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Huntington's disease is an inherited neurodegenerative disease caused by a mutation in the huntingtin gene. Its expressed product, mutated huntingtin protein, aggregates in the cell and affects wide range of cellular processes, including proteostasis mechanisms. Autophagy, classified among the mechanisms of cellular degradation and recycling of proteins and other cellular components, is one of the processes disrupted by the presence of mutated huntingtin. The goal of the project was to prepare a tandem genetic construct (fusion protein LC3-EGFP-mCherry) using chemically competent bacteria by molecular cloning methods, transfect it into prepared human lines carrying both normal and mutated huntingtin, and thus modify the lines to transiently, and if possible also constantly express this sensor of autophagy activity. Such modified lines allow monitoring the effect of the presence of normal and mutated HTT on the course of autophagy and also provide experimentally suitable conditions for influencing autophagy in these cells using inhibitors and activators from the group of small molecules. Two types of LC3-GFP-mCherry/RFP fusion proteins were successfully prepared by molecular cloning and transiently introduced by lipofection into the control ARPE19 lines. Expression of the constructs in human H9 NSC...

OF THE BACHELOR THESIS This thesis deals with the issue of nutritional disorders in Huntington's disease. Huntington's disease is an incurable, neurodegenerative, autosomal dominant inherited disease. The prevalence of Huntington's disease in Western society is 10.6-13.7 / 100,000 individuals. The disease is manifested by disorders of free motor control, speech, swallowing, postural instability, cognitive deterioration and various behavioral changes. An integral part of the disease is unwanted weight loss. In the various stages of the disease, the causes of the development of nutritional disorders may differ and combine with each other. In the theoretical part, the chapters are devoted to the etiology and pathogenesis, pathology, clinical manifestations, forms and stages of the disease, nutritional disorders and their classification, methods of nutritional status examination and specific causes of nutritional disorders that occur in each stage of the disease. A special chapter focuses on the classification of nutritional disorders according to the European Society for Clinical Nutrition and Metabolism, which was applied in the research. The practical part aims to examine the nutritional status and evaluate eating habits in a group of patients with Huntington's disease through clinical and...

Members of the large family of G proteins and their coupled receptors are involved in a variety of transduction processes the cell uses to respond to a received signal. Depending on their specific structure and function, they influence a wide range of effector molecules. A large body of research has shown that many neurodegenerative diseases have a negative impact on the signal pathways controlled by G proteins. Due to ageing population, neurodegenerative diseases are currently imposing a risk for growing numbers of people. The sequelae observed in the pathological development of such diseases include especially changes in membrane receptors representation or receptor uncoupling from G protein, which inhibits G subunits activation. The undesirable inhibition or over-stimulation of G proteins results in the increase or decrease in effector activity, which subsequently impacts the production of second messengers and the activity of subsequent members of the signal cascade. As a result, these alterations lead to an increase in intracellular concentration of Ca2+ ions, which then influence receptors responsible for excitotoxicity, and contribute to apoptosis and necrosis of neuronal population. The thesis summarizes the defects of signalling pathways controlled by trimeric G proteins in association...

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...

Due to the aging of the population, there is an increasing incidence of neurodegenerative diseases. In clinical practice there is a need to for a cheap and noninvasive method for screening and early diagnosis of neurodegenerative disorders. To this end, markers of disease progression and prognosis must be determined. EEG correlates provide information that can be used in the diagnosis and prognosis of neurodegenerative disorders. Individual diseases have their specific EEG abnormalities that are closely related to different stages of the disease. Individual illnesses - Alzheimer's disease, Parkinson's disease, Huntington's disease have their specific changes in the basic rhythms of the brain that correlate with motor and cognitive changes. This work focuses on the quantitative (qEEG) correlates of the above-mentioned diseases. Key words: brain, neural activity, EEG, quantitative EEG analysis, biomarker, connectivity, neurodegeneration, Alzheimer's disease, Parkinson's disease, Huntington's disease.

Complex functions of the basal ganglia are affected by numerous sensory and sensitive stimuli. In our studies, we investigated parameters of sense of smell and vision in neurodegenerative diseases of the basal ganglia - Parkinson's disease (PD) and Huntington's disease (HD). In the first study, we use Odourized Markers Test (OMT) to determine its applicability in PD patients, and to determine whether it distinguishes olfactory disorders between neurodegenerative and other disorders. Results show that OMT is applicable for PD patients and comparable to Sniffin' Sticks as it demonstrates gains of lower scores in PD patients compared to healthy subjects, but they do not differentiate other etiology of olfactory disorders. In the next study, we tested the pleasantness of odor stimulants in PD patients using New test of odor pleasantness (NTOP). We investigated suitability and validity of its use. We found that PD patients had lower odor rating score compared to healthy group correlated with Sniffin' Sticks and OMT. In the following study, we examined whether PD patients with visual hallucinations (PDH+) have structural retinal changes measured by optical coherence tomography (OCT) and functional retinal changes examined by 2,5% contrast sensitivity test compared to PD patients without hallucinations...

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

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...

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 is a neurodegenerative disease affecting the nervous system. It is caused by the mutation of the huntingtin protein coding gene. The mutation is manifested by the multiplication of CAG triplets. Huntingtin is present more in the cytoplasm. It interacts with many proteins and has roles also in transcription and cell transport. Huntingtin also participates in correct regulation of embryonic development and development of nerve tissue. Mutant huntingtin causes oxidative stress, mitochondrial biogenesis and OXPHOS disorders. Diagnosis of Huntington's disease is based on a laboratory examination of the presence of an allele predisposing to the disease. Clinical imaging is also an important part of diagnostics. The patient exhibits uncontrollable choreatic body movements and dementia. The aim of the thesis was to describe the main characteristics of Huntington's disease with the focus of HD on mitochondrial energy metabolism. In the practical part, the aim was to analyze mitochondrial respiration by high resolution polarography in a set of 3 fibroblast lines from patients with Huntington's disease. Polarographic analyses showed that respiration of HD fibroblasts was decreased in comparison with controls. We found that complex II of oxidative phosphorylation was most affected in HD...

The Role of the Occupational Therapist in the Treatment of Patients with Huntington's Disease Abstract: This thesis is focused on the possible potential of occupational therapy in patients with Huntington's disease, a neurodegenerative brain disease. Currently, Huntington's disease is incurable, but there are some therapeutic methods and approaches which have positive influence on a progress of the disease. Also, early intervention of occupational therapy is needed and the occupational therapist is an irreplaceable specialist as a part of an interdisciplinary team. However, in the Czech Republic there is a little experience with occupational therapy in patients with Huntington's disease. The thesis consists of a theoretical part and a practical part. The theoretical part provides basic information about the condition and by having used specialized foreign literature it also focuses on the opportunities and goals of occupational therapy in patients with Huntington's disease. In the practical part, questionnaires which were distributed among patients and patient care providers were evaluated. Results of the questionnaires provide besides other things a brief outline of patient's problematic areas. Occupational therapy evaluation of patients with Huntington's disease includes the Montreal Cognitive Assessment...

Huntington's disease (HD) is an autosomal dominant hereditary neurodegenerative disease characterized by motor, cognitive and behavioral disorders. HD is caused by expansion of CAG triplet (cytosine-adenosine-guanine) located in a gene on the short arm of the fourth chromosome. This expansion encodes an aberrant polyglutamine chain in the protein huntingtin. Physiological and mutated huntingtin (in case of HD) are expressed in almost all tissues and influences many cellular functions. The prevalence of HD in population is about 1 per 10.000. The disease is currently incurable and its mechanisms are not sufficiently understood. Besides affecting the central nervous system HD also affects peripheral tissues, including skeletal muscles. HD disrupts mitochondrial function and damages oxidative phosphorylation system, which has the task of producing energy in the form of ATP in cells. Research of transgenic minipig model for HD could help elucidate the mechanisms of disease's pathogenesis and potential therapeutic strategy. In this diploma thesis, immunodetection with help of specific antibodies to detect changes in amount of 14 selected mitochondrial proteins in skeletal muscle tissue of three age groups of transgenic HD minipigs - 24, 36 and 48 months old was used. Gradual progression in reduced...

The bachelor thesis deals with the importance of nitric oxide in the development of neurodegenerative diseases. Nitric oxide is a biological active gas which affects, among other things, many functions of the central nervous system. Its activity is dependent on the concentration in small amounts has a positive effect, while overproduction contributes significantly to the development of neurodegenerative diseases. In my work, I deal with the negative influence of nitric oxide in the development of epileptic seizures, Parkinson's disease, Alzheimer's disease and Huntington's disease. In conclusion, I mentioned the possibility of determining the amount of nitric oxide concentration. Keywords Nitric oxide, ROS, RNS, neurodegenerative diseases, epileptic seizures, Parkinson's disease, Alzheimer's disease, Huntington's disease

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...

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 inherited autosomal dominant neurodegenerative disorder caused by an unstable expansion of the CAG repeat sequence within the huntingtin gene. Huntingtin associates with ubiquitin-proteasome system that ensures degradation of particular proteins including β-catenin which is an important molecule whose equilibrated degradation is necessary for the proper functioning of the Wnt signaling pathway. The binding of β-catenin to the destruction complex is altered in HD, leading to the toxic stabilization of β-catenin. The main goal of my thesis was to determine whether the accumulation of β-catenin due to the presence of mutant huntingtin is also characteristic of Liběchov minipigs, a large animal model of Huntington's disease stably expressing N-truncated human mutant huntingtin. Using immunoblot and specific antibodies, we have revealed age-dependent accumulation of mutant huntingtin in transgenic minipigs. Unlike endogenous huntingtin, no decrease of the level of mutant huntingtin was observed in the striatum of transgenic animals. Surprisingly, this was followed by a decrease of phosphorylated β-catenin. Nevertheless, our results demostrate the accumulation of β-catenin in mesenchymal stem cells isolated from the oldest boars during ontogenesis. Furthermore, we have revealed a...

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...