No description available.

No description available.

The complex heterogeneity of schizophrenia has proved difficult to replicate in preclinical animal models. Of the many molecular targets implicated with schizophrenia, this thesis focuses on synapsin II - a pre-synaptic protein critical for neurotransmission and synaptogenesis; and parvalbumin - a calcium-binding protein found in interneurons of the dorsolateral prefrontal cortex (DLPFC) and the striatum (STR). Patients with schizophrenia display reduced levels of synapsin II mRNA in the DLPFC, while decreased activation of parvalbumin neurons in the same region has resulted in schizophrenia-like cognitive deficits. Knockdown of synapsin II in the medial prefrontal cortex (mPFC) of neonate and adult rats has previously induced schizophrenia-like alterations. However, there are concerns that must be addressed before novel animal models of schizophrenia can be developed using reductions in synapsin II. This thesis was designed to 1) eliminate maternal separation (MS) between post-natal days (PD) 14-23, which correlates with a neurodevelopmental synapsin II model, as a means of inducing schizophrenia-like behaviours; 2) reassess the use of fully and partially phosphorothioated first-generation antisense oligonucleotides to reduce synapsin II levels, and 3) evaluate parvalbumin expression in the STR following synapsin II knockdown. Results from this study indicate 1) a 36 hour MS regimen during PD 14-23 did not cause behavioural changes bearing resemblance to schizophrenia; 2) oligonucleotide sequences stabilized completely with phosphorothioate bonds were insufficient in reducing synapsin II levels and caused localized necrosis, while partially modified sequences induced a slight knockdown effect without cell death; and 3) levels of striatal parvalbumin expression were decreased in rats receiving the partially, but not fully, modified antisense sequences. The findings strengthen the face validity and safety profile of the synapsin II knockdown model. Novel evidence has also been provided for the role of parvalbumin in the striatum and suggests its influence on cognitive dysfunction in schizophrenia. / Thesis / Master of Science (MSc)

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Algal blooms caused by toxigenic cyanobacterial species are an increasing economic burden globally, as high anthropogenic inputs of nitrogen and phosphorous, coupled with rising levels of atmospheric CO2, promote eutrophication and enhance bloom proliferation. Of the freshwater bloom forming species, Microcystis aeruginosa has garnered the most attention due to the production of toxic secondary metabolites known as microcystins. These cyclic peptides are potent eukaryotic protein phosphatase 1 and 2A inhibitors, and can induce hepatic damage if concentration levels reach above the World Health Organization level of 1 µg/L. Current mitigation strategies of water column disruption or by use of broad acting chemicals, are limited in their range and may cause unwanted off target effects to the surrounding biota. Antisense oligonucleotides are short single-stranded DNA polymers that hybridize with transcribed mRNA, and suppress translation of protein products through steric hindrance of ribosomes, or by RNAse H degradation of the DNA/RNA bound complex. While antisense oligonucleotide applications have proven successful in the pharmaceutical industry, their potential remains largely unexplored in environmental contexts. For this reason, we investigated the knockdown of microcystin synthetase gene cluster mcyE in M. aeruginosa. We found that ionic charge neutralization coupled with heat shock were effective chemical competence based methods for delivery, mcyE transcript abundance in cells treated with phosphodiester linked antisense oligonucleotides significantly decreased in RT-qPCR analysis, and production of intracellular microcystin significantly decreased over a 24 hour period (-1.9 fg/cell). This work demonstrates a novel proof of concept for the potential use of exogenous antisense oligonucleotides to target M. aeruginosa in harmful algal bloom occurrences.

No description available.

No description available.

The mdx mouse is widely used as a model for Duchenne Muscular Dystrophy, a fatal X-linked disease caused by a deficiency of the sub-sarcolemmal protein, dystrophin. This dissertation reports characterisation of the features of dystrophy in the mdx mouse, including parameters such as electrophysiological and contractile properties of dystrophic cardiac tissue, quantitative evaluation of kyphosis throughout the mdx lifespan, and contractile properties of respiratory and paraspinal muscles. Following these characterisation studies, the efficacy of antisense oligonucleotides (AOs) to induce alternative mRNA splicing in mdx skeletal muscles (diaphragm and paraspinal muscles) was evaluated. The left atria of younger (<6 weeks) and older (>15 months) mdx mice showed consistently lower basal forces and responsiveness to increased calcium, while action potential duration was significantly shorter in young mice (3 weeks) and older mice (9 and 12 months) (P<0.05). Cardiac fibrosis increased with age in mdx atria and ventricles and was elevated in young (6-8 weeks) and old (15 months) mdx compared to control mice (P<0.01). This study provided insights into DMD cardiomyopathy, and suggested that very young or old mdx mice provide the most useful models. Mdx mice show thoracolumbar kyphosis like boys with Duchenne Muscular Dystrophy. A novel radiographic index, the Kyphotic Index (KI), was developed and showed that mdx mice are significantly more kyphotic from 9 months of age, an effect maintained until 17 months (P<0.05). At 17 months, the paraspinal and respiratory muscles (latissimus dorsi, diaphragm and intercostal muscles) are significantly weaker and more fibrotic (P<0.05). Administration of AOs at four sites within the diaphragm at 4 and 5 months of age significantly increased twitch and tetanic forces compared to sham treated mdx (P<0.05). However, no difference in collagen was evident and dystrophin was not detected, possibly due to the low concentration of AO utilised. This study suggested that AOs can provide functional improvement in treated skeletal muscles. Monthly injections with AOs into the paraspinal muscles from 2 months to 18 months of age alleviated kyphosis, without significantly altering twitch and tetanic forces of latissimus dorsi, diaphragm and intercostal muscles. There was evidence of less fibrosis in diaphragm and latissimus dorsi muscles (P<0.05) and reduced central nucleation of the latissimus dorsi and intercostal muscles (P<0.05). Again, dystrophin was not detected by immunoblot. These studies indicate that very young and old mdx mice display previously uncharacterised dystrophic features, and are useful models for testing new therapies such as AOs. Low doses of AOs were shown to be safe and efficacious for long-term use, however there remains a need for testing higher concentrations and improved delivery strategies.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

La maladie de Huntington (MH) est causée par une expansion de répétitions CAG sur l’exon 1 du gène huntingtine (htt), codant pour une protéine mutée. Il a été montré que la diminution d’expression de cette protéine est une piste thérapeutique très prometteuse. Dans ce projet, nous avons étudié et comparé trois approches dites «antisens» : une stratégie allèle non-spécifique, visant à diminuer de manière générale l’expression de htt ; une stratégie allèle spécifique ciblant les répétitions CAG afin d’impacter préférentiellement l’allèle muté ; et enfin une stratégie de saut d’exon permettant d’enlever des sites de clivage à l’origine d’une forme raccourcie et toxique de la protéine htt. Nous avons évalué ces approches grâce à deux outils différents : les tricyclo-DNA (TcDNA), qui sont une nouvelle classe d’oligonucléotides antisens (AON) plus performante que les chimies précédentes, et le système U7snRNA vectorisé, permettant d’induire une expression stable des séquences antisens. Dans un premier temps, ces différentes molécules ont été évaluées in vitro dans des lignées de fibroblastes de patients en quantifiant le niveau d’ARNm et de protéines htt par RTqPCR et Western blot respectivement. Par la suite, les séquences les plus efficaces in vitro ont été sélectionnées et les AON et AAV-U7snRNA correspondants ont été injectés en intracérébroventriculaire (ICV) dans un modèle murin de la MH (souris YAC128). Les résultats les plus encourageants ont été obtenus avec le TcDNA-NS (pour allèle Non Spécifique), permettant une diminution significative de l’expression de htt dans le cortex, l’hippocampe et le striatum 2 et 6 semaines après une injection ICV. Ces résultats prometteurs suggèrent le potentiel des TcDNA comme nouvel outil thérapeutique pour la MH. / Huntington’s disease (HD) is caused by a CAG repeat expansion in the exon 1 of huntingtin gene (htt), encoding for a mutant protein. It has been shown that the silencing/down regulation of huntingtin protein is a promising therapeutic lead. In this project, I have explored and compared three strategies using the antisense approach: a non-allele specific strategy, aiming to silence the global expression of htt; an allele specific strategy targeting CAG repeats to silence preferentially the mutant allele; and an exon-skipping strategy in order to remove cleavage sites which originally cause a shorter and toxic form of the htt protein. These strategies have been evaluated using two different tools: tricyclo-DNA (TcDNA), a new class of antisense oligonucleotides (AON) more efficient than the previous chemistries, and a vectorized approach using U7snRNA system allowing a stable expression of antisense sequences. Firstly, these different molecules have been assessed in vitro in HD fibroblasts quantifying mRNA and htt protein levels with RTqPCR and Western blot respectively. Subsequently, the most efficient sequences have been selected and intracerebroventricular (ICV) injections have been performed with corresponding AON and AAV-U7snRNA in a HD mouse model (YAC128). The most encouraging results have been obtained with the TcDNA-NS (for Non Specific allele), allowing a significant decrease of htt expression in cortex, hippocampus and striatum 2 and 6 weeks after ICV injection. These promising results suggest the potential of TcDNA as a new therapeutic tool for HD.