Huntington's disease, an autosomal dominant neurodegenerative disease, affects the cell in several toxical ways. One of them is accumulation of protein aggregates in cytoplasma, which could become a serious problem especially for long-lived cells such as neurons. Autophagy (macroautophagy) is an important catabolic pathway, crucial for cell survival. If fully functional, it should eliminate protein aggregates and reduce the toxic effect on the cell. However, recent works show that this pathway might be defective, most probably in the cytoplasmic cargo recognition. In my work I used a transgenic miniature pig model of Huntington's disease to verify the hypothesis of autophagical dysfunction in individuals suffering from Huntington's disease. I studied levels of autophagosomal markers - LC3 and p62 in mesenchymal stem cells after different autophagy stimulation treatments, and ammonium chloride was found the most effective. In addition I evaluated the effect of age of the animals on autophagic function, but no significant changes were identified, even if animal genotype was considered. Moreover I had an opportunity to study proteins levels in three porcine brain tissues - cortex, cerebellum and striatum. Even though there is no significant diference, we can observe a trend of LC3 II and p62 increase in...

Adenosine is a ubiquitous metabolite with multiple physiological functions in organisms. In this thesis, I studied the effect of extracellular adenosine on Huntington´s disease (HD) model Drosophila melanogaster. I show that extracellular Adenosine signaling mitigates HD pathology by observing three main types of symptoms of the disease in Drosophila. The results suggest that the mechanism involves Drosophila melanogaster adenosine receptor signaling.

The Huntington disease (HD) is a hereditary neuro-degenerative disorder caused by a mutation of the huntigtin gene that codes a protein of the same name. The mutated form of the huntigtin gene plays its part in many pathological interactions and influences a number of cellular mechanisms, including the immune system that could serve as a modifier of the neuropathology of the disease. The cells of the monocyte-macrophage system express cytokines whose production changes in relation to the activation of the cell. The presence of the mutated huntingtin protein in these cells renders them hyper-responsive to immunity incentives leading to changes in the production of cytokines. These differences are discernible a few years prior to the appearance of the symptoms. Therefore, the changes in the levels of certain cytokines could serve as appropriate biomarkers for monitoring of the onset of the disease and its progression. The HD pathogenesis includes an inflammation of the central neutral system. Inflammatory changes in peripheral tissues could reflect inflammatory processes in the central neural system. A miniature TgHD pig could represent an appropriate model organism for studying of the impact of the mHtt on the immune system. This model enables to observe a slow progression of the disease. Changes in...

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...

Huntington's disease is a dominantly-inherited autosomal neurodegenerative disease manifested by disorders of motility, cognitive function, behaviour, and weight loss, which is conditioned multifactorially. The aim of the study was to determine whether there are eating disorders in Huntington's disease, as well as its etiology and severity. Neurological scaling, anthropometric examinations, evaluation of three-day diet records, measurements with a manual dynamometer, bioimpedance analyses, indirect colorimetry and predictions of energy expenditure were performed on 10 patients. Algorithms were applied for the diagnosis of sarcopenia and malnutrition. Unwanted weight loss was observed in all patients and 4 out of 10 showed malnutrition. No difference was found between the values of measured resting metabolism and calculated according to the predictive equation. However, it has been shown that strict nutritional recommendations based on this data can be misleading for some patients with HN, as real energy consumption can be significantly higher. All our patients had a positive energy balance. A new diagnostic algorithm for the early diagnosis of sarcopenia has proven its worth. Using bioimpedance analysis and examination of the force of the handshake, we identified possible sarcopenia and already-present...

Stable porcine ES cell lines have not been succesfully established yet. Ability to selfrenew or to differentiate has been limited in different porcine ES-like cell lines so far. PiPSCs represent an alternative to pESCs. PiPSCs can be generated by reprogramming of somatic cells by introduction of several transcription factors on viral vectors and were established by several groups. However, the majority of piPS cell lines depend on transgene expression because of incomplete reprogramming and weak activation of endogenous pluripotency genes. Transgene expression can infuence differentiation potential of piPSCs. Therefore, we have used integrative and reexcisable PiggyBac transposons to generate viral free piPSCs. At the same time, small molecules (low-molecular inhibitors) with potential to increase reprogramming efficiency and to activate endogenous pluripotency genes were used in the reprogramming media. This strategy has a potential for generation of naive piPSCs. Successful excision of transgenes would generate transgene-free piPSCs with uncompromised differentiation potential. Pig (Sus Scrofa) is at the same time an important animal model in preclinical stage research of the diseases. Somatic cells used for generation of piPSCs were isolated from pigs carrying mutated huntingtin. Integration of the...

Huntington's disease (HD) is an autosomal dominant inherited neuro-psychiatric disease with usual onset in the middle age. The mutation, located on the short shoulder of chromosome 4, is an expansion of a nucleotide triplet, containing cytosine, adenine, guanine (CAG), with critical limit of 40+ repetitions. The principal symptoms include motor symptoms (chorea, dystonia, disorders of voluntary movements), progressive cognitive deterioration and neuropsychiatric symptoms (behaviour disorders, affective symptoms and so on). The clinical diagnosis is confirmed by a genetic test, which may also be carried out presymptomatically in offsprings of the diseased person. The objective of the 1st study consisted in the characterization of differences in psychiatric examination and neuropsychological testing among the people at risk (PAR), in whom it was recommended to delay the test, and people at risk, who were recommended to continue in the so-called predictive protocol. The total of 52 people have been examined (32 females, 20 males). In addition to the common psychiatric examination we have also administered the Eysenck Personality Questionnaire (EPQ-A), self-rating scale of general psychopathology (SCL- 90), three short cognitive tests - Trail making test, test of Verbal fluency and...

This diploma thesis explores problematic areas of patients with Huntington's disease in their performance during activities of daily living (ADLs) from the perspective of patients and their caregivers. The aim of the research was also to assess a possible correlation between cognitive impairment and the patient's performance in ADL. Twenty-five patients with their caregivers met the selection criteria for the research. There were used standardized assessment methods available in Czech: the Montreal Cognitive Assessment (MoCA), the Canadian Occupational Performance Measure (COPM) and the questionnaire for caregivers called Bristol Activities of Daily Living Scale (BADLS-CZ). The statistical analyses consisted of methods of the nonparametric statistics, qualitative analysis was processed by data categorizing. Caregivers reported more problematic areas in ADLs which was significantly confirmed in the statistical hypothesis testing (p <0,05). A significant correlation was seen between the results of the questionnaire and the results of the MoCA assessment (rSp = -0,620; p <0,05). For various reasons, patients with Huntington's disease did not mention as many problematic areas in performing ADL as their caregivers. Therefore, it is appropriate in clinical practice to supplement the assessment of the patient's...