In vitro activity of pineapple extracts (Ananas comosus, Bromeliaceae) on Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Measures to control the cattle tick, Rhipicephalus (Boophilus) microplus, based only on chemical products are becoming unsustainable, mainly because of the development of resistance. The objective of this study was to test the effect of the aqueous extract of pineapple skin (AEPS) and bromelain extracted from the stem (Sigma-Aldrich®, B4882) on engorged females and larvae of R. (B.) microplus in vitro. These substances were diluted in water and evaluated at eight concentrations. Engorged females were collected and distributed in groups of 10, with three repetitions for each treatment. After immersion in the solutions, the females were placed in an incubator for observation of survival, oviposition and larval hatching. The larval packet method was used, also with three repetitions with about 100 larvae each. The packets were incubated and the readings were performed after 24h. The estimated reproduction and efficacy of the solutions were calculated. The LC50 and LC90 were estimated using the Probit procedure of the SAS program. The eight concentrations were compared within each treatment by the Tukey test. For the experiment with engorged females, the most effective concentrations were 125, 250 and 500mg/mL: 33%, 48% and 59% for the AEPS and 27%, 51% and 55% for the bromelain. The LC50 and LC90 values were, respectively, 276 and 8691mg/mL for AEPS and 373 and 5172mg/mL for bromelain. None of the dilutions tested was effective against the larvae of R. (B.) microplus. This is the first report of the action of pineapple extracts or their constituents on cattle ticks. The results demonstrate that further studies regarding composition of tick cuticle, with evaluation of other solvents and formulations, should be conducted seeking to enhance the effect of pineapple extracts and compounds against this ectoparasite. © 2013 Elsevier Inc.

Genotoxic evaluation of the antimalarial drugs artemisinin and artesunate in human HepG2 cells and effects on CASP3 and SOD1 gene expressions

The malaria treatment recommended by the World Health Organization involves medicines derived from artemisinin, an active compound extracted from the plant Artemisia annua, and some of its derivatives, such as artesunate. Considering the lack of data regarding the genotoxic effects of these compounds in human cells, the objective of this study was to evaluate the cytotoxicity and genotoxicity, and expressions of the CASP3 and SOD1 genes in a cultured human hepatocellular liver carcinoma cell line (HepG2 cells) treated with artemisinin and artesunate. We tested concentrations of 2.5, 5, 7.5, 10, and 20 μg/mL of both substances with a resazurin cytotoxicity assay, and the concentrations used in the genotoxicity experiments (2.5, 5, and 10 μg/mL) and gene expression analysis (5 mg/mL) were determined. The results of the comet assay in cells treated with artemisinin and artesunate showed a significant dosedependent increase (P < 0.001) in the number of cells with DNA damage at all concentrations tested. However, the gene expression analysis revealed no significant change in expression of CASP3 or SOD1. Our data showed that although artemisinin and artesunate exhibited genotoxic effects in cultured HepG2 cells, they did not significantly alter expression of the CASP3 and SOD1 genes at the doses tested. ©FUNPEC-RP.

Evaluation of estrogenic, antiestrogenic and genotoxic activity of nemorosone, the major compound found in brown Cuban propolis

Background: Brown propolis is the major type of propolis found in Cuba; its principal component is nemorosone, the major constituent of Clusia rosea floral resins. Nemorosone has received increasing attention due to its strong in vitro anti-cancer action. The citotoxicity of nemorosone in several human cancer cell lines has been reported and correlated to the direct action it has on the estrogen receptor (ER). Breast cancer can be treated with agents that target estrogen-mediated signaling, such as antiestrogens. Phytoestrogen can mimic or modulate the actions of endogenous estrogens and the treatment of breast cancer with phytoestrogens may be a valid strategy, since they have shown anti-cancer activity.Methods: The aim of the present investigation was to assess the capacity of nemorosone to interact with ERs, by Recombinant Yeast Assay (RYA) and E-screen assays, and to determine by comet assay, if the compound causes DNA-damaging in tumoral and non-tumoral breast cells.Results: Nemorosone did not present estrogenic activity, however, it inhibited the 17-β-estradiol (E2) action when either of both methods was used, showing their antiestrogenicity. The DNA damage induced by the benzophenone in cancer and normal breast cells presented negative results.Conclusion: These findings suggest that nemorosone may have therapeutic application in the treatment of breast cancer. © 2013 Camargo et al.; licensee BioMed Central Ltd.

Predição de sombras de edifícios sobre vias urbanas com base em um modelo digital de elevações e dados de imagens aéreas de alta-resolução

Pós-graduação em Ciências Cartográficas - FCT

Calcium hydroxide root canal sealers - Histopathologic evaluation of apical and periapical repair after endodontic treatment

Eighty root canals of the premolars of 4 dogs, with vital pulp, were instrumented and filled during the same session with the Sealapex, CRCS, Sealer 26, and Apexit sealers, the animals were sacrificed 180 days after root canal filling and their maxillae and mandibles were removed and fixed in 10% formalin, After routine histologic processing, the sections were stained with hematoxylin-eosin and Mallory trichrome, Histopathologic analysis showed that Sealapex was the sealer that best permitted the deposition of mineralized tissue at the apical level and was the only sealer that provided complete sealing (37.5% of cases), With the use of Sealapex, no inflammatory infiltrate occurred and there was no reabsorption of mineralized tissues, In contrast, partial sealing and a moderate inflammatory infiltrate occurred with the use of CRCS, When Apexit and Sealer 26 were used the absence of sealing was frequent and active reabsorption of mineralized tissues occurred in most cases, the inflammatory infiltrate predominating with the use of Apexit was of the severe type, whereas with the use of Sealer 26 the inflammatory infiltrate was mild or absent.

Altered Oxygen Metabolism Associated to Neurogenesis of Induced Pluripotent Stem Cells Derived From a Schizophrenic Patient

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts, perceptions. and emotions, usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however, most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells, derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient, presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS), when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia, contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening.

Simulation of visible light communications in vehicular networks

The rapid development in the field of lighting and illumination allows low energy consumption and a rapid growth in the use, and development of solid-state sources. As the efficiency of these devices increases and their cost decreases there are predictions that they will become the dominant source for general illumination in the short term. The objective of this thesis is to study, through extensive simulations in realistic scenarios, the feasibility and exploitation of visible light communication (VLC) for vehicular ad hoc networks (VANETs) applications. A brief introduction will introduce the new scenario of smart cities in which visible light communication will become a fundamental enabling technology for the future communication systems. Specifically, this thesis focus on the acquisition of several, frequent, and small data packets from vehicles, exploited as sensors of the environment. The use of vehicles as sensors is a new paradigm to enable an efficient environment monitoring and an improved traffic management. In most cases, the sensed information must be collected at a remote control centre and one of the most challenging aspects is the uplink acquisition of data from vehicles. My thesis discusses the opportunity to take advantage of short range vehicle-to-vehicle (V2V) and vehicle-to-roadside (V2R) communications to offload the cellular networks. More specifically, it discusses the system design and assesses the obtainable cellular resource saving, by considering the impact of the percentage of vehicles equipped with short range communication devices, of the number of deployed road side units, and of the adopted routing protocol. When short range communications are concerned, WAVE/IEEE 802.11p is considered as standard for VANETs. Its use together with VLC will be considered in urban vehicular scenarios to let vehicles communicate without involving the cellular network. The study is conducted by simulation, considering both a simulation platform (SHINE, simulation platform for heterogeneous interworking networks) developed within the Wireless communication Laboratory (Wilab) of the University of Bologna and CNR, and network simulator (NS3). trying to realistically represent all the wireless network communication aspects. Specifically, simulation of vehicular system was performed and introduced in ns-3, creating a new module for the simulator. This module will help to study VLC applications in VANETs. Final observations would enhance and encourage potential research in the area and optimize performance of VLC systems applications in the future.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of synthetic cannabinoids of the aminoalkylindole type and methanandamide in serum and its application to forensic samples

After the discovery of synthetic cannabimimetic substances in 'Spice'-like herbal mixtures marketed as 'incense' or 'plant fertilizer' the active compounds have been declared as controlled substances in several European countries. As expected, a monitoring of new herbal mixtures which continue to appear on the market revealed that shortly after control measures have been taken by legal authorities, other compounds were added to existing mixtures and to new products. Several compounds of the aminoalkylindole type have been detected so far in herbal mixtures but still their consumption cannot be detected by commonly used drug-screening procedures, encouraging drug users to substitute cannabis with those products. There is a increasing demand on the part of police authorities, hospitals and psychiatrists for detection and quantification of synthetic cannabinoids in biological samples originating from psychiatric inpatients, emergency units or assessment of fitness to drive. Therefore, a liquid chromatography-tandem mass spectrometry method after liquid-liquid extraction for the quantitation of JWH-015, JWH-018, JWH-073, JWH-081, JWH 200, JWH-250, WIN 55,212-2 and methanandamide and the detection of JWH-019 and JWH-020 in human serum has been developed and fully validated according to guidelines for forensic toxicological analyses. The method was successfully applied to 101 serum samples from 80 subjects provided by hospitals, detoxification and therapy centers, forensic psychiatric centers and police authorities. Fifty-seven samples or 56.4% were found positive for at least one aminoalkylindole. JWH-019, JWH-020, JWH-200, WIN 55,212-2 and methanandamide were not detected in any of the analyzed samples.

Treatment of echinococcosis: albendazole and mebendazole - what else?

The search for novel therapeutic options to cure alveolar echinococcosis (AE), due to the metacestode of Echinococcus multilocularis, is ongoing, and these developments could also have a profound impact on the treatment of cystic echinococcosis (CE), caused by the closely related Echinococcus granulosus s.l. Several options are being explored. A viable strategy for the identification of novel chemotherapeutically valuable compounds includes whole-organism drug screening, employing large-scale in vitro metacestode cultures and, upon identification of promising compounds, verification of drug efficacy in small laboratory animals. Clearly, the current focus is targeted towards broad-spectrum anti-parasitic or anti-cancer drugs and compound classes that are already marketed, or that are in development for other applications. The availability of comprehensive Echinococcus genome information and gene expression data, as well as significant progress on the molecular level, has now opened the door for a more targeted drug discovery approach, which allows exploitation of defined pathways and enzymes that are essential for the parasite. In addition, current in vitro and in vivo models that are used to assess drug efficacy should be optimized and complemented by methods that give more detailed information on the host-parasite interactions that occur during drug treatments. The key to success is to identify, target and exploit those parasite molecules that orchestrate activities essential to parasite survival.

Force-controlled patch clamp of beating cardiac cells.

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

Mechanism of methylation gene reactivation in human cancer cells

Epigenetic silencing of tumor suppressor genes by DNA hypermethylation at promoter regions is a common event in carcinogenesis and tumor progression. Abrogation of methylation and reversal of epigenetic silencing is a very potent way in cancer treatment. However, the reactivation mechanisms are poorly understood. In this study, we first developed a cell line model system named YB5, derived from SW48 cancer cell line, which bears one copy of stably integrated EGFP gene on Chromosome 1p31.1 region. The GFP gene expression is transcriptionally silenced due to the hypermethylated promoter CMV. However, the GFP expression can be restored using demethylating agent 5-aza-2' deoxycytidine (DAC), and detected by FACS and fluorescent microscopy. Using this system, we observed the heterogeneous reactivation induced by DAC treatment. After flow sorting, GFP negative cells exhibited similar level of incomplete demethylation compared to GFP positive cells on repetitive LINE1 element, tumor suppressor genes such as P16, CDH13, and RASSF1a, and CMV promoter as well. However, the local chromatin of CMV-GFP locus altered to an open structure marked by high H3 lysine 9 acetylation and low H3 lysine 27 tri-methylation in GFP positive cells, while the GFP negative cells retained mostly the original repressive marks. Thus, we concluded that DAC induced DNA hypomethylation alone does not directly determine the level of re-expression, and the resetting of the local chromatin structure under hypomethylation environment is required for gene reactivation. Besides, a lentivirus vector-based shRNA screening was performed using the YB5 system. Although it is the rare chance that vector lands in the neighboring region of GFP, we found that the exogenous vector DNA inserted into the upstream region of GFP gene locus led to the promoter demethylation and reactivated the silenced GFP gene. Thus, epigenetic state can be affected by changing of the adjacent nucleic acid sequences. Further, this hypermethylation silenced system was utilized for epigenetic drug screening. We have found that DAC combined with carboplatin would enhance the GFP% yield and increase expression of other tumor suppressor genes than DAC alone, and this synergistic effect may be related to DNA repair process. In summary, these studies reveal that reversing of methylation silencing requires coordinated alterations of DNA methylation, chromatin structure, and local microenvironment. ^

Human midbrain precursors activate the expected developmental genetic program and differentiate long-term to functional A9 dopamine neurons in vitro. Enhancement by Bcl-XL

Understanding the molecular programs of the generation of human dopaminergic neurons (DAn) from their ventral mesencephalic (VM) precursors is of key importance for basic studies, progress in cell therapy, drug screening and pharmacology in the context of Parkinson's disease. The nature of human DAn precursors in vitro is poorly understood, their properties unstable, and their availability highly limited. Here we present positive evidence that human VM precursors retaining their genuine properties and long-term capacity to generate A9 type Substantia nigra human DAn (hVM1 model cell line) can be propagated in culture. During a one month differentiation, these cells activate all key genes needed to progress from pro-neural and prodopaminergic precursors to mature and functional DAn. For the first time, we demonstrate that gene cascades are correctly activated during differentiation, resulting in the generation of mature DAn. These DAn have morphological and functional properties undistinguishable from those generated by VM primary neuronal cultures. In addition, we have found that the forced expression of Bcl-XL induces an increase in the expression of key developmental genes (MSX1, NGN2), maintenance of PITX3 expression temporal profile, and also enhances genes involved in DAn long-term function, maintenance and survival (EN1, LMX1B, NURR1 and PITX3). As a result, Bcl-XL anticipates and enhances DAn generation.

VISIONS Vehicular communication Improvement : solution based on IMS Operational Nodes and Services

Digital services and communications in vehicular scenarios provide the essential assets to improve road transport in several ways like reducing accidents, improving traffic efficiency and optimizing the transport of goods and people. Vehicular communications typically rely on VANET (Vehicular Ad hoc Networks). In these networks vehicles communicate with each other without the need of infrastructure. VANET are mainly oriented to disseminate information to the vehicles in certain geographic area for time critical services like safety warnings but present very challenging requirements that have not been successfully fulfilled nowadays. Some of these challenges are; channel saturation due to simultaneous radio access of many vehicles, routing protocols in topologies that vary rapidly, minimum quality of service assurance and security mechanisms to efficiently detect and neutralize malicious attacks. Vehicular services can be classified in four important groups: Safety, Efficiency, Sustainability and Infotainment. The benefits of these services for the transport sector are clear but many technological and business challenges need to be faced before a real mass market deployment. Service delivery platforms are not prepared for fulfilling the needs of this complex environment with restrictive requirements due to the criticism of some services To overcome this situation, we propose a solution called VISIONS “Vehicular communication Improvement: Solution based on IMS Operational Nodes and Services”. VISIONS leverages on IMS subsystem and NGN enablers, and follows the CALM reference Architecture standardized by ISO. It also avoids the use of Road Side Units (RSUs), reducing complexity and high costs in terms of deployment and maintenance. We demonstrate the benefits in the following areas: 1. VANET networks efficiency. VISIONS provide a mechanism for the vehicles to access valuable information from IMS and its capabilities through a cellular channel. This efficiency improvement will occur in two relevant areas: a. Routing mechanisms. These protocols are responsible of carrying information from a vehicle to another (or a group of vehicles) using multihop mechanisms. We do not propose a new algorithm but the use of VANET topology information provided through our solution to enrich the performance of these protocols. b. Security. Many aspects of security (privacy, key, authentication, access control, revocation mechanisms, etc) are not resolved in vehicular communications. Our solution efficiently disseminates revocation information to neutralize malicious nodes in the VANET. 2. Service delivery platform. It is based on extended enablers, reference architectures, standard protocols and open APIs. By following this approach, we reduce costs and resources for service development, deployment and maintenance. To quantify these benefits in VANET networks, we provide an analytical model of the system and simulate our solution in realistic scenarios. The simulations results demonstrate how VISIONS improves the performance of relevant routing protocols and is more efficient neutralizing security attacks than the widely proposed solutions based on RSUs. Finally, we design an innovative Social Network service based in our platform, explaining how VISIONS facilitate the deployment and usage of complex capabilities. RESUMEN Los servicios digitales y comunicaciones en entornos vehiculares proporcionan herramientas esenciales para mejorar el transporte por carretera; reduciendo el número de accidentes, mejorando la eficiencia del tráfico y optimizando el transporte de mercancías y personas. Las comunicaciones vehiculares generalmente están basadas en redes VANET (Vehicular Ad hoc Networks). En dichas redes, los vehículos se comunican entre sí sin necesidad de infraestructura. Las redes VANET están principalmente orientadas a difundir información (por ejemplo advertencias de seguridad) a los vehículos en determinadas zonas geográficas, pero presentan unos requisitos muy exigentes que no se han resuelto con éxito hasta la fecha. Algunos de estos retos son; saturación del canal de acceso de radio debido al acceso simultáneo de múltiples vehículos, la eficiencia de protocolos de encaminamiento en topologías que varían rápidamente, la calidad de servicio (QoS) y los mecanismos de seguridad para detectar y neutralizar los ataques maliciosos de manera eficiente. Los servicios vehiculares pueden clasificarse en cuatro grupos: Seguridad, Eficiencia del tráfico, Sostenibilidad, e Infotainment (información y entretenimiento). Los beneficios de estos servicios para el sector son claros, pero es necesario resolver muchos desafíos tecnológicos y de negocio antes de una implementación real. Las actuales plataformas de despliegue de servicios no están preparadas para satisfacer las necesidades de este complejo entorno con requisitos muy restrictivos debido a la criticidad de algunas aplicaciones. Con el objetivo de mejorar esta situación, proponemos una solución llamada VISIONS “Vehicular communication Improvement: Solution based on IMS Operational Nodes and Services”. VISIONS se basa en el subsistema IMS, las capacidades NGN y es compatible con la arquitectura de referencia CALM estandarizado por ISO para sistemas de transporte. También evita el uso de elementos en las carreteras, conocidos como Road Side Units (RSU), reduciendo la complejidad y los altos costes de despliegue y mantenimiento. A lo largo de la tesis, demostramos los beneficios en las siguientes áreas: 1. Eficiencia en redes VANET. VISIONS proporciona un mecanismo para que los vehículos accedan a información valiosa proporcionada por IMS y sus capacidades a través de un canal de celular. Dicho mecanismo contribuye a la mejora de dos áreas importantes: a. Mecanismos de encaminamiento. Estos protocolos son responsables de llevar información de un vehículo a otro (o a un grupo de vehículos) utilizando múltiples saltos. No proponemos un nuevo algoritmo de encaminamiento, sino el uso de información topológica de la red VANET a través de nuestra solución para enriquecer el funcionamiento de los protocolos más relevantes. b. Seguridad. Muchos aspectos de la seguridad (privacidad, gestión de claves, autenticación, control de acceso, mecanismos de revocación, etc) no están resueltos en las comunicaciones vehiculares. Nuestra solución difunde de manera eficiente la información de revocación para neutralizar los nodos maliciosos en la red. 2. Plataforma de despliegue de servicios. Está basada en capacidades NGN, arquitecturas de referencia, protocolos estándar y APIs abiertos. Siguiendo este enfoque, reducimos costes y optimizamos procesos para el desarrollo, despliegue y mantenimiento de servicios vehiculares. Para cuantificar estos beneficios en las redes VANET, ofrecemos un modelo de analítico del sistema y simulamos nuestra solución en escenarios realistas. Los resultados de las simulaciones muestran cómo VISIONS mejora el rendimiento de los protocolos de encaminamiento relevantes y neutraliza los ataques a la seguridad de forma más eficientes que las soluciones basadas en RSU. Por último, diseñamos un innovador servicio de red social basado en nuestra plataforma, explicando cómo VISIONS facilita el despliegue y el uso de las capacidades NGN.

Magnetic Devices and Techniques for the Study of Viscoelasticity of Biomaterials and Myocardial Forces

Thesis (Ph.D.)--University of Washington, 2016-06

Microspheres as a vehicle for biomolecule delivery to neural stem cells

Neural stem cells (NSC) are a valuable model system for understanding the intrinsic and extrinsic controls for self-renewal and differentiation choice. They also offer a platform for drug screening and neurotoxicity studies, and hold promise for cell replacement therapies for the treatment of neurodegenerative diseases. Fully exploiting the potential of this experimental tool often requires the manipulation of intrinsic cues of interest using transfection methods, to which NSC are relatively resistant. In this paper, we show that mouse and human NSC readily take up polystyrene-based microspheres which can be loaded with a range of chemical or biological cargoes. This uptake can take place in the undifferentiated stage without affecting NSC proliferation and their capacity to give rise to neurons and glia. We demonstrate that ß-galactosidase-loaded microspheres could be efficiently introduced into NSC with no apparent toxic effect, thus providing proof-of-concept for the use of microspheres as an alternative biomolecule delivery system.