Evaluation of femur of orchiectomized guinea pigs by bone densitometry using dual-energy x-ray absortiometry (DXA) and mechanical testing

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeitos das antocianinas, ácidos hidroxinâmicos e vitamina C sobre a biometria, composição corporal e qualidade óssea de ratos Wistar

Pós-graduação em Medicina Veterinária - FCAV

EFFECTS OF A BIRD HAZARD REDUCTION FORCE ON REDUCING BIRD/AIRCRAFT STRIKE HAZARDS AT THE ATLANTIC CITY INTERNATIONAL AIRPORT, NJ

Bird-aircraft strikes at the Atlantic City International Airport (ACY) increased from 18 in 1989 to 37 in 1990. The number of bird-aircraft strikes involving gulls (Larus spp.) during this time rose from 6 to 27, a 350% increase. The predominant species involved in bird strikes was the laughing gull (L. atricilla). Pursuant to an interagency agreement between the U.S. Department of Transportation (USDOT), Federal Aviation Administration (FAA) and the U.S. Department of Agriculture (USDA)l Animal and Plant Health Inspection Service (APHIS)/Animal Damage Control (ADC), ADC established a Emergency/Experimental Bird Hazard Reduction Force (BHFF) at ACY in 1991. An Environmental Assessment (EA) and Finding of No Significant Impact (FONSI) for the 1991 Emergency/Experimental BHRF was executed and signed by the FAA on 19 May 1991. The BHRF was adopted at this time by the FAA Technical Center as an annual program to reduce bird strikes at ACY. The BHRF goals are to minimize or eliminate the incidence of bird-aircraft strikes and runway closures due to increased bird activities. A BHRF team consisting of ADC personnel patrolled ACY for 95 days from 26 May until 28 August 1992, for a total of 2,949 person-hours. The BHRF used a combination of pyrotechnics, amplified gull distress tapes and live ammunition to harass gulls away from the airport from dawn to dusk. Gullaircraft strikes were reduced during BHRF operations in 1992 by 86% compared to gull strikes during summer months of 1990 when there was not a BHRF team. Runway closures due to bird activity decreased 100% compared to 1990 and 1991 closures. The BHRF should continue at ACY as long as birds are a threat to human safety and aircraft operations.

Chronic Intermittent Hypoxia Depresses Afferent Neurotransmission in NTS Neurons by a Reduction in the Number of Active Synapses

Long-term synaptic plasticity has been recently described in brainstem areas associated to visceral afferent sensory integration. Chronic intermittent hypoxia (CIH), an animal model for studying obstructive sleep apnea in humans, depresses the afferent neurotransmission in nucleus tractus solitarii (NTS) neurons, which affect respiratory and autonomic regulation. Here we identified the synaptic mechanisms of CIH-induced depression of the afferent neurotransmission in NTS neurons in juvenile rats. We verified that CIH reduced the amplitude of both NMDA and non-NMDA glutamatergic excitatory currents (eEPSCs) evoked by tractus solitarii stimulation (TS-eEPSC) of second-order neurons in the NTS. No changes were observed in release probability, evidenced by absence of any CIH-elicited effects on short-term depression and failures in EPSCs evoked in low calcium. CIH also produced no changes in TS-eEPSC quantal size, since the amplitudes of both low calcium-evoked EPSCs and asynchronous TS-eEPSCs (evoked in the presence of Sr2+) were unchanged. Using single TS afferent fiber stimulation in slices from control and CIH rats we clearly show that CIH reduced the quantal content of the TS-eEPSCs without affecting the quantal size or release probability, suggesting a reduction in the number of active synapses as the mechanism of CIH induced TS-eEPSC depression. In accordance with this concept, the input-output relationship of stimulus intensity and TS-eEPSC amplitude shows an early saturation in CIH animals. These findings open new perspectives for a better understanding of the mechanisms underlying the synaptic plasticity in the brainstem sensory neurons under challenges such as those produced by CIH in experimental and pathological conditions.

Progress in animal experimentation ethics. A case study from a brazilian medical school and from the international medical literature

PURPOSE: This study describes in Brazil and in the global biomedical community the time course of the development of animal research welfare guidelines. METHODS: The database of the Ethics Committee of the Faculty of Medicine of Ribeirao Preto (EC/FMRP-USP), Brazil, was surveyed since its inception in 2002 as the regulations became more stringent to provide better protection of animal research welfare at this institution. Medline database was evaluated to identify the number of publications in the period between 1968 and 2008 that used research animals and were in compliance with established ethics guidelines. RESULTS: The EC/FMRP-USP evaluated 979 projects up until 2009. Most of the applications came from Department of Physiology and the most frequently requested species was the rat. In 2004, national research funding agencies started to request prior approval from institutional review ethics committees prior to application review and this requirement became federal law in Brazil in 2008. The analysis of international publications revealed a relative reduction in studies involving research animals (18% in 1968 to 7.5% in 2008). CONCLUSIONS: The present work showed that in the last four decades major changes occurred in the guidelines dictating use of research animals occurred and they are being adopted by developing countries. Moreover, animal welfare concern in the scientific community preceded the introduction of journal guidelines for this purpose. Furthermore, in Brazil it was anticipated that laws were needed to protect animal research welfare from being not upheld.

Gametic phase disequilibrium between the syntenic multiallelic HTG4 and HMS3 markers widely used for parentage testing in Thoroughbred horses

Validation of parentage and horse breed registries through DNA typing relies on estimates of random match probabilities with DNA profiles generated from multiple polymorphic loci. Of the twenty-seven microsatellite loci recommended by the International Society for Animal Genetics for parentage testing in Thoroughbred horses, eleven are located on five chromosomes. An important aspect in determining combined exclusion probabilities is the ascertainment of the genetic linkage status of syntenic markers, which may affect reliable use of the product rule in estimating random match probabilities. In principle, linked markers can be in gametic phase disequilibrium (GD). We aimed at determining the extent, by frequency and strength, of GD between the HTG4 and HMS3 multiallelic loci, syntenic on chromosome 9. We typed the qualified offspring (n (1) = 27; n (2) = 14) of two Quarter Bred stallions (registered by the Brazilian Association of Quarter Horse Breeders) and 121 unrelated horses from the same breed. In the 41 informative meioses analyzed, the frequency of recombination between the HTG4 and HMS3 loci was 0.27. Consistent with genetic map distances, this recombination rate does not fit to the theoretical distribution for independently segregated markers. We estimated sign-based D' coefficients as a measure of GD, and showed that the HTG4 and HMS3 loci are in significant, yet partial and weak, disequilibrium, with two allele pairs involved (HTG4*M/HMS3*P, D'(+) = 0.6274; and HTG4*K/HMS3*P, D'(-) = -0.6096). These results warn against the inadequate inclusion of genetically linked markers in the calculation of combined power of discrimination for Thoroughbred parentage validation.

Cardiometabolic risk reduction through lifestyle intervention programs in the Brazilian public health system

Public health strategies to reduce cardiovascular morbidity and mortality should focus on global cardiometabolic risk reduction. The efficacy of lifestyle changes to prevent type 2 diabetes have been demonstrated, but low-cost interventions to reduce cardiometabolic risk in Latin-America have been rarely reported. Our group developed 2 programs to promote health of high-risk individuals attending a primary care center in Brazil. This study compared the effects of two 9-month lifestyle interventions, one based on medical consultations (traditional) and another with 13 multi-professional group sessions in addition to the medical consultations (intensive) on cardiometabolic parameters. Adults were eligible if they had pre-diabetes (according to the American Diabetes Association) and/or metabolic syndrome (International Diabetes Federation criteria for Latin-America). Data were expressed as means and standard deviations or percentages and compared between groups or testing visits. A p-value < 0.05 was considered significant. Results: 180 individuals agreed to participate (35.0% men, mean age 54.7 ± 12.3 years, 86.1% overweight or obese). 83 were allocated to the traditional and 97 to the intensive program. Both interventions reduced body mass index, waist circumference and tumor necrosis factor-α. Only intensive program reduced 2-hour plasma glucose and blood pressure and increased adiponectin values, but HDL-cholesterol increased only in the traditional. Also, responses to programs were better in intensive compared to traditional program in terms of blood pressure and adiponectin improvements. No new case of diabetes in intensive but 3 cases and one myocardial infarction in traditional program were detected. Both programs induced metabolic improvement in the short-term, but if better results in the intensive are due to higher awareness about risk and self-motivation deserves further investigation. In conclusion, these low-cost interventions are able to minimize cardiometabolic risk factors involved in the progression to type 2 diabetes and/or cardiovascular disease.

Progress in animal experimentation ethics: a case study from a Brazilian medical school and from the international medical literature

PURPOSE: This study describes in Brazil and in the global biomedical community the time course of the development of animal research welfare guidelines. METHODS: The database of the Ethics Committee of the Faculty of Medicine of Ribeirao Preto (EC/FMRP-USP), Brazil, was surveyed since its inception in 2002 as the regulations became more stringent to provide better protection of animal research welfare at this institution. Medline database was evaluated to identify the number of publications in the period between 1968 and 2008 that used research animals and were in compliance with established ethics guidelines. RESULTS: The EC/FMRP-USP evaluated 979 projects up until 2009. Most of the applications came from Department of Physiology and the most frequently requested species was the rat. In 2004, national research funding agencies started to request prior approval from institutional review ethics committees prior to application review and this requirement became federal law in Brazil in 2008. The analysis of international publications revealed a relative reduction in studies involving research animals (18% in 1968 to 7.5% in 2008). CONCLUSIONS: The present work showed that in the last four decades major changes occurred in the guidelines dictating use of research animals occurred and they are being adopted by developing countries. Moreover, animal welfare concern in the scientific community preceded the introduction of journal guidelines for this purpose. Furthermore, in Brazil it was anticipated that laws were needed to protect animal research welfare from being not upheld.

Adaptations of the aging animal to exercise: role of daily supplementation with melatonin

The pineal gland, through melatonin, seems to be of fundamental importance in determining the metabolic adaptations of adipose and muscle tissues to physical training. Evidence shows that pinealectomized animals fail to develop adaptive metabolic changes in response to aerobic exercise and therefore do not exhibit the same performance as control-trained animals. The known prominent reduction in melatonin synthesis in aging animals led us to investigate the metabolic adaptations to physical training in aged animals with and without daily melatonin replacement. Male Wistar rats were assigned to four groups: sedentary control (SC), trained control (TC), sedentary treated with melatonin (SM), and trained treated with melatonin (TM). Melatonin supplementation lasted 16 wk, and the animals were subjected to exercise during the last 8 wk of the experiment. After euthanasia, samples of liver, muscle, and adipose tissues were collected for analysis. Trained animals treated with melatonin presented better results in the following parameters: glucose tolerance, physical capacity, citrate synthase activity, hepatic and muscular glycogen content, body weight, protein expression of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and protein kinase activated by adenosine monophosphate (AMPK) in the liver, as well as the protein expression of the glucose transporter type 4 (GLUT4) and AMPK in the muscle. In conclusion, these results demonstrate that melatonin supplementation in aging animals is of great importance for the required metabolic adaptations induced by aerobic exercise. Adequate levels of circulating melatonin are, therefore, necessary to improve energetic metabolism efficiency, reducing body weight and increasing insulin sensitivity.

The impact of swine welfare on some qualitative traits of meat and long cured animal derived products

The present dissertation collects the results of three different research trials which have the common aim to understand the effects of swine welfare (both at farm level and during transport) on the main fresh and dry-cured meat characteristics. The first trial was carried out in order to compare the effects of illumination regimes differing in light duration or light intensity on meat and ham quality of Italian heavy pigs. The results of this trial support the conclusion that, within a moderate range of light intensity and given an appropriate dark period for animal rest, an increase of light duration or intensity above the minimum mandatory levels has no negative impact on carcass composition, meat or long-cured hams quality. The second trial was designed with the aim to investigate the effects of water restriction on growth traits, animal welfare and meat and ham quality of liquid-fed heavy pigs. Overall, the parameters analyzed as concerns growth rate, behavioural traits, blood, as well as carcass, fresh meat and cured hams quality were not affected by the absence of fresh drinking water. However, since liquid feeding did not suppress drinker use or drinker manipulation in the experimental groups, water restriction does not appear to be an applicable method to obtain a reduction of water waste. The third trial, which was carried out in Canada, tested the effectiveness of water sprinkling market-weight pigs (115±10Kg BW) before and after transport in reducing the heat stress experienced under commercial transport conditions. Our results show that the water sprinkling protocol proposed may reduce heat stress during transport and improve pork quality, particularly in specific trailer compartments. This body of research supports the general conclusion that swine welfare could be improved in different scenarios through simple and cost-effective means, without negatively affecting the quality of the main animal-derived products.

Entwicklung eines pharmakologischen Modells zur Prüfung immuntherapeutischer Antikörper mit dreidimensionalen Tumorzellkulturen in vitro

In den letzten Jahren hat die Tumorbehandlung mit immunologischen Präparaten an Bedeutung gewonnen. Der allgemeine Ablauf der Testung eines Arzneimittelkandidaten sieht vor, zunächst in Zellkulturversuchen und Tierversuchen Wirkweise und Sicherheit, sowie voraussichtliche Abbauwege und mögliche Gefahren so beurteilen zu können, dass sie für einen Einsatz im Menschen in Frage kommen. Zur präklinischen in vitro-Testung werden dabei in der Regel Monolayer-Zellkulturen oder Einzelzellsuspensionen eingesetzt. Der Einsatz von 3D-Zellkulturmodellen, welche den Aufbau von Mikrometastasen oder intervaskuläre Areale in Tumoren exakter widerspiegeln, führt zu wesentlich besseren Voraussagen bezüglich der klinischen Wirksamkeit neuer Präparate. Das Ziel dieser Arbeit war daher die Entwicklung und Anwendung eines neuen 3D-Zellkulturbasierten Systems zur Testung trifunktionaler bispezifischer Antikörper für die Tumorbehandlung, welches sich auch auf andere vergleichbare Präparate übertragen lässt.rnIn meiner Arbeit konnte ich mehrere humane Tumorzelllinien definieren, mit denen es gelang, stabile Co-Kulturen von Multi Cellular Tumour Spheroids (MCTS) mit Peripheral Blood Mononuclear Cells (PBMC) in miniaturisierten Spinner-Flaschen zu etablieren. Spinner-Flaschen, in denen die im Kulturmedium befindlichen Immunzellen, MCTS und Therapeutika ständig frei zirkulieren, sind besonders für eine wirklichkeitsnahe Nachbildung der in vivo-Simulation mit disseminierten Tumorzellen oder mit malignem Aszites geeignet. Diese Art der Kultivierung erlaubte Beobachtungszeiten von ≥20 Tagen für eine große Bandbreite Analysemethoden. Zu den mit dem erstellten Protokoll standardmäßig durchführbaren Analysemethoden zählen unter anderem immunhistochemische Färbungen an Sphäroid-Gefrierschnitten, Vitalitätstest, Untersuchung der Plattierungs-Effizienz, Bestimmung der Sphäroidvolumina, Zytokinbestimmungen aus dem Medienüberstand mit Cytokine Bead Arrays, PCR-Analysen immunzellspezifischer Antigene, sowie durchflusszytometrische Analysen. Diese Methodenkombination erlaubt einen sehr detaillierten Einblick in die Wirkweise und Effizienz neuer Immuntherapeutika aus verschiedensten Blickwinkeln und stellt ein reproduzierbares Testsystem zur präklinischen Testung von Immuntherapeutika dar, das zukünftig als Bindeglied zwischen Monolayer-Zellkulturen und klinischen Prüfungen einen festen Platz einnehmen könnte.rnMit dem beschriebenen 3D-Zellkultur-System wurden in der vorliegenden Arbeit die trifunktionalen bispezifischen Antikörper catumaxomab (unter dem Handelsnamen Removab® für die Behandlung maligner Ascites zugelassen) und ertumaxomab (derzeit in klinischen Prüfungen) hinsichtlich ihrer Wirkweise untersucht. Die Antikörper besitzen im Gegensatz zu herkömmlichen monoklonalen Antikörpern zwei verschiedene Bindungsarme, einer gegen CD3 auf T-Zellen, der zweite gegen EpCAM respektive Her2/neu - beides weit verbreitete Tumorantigene - gerichtet. An ihrem Fc-Teil besitzen sie eine dritte Bindungskapazität, über welche sie an Fcγ RI, -IIa und -III positive akzessorische Zellen binden. Diese Kombination ermöglicht theoretisch die Ausbildung eines Tri-Zell-Komplexes aus T-Zelle, Tumorzelle und akzessorischer Zelle. Dies stellt eine wirkungsvolle Therapieoption unter Ausnutzung der körpereigenen, immunologischen Abwehr dar. rnIm Rahmen dieser Arbeit wurde gezeigt, dass beide Antikörper eine Größenreduktion der Sphäroide mit den entsprechenden Tumorantigenen in gleichem Maße bewirkten und die Plattierungseffizienz durch ertumaxomab dosisabhängig reduziert wurde. Mit dem erstellten Testsystem konnte der Wirkmechanismus von catumaxomab auf Sphäroide der Zelllinie FaDu (Kopf-Hals-Plattenepithelkarzinom) detaillierter gezeigt werden: catumaxomab wirkte dosisabhängig auf die Reduktion der Sphäroidvolumina und die zunehmende Infiltration von CD45+ Zellen, die als T-, NK- und/oder dendritische Zellen identifiziert wurden. Des Weiteren rief die catumaxomab-Gabe eine verstärkte Ausschüttung der Zytokine IL-2, IFN-γ und TNF-α hervor. Diese Ergebnisse sprechen dafür, dass catumaxomab die zelluläre Immunantwort aktiviert.rnDie Standard-Tumorbehandlung beinhaltet die Gabe von Chemotherapeutika. Oft werden dafür Zytostatika mit dem unerwünschten Nebeneffekt auch gesunde proliferierende Zellen anzugreifen verwendet. Dies kann prinzipiell auch die Wirksamkeit der Antikörper-Therapie beeinflussen. Aus diesem Grund wurden in dieser Arbeit zusätzlich vergleichende Kombinations-Versuche mit catumaxomab und einem gängigen Zytostatikum - Cisplatin - durchgeführt. Mit Untersuchungen der Sphäroidvolumina, Vitalitätstests und Plattierungseffizienz konnte gezeigt werden, dass die Wirkung von catumaxomab bei gleichzeitiger Anwendung beider Therapeutika aufrecht erhalten bleibt und diese sogar additiv verstärkt wird. Eine Kombinationstherapie im Menschen ist daher denkbar.rnrn

Development and in vitro testing of liposomal gadolinium-formulations for neutron capture therapy of glioblastoma multiforme

For the improvement of current neutron capture therapy, several liposomal formulations of neutron capture agent gadolinium were developed and tested in a glioma cell model. Formulations were analyzed regarding physicochemical and biological parameters, such as size, zeta potential, uptake into cancer cells and performance under neutron irradiation. The neutron and photon dose derived from intracellular as well as extracellular Gd was calculated via Monte Carlo simulations and set in correlation with the reduction of cell survival after irradiation. To investigate the suitability of Gd as a radiosensitizer for photon radiation, cells were also irradiated with synchrotron radiation in addition to clinically used photons generated by linear accelerator.rnIrradiation with neutrons led to significantly lower survival for Gd-liposome-treated F98 and LN229 cells, compared to irradiated control cells and cells treated with non-liposomal Gd-DTPA. Correlation between Gd-content and -dose and respective cell survival displayed proportional relationship for most of the applied formulations. Photon irradiation experiments showed the proof-of-principle for the radiosensitizer approach, although the photon spectra currently used have to be optimized for higher efficiency of the radiosensitizer. In conclusion, the newly developed Gd-liposomes show great potential for the improvement of radiation treatment options for highly malignant glioblastoma.rn

Testing of subgrid scale (SGS) models for large-eddy simulation (LES) of turbulent channel flow

Sub-grid scale (SGS) models are required in order to model the influence of the unresolved small scales on the resolved scales in large-eddy simulations (LES), the flow at the smallest scales of turbulence. In the following work two SGS models are presented and deeply analyzed in terms of accuracy through several LESs with different spatial resolutions, i.e. grid spacings. The first part of this thesis focuses on the basic theory of turbulence, the governing equations of fluid dynamics and their adaptation to LES. Furthermore, two important SGS models are presented: one is the Dynamic eddy-viscosity model (DEVM), developed by \cite{germano1991dynamic}, while the other is the Explicit Algebraic SGS model (EASSM), by \cite{marstorp2009explicit}. In addition, some details about the implementation of the EASSM in a Pseudo-Spectral Navier-Stokes code \cite{chevalier2007simson} are presented. The performance of the two aforementioned models will be investigated in the following chapters, by means of LES of a channel flow, with friction Reynolds numbers $Re_\tau=590$ up to $Re_\tau=5200$, with relatively coarse resolutions. Data from each simulation will be compared to baseline DNS data. Results have shown that, in contrast to the DEVM, the EASSM has promising potentials for flow predictions at high friction Reynolds numbers: the higher the friction Reynolds number is the better the EASSM will behave and the worse the performances of the DEVM will be. The better performance of the EASSM is contributed to the ability to capture flow anisotropy at the small scales through a correct formulation for the SGS stresses. Moreover, a considerable reduction in the required computational resources can be achieved using the EASSM compared to DEVM. Therefore, the EASSM combines accuracy and computational efficiency, implying that it has a clear potential for industrial CFD usage.

Physician speciality and pain reduction in patients with depressive symptoms under treatment with venlafaxine

Excessive pain perception may lead to unnecessary diagnostic testing or invasive procedures resulting in iatrogenic complications and prolonged disability. Naturalistic studies on patients with chronic pain and depressive symptoms investigating the impact of medical speciality on treatment outcome in a primary care setting are lacking.

Preclinical testing of strategies for therapeutic targeting of human T-cell trafficking in vivo

Naive T cells are migratory cells that continuously recirculate between blood and lymphoid tissues. Antigen-specific stimulation of T cells within the lymph nodes reprograms the trafficking properties of T cells by inducing a specific set of adhesion molecules and chemokine receptors on their surface which allow these activated and effector T cells to effectively and specifically home to extralymphoid organs. The observations of organ-specific homing of T cells initiated the development of therapeutic strategies targeting adhesion receptors for organ-specific inhibition of chronic inflammation. As most adhesion receptors have additional immune functions besides mediating leukocyte trafficking, these drugs may have additional immunomodulatory effects. Therapeutic targeting of T-cell trafficking to the central nervous system is the underlying concept of a novel treatment of relapsing remitting multiple sclerosis with the humanized anti-alpha-4-integrin antibody natalizumab. In this chapter, we describe a possible preclinical in vivo approach to directly visualize the therapeutic efficacy of a given drug in inhibiting T-cell homing to a certain organ at the example of the potential of natalizumab to inhibit the trafficking of human T cells to the inflamed central nervous system in an animal model of multiple sclerosis.