The role of P16ink4a and P53 immunostaining in predicting recurrence of HG-CIN after conization treatment

Objective: Io evaluate the expression of p16INK4a and p53 biomarkers in conization specimens from patients with high grade cervical intraepithelial neoplasia (HG-CIN), correlating them with the ability to predict the recurrence. Methods : we conducted a retrospective study of patients with HG-CIN in cervical biopsy treated with conization between January 1999 and January 2006 who had a minimum follow-up of 18 months. The expression of the p16 and p53 was assessed by tissue microarrays and correlated with disease recurrence. For analysis, we used the test of proportions (chi-square), considering value p<0.05, 95% CI and calculations of sensitivity, specificity and accuracy of these immunomarkers in predicting recurrence. Results : the series comprised 83 patients aged between 16 and 86 years (35±11.7), divided into two groups: 30 with HG-CIN recurrence (study group) and 53 without recurrence (control group). Mean age, parity, smoking and conization technique were similar in both groups. The p53 expression was present in 43% of the study group and 57% of the control group, and the p16 was present in 43% of the study group and in 57% of the control group (p>0.05). p53 had a positive predictive value (PPV) of 42% and negative predictive value (NPV) of 73%, sensitivity 70%, specificity of 47% and accuracy of 59%. The p16, PPV 42%, NPV 72%, sensitivity 66%, specificity of 49% and accuracy of 56%. Conclusion : immunohistochemistry expression of p53 and p16 showed low sensitivity and low specificity as predictors of HG-CIN recurrence after conization treatment.

Accuracy of sonography and hysteroscopy in the diagnosis of premalignant and malignant polyps in postmenopausal women

PURPOSE: To evaluate the accuracy of sonographic endometrial thickness and hysteroscopic characteristics in predicting malignancy in postmenopausal women undergoing surgical resection of endometrial polyps. METHODS: Five hundred twenty-one (521) postmenopausal women undergoing hysteroscopic resection of endometrial polyps between January 1998 and December 2008 were studied. For each value of sonographic endometrial thickness and polyp size on hysteroscopy, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated in relation to the histologic diagnosis of malignancy. The best values of sensitivity and specificity for the diagnosis of malignancy were determined by the Receiver Operating Characteristic (ROC) curve. RESULTS: Histologic diagnosis identified the presence of premalignancy or malignancy in 4.1% of cases. Sonographic measurement revealed a greater endometrial thickness in cases of malignant polyps when compared to benign and premalignant polyps. On surgical hysteroscopy, malignant endometrial polyps were also larger. An endometrial thickness of 13 mm showed a sensitivity of 69.6%, specificity of 68.5%, PPV of 9.3%, and NPV of 98% in predicting malignancy in endometrial polyps. Polyp measurement by hysteroscopy showed that for polyps 30 mm in size, the sensitivity was 47.8%, specificity was 66.1%, PPV was 6.1%, and NPV was 96.5% for predicting cancer. CONCLUSIONS: Sonographic endometrial thickness showed a higher level of accuracy than hysteroscopic measurement in predicting malignancy in endometrial polyps. Despite this, both techniques showed low accuracy for predicting malignancy in endometrial polyps in postmenopausal women. In suspected cases, histologic evaluation is necessary to exclude malignancy.

Uterine Doppler velocimetry of the uterine arteries in the second and third trimesters for the prediction of gestational outcome

PURPOSE: The aim of this longitudinal study was to investigate the value of uterine artery Doppler sonography during the second and third trimesters in the prediction of adverse pregnancy outcome in low-risk women. METHODS: From July 2011 to August 2012, a total of 205 singleton pregnant women presenting at our antenatal clinic were enrolled in this prospective study and were assessed for baseline demographic and obstetric data. They underwent ultrasound evaluation at the time of second and third trimesters, both included Doppler assessment of bilateral uterine arteries to determine the values of the pulsatility index (PI) and resistance index (RI) and presence of early diastolic notch. The endpoint of this study was assessing the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Doppler ultrasonography of the uterine artery, for the prediction of adverse pregnancy outcomes including preeclampsia, stillbirth, placental abruption and preterm labor. RESULTS: The mean age of cases was 26.4±5.11. The uterine artery PI and RI values for both second (PI: 1.1±0.42 versus 1.53±0.59, p=0.002; RI: 0.55±0.09 versus 0.72±0.13, p=0.000 respectively) and third-trimester (PI: 0.77±0.31 versus 1.09±0.46, p=0.000; RI: 0.46±0.10 versus 0.60±0.14, p=0.010 respectively) evaluations were significantly higher in patients with adverse pregnancy outcome than in normal women. Combination of PI and RI >95th percentile and presence of bilateral notch in second trimester get sensitivity and specificity of 36.1 and 97% respectively, while these measures were 57.5 and 98.2% in third trimester. CONCLUSIONS: According to our study, it seems that uterine artery Doppler may be a valuable tool for the prediction of a variety of adverse outcomes in second and third trimesters.

Co-infection by porcine circovirus type 2 and porcine parvovirus in aborted fetuses and stillborn piglets in southern Brazil

Porcine circovirus types 1 and 2 (PCV1, PCV2) and porcine parvovirus (PPV) are widespread in pig populations around the world. Nevertheless, only PCV2 has been associated with different clinical syndromes, thus representing a major problem to the pig industry. The association of cases of swine abortions and stillborns with PCV1 and PCV2 and PPV was studied retrospectively (2005-2007). Additional pathogens were also investigated in lesioned fetuses. The studied litters included stillborn piglets and several mummified fetuses of varied sizes. Ventricular dilatation, myocardial pale areas, and mesocolic edema were the gross lesions. Escherichia coli was detected as co-infecting with PCV2 the cases in which mesocolic edema was seen. Microscopic lesions included non-suppurative myocarditis, myocardial necrosis and fibrosis, mineralization foci and intranuclear inclusion bodies in cardiomyocytes, and interstitial mononuclear pneumonia. Samples from 7 (5.78 per cent) of 121 aborted fetuses and stillborn piglets had lesions consistent with a viral cause and showed both positive anti-PCV2 immunostaining as well as PCV2-PCR. In samples from 3 (2.47 per cent) of these 7 fetuses, co-infection with PPV was confirmed by Nested-PCR. Both viruses were detected in fetuses at different stages of gestation. Viral antigens of PCV2 were detected by immunohistochemistry mainly in macrophages and myocytes. PCV1 individually was not detected in any of these affected fetuses, but it was associated with PCV2 and/or PPV in some of them. These findings indicate that PCV2 alone or in association with PPV should be kept in mind when investigating causes of infectious abortion in pigs in Brazil.

Interstitial nephritis of slaughtered pigs in the State of Mato Grosso, Brazil

This study evaluated histological lesions in kidney samples from pigs with nephritis in two slaughterhouses in the State of Mato Grosso, Brazil. Four hundred samples were subjected to histology, anti-porcine circovirus type 2 (PCV2) immunohistochemistry (IHC), anti-Leptospira sp. immunofluorescence (IF), and polymerase chain reaction (PCR) for PCV2, porcine parvovirus (PPV), and Torque teno virus type 1 and 2 (TTV1, TTV2) detection. Histological lesions were found in 81% of the samples, and mononuclear interstitial nephritis was the most frequent lesion (77.50%). A follicular pattern was observed in 40.97% of the interstitial nephritis lesions. PCV2, PPV, TTV1, and TTV2 were identified in the kidneys by PCR in 27.25%, 28.50%, 94%, and 87.5% of the samples, respectively. Leptospira sp. was not detected through IF. Infection by PCV2 (PCR) and the presence of histological lesions (P=0.008) and giant cells (P=0.0016) were significantly associated. An association was observed between the TTV2-TTV1 co-infection (P<0.0001) and the risk for pathogenesis. These findings indicated that PCV2, PPV, TTV1, and TTV2 were widely distributed among pigs in the local farms and that the presence of these agents should be considered in the differential diagnosis of kidneys with interstitial nephritis in pigs.

Effects of cytochrome P450 enzyme inhibitors and inducers on the metabolism of S-ketamine

The human body eliminates foreign compounds primarily by metabolizing them to hydrophilic forms to facilitate effective excretion through the kidneys. Cytochrome P450 (CYP) enzymes in the liver and intestine contribute to the metabolism of many drugs. Pharmacokinetic drugdrug interactions occur if the activity of CYPs are inhibited or induced by another drug. Prescribing multiple drugs to the improve effectiveness of therapy or to treat coexisting diseases is a common practice in clinical medicine. Polypharmacy predisposes patients to adverse effects because of the profound unpredictability in CYP enzymatic-mediated drug metabolism. S-ketamine is a phencyclidine derivative which functions as an antagonist of the N-methyl-Daspartate (NMDA) receptor in the central nervous system. It is a unique anaesthetic producing “dissociative anaesthesia” in high doses and analgesia in low doses. Studies with human liver microsomes suggest that ketamine is metabolized primarily via CYP3A4 and CYP2B6 enzymes. In this thesis, in healthy volunteers, randomized and controlled cross-over studies were conducted to investigate the effects of different CYP inducers and inhibitors on the pharmacokinetics and pharmacodynamics of oral and intravenous S-ketamine. The plasma concentrations of ketamine and its metabolite, norketamine, were determined at different timepoints over a 24 hour period. Other pharmacodynamic variables were examined for 12 hours. Results of these studies showed that the inhibition of the CYP3A4 pathway by clarithromycin or grapefruit juice increased the exposure to oral S-ketamine by 2.6- and 3.0-fold. Unexpectedly, CYP3A4 inhibition by itraconazole caused no significant alterations in the plasma concentrations of oral S-ketamine. CYP3A4 induction by St. John´s wort or rifampicin decreased profoundly the concentrations of oral S-ketamine. However, after rifampicin, there were no significant differences in the plasma concentrations of S-ketamine when it was administered intravenously. This demonstrated that rifampicin inhibited the metabolism of Sketamine at the intestinal level. When CYP2B6 was inhibited by ticlopidine, there was a 2.4- fold increase in the exposure of S-ketamine. These studies demonstrated that low dose oral Sketamine is metabolized both via CYP3A4 and CYP2B6 pathways. The concomitant use of drugs that affect CYP3A4 or CYP2B6, during oral S-ketamine treatment, may cause clinically significant drug-drug interactions.

Auscultatory and electrocardiographic characteristics of Crioulo horses

In order to determine auscultatory and electrocardiographic characteristics of Crioulo horses, one hundred animals ranging between one and twenty-six years of age (21 stallions, nine geldings, 27 pregnant mares e 43 not pregnant mares) were evaluated. The cardiac auscultation was performed during the clinical examination of the cardiovascular system, evaluating frequency, rate, normal and abnormal heart sounds (heart murmurs). The electrocardiographic examination followed the bipolar base-apex derivative system with animals at rest, by using an ECG-PC TEB equipment. The cardiac frequency, heart rate, morphology, duration, wave and complex amplitudes and interval durations were determined. The results were submitted to ANOVA and Tukey tests with an error probability of 5%. The cardiac auscultation revealed presence of functional systolic and diastolic murmur (10.00%) and systolic murmur compatible with tricuspid regurgitation besides normal heart sounds S1 (100.0%), S2 (100.0%), S3 (19.0%) and S4 (34.0%). The cardiac frequency obtained the average of 43.64 bpm, observing significative differences in relation to sexual and age factors and training level. The sinus rhythm was the most frequent (57.00%), followed by sinus tachycardia (38.00%) and sinus arrhythmia (5.00%), being observed rhythm disturbances in 16% of tracings. The P and T waves were observed more frequently in their forms P bifida positive (95.00%) and biphasic T (91.00%), being variable at tracing. There were also observed Q waves in 12.00% of the tracings. Thus, it was concluded that the auscultatory characteristics of Crioulo horses are according to the described in the literature for the species and the sexual factor, category, age factor and training level can influence some electrocardiographic parameters.

Estimation of aircraft aerodynamic derivatives using Extended Kalman Filter

Design of flight control laws, verification of performance predictions, and the implementation of flight simulations are tasks that require a mathematical model of the aircraft dynamics. The dynamical models are characterized by coefficients (aerodynamic derivatives) whose values must be determined from flight tests. This work outlines the use of the Extended Kalman Filter (EKF) in obtaining the aerodynamic derivatives of an aircraft. The EKF shows several advantages over the more traditional least-square method (LS). Among these the most important are: there are no restrictions on linearity or in the form which the parameters appears in the mathematical model describing the system, and it is not required that these parameters be time invariant. The EKF uses the statistical properties of the process and the observation noise, to produce estimates based on the mean square error of the estimates themselves. Differently, the LS minimizes a cost function based on the plant output behavior. Results for the estimation of some longitudinal aerodynamic derivatives from simulated data are presented.

Modelling Long-Run Relationship between Spot and Future Prices of Different Commodities

The purpose of this study is to investigate whether there exists any kind of relationship between the spot and future prices of the different commodities or not. Commodities like cocoa, coffee, crude oil, gold, natural gas and silver are considered from January 3, 2000 to December 31, 2012. For this purpose, ADF test and KPSS test are used in testing the stationarity whereas Johansen Cointegration test is used in testing the long-run relationship. Johansen co-integration test exhibits that there at least 5 co-integrating pairs out of 6 except crude oil. Moreover, the result of Granger Causality supports the fact that if two or more than two time series tend to be co-integrated there exists either uni-directional or bi-directional relationship. However, our results reveled that although there exists the co-integration between the variable, one might not granger causes another .VAR model is also used to measure the proportion of effects. These findings will help the derivative market and arbitragers in developing the strategies to gain the maximum profit in the financial market.

Selective inhibition of human tankyrases

Tankyrases belong to the Diphtheria toxin-like ADP-ribosyltransferase (ARTD) enzyme superfamily, also known as poly(ADP-ribose) polymerases (PARPs). They catalyze a covalent post-translational modification reaction where they transfer ADP-ribose units from NAD+ to target proteins. Tankyrases are involved in many cellular processes and their roles in telomere homeostasis, Wnt signaling and in several diseases including cancers have made them interesting drug targets. In this thesis project, selective inhibition of human tankyrases was studied. A homogeneous fluorescence-based assay was developed to screen the compound libraries. The assay is inexpensive, operationally easy, and performs well according to the statistical analysis. Assay suitability was confirmed by screening a natural product library. Flavone was identified as the most potent inhibitor in the library and this motivated us to screen a larger flavonoid library. Results showed that flavones were indeed the best inhibitor of tankyrases among flavonoids. To further study the structure-activity relationship, a small library of flavones containing single substitution was screened and potency measurements allowed us to generate structure-activity relationship. Compounds containing substitutions at 4´-position were more potent in comparison to other substitutions, and importantly, hydrophobic groups improved isoenzyme selectivity as well as the potency. A flavone derivative containing a hydrophobic isopropyl group (compound 22), displayed 6 nM potency against TNKS1, excellent isoenzyme selectivity and Wnt signaling inhibition. Protein interactions with compounds were studied by solving complex crystal structures of the compounds with TNKS2 catalytic domain. A novel tankyrase inhibitor (IWR-1) was also crystallized in complex with TNKS2 catalytic domain. The crystal structure of TNKS2 in complex with IWR-1 showed that the compound binds to adenosine site and it was the first known ARTD inhibitor of this kind. To date, there is no structural information available about the substrate binding with any of the ARTD family members; therefore NAD+ was soaked with TNKS2 catalytic domain crystals. However, analysis of crystal structure showed that NAD+ was hydrolyzed to nicotinamide. Also, a co-crystal structure of NAD+ mimic compound, EB-47, was solved which was used to deduce some insights about the substrate interactions with the enzyme. Like EB-47, other ARTD1 inhibitors were also shown to inhibit tankyrases. It indicated that selectivity of the ARTD1 inhibitors should be considered as some of the effects in cells could come from tankyrase inhibition. In conclusion, the study provides novel information on tankyrase inhibition and presents new insight into the selectivity and potency of compounds.

Holstein white coat color and performance: phenotypic, genetic and environmental correlations

Correlations of measures of percentages of white coat color, five measures of production and two measures of reproduction were obtained from 4293 first lactation Holsteins from eight Florida dairy farms. Percentages of white coat color were analyzed as recorded and transformed by an extension of Box-Cox procedures. Statistical analyses were by derivative-free restricted maximum likelihood (DFREML) with an animal model. Phenotypic and genetic correlations of white percentage (not transformed) were with milk yield, 0.047 and 0.097; fat yield, 0.002 and 0.004; fat percentage, -0.047 and -0.090; protein yield, 0.024 and 0.048; protein percentage, -0.070 and -0.116; days open, -0.012 and -0.065; and calving interval, -0.007 and -0.029. Changes in magnitude of correlations were very small for all variables except days open. Genetic and phenotypic correlations of transformed values with days open were -0.027 and -0.140. Modest positive correlated responses would be expected for white coat color percentage following direct selection for milk, fat, and protein yields, but selection for fat and protein percentages, days open, or calving interval would lead to small decreases.

Immunization against the colonization factor antigen I of enterotoxigenic Escherichia coli by administration of a bivalent Salmonella typhimurium aroA strain

An expression plasmid (pCFA-1) carrying the cfaB gene that codes for the enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin colonization factor antigen I (CFA/I) subunit was constructed and used to transform a derivative of the attenuated Salmonella typhimurium aroA vaccine strain SL3261 carrying an F'lacIq. Treatment of the transformed strain with isopropyl-ß-D-thiogalactopyranoside (IPTG) resulted in elevated in vitro expression of the CFA/I subunit. Although flagellar function and lipopolysaccharide (LPS) synthesis were similar in both the parental and the recombinant strains, spleen colonization was reduced in the recombinant strain. All BALB/c mice parenterally inoculated with the recombinant strain developed significant anti-CFA/I and anti-LPS serum antibody titers (P<0.05). Moreover, 2 of 5 mice orally inoculated with the engineered Salmonella strain developed anti-CFA/I intestinal IgA (P>0.05) while 4/5 of the same mice developed anti-LPS IgA (P<0.05). The results indicate that the vaccine strain elicited an antibody response against the bacterial host both after oral and intravenous immunization while the response against the CFA/I antigen was significant only after inoculation by the intravenous route

Further analysis of open-respirometry systems: an a-compartmental mechanistic approach

A system is said to be "instantaneous" when for a given constant input an equilibrium output is obtained after a while. In the meantime, the output is changing from its initial value towards the equilibrium one. This is the transient period of the system and transients are important features of open-respirometry systems. During transients, one cannot compute the input amplitude directly from the output. The existing models (e.g., first or second order dynamics) cannot account for many of the features observed in real open-respirometry systems, such as time lag. Also, these models do not explain what should be expected when a system is speeded up or slowed down. The purpose of the present study was to develop a mechanistic approach to the dynamics of open-respirometry systems, employing basic thermodynamic concepts. It is demonstrated that all the main relevant features of the output dynamics are due to and can be adequately explained by a distribution of apparent velocities within the set of molecules travelling along the system. The importance of the rate at which the molecules leave the sensor is explored for the first time. The study approaches the difference in calibrating a system with a continuous input and with a "unit impulse": the former truly reveals the dynamics of the system while the latter represents the first derivative (in time) of the former and, thus, cannot adequately be employed in the apparent time-constant determination. Also, we demonstrate why the apparent order of the output changes with volume or flow.

DNA topoisomerase inhibitors: biflavonoids from Ouratea species

Topoisomerase inhibitors are agents with anticancer activity. 7"-O-Methyl-agathisflavone (I) and amentoflavone (II) are biflavonoids and were isolated from the Brazilian plants Ouratea hexasperma and O. semiserrata, respectively. These biflavonoids and the acetyl derivative of II (IIa) are inhibitors of human DNA topoisomerases I at 200 µM, as demonstrated by the relaxation assay of supercoiled DNA, and only agathisflavone (I) at 200 µM also inhibited DNA topoisomerases II-alpha, as observed by decatenation and relaxation assays. The biflavonoids showed concentration-dependent growth inhibitory activities on Ehrlich carcinoma cells in 45-h culture, assayed by a tetrazolium method, with IC50 = 24 ± 1.4 µM for I, 26 ± 1.1 µM for II and 10 ± 0.7 µM for IIa. These biflavonoids were assayed against human K562 leukemia cells in 45-h culture, but only I showed 42% growth inhibitory activity at 90 µM. Our results suggest that biflavonoids are targets for DNA topoisomerases and their cytotoxicity is dependent on tumor cell type.

Plasma free and total carnitine measured in children by tandem mass spectrometry

Free and total carnitine quantification is important as a complementary test for the diagnosis of unusual metabolic diseases, including fatty acid degradation disorders. The present study reports a new method for the quantification of free and total carnitine in dried plasma specimens by isotope dilution electrospray tandem mass spectrometry with sample derivatization. Carnitine is determined by looking for the precursor of ions of m/z = 103 of N-butylester derivative, and the method is validated by comparison with radioenzymatic assay. We obtained an inter- and intra-day assay coefficient of variation of 4.3 and 2.3, respectively. Free and total carnitine was analyzed in 309 dried plasma spot samples from children ranging in age from newborn to 14 years using the new method, which was found to be suitable for calculating reference age-related values for free and total carnitine (less than one month: 19.3 ± 2.4 and 23.5 ± 2.9; one to twelve months: 28.8 ± 10.2 and 35.9 ± 11.4; one to seven years: 30.7 ± 10.3 and 38.1 ± 11.9; seven to 14 years: 33.7 ± 11.6, and 43.1 ± 13.8 µM, respectively). No difference was found between males and females. A significant difference was observed between neonates and the other age groups. We compare our data with reference values in the literature, most of them obtained by radioenzymatic assay. However, this method is laborious and time consuming. The electrospray tandem mass spectrometry method presented here is a reliable, rapid and automated procedure for carnitine quantitation.