Intervertebral discs (IVD) degenerate earlier than many other musculoskeletal tissues and will continue to degenerate with aging. IVD degeneration affects up to 80 percent of the adult population and is a major contributing factor to low back pain. Anti-sclerostin antibody is an FDA-approved treatment for osteoporosis in postmenopausal women at high-risk for fracture and, as a systemic stimulant of the Wnt/LRP5/b-Catenin signaling pathway, may impact the IVD. Stabilization of b-Catenin in the IVD increases Wnt signaling and is anabolic to the extracellular matrix (ECM), while deletion of b-catenin or LRP5 decreases Wnt signaling and is catabolic to the ECM. Here, we hypothesized that a reduction of Sost would stimulate ECM anabolism. Lumbar and caudal (tail) IVD and vertebrae of Sost KO and WT (wildtype) mice (n=8 each) were harvested at 16 weeks of age and tested by MRI, histology, immunohistochemistry, Western Blot, qPCR, and microCT. Compared to WT, Sost KO reduced sclerostin protein and Sost gene expression. Next, Sost KO increased the hydration of the IVD and the proteoglycan stain in the nucleus pulposus and decreased the expression of genes associated with IVD degeneration, e.g., heat shock proteins. However, deletion of Sost was compensated by less unphosphorylated (active) b-Catenin protein in the cell nucleus, upregulation of Wnt signaling inhibitors Dkk1 and sFRP4, and catabolic ECM gene expression. Consequently, notochordal and early chondrocyte-like cells (CLCs) were replaced by mature CLCs. Overall, Sost deletion increased hydration and proteoglycan protein content, but activated a compensatory suppression of Wnt signaling that may trigger chondrogenesis and may potentially be iatrogenic to the IVD in the long-term.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Gabriela Kocurová Supervisor: Prof. MUDr. Jaroslav Dršata, CSc. Title of diploma thesis: Investigation of proteolytic enzymes expression in different tissues at the transgenic animal model of Huntington's disease by means of biochemical and immunohistochemical methods Background: Huntington's disease (HD) is a neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the huntingtin (Htt) protein. Because it is known that mutant Htt and especially its small proteolytic fragments are toxic to neurons (particularly those in the striatum and cortex), it has been suggested that proteolysis of mutant huntingtin (mHtt) might play an important role in HD pathogenesis. Therefore, the aim of the present study was to examine the expression of endogenous and mtHtt and possible participation of the proteolytic enzymes from the group of caspases, matrix metalloproteinases (MMPs), kallikreins (KLKs) and calpains in HD pathology of brain tissue. Methods: In this study we used WT and TgHD minipigs for N-terminal part of the human mtHtt (548aaHTT-145Q, both F2 generation, age 36 months; F3 generation, age 48 months in additional experiment), R6/2 mice were used as...

Huntington's disease (HD) belongs to neurodegenerative disorders. It is a monogenic disease caused by trinucleotic CAG expansion in exon 1 of gene coding protein huntingtin. Even though the cause of HD is known since 1993, the pathophysiology and cure for HD reminds to be found. The animal models are being used for better understanding of HD. The most common animal models for HD are rodents, especially mice but it was also important to create large animal models, which will be more like human. Therefore, TgHD minipig was created in Academic of Science in Liběchov in 2009. This model was created by microinjection of lentiviral vector carrying N-terminal part of human HTT with 124 repetitive CAG in exon 1. This model is viable and in every generation, is part of the offspring transgenic. In this thesis, I specialized to biochemical and behavioral changes of this model. I compared transgenic and wild type siblings. I found that biochemical changes are manifested mostly by increased level of mtHtt fragments in testes and brain. In behavioral part of this thesis I established new methods for testing behavioral changes in this model. The introduced methods showed some changes between wild type and transgenic animals at the tested ages but these changes were not significant due to the low number of...

Huntington's disease (HD) is inherited fatal disorder caused by CAG triplet expansions in the huntingtin gene resulting in the expression of mutated huntingtin protein (mtHtt). The main symptoms of HD are neurodegeneration, osteoporosis, testicular degeneration, loss of muscle tissue and heart muscle malfunction, weight loss, metabolic changes, and sleeping disturbances. Since huntingtin protein (Htt) has a role in several biological processes, many molecular mechanisms, like oxidative stress, mitochondrial dysfunction, DNA-damage, and others, are affected by mtHtt. However, its exact pathogenic mechanisms in HD are still not well understood. Transgenic minipig model of HD (TgHD) serves an opportunity to isolate unlimited number of primary cells and unlike primary cells obtained from HD patients, often in the late stages of the disease, the TgHD minipig model allows to monitor molecular changes occurring gradually with age and progression of the disease. Thus, TgHD minipig model and primary cells isolated from it play an important role in investigating and understanding the underlying mechanistic cause of HD. We focused on molecular and cellular changes in primary cells isolated from TgHD minipigs and their wild type (WT) controls at different ages (24, 36, and 48 months). In mesenchymal stem cells...

Synthetic N-methoxybenzyl ("NBOMe") phenylethylamine derivatives were developed as highly potent agonists for the research of 5-hydroxytryptamine receptors, however they have become available on the black market in the past few years. They are sought by recreational users as "legal LSD", in worse cases the replacement is unsuspected and these substances are a cause of serious intoxication with signs of serotonin toxicity. At this moment, there is very little data available, but their low price, easy access via the internet and legal status make these substances an interesting target for recreational users and a big challenge for scientists and for the lawmakers. 2CBFly-NBOMe belongs to this group. There is no information about its recreational use for now, but thiat is probably only a matter of time. The aim of this study is to determine acute effects of 2CBFly-NBOMe in Wistar rats. Active substance significantly reduced locomotor activity of the animals and caused higher anxiety, it also disrupted sensorimotor gating and lowered body temperature of animals. Pharmacokinetic profile was measured only in the blood serum, the highest concentrations of 2CBFly-NBOMe were detected 30-60 minutes after the administration of hallucinogen. This paper provides first information on the effect of 2CBFly-NBOMe on...

Intrastriatal injections of L-Pyroglutamate (L-PGA) in mice produced behavioral and neuropathological effects that resemble in part the kainate-injected rat striatal model of Huntington's Disease (HD). The behavioral responses induced after unilateral injections of L-PGA included circling, postural asymmetry of head and trunk and possible dyskinesias. The neuropil in the injected striatum contained dilated profiles, degenerating neurons and oligodendroglia, and numerous phagocytic microglial-like cells. A dose response relation existed. The size of the lesion (expressed as a percent volume of the striatum destroyed) ranged from 1±0.18% at 0.02 μmoles to 20.2±3.97% at 200 μmoles L-PGA (pH=7.3). L-PGA is a weak neurotoxin when compared to kainic acid. Several factors raise interest in the possible role of L-PGA in HD, including the recently reported elevated plasma levels of L-PGA in some HD patients [51,52], and these are considered in the discussion.

The recent discovery that T cells recognize different sets of herpes simplex virus type 1 and type 2 epitopes from seropositive symptomatic and asymptomatic individuals might lead to a fundamental immunologic advance in vaccine development against herpes infection and diseases. The newly introduced needle-free mucosal (i.e., topical ocular and intravaginal) lipopeptide vaccines provide a novel strategy that might target ocular and genital herpes and possibly provide 'heterologous protection' from HIV-1. Indeed, mucosal self-adjuvanting lipopeptide vaccines are easy to manufacture, simple to characterize, extremely pure, cost-effective, highly immunogenic and safe. In this review, we bring together recent published and unpublished data that illuminates the status of epitope-based herpes vaccine development and present an overview of our recent approach to an 'asymptomatic epitope'-based lipopeptide vaccine.

La maladie d’Alzheimer (MA) est actuellement conceptualisée comme un continuum : la démence représentant la conséquence clinique d’une longue période où s’accumule des éléments pathologiques dans le cerveau d’individus indemnes de symptômes comportementaux. Les futures thérapies devront idéalement débuter avant l’apparition des symptômes mais le manque actuel d’outils reproduisant avec pertinence cette phase préclinique empêche leurs développements. Dans ce travail de thèse, nous avons d’abord levé ce verrou technologique. Un nouveau modèle a été créé dans lequel la production de peptides Aβs solubles en quantité faible, mais néanmoins suffisante pour induire une hyper-phosphorylation des protéines Tau, perturbe la fonction cognitive bien avant l’apparition d’éléments caractéristiques de la MA (plaques amyloïdes, dégénérescences neurofibrillaires, inflammation). Cette avancée technologique, nous a ensuite permis d’évaluer l’implication de la protéine kinase DYRK1A et le potentiel thérapeutique de molécules modulant ses fonctions dans l’ensemble de la pathologie Alzheimer. Nos résultats démontrent que l’inhibition de son activité kinase réduit l’hyper-phosphorylation des protéines Tau et améliore la fonction mnésique chez notre animal modélisant la phase préclinique de la MA. A contrario, nous démontrons que DYRK1A est sensible à un clivage protéolytique dans le cerveau de patients atteints de démence Alzheimer et acquiert de nouvelles fonctions biologiques. Dans ce contexte, la prévention de ce clivage réduit l’inflammation et restaure les déficits cognitifs chez la souris modélisant la phase clinique de la MA. En ciblant différentes phases de la MA, ces données ouvrent donc la voie à la médecine personnalisée et à des stratégies de traitement plus ciblées. / Current view conceptualizes Alzheimer’s disease (AD) as a continuum, with dementia representing the clinical outcome of a long period of cumulative pathological events in the brain of individual with free cognitive symptoms. New therapies for AD should ideally be started before the onset of symptoms but the lack of suitable tools mimicking preclinical stage of AD limits their future evaluations. In this work, we break this technological limitation. A new animal model have been developed in which a small amount of soluble Aβs forms able to induce hyper-phosphorylation of Tau is sufficient to disturb cognitive function long before classical lesions occur (amyloid plaques, neurofibrillary tangles and inflammation). This technological breakthrough allows us to evaluate involvement of the protein kinase DYRK1A and therapeutic potential of molecules modifying its functions in different stages of AD. Our results demonstrate that inhibition of its kinase activity reduces hyper-phosphorylation of Tau proteins and alleviates memory function in our preclinical AD-like animal model. In contrast, we provide evidences that DYRK1A undergoes a cleavage in brain of patient with clinical AD and gains new biological functions. Prevention of this proteolysis reduces inflammation and restores cognitive impairments in a clinical AD-like mice model. By targeting distinct phases of the disease, these data open avenue for personalized medicine and more-targeted treatment strategies.

Enterovirus D68 (EV-D68) virus has become more prevalent over the last 15 to 20 years. EV-D68 attacks the respiratory system and can cause severe disease in individuals who have underlying respiratory problems. There have also been reports of individuals with EV-D68 showing signs of neurological system problems and acute flaccid paralysis. Because of the increase in patients with EV-D68 and also the potential for neurological disease, an animal model is needed to study the disease and to evaluate experimental therapies for EV-D68 infection. To develop the animal model, 4-week old AG129 mice that lack alpha and beta interferon receptors, making them immunosuppressed, were used. The mice were infected with EV-D68 by the intranasal route to allow infection of the lungs. On day-3 post-infection the mice were euthanized and lungs were removed and homogenized for collection of virus. The newly collected virus was then used to infect another set of mice. This procedure was repeated 30 times. As passage number increased so did the amount of virus that was collected from the lungs of mice. The virus titer increased 320-fold between mouse passage 0 to 30. At the end of the thirtieth passage, multiple tissues (lungs, liver, kidney, spleen, blood, brain, spinal cord and leg muscle) were collected from infected mice over several days and titered to demonstrate how quickly the virus spread to various tissues within the mouse. The virus replicated most rapidly in the lungs and remained in the lungs longer than the other tissues evaluated. However, large quantities of virus were found in all tissues evaluated. Finally, several experimental antiviral compounds were evaluated: rupintrivir, pleconaril, ribavirin, enviroxime and guanidine, all of which showed therapeutic potential in cell culture. In the animal model rupintrivir, pleconaril, ribavirin and enviroxime did not show any therapeutic effect. Only guanidine reduced the amount of virus that was found in the lungs as well as in whole blood.

Contents of the present thesis were published in the following articles. 1. Suzuki S, Mori K, Higashino A, Iwasaki Y, Yasutomi Y, Maki N, Akari H. 2016. Persistent replication of a hepatitis C virus genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey. Microbiol Immunol 60:26-34. Copyright © 1999-2016 John Wiley & Sons, Inc.2. Yoshida T, Suzuki S, Iwasaki Y, Kaneko A, Saito A, Enomoto Y, Higashino A, Watanabe A, Suzuki J, Inoue K, Kuroda T, Takada M, Ito R, Ito M, Akari H. 2013. Efficient in vivo depletion of CD8+ T lymphocytes in common marmosets by novel CD8 monoclonal antibody administration. Immunol Lett 154:12-17.Copyright © 2013 Elsevier B. V. / 京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19546号 / 理博第4206号 / 新制||理||1604(附属図書館) / 32582 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 明里 宏文, 教授 岡本 宗裕, 教授 中村 克樹 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM