Validation of neuronal inclusions as a biomarker for pre-clinical trials in a mouse model of Machado-Joseph disease

Dissertação de mestrado em Ciências da SaúdePolyglutamine disorders are a class of nine inherited neurodegenerative disorders caused by expansion of a CAG repeat within otherwise unrelated genes. Polyglutamine expansion causes aberrant protein misfolding that leads to formation of protein aggregation and neuronal loss. Machado-Joseph disease (MJD) is included in this group of disorders, being caused by expansion of a CAG tract in the C-terminal region of the protein Ataxin-3 (ATXN3). MJD is characterized by the formation of intranuclear neuronal inclusions (NIs) and nervous system dysfunction, but the mechanism of this ATXN3- mediated dysfunction is still unsolved. So far, some symptoms can be controlled with specific treatments, but there is no effective therapy for MJD. Our team previously established a new MJD mouse model expressing human ATXN3 with 135 glutamines – CMVMJD135 – that presents phenotypic, pathologic and genetic similarities with the human disorder, namely several reliable and quantifiable phenotypic markers that can be used in therapeutic trials. The NFIs are a pathological hallmark of this disease and have been found in this mouse model, as happens in human patients. Many studies related with therapeutic targets in polyQ diseases reveal phenotype amelioration without mention to neuropathology, namely inclusion load. Immunohistochemistry was performed to evaluate the relevance of inclusion load assessment in pre-clinical trials using a mouse model of MJD. We evaluated the utility of using this biomarker in therapeutic trials using three pharmacological compounds: creatine, 17-DMAG and valproic acid. We have found a significant reduction in inclusion load in the lateral reticular nucleus (LRT) region and a tendency towards a reduction of neuronal inclusions in the facial nuclei (7N) region of CMVMJD135 mice treated with creatine. The chronic treatment with creatine also lead to an amelioration of the motor phenotype observed in these animals, namely an increase in muscle strength and a better motor performance in the motor swimming test. Another compound, 17-DMAG did not show any differences between treated and non-treated transgenic mice group regarding NIs in the LRT and 7N brain regions, although a phenotype amelioration was observed in several behavioral paradigms. CMVMJD135 mice treated with Valproic acid exhibit a tendency towards a reduction of neuronal inclusions load in the LRt but no reduction in inclusion load in the 7N brain region. Globally our results suggest that there is no direct correlation between the aggregate load in these brain regions of the CMVMJD135 mice and the phenotypic effect observed upon chronic treatment with different compounds.As doenças de poliglutaminas são um conjunto de doenças neurodegenerativas hereditárias causadas pela expansão de um tripleto de CAG nas regiões codificantes dos respetivos genes causadores. A expansão do tripleto CAG leva à formação de proteínas que possuem uma expansão anormal de uma sequência de poliglutamina, causando o seu desarranjo conformacional, podendo estas proteínas agregar e conduzir à morte neuronal num estadio mais avançado da doença. A doença de Machado-Joseph (DMJ) inclui-se neste grupo de doenças, sendo caracterizada por uma expansão de glutaminas localizada na região Cterminal da proteína envolvida na DMJ, a ataxina-3 (ATXN3). São características comuns a todas as doenças de poliglutaminas a presença de inclusões proteicas intranucleares nos neurónios (IINs) e a disfunção neuronal. No entanto, permanece por esclarecer a relevância das IINsna patogénese da DMJ. Até à data, embora seja possível controlar alguma da sintomatologia observada nos doentes, não existe um tratamento eficaz para a DMJ. No presente estudo, utilizou-se um modelo de ratinho para a DMJ que foi previamente gerado no nosso laboratório, e que apresenta várias características semelhantes às da doença humana, quer a nível genético e fenotípico, quer a nível neuropatológico. Este modelo apresenta características passíveis de quantificação, permitindo o seu uso em ensaios pre-clínicos. As IINs são uma característica chave da DMJ e foram detetadas nos cérebros deste modelo. Vários ensaios pré-clínicos recorrendo a várias estratégias terapêuticas e a diferentes modelos de ratinho, demonstram uma melhoria do fenótipo observado em cada modelo, no entanto, a referência ao efeito dos compostos utilizados na quantidade de agregados proteicos não é clara. Numa tentativa de esclarecer a relação entre a melhoria no fenótipo e a quantidade de IINs nos cérebros dos ratinhos CMVMJD135, foi efetuado um estudo de quantificação das IINs em algumas regiões do cérebro relevantes na DMJ, após o tratamento crónico dos ratinhos com três compostos: creatina, 17-DMAG e ácido valpróico. Foi possível observar uma redução significativa da densidade de IINs no LRT e uma tendência para o mesmo resultado no 7N nos animais tratados com creatina, quando comparados com animais veículo. Assim, e tendo em conta que a creatina apresentou um efeito benéfico nos sintomas motores e também melhorou a perda de força muscular dos animais transgénicos, pode dizer-se que existe uma correlação direta entre densidade de IINs positivos para a ATXN3 e o efeito positivo no fenótipo destes animais. Em contraste, apesar de o 17-DMAG ter tido um efeito positivo no fenótipo dos animais, não foi observada uma redução da densidade de IINs nos núcleos LRT e 7N. Por fim, o ácido valpróico demonstrou ter um efeito muito ligeiro e tardio no fenótipo dos animais, não tendo sido observada uma redução significativa da densidade dasIINs nas áreas acima referidas. Globalmente, estes resultados demonstram que não existe uma relação direta entre densidade de IINs nestas regiões e a melhoria a nível fenotípico dos animais transgénicos para a DMJ

Test anxiety levels of board exam going students in Tamil Nadu, India

The latest report by the National Crime Records Bureau has positioned Tamil Nadu as the Indian state with highest suicide rate. At least in part, this is happening due to exam pressure among adolescents, emphasizing the imperative need to understand the pattern of anxiety and various factors contributing to it among students. The present study was conducted to analyze the level of state anxiety among board exam attending school students in Tamil Nadu, India. A group of 100 students containing 50 boys and 50 girls from 10th and 12th grades participated in the study and their state anxiety before board exams was measured by Westside Test Anxiety Scale. We found that all board exam going students had increased level of anxiety, which was particularly higher among boys and 12th standard board exam going students. Analysis of various demographic variables showed that students from nuclear families presented higher anxiety levels compared to their desired competitive group. Overall, our results showing the prevalence of state anxiety among board exam going students in Tamil Nadu, India, support the recent attempt taken by Tamil Nadu government to improve student's academic performance in a healthier manner by appointing psychologists in all government schools

Signaling pathways influencing tumor microenvironment and their exploitation for targeted drug delivery

In the recent years, the "tumor microenvironment" has been receiving growing attention due to its involvement in neoplastic transformation, tumor growth, invasion, and protection of tumor cells from host immune response. All these events are facilitated by chemical signals produced by the tumor as well as the surrounding stromal cells. This review is divided into two main parts in which the first part discusses the receptor tyrosine kinase (RTK)-mediated growth factor signaling, steroid hormone (SH) signaling, ancient signaling pathways, and other molecules that are involved in tumorigenesis and how they interact with each other to create a complex tumor microenvironment. In the second part, we bring together the recent nanocarrier-mediated drug delivery approaches to target the signaling pathways/molecules present in the tumor microenvironment.Foundation for Science and Technology (FCT) [(SFRH/BPD/89493/2012]; FCT [SFRH/BD/72809/2010]; Portuguese Government; FCT national funds (PIDDAC) [PTDC/AGR-GPL/119211/2010, PEst-C/AGR/UI4033/2011]; European Fund for Regional Development (FEDER) through COMPETE Operational Programme Competitive Factors (POFC)info:eu-repo/semantics/publishedVersio

Delivery as nanoparticles reduces imatinib mesylate-induced cardiotoxicity and improves anticancer activity

Clinical effectiveness of imatinib mesylate in cancer treatment is compromised by its off-target cardiotoxicity. In the present study, we have developed physically stable imatinib mesylate-loaded poly(lactide-co-glycolide) nanoparticles (INPs) that could sustainably release the drug, and studied its efficacy by in vitro anticancer and in vivo cardiotoxicity assays. MTT (methylthiazolyldiphenyl-tetrazolium bromide) assay revealed that INPs are more cytotoxic to MCF-7 breast cancer cells compared to the equivalent concentration of free imatinib mesylate. Wistar rats orally administered with 50 mg/kg INPs for 28 days showed no significant cardiotoxicity or associated changes. Whereas, increased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels, and reduced white blood cell, red blood cell, and hemoglobin content were observed in the animals administered with free drug. While the histological sections from hearts of animals that received INPs did not show any significant cardiotoxic symptoms, loss of normal architecture and increased cytoplasmic vacuolization were observed in the heart sections of animals administered with free imatinib mesylate. Based on these results, we conclude that nano-encapsulation of imatinib mesylate increases its efficacy against cancer cells, with almost no cardiotoxicity