Algorithm for the Parkinson’s disease behavioural models characterization using a biosensor

Dissertação para obtenção do Grau de Mestre em Engenharia Biomédica

Dissecting gastrointestinal dysfunction and inflammation in a “pre-motor” rodent model of Parkinson’s disease

Research on Parkinson’s disease (PD) has mainly focused on the degeneration of the dopaminergic neurons of nigro-striatal (NS) pathway; also, post-mortem studies have demonstrated that the noradrenergic and the serotonergic transmitter systems are also affected (Jellinger, 1999). Degeneration of these neuronal cell bodies is generally thought to start prior to the loss of dopaminergic neurons in the NS pathway and precedes the appearance of the motor symptoms that are the “hallmark” of PD. Gastrointestinal (GI) motility is often disturbed in PD, manifesting chiefly as impaired gastric emptying and constipation. These GI dysfunction symptoms may be the result of a loss in noradrenergic and serotonergic innervation. GI deficits were evaluated using an organ bath technique. Groups treated with different combinations of neurotoxins (6-OHDA alone, 6-OHDA + pCA or 6-OHDA + DSP-4) presented significant differences in gut contractility compared to control groups. Since a substantial body of literature suggests the presence of an inflammatory process in parkinsonian state (Whitton, 2007), changes in pro-inflammatory cytokines in the gut were assessed using a cytokine microarray. It has been found in this work that groups with a combined dopaminergic and noradrenergic lesion have a significant increase in both expressions of IL-13 and VEGF. IL-6 also shows a decrease in treatment groups; however this decrease did not reach statistical significance. The therapeutic value of Exendin-4 (EX-4) was evaluated. It has been previously demonstrated that EX-4, a glucagon-like peptide-1 receptor (GLP-1R) agonist, is neuroprotective in rodent models of PD (Harkavyi et al., 2008). In this thesis it has been found that EX-4 was able to reverse a decrease in gut contractility obtained through intracerebral bilateral 6-OHDA injection. Although more studies are required, EX-4 could be used as a possible therapy for the GI symptoms prominent in the early stages of PD.

Comparison of different techniques for mouse transgenesis

RESUMO: O uso de ratinhos transgénicos em neurociências aumentou consideravelmente nos últimos anos devido ao crescente interesse em compreender o cérebro e a necessidade de solucionar situações clínicas do foro neurológico e psiquiátrico. Para esse efeito, diferentes métodos de produção de animais transgénicos têm sido testados. O objectivo desta tese foi comparar métodos de integração aleatória de um transgene no genoma de ratinhos em termos de eficiência, estabilidade da integração do transgene, número de animais e de horas de trabalho necessárias para cada método. Assim, foi comparado o método mais utilizado - microinjecção pronuclear (PNMI) - com duas outras técnicas cujo desempenho foi considerado promissor – a transferência génica através dos testículos por electroporação e transfecção por lentivírus in vivo. As três técnicas foram realizadas usando um gene repórter sob o controlo de um promotor constitutivo, e depois reproduzidas usando um gene de interesse de modo a permitir obtenção de um animal capaz de ser usado em experimentação laboratorial. O transgene de interesse utilizado codifica uma proteína de fusão correspondendo a uma variante da rodopsina (channelrhodopsin) fundida à proteína enhanced yellow fluorescente protein ((EYFP) resultando num produto designado ChR2-EYFP. Este animal transgénico apresentaria expressão deste canal iónico apenas em células dopaminergicas, o que, com manipulação optogenética, tornaria possivel a activação especifica deste grupo de neurónios e, simultaneamente, a observação do impacto desta manipulação no comportamento num animal em livre movimento. Estas ferramentas são importantes na investigação básica em neurociências pois ajudam a esclarecer o papel de grupos específicos de neurónios e compreender doenças como a doença de Parkinson ou a esquizofrenia onde a função de certos tipos de neurónios de encontra alterada. Quando comparados os três métodos realizados verifica-se que usando um gene repórter PMNI resulta em 31,3% de, a de animais transgénicos obtidos, a electroporação de testículos em 0% e a injecção de lentivírus em 0%. Quando usado o gene de interesse, os resultados obtidos são, respectivamente, 18,8%, 63,9% e 0%.--------------ABSTRACT: The use of transgenic mice is increasing in all fields of research, particularly in neuroscience, due to the widespread need of animal models to solve neurological and psychiatric medical conditions. Different methodologies have been tested in the last decades in order to produce such transgenic animals. The ultimate goal of this thesis is to compare different methods of random integration of a transgene in the genome of mice in terms of efficiency, stability of the transgene integration, number of animals required and the labour intensity of each technique. We compared the most used method – pronuclear microinjection (PNMI) – with two other promising techniques – Testis Mediated Gene Transfer (TMGT) by electroporation and in vivo lentiviral transfection. The three techniques were performed using a reporter gene – green fluorescent protein (GFP), whose transcription was driven by the constitutive cytomegalovirus (CMV) promoter. These three techniques were later reproduced using the tyrosine hydroxylase promoter (TH) and the neuronal manipulator, channelrhodopsin-2 fused to the enhanced yellow fluorescent reporter protein (ChR2-EYFP). The transgenic animal we sough to produce would express the light driven channel only in dopaminergic cells, making possible to specifically activate this group of neurons, while simultaneously observe the behaviour in a freely moving animal. This is a very important tool in basic neuroscience research since it helps to clarify the role of specific groups of neurons, map circuits in the brain, and consequently understand neurological diseases such as Parkinson’s disease or schizophrenia, where the function of certain types of neurons is affected. When comparing the three methods, it was verified that using a reporter gene PNMI resulted in 31.3% of transgenic mice obtained, testis electroporation in 0% and lentiviral injection in 0%. When using the gene of interest, the results obtained were, respectively, 18.8%, 63.9% and 0%.

Modulation of α-synuclein aggregation and toxicity

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Anti-apolipoprotein A-I antibodies in the pathogenesis of Alzheimer disease : project plan

RESUMO: A doença de Alzheimer (AD) é a forma mais comum de demência em todo o mundo e sua prevalência deverá duplicar até 2050. Os mecanismos precisos responsáveis pela AD são desconhecidas mas as características histopatológicas estão bem caracterizadas. A hipótese mais importante para a perda neuronal e declínio cognitivo na AD é a cascata amilóide que indica que AD é o resultado da sobreprodução de beta amilóide (Aβ) e / ou remoção ineficaz; a acumulação do BA no cérebro seria o passo crítico na patogénese da AD. Actualmente, a identificação de proteínas que se ligam ao Aβ e modulam a sua agregação e neurotoxicidade pode proporcionar a base para novas abordagens terapêuticas. A apolipoproteína AI (ApoA-I), o principal componente das HDL humanas, interage com o domínio extracelular da proteína precursora de amilóide (APP), bem como com o Aβ. Estudos epidemiológicos têm mostrado uma diminuição acentuada da ApoA-I plasmática em doentes com AD, com uma correlação inversa entre o nível de ApoA-I e o risco de AD. Este trabalho pretende apresentar um projecto que tem como objectivo investigar se os anticorpos anti-apo AI podem impedir a formação de complexos Aβ / ApoA-I, bloqueando o efeito protector da ApoA-I. A hipótese baseia-se na possibilidade dos doentes com AD terem anticorpos anti-ApoA-I plasmáticos e de estes poderem interferir com a formação do complexo no LCR.------- ABSTRACT:Alzheimer’s disease (AD) is the most common form of dementia world-wide and its prevalence is expected to double by the year 2050. The precise mechanisms responsible for AD are unknown but the histopathologic features are well-characterised. The most compelling hypothesis for neuronal loss and cognitive decline in AD is the amyloid cascade hypothesis which states that AD is the result of amyloid beta (Aβ) overproduction and/or ineffective clearance and its accumulation in the brain would be the critical step in AD pathogenesis. Currently, identification of proteins that bind Aβ and modulate its aggregation and neurotoxicity could provide the basis for novel treatment approaches. Apolipoprotein A-I (ApoA-I), the main constituent of human HDL, ApoA-I interacts with the extracellular domain of amyloid precursor protein (APP), as well as with Aβ itself. Epidemiological studies have shown a marked decrease of plasma ApoA-I levels in AD patients, with an inverse correlation between the ApoA-I level and the risk of AD. This work intends to present a project that aims to investigate if anti-ApoA-I antibodies may prevent the formation of the Aβ /ApoA-I complex and by doing so blocking the protective effect of ApoA-I in AD. We base the hypothesis on the possibility that patients with AD might have anti-ApoA-I antibodies in plasma and that these can interfere with the complex formation in the cerebrospinal fluid (CSF).

Cerebral vasculopathy in children with sickle cell disease A study of genetic modulators of the disease

Sickle cell disease (SCD) is a genetic disorder with recessive transmission, caused by the mutation HBB:c.20A>T. It originates hemoglobin S that forms polymers inside the erythrocyte, upon deoxygenation, deforming it and ultimately leading to premature hemolysis. The disease presents with high heterogeneity of clinical manifestations, the most devastating of which, ischemic stroke, occurs in 11% of patients until 20 years of age. In this study, we tried to identify genetic modifiers of risk and episodes of stroke by studying 66 children with SCD, grouped according to the degree of cerebral vasculopathy (Stroke, Risk and Control). Association studies were performed between the three phenotypic groups and hematological and biochemical parameters of patients, as well as with 23 polymorphic regions in genes related to vascular cell adhesion (VCAM-1, THBS-1 and CD36), vascular tonus (NOS3 and ET-1) and inflammation (TNF-α and HMOX-1). Relevant data was collected from patient’s medical records. Known genetic modulators of SCD (beta-globin cluster haplotype and HBA and BCL11A genotypes) and putative genetic modifiers of cerebral vasculopathy were characterized. Differences in their distribution among groups were assessed. VCAM-1 rs1409419 allele C and NOS3 rs207044 allele C were associated to stroke events, while VCAM-1 rs1409419 allele T was found to be protective. Alleles 4a and 4b of NOS3 27 bp VNTR appeared to be respectively associated to stroke risk and protection. HMOX-1 longer STRs seemed to predispose to stroke. Higher hemoglobin F levels were found in Control group, as a result of Senegal haplotype or of BCL11A rs11886868 allele T, and higher lactate dehydrogenase levels, marker of hemolysis, were found in Risk group. Molecular mechanisms underlying the modifier functions of the relevant genetic variants are discussed.