. The ultrasound findings were similar to those available in the literature on domestic dogs, which were used for comparison as a result of the lack of published data regarding maned wolves. The latter species showed characteristics closely resembling those of the former, differing in the spleen and left renal cortex echogenicities, in the appearance of the prostatic and testicular regions and in the hepatic portal vein morphology. In the current study, the biometric values were similar to those previously published; however, no data regarding thoracic perimeter, modified crown-rump length or thoracic depth were found in the literature for this Canidae species. Statistical analysis showed the existence of a strong negative correlation between the modified crown-rump length and left renal length, between the modified crown-rump length and the right renal volume, between the thoracic perimeter and the height at the cranial pole of the left adrenal gland and between the thoracic perimeter and the height at the caudal pole of the left adrenal gland. Laboratory findings, including segmented neutrophil, eosinophil, monocyte and lymphocyte counts and the serum levels of glucose, ALT, alkaline phosphatase, urea, total protein, globulin, creatine phosphokinase, triglyceride, sodium, phosphate, potassium and chloride, were inconsistent with values found by other authors. The ultrasound is a diagnostic imaging method that must be further explored in the medicine of wild animals; therefore, additional research in this area is required.

Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

Characterization of Lubricating Oil Using Ultrasound

This paper presents an ultrasonic method to measure small concentrations of water in lubricating oil. It uses an ultrasonic measurement cell composed by a piezoceramic emitter (5 and 10 MHz), and a large aperture PVDF receiver that eliminates diffraction effects. The propagation velocity, attenuation coefficient and density of several samples of water-in-oil emulsion were measured. The concentrations of water of the samples were in the range of 0 to 5% in volume, and the results showed that these low concentrations can be discriminated within a resolution of 0.2% in the studied range, using the measurement of the propagation velocity.

Spontaneous Focusing on Quantitative Relations and the Development of Rational Number Conceptual Knowledge

The aim of the present set of studies was to explore primary school children’s Spontaneous Focusing On quantitative Relations (SFOR) and its role in the development of rational number conceptual knowledge. The specific goals were to determine if it was possible to identify a spontaneous quantitative focusing tendency that indexes children’s tendency to recognize and utilize quantitative relations in non-explicitly mathematical situations and to determine if this tendency has an impact on the development of rational number conceptual knowledge in late primary school. To this end, we report on six original empirical studies that measure SFOR in children ages five to thirteen years and the development of rational number conceptual knowledge in ten- to thirteen-year-olds. SFOR measures were developed to determine if there are substantial differences in SFOR that are not explained by the ability to use quantitative relations. A measure of children’s conceptual knowledge of the magnitude representations of rational numbers and the density of rational numbers is utilized to capture the process of conceptual change with rational numbers in late primary school students. Finally, SFOR tendency was examined in relation to the development of rational number conceptual knowledge in these students. Study I concerned the first attempts to measure individual differences in children’s spontaneous recognition and use of quantitative relations in 86 Finnish children from the ages of five to seven years. Results revealed that there were substantial inter-individual differences in the spontaneous recognition and use of quantitative relations in these tasks. This was particularly true for the oldest group of participants, who were in grade one (roughly seven years old). However, the study did not control for ability to solve the tasks using quantitative relations, so it was not clear if these differences were due to ability or SFOR. Study II more deeply investigated the nature of the two tasks reported in Study I, through the use of a stimulated-recall procedure examining children’s verbalizations of how they interpreted the tasks. Results reveal that participants were able to verbalize reasoning about their quantitative relational responses, but not their responses based on exact number. Furthermore, participants’ non-mathematical responses revealed a variety of other aspects, beyond quantitative relations and exact number, which participants focused on in completing the tasks. These results suggest that exact number may be more easily perceived than quantitative relations. As well, these tasks were revealed to contain both mathematical and non-mathematical aspects which were interpreted by the participants as relevant. Study III investigated individual differences in SFOR 84 children, ages five to nine, from the US and is the first to report on the connection between SFOR and other mathematical abilities. The cross-sectional data revealed that there were individual differences in SFOR. Importantly, these differences were not entirely explained by the ability to solve the tasks using quantitative relations, suggesting that SFOR is partially independent from the ability to use quantitative relations. In other words, the lack of use of quantitative relations on the SFOR tasks was not solely due to participants being unable to solve the tasks using quantitative relations, but due to a lack of the spontaneous attention to the quantitative relations in the tasks. Furthermore, SFOR tendency was found to be related to arithmetic fluency among these participants. This is the first evidence to suggest that SFOR may be a partially distinct aspect of children’s existing mathematical competences. Study IV presented a follow-up study of the first graders who participated in Studies I and II, examining SFOR tendency as a predictor of their conceptual knowledge of fraction magnitudes in fourth grade. Results revealed that first graders’ SFOR tendency was a unique predictor of fraction conceptual knowledge in fourth grade, even after controlling for general mathematical skills. These results are the first to suggest that SFOR tendency may play a role in the development of rational number conceptual knowledge. Study V presents a longitudinal study of the development of 263 Finnish students’ rational number conceptual knowledge over a one year period. During this time participants completed a measure of conceptual knowledge of the magnitude representations and the density of rational numbers at three time points. First, a Latent Profile Analysis indicated that a four-class model, differentiating between those participants with high magnitude comparison and density knowledge, was the most appropriate. A Latent Transition Analysis reveal that few students display sustained conceptual change with density concepts, though conceptual change with magnitude representations is present in this group. Overall, this study indicated that there were severe deficiencies in conceptual knowledge of rational numbers, especially concepts of density. The longitudinal Study VI presented a synthesis of the previous studies in order to specifically detail the role of SFOR tendency in the development of rational number conceptual knowledge. Thus, the same participants from Study V completed a measure of SFOR, along with the rational number test, including a fourth time point. Results reveal that SFOR tendency was a predictor of rational number conceptual knowledge after two school years, even after taking into consideration prior rational number knowledge (through the use of residualized SFOR scores), arithmetic fluency, and non-verbal intelligence. Furthermore, those participants with higher-than-expected SFOR scores improved significantly more on magnitude representation and density concepts over the four time points. These results indicate that SFOR tendency is a strong predictor of rational number conceptual development in late primary school children. The results of the six studies reveal that within children’s existing mathematical competences there can be identified a spontaneous quantitative focusing tendency named spontaneous focusing on quantitative relations. Furthermore, this tendency is found to play a role in the development of rational number conceptual knowledge in primary school children. Results suggest that conceptual change with the magnitude representations and density of rational numbers is rare among this group of students. However, those children who are more likely to notice and use quantitative relations in situations that are not explicitly mathematical seem to have an advantage in the development of rational number conceptual knowledge. It may be that these students gain quantitative more and qualitatively better self-initiated deliberate practice with quantitative relations in everyday situations due to an increased SFOR tendency. This suggests that it may be important to promote this type of mathematical activity in teaching rational numbers. Furthermore, these results suggest that there may be a series of spontaneous quantitative focusing tendencies that have an impact on mathematical development throughout the learning trajectory.

Leaf blade quantitative anatomy of sugarcane cultivars and clones

The objective of this study was to make a quantitative assess of the anatomic characteristics of leaf blade of the sugarcane cultivars RB855113, SP80-1842, SP80-1816, RB867515 and clone RB957689 presenting different sensitivity to the mixture of sodium trifloxysulfuron + ametryn herbicides. Compared to the other cultivars assessed, RB855113 cultivar, considered more sensitive to the herbicide mixture, presented relevant differences such as greater proportion of bulliform cells, greater tissue proportion in the transverse section of the leaf blade, greater stomata and trichome density on both surfaces, thinner epidermis on the adaxial surface and length of stomata on both surfaces. The external paraclinal wall of the bulliform cells was thinner than in the common epidermis cells in all the genotypes on the adaxial and abaxial surfaces. Multivariate analysis of the data on the variables considered most relevant to explain the herbicide penetration singled out the sensitive RB855113 from the other materials. Such characteristics can explain the greater penetration, and consequently, greater sensitivity of this cultivar to the sodium trifloxysulfuron + ametryn mixture.

Quantitative variables applied to phenological studies of Sargassum vulgare C. Agardh (Phaeophyceae - Fucales) from Ilha Grande Bay, State of Rio de Janeiro

The suitability of quantitative variables for phenological studies was evaluated in a population of the brown seaweed Sargassum vulgare from "Praia das Gordas", Angra dos Reis, Ilha Grande Bay, state of Rio de Janeiro. From June 1998 to May 1999, twenty adult individuals were randomly sampled at bimonthly intervals. Fifteen variables related to the vegetative and reproductive development of perennial and non-perennial parts of the individuals were quantified. Variables related to the non-perennial parts were more useful than those related to the perennial parts, because they showed a clear variation over the year. Vegetative development declined from June to October, and increased from October to February, when maximum median values of thallus height, total dry mass, non-perennial parts dry mass, and degree of branching were reached. This pattern coincided with those described for other species of the genus from warm temperate regions. Thallus height, a usually employed character in other phenological studies of Sargassum, showed lower coefficient of variation (53.2%) than those related to dry mass (72.0% to 182.3%). Peak of reproduction occurred from June to August, according to the following variables: fertile primary lateral branches number and dry mass and receptacles dry mass. Non-perennial parts dry mass and receptacles dry mass are recommended for phenological studies of S. vulgare. This methodological procedure avoids the sampling of the whole individual and warrants its regeneration from the perennial parts.

Quantitative Analysis of Novel Prostate Cancer Markers in Tissue

Prostate cancer is a heterogeneous disease affecting an increasing number of men all over the world, but particularly in the countries with the Western lifestyle. The best biomarker assay currently available for the diagnosis of the disease, the measurement of prostate specific antigen (PSA) levels from blood, lacks specificity, and even when combined with invasive tests such as digital rectal exam and prostate tissue biopsies, these methods can both miss cancers, and lead to overdiagnosis and subsequent overtreatment of cancers. Moreover, they cannot provide an accurate prognosis for the disease. Due to the high prevalence of indolent prostate cancers, the majority of men affected by prostate cancer would be able to live without any medical intervention. Their latent prostate tumors would not cause any clinical symptoms during their lifetime, but few are willing to take the risk, as currently there are no methods or biomarkers to reliably differentiate the indolent cancers from the aggressive, lethal cases that really are in need of immediate medical treatment. This doctoral work concentrated on validating 12 novel candidate genes for use as biomarkers for prostate cancer by measuring their mRNA expression levels in prostate tissue and peripheral blood of men with cancer as well as unaffected individuals. The panel of genes included the most prominent markers in the current literature: PCA3 and the fusion gene TMPRSS2-ERG, in addition to BMP-6, FGF-8b, MSMB, PSCA, SPINK1, and TRPM8; and the kallikrein-related peptidase genes 2, 3, 4, and 15. Truly quantitative reverse-transcription PCR assays were developed for each of the genes for the purpose, time-resolved fluorometry was applied in the real-time detection of the amplification products, and the gene expression data were normalized by using artificial internal RNA standards. Cancer-related, statistically significant differences in gene transcript levels were found for TMPRSS2-ERG, PCA3, and in a more modest scale, for KLK15, PSCA, and SPINK1. PCA3 RNA was found in the blood of men with metastatic prostate cancer, but not in localized cases of cancer, suggesting limitations for using this method for early cancer detection in blood. TMPRSS2-ERG mRNA transcripts were found more frequently in cancerous than in benign prostate tissues, but they were present also in 51% of the histologically benign prostate tissues of men with prostate cancer, while being absent in specimens from men without any signs of prostate cancer. PCA3 was shown to be 5.8 times overexpressed in cancerous tissue, but similarly to the fusion gene mRNA, its levels were upregulated also in the histologically benign regions of the tissue if the corresponding prostate was harboring carcinoma. These results indicate a possibility to utilize these molecular assays to assist in prostate cancer risk evaluation especially in men with initially histologically negative biopsies.

Modelling of ultrasound assisted mixing in Newtonian and non-Newtonian liquids

The purpose of this work is to obtain a better understanding of behaviour of possible ultrasound appliance on fluid media mixing. The research is done in the regard to Newtonian and non-Newtonian fluids. The process of ultrasound appliance on liquids is modelled in COMSOL Multiphysics software. The influence of ultrasound using is introduced as waveform equation. Turbulence modelling is fulfilled by the k-ε model in Newtonian fluid. The modeling of ultrasound assisted mixing in non-Newtonian fluids is based on the power law. To verify modelling results two practical methods are used: Particle Image Velocimetry and measurements of mixing time. Particle Image Velocimetry allows capturing of velocity flow field continuously and presents detailed depiction of liquid dynamics. The second way of verification is the comparison of mixing time of homogeneity. Experimentally achievement of mixing time is done by conductivity measurements. In modelling part mixing time is achieved by special module of COMSOL Multiphysics – the transport of diluted species. Both practical and modelling parts show similar radial mechanism of fluid flow under ultrasound appliance – from the horn tip fluid moves to the bottom and along the walls goes back. Velocity profiles are similar in modelling and experimental part in the case of Newtonian fluid. In the case of non-Newtonian fluid velocity profiles do not agree. The development track of ultrasound-assisted mixing modelling is presented in the thesis.

Quantitative features of Cedrela odorata L. wood (Meliaceae)

Cedrela odorata L. (Meliaceae) occurs in the Atlantic forest, Amazon rain forest, riparian forest of the country, and wetlands, which demand species adapted to their water conditions. Studies in ecological wood anatomy demonstrated that weather factors' variations have direct influence on the wood anatomical structure and that the fragmentation of the natural habitats is a direct cause of the edge effect which alters the abiotic aspects of the location, interfering consequently in its vegetation. A comparative analysis of 20 anatomical quantitative features of the wood structure was performed in populations of Cedrela odorata growing inside and on the edge of the swamp forest and granulometric analysis was made on the soil. The quantitative data were submitted to the Mann-Whitney's nonparametric test, presenting a statistically significant value decrease in the eleven wood features mean for the specimens growing in the edge of swamp forest.

Quantitative receptor radioautography in the study of receptor-receptor interactions in the nucleus tractus solitarii

The nucleus tractus solitarii (NTS) in the dorsomedial medulla comprises a wide range of neuropeptides and biogenic amines. Several of them are related to mechanisms of central blood pressure control. Angiotensin II (Ang II), neuropeptide Y (NPY) and noradrenaline (NA) are found in the NTS cells, as well as their receptors. Based on this observation we have evaluated the modulatory effect of these peptide receptors on a2-adrenoceptors in the NTS. Using quantitative receptor radioautography, we observed that NPY and Ang II receptors decreased the affinity of a2-adrenoceptors for their agonists in the NTS of the rat. Cardiovascular experiments agreed with the in vitro data. Coinjection of a threshold dose of Ang II or of the NPY agonists together with an ED50 dose of adrenergic agonists such as NA, adrenaline and clonidine counteracted the depressor effect produced by the a2-agonist in the NTS. The results provide evidence for the existence of an antagonistic interaction between Ang II at1 receptors and NPY receptor subtypes with the a2-adrenoceptors in the NTS. This receptor interaction may reduce the transduction over the a2-adrenoceptors which can be important in central cardiovascular regulation and in the development of hypertension