Efeito do GQ-02, derivado de tiazolidinadionas, sobre a aquisição de fenótipo termogênico pelo adipócito in vivo e em cultura de células

Dissertação (mestrado)—Universidade de Brasília, Faculdade em Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2016.

Contribution to the study of prognostic factors in canine oral squamous cell carcinoma

Tese de Doutoramento em Ciências Veterinárias, na Especialidade de Clínica

Screening natural resources for mineralogenic and osteogenic bioactivities using in vitro and in vivo fish systems

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Role of Ccbe1 during cardiac differentiation of mouse ESCs

Tese de Doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Eficacia de las terapias celular y génica como agentes inmunomoduladores en el tratamiento de la esclerosis múltiple

El interferón beta (IFNb) ha sido uno de los fármacos más utilizados en el tratamiento de la esclerosis múltiple (EM), gracias a su efecto inmunomodulador, antiproliferativo y antiviral. Sin embargo, existe un porcentaje de pacientes que responden de forma subóptima al tratamiento, sugiriendo la necesidad de buscar alternativas terapéuticas innovadoras. En este contexto se ha observado que las células madre mesenquimales derivadas del tejido adiposo (AdMSCs) presentan capacidad inmunomoduladora, neuroprotectora y regeneradora del tejido dañado en ensayos preclínicos con el modelo animal más frecuentemente usado en el estudio de la EM, la Encefalomielitis Autoinmune Experimental (EAE), lo que las hace buenas candidatas como terapia alternativa para la EM. Además, su capacidad de migración hacia el tejido dañado les confiere potencial para ser utilizadas como transportadoras de factores terapéuticos hacia las zonas lesionadas del SNC. Por ello, nos planteamos evaluar la eficacia terapéutica de las terapias celular con AdMSCs y génica con AdMSCs modificadas genéticamente para producir IFNb, en modelos de EAE. Para llevarlo a cabo se realizó la caracterización de la población de AdMSCs de la cepa de ratón SJL/jCrL (SJL-AdMSCs), usando como control las AdMSCs de la cepa C57BL/6, ampliamente caracterizadas en la literatura, la generación de líneas de AdMSCs secretoras de IFNb (AdMSCs-IFNb) mediante lentivirus y su posterior caracterización y comparación con las mismas células sin transducir, la evaluación de los efectos de las terapias celulares autóloga, alogénica y génica en los modelos de EAE crónico progresivo (EAE-CP) y remitente recurrente (EAE-RR) y el estudio de la migración de las AdMSCs administradas como terapia autóloga y de las AdMSCs-IFNb. Los resultados obtenidos en cada uno de los objetivos planteados nos condujeron a una serie de conclusiones: las SJL-AdMSCs aisladas, cultivadas y expandidas bajo nuestras condiciones experimentales, cumplen los criterios mínimos determinados para ser consideradas células madre mesenquimales. Además, estas células presentan eficacia clínica y efectos neuroinmunomoduladores al ser utilizadas como transplantes autólogos y alogénicos en animales con EAE-RR y EAE-CP respectivamente. Por otro lado, las SJL-AdMSCs constituyen una población apta para dar soporte al desarrollo de la terapia génica, ya que la alteración de su material genético por la inserción del IFNb no supone la modificación de sus propiedades biológicas ni funcionales en estudios preclínicos en modelos de EM. Estas AdMSCs-IFNb, constituyen una línea de células mesenquimales de crecimiento estable que produce elevados niveles de IFNb de forma constitutiva. Además, los transplantes con AdMSCs-IFNb son eficaces como tratamiento terapéutico en animales con EAE-RR y EAE-CP al modular tanto la sintomatología como los procesos inflamatorios y neurodegenerativos propios de la enfermedad. Sin embargo, los resultados no permiten discriminar si los efectos observados son debidos a las propiedades del inmunomodulador secretado, a las propias células mesenquimales o a la acción conjunta de ambos. En último lugar, la migración celular de las AdMSCs autólogas se potencia por los estados de inflamación activa en ambos modelos de EAE, mostrando una amplia biodistribución celular. La localización prioritaria fue inicialmente en pulmones y, posteriormente en zona de órganos linfáticos, como hígado y bazo, y del SNC a nivel de la médula espinal. La señal bioluminiscente de las AdMSCs-IFNb en el modelo EAE-CP es mayor que la emitida por las células de la terapia autóloga. Sin embargo, la migración de las células transfectadas no aparece fuertemente influenciada por los procesos proinflamatorios. En el modelo EAE-RR estas diferencias entre terapias son incluso más moderadas. Las áreas donde se registra señal son similares a las de las células autólogas, apareciendo principalmente en zonas correspondientes a pulmones, hígado, bazo y médula espinal a lo largo del tiempo experimental.

Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.

Preclinical animal models for segmental bone defect research and tissue engineering

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, however they are limited in access and availability and associated with donor site morbidity, haemorrhage, risk of infection, insufficient transplant integration, graft devitalisation, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. Analysing the tissue engineering literature it can be concluded that bone regeneration has become a focus area in the field. Hence, a considerable number of research groups and commercial entities work on the development of tissue engineered constructs for bone regeneration. However, bench to bedside translations are still infrequent as the process towards approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. In translational orthopaedic research, the utilisation of large preclinical animal models is a conditio sine qua non. Consequently, to allow comparison between different studies and their outcomes, it is essential that animal models, fixation devices, surgical procedures and methods of taking measurements are well standardized to produce reliable data pools as a base for further research directions. The following chapter reviews animal models of the weight-bearing lower extremity utilized in the field which include representations of fracture-healing, segmental bone defects, and fracture non-unions.

Inhibition of p38 pathway leads to OA-like changes in a rat animal model

Objectives The p38 mitogen-activated protein kinase (MAPK) signal transduction pathway is involved in a variety of inflammatory responses, including cytokine generation, cell differentiation proliferation and apoptosis. Here, we examined the effects of systemic p38 MAPK inhibition on cartilage cells and osteoarthritis (OA) disease progression by both in vitro and in vivo approaches. Methods p38 kinase activity was evaluated in normal and OA cartilage cells by measuring the amount of phosphorylated protein. To examine the function of p38 signaling pathway in vitro, normal chondrocytes were isolated and differentiated in the presence or absence of p38 inhibitor; SB203580 and analysed for chondrogenic phenotype. Effect of systemic p38 MAPK inhibition in normal and OA (induced by menisectomy) rats were analysed by treating animals with vehicle alone (DMS0) or p38 inhibitor (SB203580). Damage to the femur and tibial plateau was evaluated by modified Mankin score, histology and immunohistochemistry. Results Our in vitro studies have revealed that a down-regulation of chondrogenic and increase of hypertrophic gene expression occurs in the normal chondrocytes, when p38 is neutralized by a pharmacological inhibitor. We further observed that the basal levels of p38 phosphorylation were decreased in OA chondrocytes compared with normal chondrocytes. These findings together indicate the importance of this pathway in the regulation of cartilage physiology and its relevance to OA pathogenesis. At in vivo level, systematic administration of a specific p38 MAPK inhibitor, SB203580, continuously for over a month led to a significant loss of proteoglycan; aggrecan and cartilage thickness. On the other hand, SB203580 treated normal rats showed a significant increase in TUNEL positive cells, cartilage hypertrophy markers such as Type 10 collagen, Runt-related transcription factor and Matrix metalloproteinase-13 and substantially induced OA like phenotypic changes in the normal rats. In addition, menisectomy induced OA rat models that were treated with p38 inhibitor showed aggravation of cartilage damage. Conclusions In summary, this study has provided evidence that the component of the p38 MAPK pathway is important to maintain the cartilage health and its inhibition can lead to severe cartilage degenerative changes. The observations in this study highlight the possibility of using activators of the p38 pathway as an alternative approach in the treatment of OA.

Tissue engineering bone - reconstruction of critical sized segmental bone defects in a large animal model

Currently, well established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, their application, however, is associated with disadvantages. These include limited access and availability, donor site morbidity and haemorrhage, increased risk of infection, and insufficient transplant integration. As a result, recent research focuses on the development of complementary therapeutic concepts. The field of tissue engineering has emerged as an important alternative approach to bone regeneration. Tissue engineering unites aspects of cellular biology, biomechanical engineering, biomaterial sciences and trauma and orthopaedic surgery. To obtain approval by regulatory bodies for these novel therapeutic concepts the level of therapeutic benefit must be demonstrated rigorously in well characterized, clinically relevant animal models. Therefore, in this PhD project, a reproducible and clinically relevant, ovine, critically sized, high load bearing, tibial defect model was established and characterized as a prerequisite to assess the regenerative potential of a novel treatment concept in vivo involving a medical grade polycaprolactone and tricalciumphosphate based composite scaffold and recombinant human bone morphogenetic proteins.

SERS based detection of barcoded gold nanoparticle assemblies from within animal tissue

We have explored the potential of deep Raman spectroscopy, specifically surface enhanced spatially offset Raman spectroscopy (SESORS), for non-invasive detection from within animal tissue, by employing SERS-barcoded nanoparticle (NP) assemblies as the diagnostic agent. This concept has been experimentally verified in a clinic-relevant backscattered Raman system with an excitation line of 785 nm under ex vivo conditions. We have shown that our SORS system, with a fixed offset of 2-3 mm, offered sensitive probing of injected QTH-barcoded NP assemblies through animal tissue containing both protein and lipid. In comparison to that of non-aggregated SERS-barcoded gold NPs, we have demonstrated that the tailored SERS-barcoded aggregated NP assemblies have significantly higher detection sensitivity. We report that these NP assemblies can be readily detected at depths of 7-8 mm from within animal proteinaceous tissue with high signal-to-noise (S/N) ratio. In addition they could also be detected from beneath 1-2 mm of animal tissue with high lipid content, which generally poses a challenge due to high absorption of lipids in the near-infrared region. We have also shown that the signal intensity and S/N ratio at a particular depth is a function of the SERS tag concentration used and that our SORS system has a QTH detection limit of 10-6 M. Higher detection depths may possibly be obtained with optimization of the NP assemblies, along with improvements in the instrumentation. Such NP assemblies offer prospects for in vivo, non-invasive detection of tumours along with scope for incorporation of drugs and their targeted and controlled release at tumour sites. These diagnostic agents combined with drug delivery systems could serve as a “theranostic agent”, an integration of diagnostics and therapeutics into a single platform.

Neurodevelopmental animal models of schizophrenia: Effects on prepulse inhibition

Epidemiological studies have shown increased incidence of schizophrenia in patients subjected to different forms of pre- or perinatal stress. However, as the onset of schizophrenic illness does not usually occur until adolescence or early adulthood, it is not yet fully understood how disruption of early brain development may ultimately lead to malfunction years later. In order to elucidate a possible role for neurodevelopmental factors in the pathogenesis of schizophrenia and to highlight potential new treatments, animal models are needed. Prepulse inhibition (PPI) is a model of sensorimotor gating mechanisms in the brain. It is disrupted in schizophrenia patients and the disruption can be reversed with atypical antipsychotics. It has been widely used in animal studies to explore central mechanisms possibly involved in schizophrenia. There has been a recent surge of behavioural and neurochemical animal studies on neurodevelopmental models, particularly on the effects of postweaning isolation, maternal separation and neonatal lesions of the hippocampus. In these models, long lasting alterations in behaviour and/or molecular changes in specific brain regions are observed, comparable to those seen in schizophrenia. The aim of this article is to critically review the available literature on such neurodevelopmental animal models with special focus on the effects on PPI and brain regions that are putatively involved in regulation of PPI.

Animal magnetism and curriculum history

This article elaborates the impact that crises of authority provoked by animal magnetism, mesmerism, and hypnosis in the 19th century had for field formation in American education. Four layers of analysis elucidate how curriculum history’s repetitive focus on public school policy and classroom practice became possible. First, the article surveys external conditions of possibility for the enactment of compulsory public schooling. Second, “internal” conditions of possibility for the formation of educational objects (e.g., types of children) are documented via the processes of différance that were generated from within the experiences of confinement. Third, the article maps how these were interpenetrated by animal magnetic debates that were lustered and planished in education’s emerging field, including impact upon behavior management practices, the contouring of expertise and authority, the role of Will in intelligence testing and child development theories, and the redefinition of public and private. Last, the article examines implications for curriculum history, whether policy- or practice-oriented, especially around the question of influence, the theorization of child mind, and philosophies of Being.

Effect of an interactive therapeutic robotic animal on engagement, mood states, agitation and psychotropic drug use in people with dementia: A cluster-randomised controlled trial protocol

Introduction: Apathy, agitated behaviours, loneliness and depression are common consequences of dementia. This trial aims to evaluate the effect of a robotic animal on behavioural and psychological symptoms of dementia in people with dementia living in long-term aged care. Methods and analysis: A cluster-randomised controlled trial with three treatment groups: PARO (robotic animal), Plush-Toy (non-robotic PARO) or Usual Care (Control). The nursing home sites are Australian Government approved and accredited facilities of 60 or more beds. The sites are located in South-East Queensland, Australia. A sample of 380 adults with a diagnosis of dementia, aged 60 years or older living in one of the participating facilities will be recruited. The intervention consists of three individual 15 min non-facilitated sessions with PARO or Plush- Toy per week, for a period of 10 weeks. The primary outcomes of interest are improvement in agitation, mood states and engagement. Secondary outcomes include sleep duration, step count, change in psychotropic medication use, change in treatment costs, and staff and family perceptions of PARO or Plush-Toy. Video data will be analysed using Noldus XT Pocket Observer; descriptive statistics will be used for participants’ demographics and outcome measures; cluster and individual level analyses to test all hypotheses and Generalised Linear Models for cluster level and Generalised Estimation Equations and/or Multi-level Modeling for individual level data. Ethics and dissemination: The study participants or their proxy will provide written informed consent. The Griffith University Human Research Ethics Committee has approved the study (NRS/03/14/HREC). The results of the study will provide evidence of the efficacy of a robotic animal as a psychosocial treatment for the behavioural and psychological symptoms of dementia. Findings will be presented at local and international conference meetings and published in peer-reviewed journals.

Better early osteogenesis of electroconductive hydroxyapatite-calcium titanate composites in a rabbit animal model

In view of the fact that bone healing can be enhanced due to external electric field application, it is important to assess the influence of the implant conductivity on the bone regeneration in vivo. To address this issue, this study reports the in vivo biocompatibility property of multistage spark plasma sintered hydroxyapatite (HA)-80 wt % calcium titanate (CaTiO3) composites and monolithic HA, which have widely different conductivity property (14 orders of magnitude difference). The ability of bone regeneration was assessed by implantation in cylindrical femoral bone defects of rabbit animal model for varying time period of 1, 4, and 12 weeks. The overall assessment of the histology results suggests that the progressive healing of bone defects around HA-80 wt % CaTiO3 is associated with a better efficacy with respect to (w.r.t) early stage neobone formation, which is histomorphometrically around 140% higher than monolithic HA. Overall, this study demonstrates that the in vivo biocompatibility property of HA-80 wt % CaTiO3 with respect to local effects after 12 weeks of implantation is not compromised both qualitatively and quantitatively, and a comparison with control implant (HA) points toward the critical role of electrical conductivity on better early stage bone regeneration. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 842-851, 2014.

Degradación in vivo de la materia seca y materia orgánica del Teocinte (Zea nicaraguensis Iltis y Benz)

Este estudio se realizó con el objetivo de determinar la degradabilidad in vivo del Teocinte (Zea nicaraguensis Iltis & Benz). El ensayo de degradabilidad fue realizado en la galera experimental de la Facultad de Ciencia Animal adscrita a la Universidad Nacional Agraria. En este ensayo se utilizaron dos vacas de raza Reyna, secas y vacías, con fistulas ruminales. Los tiempos de incubación (0, 0.5, 1, 3, 6, 9, 12, 24, 48, 72, 96 horas) fueron considerados los tratamientos (11) por 4 réplicas por cada tiempo. Las variables evaluadas fueron: degradación de materia seca (DMS) y degradación de materia orgánica (DMO). Para la determinación de las variables, se utilizó el modelo exponencial descrito por Orskov and McDonald (1979). Como resultados finales de la Degradabilidad de la MS se obtuvo una fracción soluble de 2.44% (a), fracción insoluble pero potencialmente degradable del 23.35% (b), una tasa de degradación de la fracción potencialmente degradada estimada del 2.61%* horas (c) y la fracción no degradable [100-(2.44 + 23.35)] del 74.05%. En la Degradabilidad de la MO la fracción soluble fue de 2.43% (a), la fracción insoluble pero potencialmente degradable de 23.35% (b), una tasa de degradación de la fracción potencialmente degradada estimada de 2.61%* horas y la fracción no degradable [100-(2.43 + 23.35)] de 74.22%. El Teocinte en la edad de corte utilizada en este experimento no tiene valores de degradabilidad compatibles con sistemas de producción animal de alta intensidad por lo que se sugiere seguir investigando en su potencialidad.