Incerteza intervalar em otimização e controle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Parâmetros genéticos para características indicadoras de eficiência reprodutiva e produtiva de ovinos da raça Santa Inês

Pós-graduação em Genética e Melhoramento Animal - FCAV

Role of the amygdala in the reinforcement omission effect

The reinforcement omission effect (ROE) has been attributed to both motivational and attentional consequences of surprising reinforcement omission. Recent evidence suggests that the basolateral complex of the amygdala is involved in motivational components related to reinforcement value, whereas the central nucleus of the amygdala is involved in the processing of the attentional consequences of surprise. This study was designed to verify whether the mechanisms involved in the ROE depend on the integrity of either the basolateral amygdala complex or central nucleus of the amygdala. The ROE was evaluated in rats with lesions of either the central nucleus or basolateral complex of the amygdala and trained on a fixed-interval schedule procedure (Experiment 1) and fixed-interval with limited hold signaled schedule procedure (Experiment 2). The results of Experiment 1 showed that sham-operated rats and rats with lesions of either the central nucleus or basolateral area displayed the ROE. In contrast, in Experiment 2, subjects with lesions of the central nucleus or basolateral complex of the amygdala exhibited a smaller ROE compared with sham-operated subjects. Thus, the effects of selective lesions of amygdala subregions on the ROE in rats depended on the training procedure. Furthermore, the absence of differences between the lesioned groups in either experiment did not allow the dissociation of attentional or motivational components of the ROE with functions of specific areas of the amygdala. Thus, results did not show a functional double-dissociation between the central nucleus and basolateral area in the ROE.

Tetracycline-inducible RNAi Knockdown of SCL (Stem Cell Leukaemia) in Mice

RNAi (RNA interference) is a powerful technology for sequence-specific targeting of mRNAs. This thesis was aimed at establishing conditions for conditional RNAi-mediated silencing first in vitro and subsequently also in transgenic mice. As a target the basic helix-loop-helix transcription factor encoding gene SCL (stem cell leukaemia also known as Tal-1 or TCL5) was used. SCL is a key regulator for haematopoietic development and ectopic expression of SCL is correlated with acute T-lymphoblastic leukaemias. Loss of SCL function studies demonstrated that ab initio deletion of SCL resulted in embryonic lethality around day E9 in gestation. To be able to conditionally inactivate SCL, RNAi technology was combined with the tetracycline-dependent regulatory system. This strategy allowed to exogenously control the induction of RNAi in a reversible fashion and consequently the generation of a completely switchable RNAi knockdown. First a suitable vector allowing for co-expression of tetracycline-controlled shRNAs (small hairpin RNAs) and constitutively active EGFP (enhanced green fluorescent protein) was generated. This novel vector, pRNAi-EGFP, was then evaluated for EGFP expression and tetracycline-mediated expression of shRNAs. Four sequences targeting different regions within the SCL mRNA were tested for their efficiency to specifically knockdown SCL. These experiments were performed in M1 murine leukaemia cells and subsequently in the HEK 293 cell line, expressing an engineered HA-tagged SCL protein. The second assay provided a solid experimental method for determining the efficiency of different SCL-siRNA knockdown constructs in tissue culture. Western blotting analyses revealed a down regulation of SCL protein for all four tested SCL-specific target sequences albeit with different knockdown efficiencies (between 25% and 100%). Furthermore, stringent tetracycline-dependent switchability of shRNA expression was confirmed by co-transfecting the SCL-specific pRNAi-EGFP vector (SCL-siRNA) together with the HA-tagged SCL expression plasmid into the HEK 293TR /T-REx cell line constitutively expressing the tetracycline repressor (TetR). These series of experiments demonstrated tight regulation of siRNA expression without background activity. To be able to control the SCL knockdown in vivo and especially to circumvent any possible embryonic lethality a transgenic mouse line with general expression of a tetracycline repressor was needed. Two alternative methods were used to generate TetR mice. The first approach was to co-inject the tetracycline-regulated RNAi vector together with a commercially available and here specifically modified T-REx expression vector (SCL-siRNA T-REx FRT LoxP mouse line). The second method involved the generation of a TetR expressor mouse line, which was then used for donating TetR-positive oocytes for pronuclear injection of the RNAi vector (SCL-siRNA T-REx mouse line). As expected, and in agreement with data from conditional Cre-controlled adult SCL knockout mice, post-transcriptional silencing of SCL by RNAi caused a shift in the maturation of red blood cell populations. This was shown in the bone marrow and peripheral blood by FACS analysis with the red blood cell-specific TER119 and CD71 markers which can be used to define erythrocyte differentiation (Lodish plot technique). In conclusion this study established conditions for effective SCL RNAi-mediated silencing in vitro and in vivo providing an important tool for further investigations into the role of SCL and, more generally, of its in vivo function in haematopoiesis and leukaemia. Most importantly, the here acquired knowledge will now allow the establishment of other completely conditional and reversible knockdown phenotypes in mice.

Compliance lebertransplantierter Patienten mit dem Immunsuppressivum Tacrolimus bei 2x täglicher im Vergleich zu 1x täglicher Einnahme

Compliance lebertransplantierter Patienten mit der immunsuppressiven Therapie ist unerlässlich für den lang-fristigen Erfolg der Lebertransplantation. Aus Non-Compliance mit der immunsuppressiven Therapie können Abstoßungsreaktionen, Organverlust oder sogar Tod resultieren. Hauptziel der vorliegenden Studie war die erstmalige Evaluation der Compliance bei Einnahme von Prograf® (zweimal tägliche Einnahme von Tacrolimus) im Vergleich zur Einnahme von Advagraf® (einmal tägliche Einnahme von Tacrolimus). Von Interesse war außerdem die Fragestellung, ob sich die Compliance bezüglich der immunsuppressiven Therapie mit dem Zeitabstand zur Transplantation verändert. rnDie Compliancemessung wurde offen mittels MEMS® (Aardex Ltd., Schweiz) durchgeführt, der Patient war also über die Compliancekontrolle informiert. Mittels MEMS® konnten Datum und Uhrzeit der Dosisentnahme dokumentiert und damit zuverlässig das gesamte Compliancemuster über im Durchschnitt 176 Tage mit der zweimal täglichen Einnahme und 188 Tage mit der einmal täglichen Einnahme pro Patient erfasst werden. 65 Patienten mit dem Basisimmunsuppressivum Prograf® wurden in die prospektive, nicht-interventionelle Studie eingeschlossen und nach Per Protokoll-Analyse konnten die Daten von 63 in Mainz lebertransplantierten Patienten ausgewertet werden (Prograf®: Gruppe 1: 15 Patienten (Pat.), Gruppe 2: 23 Pat., Gruppe 3: 22 Pat., Drop-outs: 3 Pat.; Advagraf®: Gruppe 1: 16 Pat., Gruppe 2: 23 Pat., Gruppe 3: 23 Pat., Drop-outs: 1 Pat.). Die Dosing Compliance (DC), definiert als Prozent der Tage, an denen der MEMS®-Behälter korrekt geöffnet und die Dosis höchstwahrscheinlich korrekt eingenommen wurde, war der primäre Zielparameter. Weitere Methoden der Compliancemessung, wie der Pill Count, mehrere Fragebögen (Selbsteinschätzung, Patientenwissen-, Morisky-, MESI-, HADS-, SF-36- und Patientenzufriedenheit-Fragebogen) sowie die Blutspiegelmessung wurden eingesetzt, um die Compliance der Patienten umfassend charakterisieren zu können. rnDer Median der DC mit der zweimal täglichen Einnahme betrug 97% bei Pat. > 6 m.p.t. < 2 y.p.t., 97% bei Pat. > 2 y.p.t. < 5 y.p.t. und 98% bei Pat. > 5 y.p.t. (p=0,931; Kruskal-Wallis-Test). Der Median der DC von Tacroli-mus bei einmal täglicher Einnahme (Advagraf®) betrug 99% bei Pat. > 6 m.p.t. < 2 y.p.t., 98% bei Pat. > 2 y.p.t. < 5 y.p.t. und 97% bei Pat. > 5 y.p.t. (p=0,158; Kruskal-Wallis-Test). Insgesamt zeigten die Patienten während des gesamten Beobachtungszeitraums von 12 Monaten eine gute Compliance für die Einnahme ihres Immun-suppressivums. Die Timing Compliance (TiC)-raten lagen auf einem niedrigeren Niveau als die Dosing- und Taking Compliance (TC)-raten. Die Complianceraten der drei Subgruppen unterschieden sich nicht signifikant. Die Patienten mit dem geringsten Abstand zur Transplantation zeigten bei beinahe allen Messmethoden die höchste Compliance im Gegensatz zur etwas geringeren Compliance der Patienten mit größerem Abstand zur Transplantation. Die während der Advagraf®-Phase mittels MEMS® gemessenen DC-, TC- und TiC-raten fielen höher aus als bei Einnahme von Prograf® (p(DC)=0,003; p(TC)=0,077; p(TiC)=0,003; Wilcoxon Vorzeichen-Rang-Test). Dieses Ergebnis untermauert die in anderen Indikationen gefundene Complianceverbesserung durch die einmal tägliche Arzneimittelgabe im Vergleich zur zweimal täglichen Gabe. Die Auswertung der Drug Holidays ergab für die Advagraf®-Phase hingegen niedrigere Complianceraten als für die Prograf®-Phase. Dieses Ergebnis ist auf die Definition des Drug Holidays (keine Arzneimitteleinnahme über 48 h) zurück zu führen. Die Chance Advagraf® einmal pro Tag zu vergessen ist doppelt so hoch, als Prograf® dreimal aufeinander fol-gend zu vergessen. Mit einer verhältnismäßigeren Definition von Drug Holidays (Einnahmepause von 72 Stun-den bei einmal täglicher Einnahme von Advagraf® entsprechend drei ausgelassenen Dosen von Prograf®) ist die Compliancerate 81%. Die Ergebnisse des Pill Counts waren sowohl bei Einnahme von Prograf® als auch von Advagraf® mit der jeweils gemessenen TC vergleichbar, was die Zuverlässigkeit der Messergebnisse bes-tätigt. rnDie zusätzlich eingesetzten Methoden verifizierten das Ergebnis der höheren Compliance mit der einmal tägli-chen Einnahme. Die während der Advagraf®-Phase beantworteten Fragebögen zeigten einen Trend zu besserer Compliance und Lebensqualität. Lediglich die Ergebnisse des MESI-Fragebogens und der Blutspiegelmessungen wichen sowohl während der Prograf®- als auch während der Advagraf®-Phase stark von den Ergebnis-sen der anderen Methoden ab. rnUnter Einbeziehung aller mittels MEMS® und Pill Count objektiv gemessenen Complianceparameter konnten während der Prograf®-Einnahme 54 von 60 Pat. (90%) und während der Advagraf®-Phase 59 von 62 Pat. (95%) als compliant eingestuft werden. Aufgrund subjektiver Compliancemessungen waren 49 von 58 Pat. (84%) während der Prograf®- und 54 von 59 Pat. (92%) während der Advagraf®-Phase als compliant einzustufen. Es wurde beobachtet, dass die zeitlich korrekte Einnahme der Morgendosis einfacher und bei Einmalgabe zu bevorzugen ist. Die wochentagsbezogene Auswertung ergab erwartungsgemäß, dass am Wochenende (Samstag und Sonntag) am häufigsten Dosen ausgelassen wurden. rnDie Umstellung von Prograf® auf Advagraf® stellte kein Problem dar. Beinahe alle Patienten waren dankbar und zufrieden mit der Reduzierung der Dosierungsfrequenz und der größeren Unabhängigkeit durch die entfallene abendliche Einnahme. Der positive Einfluss der geringeren Dosierungshäufigkeit auf die Langzeitcompliance der Patienten, ist ein hinreichender Grund die Entwicklung von Formulierungen zur einmal täglichen Ein-nahme für weitere Immunsuppressiva zu fordern. Insbesondere bei den häufig eingesetzten Kombinationstherapien von Immunsuppressiva würde der Effekt der Complianceverbesserung noch verstärkt werden, wenn alle eingesetzten Immunsuppressiva zur einmal täglichen Gabe geeignet wären.

Evaluation of p24-based antiretroviral treatment monitoring in pediatric HIV-1 infection: prediction of the CD4+ T-cell changes between consecutive visits

Worldwide, 700,000 infants are infected annually by HIV-1, most of them in resource-limited settings. Care for these children requires simple, inexpensive tests. We have evaluated HIV-1 p24 antigen for antiretroviral treatment (ART) monitoring in children. p24 by boosted enzyme-linked immunosorbent assay of heated plasma and HIV-1 RNA were measured prospectively in 24 HIV-1-infected children receiving ART. p24 and HIV-1 RNA concentrations and their changes between consecutive visits were related to the respective CD4+ changes. Age at study entry was 7.6 years; follow-up was 47.2 months, yielding 18 visits at an interval of 2.8 months (medians). There were 399 complete visit data sets and 375 interval data sets. Controlling for variation between individuals, there was a positive relationship between concentrations of HIV-1 RNA and p24 (P < 0.0001). While controlling for initial CD4+ count, age, sex, days since start of ART, and days between visits, the relative change in CD4+ count between 2 successive visits was negatively related to the corresponding relative change in HIV-1 RNA (P = 0.009), but not to the initial HIV-1 RNA concentration (P = 0.94). Similarly, we found a negative relationship with the relative change in p24 over the interval (P < 0.0001), whereas the initial p24 concentration showed a trend (P = 0.08). Statistical support for the p24 model and the HIV-1 RNA model was similar. p24 may be an accurate low-cost alternative to monitor ART in pediatric HIV-1 infection.

The role of epistasis in the manifestation of heterosis: a systems-oriented approach

Heterosis is widely used in breeding, but the genetic basis of this biological phenomenon has not been elucidated. We postulate that additive and dominance genetic effects as well as two-locus interactions estimated in classical QTL analyses are not sufficient for quantifying the contributions of QTL to heterosis. A general theoretical framework for determining the contributions of different types of genetic effects to heterosis was developed. Additive x additive epistatic interactions of individual loci with the entire genetic background were identified as a major component of midparent heterosis. On the basis of these findings we defined a new type of heterotic effect denoted as augmented dominance effect di* that comprises the dominance effect at each QTL minus half the sum of additive x additive interactions with all other QTL. We demonstrate that genotypic expectations of QTL effects obtained from analyses with the design III using testcrosses of recombinant inbred lines and composite-interval mapping precisely equal genotypic expectations of midparent heterosis, thus identifying genomic regions relevant for expression of heterosis. The theory for QTL mapping of multiple traits is extended to the simultaneous mapping of newly defined genetic effects to improve the power of QTL detection and distinguish between dominance and overdominance.

Cartilage T2 assessment at 3-T MR imaging: in vivo differentiation of normal hyaline cartilage from reparative tissue after two cartilage repair procedures--initial experience

PURPOSE: To prospectively compare cartilage T2 values after microfracture therapy (MFX) and matrix-associated autologous chondrocyte transplantation (MACT) repair procedures. MATERIALS AND METHODS: The study had institutional review board approval by the ethics committee of the Medical University of Vienna; informed consent was obtained. Twenty patients who underwent MFX or MACT (10 in each group) were enrolled. For comparability, patients of each group were matched by mean age (MFX, 40.0 years +/- 15.4 [standard deviation]; MACT, 41.0 years +/- 8.9) and postoperative interval (MFX, 28.6 months +/- 5.2; MACT, 27.4 months +/- 13.1). Magnetic resonance (MR) imaging was performed with a 3-T MR imager, and T2 maps were calculated from a multiecho spin-echo measurement. Global, as well as zonal, quantitative T2 values were calculated within the cartilage repair area and within cartilage sites determined to be morphologically normal articular cartilage. Additionally, with consideration of the zonal organization, global regions of interest were subdivided into deep and superficial areas. Differences between cartilage sites and groups were calculated by using a three-way analysis of variance. RESULTS: Quantitative T2 assessment of normal native hyaline cartilage showed similar results for all patients and a significant trend of increasing T2 values from deep to superficial zones (P < .05). In cartilage repair areas after MFX, global mean T2 was significantly reduced (P < .05), whereas after MACT, mean T2 was not reduced (P > or = .05). For zonal variation, repair tissue after MFX showed no significant trend between different depths (P > or = .05), in contrast to repair tissue after MACT, in which a significant increase from deep to superficial zones (P < .05) could be observed. CONCLUSION: Quantitative T2 mapping seems to reflect differences in repair tissues formed after two surgical cartilage repair procedures. (c) RSNA, 2008.

Differentiating normal hyaline cartilage from post-surgical repair tissue using fast gradient echo imaging in delayed gadolinium-enhanced MRI (dGEMRIC) at 3 Tesla

The purpose was to evaluate the relative glycosaminoglycan (GAG) content of repair tissue in patients after microfracturing (MFX) and matrix-associated autologous chondrocyte transplantation (MACT) of the knee joint with a dGEMRIC technique based on a newly developed short 3D-GRE sequence with two flip angle excitation pulses. Twenty patients treated with MFX or MACT (ten in each group) were enrolled. For comparability, patients from each group were matched by age (MFX: 37.1 +/- 16.3 years; MACT: 37.4 +/- 8.2 years) and postoperative interval (MFX: 33.0 +/- 17.3 months; MACT: 32.0 +/- 17.2 months). The Delta relaxation rate (DeltaR1) for repair tissue and normal hyaline cartilage and the relative DeltaR1 were calculated, and mean values were compared between both groups using an analysis of variance. The mean DeltaR1 for MFX was 1.07 +/- 0.34 versus 0.32 +/- 0.20 at the intact control site, and for MACT, 1.90 +/- 0.49 compared to 0.87 +/- 0.44, which resulted in a relative DeltaR1 of 3.39 for MFX and 2.18 for MACT. The difference between the cartilage repair groups was statistically significant. The new dGEMRIC technique based on dual flip angle excitation pulses showed higher GAG content in patients after MACT compared to MFX at the same postoperative interval and allowed reducing the data acquisition time to 4 min.

Recurrence characteristics in European patients with ocular toxoplasmosis

BACKGROUND: The risk of function loss after each episode of ocular toxoplasmosis (OT) supports efforts to improve our understanding of the disease. Patients and methods: 139 patients with OT were contacted retrospectively and requested to complete a questionnaire addressing course and activity of their disease. This information was compared with that retrieved from their medical records. Sixty-three patients completed the questionnaire and were included in the study. They were allocated according to their median age to one of two groups (group 1: <20.9 years; group 2: >or=20.9 years). RESULTS: The mean reported age at the time of first ocular manifestation was 23.9 (median 20.9, range 0 to 70.5; SD 12.9) years. The clinical diagnosis was made 3.5 years later (p = 0.0008). The follow-up time was 6.5 (median 5.0; range 0.5 to 49.9; SD 7.6) years. The recurrence rate was higher in patients below 20.9 years (66%; n = 35) than in older patients (39%; n = 28; chi(2) test, p<0.05). Patients reporting only one episode were older at first manifestation (29.6 (median 25.6; range 10.6 to 70.5; SD 14.3) years; n = 29) than those reporting two episodes (17.9 (median 19.5; range 5.9 to 33.9; SD 7.8) years; n = 15 (p<0.05)). The proportion of patients who developed a recurrence was 54-63% after each episode without a tendency to enlarge, and the interval between successive episodes remained stable between 1.0 and 1.7 years for the first three recurrences. CONCLUSION: Younger OT patients carry a higher risk of developing a recurrence than older ones. After each episode, two-thirds of all OT patients will develop another one.

ROBREG: Stata module providing robust regression estimators

robreg provides a number of robust estimators for linear regression models. Among them are the high breakdown-point and high efficiency MM-estimator, the Huber and bisquare M-estimator, and the S-estimator, each supporting classic or robust standard errors. Furthermore, basic versions of the LMS/LQS (least median of squares) and LTS (least trimmed squares) estimators are provided. Note that the moremata package, also available from SSC, is required.

Deep Into the Fibers! Postmortem Diffusion Tensor Imaging in Forensic Radiology.

PURPOSE In traumatic brain injury, diffusion-weighted and diffusion tensor imaging of the brain are essential techniques for determining the pathology sustained and the outcome. Postmortem cross-sectional imaging is an established adjunct to forensic autopsy in death investigation. The purpose of this prospective study was to evaluate postmortem diffusion tensor imaging in forensics for its feasibility, influencing factors and correlation to the cause of death compared with autopsy. METHODS Postmortem computed tomography, magnetic resonance imaging, and diffusion tensor imaging with fiber tracking were performed in 10 deceased subjects. The Likert scale grading of colored fractional anisotropy maps was correlated to the body temperature and intracranial pathology to assess the diagnostic feasibility of postmortem diffusion tensor imaging and fiber tracking. RESULTS Optimal fiber tracking (>15,000 fiber tracts) was achieved with a body temperature at 10°C. Likert scale grading showed no linear correlation (P > 0.7) to fiber tract counts. No statistically significant correlation between total fiber count and postmortem interval could be observed (P = 0.122). Postmortem diffusion tensor imaging and fiber tracking allowed for radiological diagnosis in cases with shearing injuries but was impaired in cases with pneumencephalon and intracerebral mass hemorrhage. CONCLUSIONS Postmortem diffusion tensor imaging with fiber tracking provides an exceptional in situ insight "deep into the fibers" of the brain with diagnostic benefit in traumatic brain injury and axonal injuries in the assessment of the underlying cause of death, considering influencing factors for optimal imaging technique.

Optimization of Optical Follow-up Strategies Based on Covariance Analysis

An in-depth study, using simulations and covariance analysis, is performed to identify the optimal sequence of observations to obtain the most accurate orbit propagation. The accuracy of the results of an orbit determination/ improvement process depends on: tracklet length, number of observations, type of orbit, astrometric error, time interval between tracklets and observation geometry. The latter depends on the position of the object along its orbit and the location of the observing station. This covariance analysis aims to optimize the observation strategy taking into account the influence of the orbit shape, of the relative object-observer geometry and the interval between observations.