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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...

Stem cells use different mechanisms of intercellular communication to modulate an immune response. Mitochondrial transfer is one of the mechanisms which induce metabolic changes, support cell survival, and change the phenotype of immune cells. Nevertheless, little is known about the mechanism used for transfer of mitochondria between different cell populations and the faith of mitochondria inside the acceptor cell. This thesis aims to describe the mechanism of transfer and the provided modulation. Factors that could affect mitochondrial transfer including reactive oxygen species production, apoptosis and mitochondria function were analyzed. And the impact of mitochondrial transfer on cell survival and mitophagy was described. The next aim was to compare the ability of mesenchymal stem cells (MSC) and Sertoli cells (SC) to transfer mitochondria, with MSC being more productive in the transfer of mitochondria than SC. Significant differences in the presence of mitochondria from donor MSC or SC in individual populations of immune cells were also detected. To explain these findings, the impact of reactive oxygen species on the transfer of mitochondria was analyzed in detail, although it wasn't confirmed. However, it needs to be highlighted that mitophagy plays an important role before and after...

Plants are sessile organisms and when attacked by microbes, they cannot easily run away. For this reason, they have developed sophisticated defensive mechanisms, that allow them to defend themselves. Since plants, unlike mammals, do not have any special immune cells, their defense takes place in each cell separately. The key moment during a microbial infection is the recognition of the microbe by the plant through its released molecular patterns (mostly proteins) associated with microbes (MAMPs). MAMPs trigger signaling cascades that lead to the secretion of antimicrobial compounds to the site of an attack. The process of autophagy is also important in the defense against microbes, which not only maintains a cellular homeostasis and controls the level of phytohormones and defense proteins in the plant cytoplasm, but also participates in the secretory activity of the cell. Recent analyzes of plant secretome have shown that plants secrete many proteins (including defensive ones) independently of the signal peptide and compartments of a conventional secretion. During exocytosis a vesicle fuses with the cytoplasmic membrane. The octameric protein complex exocyst and SNARE proteins take part in this process. The exocyst complex is highly diversified in plants - especially it's EXO70 subunit, which is...

Name and surname of the author: Bc. Pavla Jelínková Institution: Third Faculty of Medicine, Charles University Title: Effect of illusory movements physiotherapy on vital and metabolic functions of patients with large burns Supervisor: MUDr. Bohumil Bakalář Year of defense: 2022 Keywords: large burns; illusory movements; proprioceptive neuromuscular facilitation; vital functions; catabolism; intensive care unit-acquired weakness The aim of this thesis was to determine how the usage of functional proprioceptive stimulation (so-called illusory movements) influenced vital and metabolic functions of patients with extensive burns. In the first part of the thesis we focus on defining the issues we encounter in patients with extensive trauma. Extensive damage not only affects metabolic and vital functions, but the extent of the skin injury changes prognosis of the injured person and the length of hospitalization. The longer patients are hospitalized in intensive care units, the more we encounter acquired muscle weakness. One of the ways to prevent this phenomenon is effective rehabilitation, which is quite challenging in burn patients. As an alternative, the usage of proprioceptive stimulation is offered. It creates illusions instead of real movements in the cerebral cortex. However, there has been no...

Oxidative phosphorylation (OXPHOS) system is best known for producing ATP in mitochondria. As a major metabolic hub, OXPHOS also supports other biological functions that are unrelated to ATP synthesis. Are these additional roles of OXPHOS unique to different cell states, such as to proliferation and quiescence, two of the most contrasting states of a mammalian cell? In this thesis we used genetic models of OXPHOS deficiency to show that in proliferating cells and in tumors OXPHOS primarily supports biosynthesis of nucleotides, whereas in quiescent cells OXPHOS provides oxidative stress resistance. Mechanistically, in growing tumors OXPHOS drives dihydroorotate dehydrogenase (DHODH), an enzyme crucial for de novo synthesis of pyrimidines. Complex II of OXPHOS also acts as a sensor of mitochondrial dysfunction and regulates pyrimidine synthesis for energy conservation. In contrast, in quiescent cells OXPHOS maintains autophagy by continuous low level ROS generation and together with the antioxidant defense provides constitutive oxidative stress protection. In summary, beyond ATP production OXPHOS has additional vital roles tailored to the particular requirements of quiescence and proliferation, with relevance for pathophysiology in cancer and vascular biology.

Study of mechanisms influencing inflammatory and neurodegenerative processes and their subsequent treatment in models of ALS and spinal cord injury The mechanisms of neurodegeneration during spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS) are complex and poorly understood, which is why it's troublesome to counteract them with effective therapies. This thesis explores the pathways of autophagy, endoplasmic reticulum (ER) stress, and the mammalian target of rapamycin (mTOR) pathway that regulates these mechanisms in models of both SCI and ALS. Upregulation of autophagy and the mTOR pathway in an in vivo contusion SCI injury model was confirmed. The mTOR inhibition led to upregulation of autophagy, reduction of inflammation, and recovery in acute SCI. Upregulated autophagy was discovered in the SOD1G93A rat model of ALS. By treating the ALS rats with human mesenchymal stem cells, prolonged survival of the animals and preservation of motor neurons (MNs) possibly occurred through modulation of autophagy. The involvement of the mTOR pathway in the degeneration of MNs was further explored in the context of astrocytes. Pleckstrin homology like domain family A member 3 (PHLDA3), a newly discovered repressor of the mTOR pathway, was found to lead to ER stress if overexpressed in astrocytes...

This bachelor thesis focuses on the issue of intermittent fasting and its impact on human health. The thesis is conceived as literary research. It presents various forms of intermittent fasting and their influence on physiological processes in the body and on specific diseases. Furthermore, this bachelor thesis focuses on negative side effects. The main aim of the thesis is to present the findings in a comprehensive form. The content of the thesis emerges mainly from foreign literature, articles and studies dealing with the issue. The work presents intermittent fasting as an alternative approach to eating with the potential to cure various diseases. It also discusses the advantages and disadvantages of other eating styles, such as a caloric restriction diet.

Exocyst is a protein complex composed of eight subunits, evolutionarily conserved in yeasts, animals, and plants. The main function of exocyst is to mediate the tethering of secretory vesicles to the plasma membrane. However, the involvement of exocyst in some other processes, especially in autophagy, has been recently discovered. Plant exocyst is specific because most of its subunits have multiple paralogs. The most diversified subunit is EXO70, which is encoded by 23 paralogous genes in Arabidopsis thaliana. In this thesis, I dealt with subunit AtEXO70E2 (AT5G61010), which has been localized to double-membrane compartments considerably reminiscent of autophagosomes. These compartments were named EXPOs (for exocyst-positive organelles) and described as a component of unconventional protein secretion pathways. There are also hints that EXO70E2 could play a role in autophagic processes. However, details of this relationship remained unexplored. For my experiments, I used stably transformed lines of A. thaliana and transiently transformed leaves of Nicotiana benthamiana. I performed numerous colocalization experiments, applied various pharmacological treatments to the studied lines, and analyzed a mutant line in the EXO70E2 gene. According to my observations, protein EXO70E2 is expressed especially...

The aim of this study was to analyse the process of apoptosis and autophagy in plasmacytoid dendritic cells and Langerhans cells infected with tick-borne encephalitis virus. Moreover, the thesis evaluates the effect of tick saliva on apoptosis and autophagy in these cells.

Autophagy is a process of degradation of the cell cytoplasm, proteins, and organelles. It appears mainly as a response to a lack of nutrients or damage of cell structures and components. Yeast Saccharomyces cerevisiae and also Komagataella phapphi are the main model organisms for the research of autophagy. Autophagy is classified by two main criteria. Firstly, we divide autophagy into macroautophagy and microautophagy. During macroautophagy, the phagophore vesicle, called the autophagosome, is created around sequestrated cargo and then fuses with the vacuole. Microautophagy is a process in which a vacuolar membrane directly surrounds and sequester degraded cargo. During non-selective (bulk) autophagy, a random non- specific portion of cytoplasm is degraded. On the other hand, selective autophagy serves as a pathway for the degradation of specific proteins and organelles. Autophagy research has nowadays a great medical significance thanks to the role of autophagy defects in a wide area of human diseases.

Vandetanib, lenvatinib and cabozantinib are inhibitors of receptor tyrosine kinases approved to treat locally advanced or metastatic thyroid gland, kidney and liver cancers. These multi- kinase inhibitors, inhibit phosphorylation of tyrosine moieties of protein, thus modulate cell signalization in cancer cells. Metabolites of vandetanib, lenvatinib and cabozantinib were detected in vitro as well as in vivo in blood and urine. Cytochromes P450 and flavin monooxygenases were identified as primary enzymes participating in metabolism of these drugs. Literature lacks information regarding pharmacological efficacy of vandetanib, lenvatinib and cabozantinib metabolites. The aim of this diploma thesis was the investigation of pharmacological efficacy of N-oxides of vandetanib, lenvatinib and cabozantinib. The viability measurement under normoxic and hypoxic conditions was employed to determined their efficacy. The expression of enzymes of the first phase of xenobiotics metabolism (CYP 450 1A1, 1B1, 3A4 a CYP 450 oxidoreductase) and receptor tyrosine kinases RET and VEGFR2, as well as mechanism of changes in their expression were investigated using western blotting and flow cytometry. High performance liquid chromatography was utilised to investigate possible metabolism of tyrosine kinase inhibitors and...

Caloric restriction has long been known in the scientific world as a life-prolonging condition, and there is growing evidence that the effect of activation of non-selective autophagy is responsible for these effects. In this thesis, the molecular mechanisms of autophagy in the state of caloric restriction will be described, as well as its beneficial effects on the aging of the organism and various health problems associated with aging and the modern way of life will be summarized. Some risks that could result from unprofessional intervention in one's own body will also be mentioned. It is hypothesized that practicing caloric restriction under the supervision of an expert brings prevention of diseases of civilization diseases and diseases associated with aging and prolongs quality of life, which would, among other things, help reduce the cost of health and social care. Keywords: Autophagy, caloric restriciton, molecular mechanisms

Autophagy is a cellular mechanism when cell gets rid of its damaged or unwanted components. These are wrapped into a double membrane and creates so-called autophagosome, which is transported to the vacuole, where its contents are degraded or recycled. Autophagy runs essentially through whole life of the plant, but if it is necessary, for example in response to stressful conditions, it is significantly intensified. It is relatively complexly regulated mechanism, which, especially in plants, still has not been completely described. Autophagy plays an important role under the influence of stress conditions. Especially, the effects of abiotic stress play an important role in plant life. Plants are immobile organisms so they must therefore develop mechanisms to enable them to survive in adverse conditions. In response to different types of stress, we can observe different roles in the involvement of autophagy, whether it is disposal of poorly folded proteins, remobilization of nutrients or antioxidant mechanism. Autophagy is also especially important for the process of stress memory and regulation.

Safety concerns arising from cytotoxic behavior of nanoparticles (NPs) in complex biological environment remain the main problem limiting NPs application in biomedicine. In this study, we have investigated cytotoxicity of NPs with different composition, shape and size, namely SiO2 NPs (SiNPs, 7-14 nm), superparamagnetic iron oxide NPs (SPIONs, 8 nm) and carboxylated multiwalled carbon nanotubes (CNTCOOHs, diameter: 60-100 nm, length: 1-2 μm). Cytotoxicity was evaluated with newly designed screening assay capable to simultaneously assess activity of cell dehydrogenases, activity of lactate dehydrogenase (LDH) released from cells into environment and number of intact cell nuclei and apoptotic bodies in human umbilical vein endothelial cell (HUVEC) culture growing in the very same well of the 96-well plate. Aforementioned attributes were subsequently utilized to obtain information about cell viability and necrotic and apoptotic aspects of cell death. Results from this "three-in-one" cell death screening (CDS) assay showed that SiNPs and CNTCOOHs evoked pronounced cytotoxic effect demonstrated as decrease of cell viability and development of apoptotic bodies formation. In contrast to this, SPIONs induced only mild cytotoxicity. Moreover, SiNPs impaired cell membrane leading to increased LDH release...

Protein homeostasis (proteostasis) plays an important role in maintaining normal cell function and viability. Neurons are particularly vulnerable to proteostasis dysregulation, resulting in damage, dysfunction, and neuronal death, as manifested in many neurodegenerative diseases. One of them is Huntington disease, hereditary neurodegeneration with autosomal dominant inheritance. Expansion of the CAG repeats in the huntingtin gene is translated into an abnormally long glutamine chain in huntingtin protein, leading to disruption of neuronal proteostasis. The primary affected area of the brain is the striatum of the basal ganglia. Disease is progressive, the onset of symptoms usually occurs in adulthood, and after many years leads to the death of the patient. Despite intensive research, disease pathology is still not fully understood, treatment is still only symptomatic and new studies, together with a deeper understanding, also raise many new questions. Through the complexity of the issue, the study of proteostasis in neurodegeneration can bring not only possible implications for therapy, but also could go deeper into the understanding of stress, memory or aging processes.

Autophagy is an essential mechanism of recycling, which represents a path leading to utilization and reorganization of intracellular components (nutrients, organelles, etc). Recent studies on adipose tissue have shown, that autophagy plays crucial role in differentiation of the adipocytes precursors and in phenotypic tissue transformations. The aim of this thesis is to summarize the characteristics of autophagy in terms of various adipose tissues as well as to present the regulation of autophagy as a potentially useful in therapy focused on obesity treatment. However, with the regulation of autophagy, there are risks that will also be considered in the present thesis. Keywords: autophagy, adipose tissue, therapy

The characteristics of many neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's disease is the accumulation of proteins and damaged organelles in the cytoplasm. Unfortunately, they are not sufficiently eliminated by autophagy. The basal autophagy, that maintains the cellular homeostasis, is disturbed in neurodegeneration. The process of autophagy becomes saturated and unable to remove all the toxic substances. Therefore, other degradation mechanisms are activated, aiming to restore the homeostasis. However, the neuronal cells are damaged under certain conditions leading to their death. The reduction in the number of neurons in specific brain areas may cause severe ataxias and dementias. Better understanding of autophagocytosis and its role v pathogenesis of neurodegenerative diseases may contribute to more effective treatment of these serious diseases in the future. Key words: autophagy, neurodegeneration, Alzheimer's disease, Parkinson's disease, Huntington's disease

Mitochondrial dysfunctions contribute to the progression of many neurodegenerative diseases, including Huntington's disease (HD). In HD, mutation in the huntingtin gene (HTT) results in the expansion of CAG repeats, causing the growth of the polyglutamine tract. This growth is responsible for the gain of toxicity function of the protein. The turnover of dysfunctional and damaged mitochondria is mediated via mitophagy - a selective form of autophagy. Additionally, mitophagy impairments have recently been described to play a key role not only in neurodegenerative diseases. The protrusion of mitophagy results in the clustering of defective mitochondria, organelles which are responsible for fulfilling the energetic demands of neural cells. The most distinctive impact of the impairment is on the striatal medium spiny neurons and results in the development of motor and cognitive dysfunctions. This thesis describes how HD affects mitophagy and reveals the biggest obstacle of mitophagy - disruption of mitochondria targeting into emerging autophagosomes caused by the abnormal interaction of mHTT and p62. Induction of mitophagy at this stage could be crucial for the future therapeutic research of HD. Generally, initiation of mitophagy could become a relevant therapeutic target for many other...

Autophagy is an evolutionarily conserved degradative pathway. Autophagy occurs constitutively at a basal level and it is involved in the recycling and turnover of damaged or superfluous organelles and proteins. It has a critical role in cellular homeostasis. Autophagy can be induced in response to starvation or other types of stress. Induction of autophagy during these conditions has a major role in protection and adaptation of the cell. Autophagy needs to be properly regulated. A wide range of diseases is associated with dysregulation of autophagy. Better understanding of autophagy mechanisms can help to develop strategies designed to modulate autophagic responses occuring in a number of diseases. This work is focused on current knowledge of main types of autophagy and how autophagy helps yeast cells to adapt. Key words: autophagy, yeast, degradative pathway, adaptation, TORC1

The mortality of cardiovascular diseases remains high and it likely tends to increase in the future. Although many ways how to increase the resistance against myocardial ischemia- reperfusion damage have been described, few of them were transferred into clinical practice. Cardioprotective effect of chronic hypoxia has been described during 60s of the last century. Its detailed mechanism has not been elucidated, but a number of components has been identified. One of these components presents protein kinase C (PKC). The role of PKC was described in detail in the mechanism of ischemic preconditioning, but its involvement in the mechanism of cardioprotection induced by chronic hypoxia remains unclear. One reason is the amount of PKC isoforms, which have often contradictory effects, and the diversity of hypoxic models used. The most frequently mentioned isoforms in connection with cardioprotection are PKCδ and PKCε. The aim of my thesis was to analyze changes in these PKC isoforms at two different cardioprotective models of hypoxia - intermittent hypobaric (IHH) and continuous normobaric hypoxia (CNH). We also examined the target proteins of PKCδ and PKCε after the adaptation to IHH, which could be involved in the mechanism of cardioprotection. These included proteins associated with apoptosis and...

Drug resistance in cancer cells is a frequent cause of breast cancer therapy failure. The aim of this thesis was to elucidate mechanisms of resistance to taxanes, that are used in therapy of various types of cancer, including breast cancer. We particularly assessed the role of autophagy and changes in βII- and βIII isotype gene expression in development of taxane resistance. As model of breast cancer we used human sensitive cell lines SK-BR-3, MCF-7 a T47-D and resistant sublines SK-BR-3-PAC/REZ a MCF-7- PAC/REZ which grow in paclitaxel concentration lethal for sensitive sublines. In cell lines SK-BR-3 and MCF-7, taxane application decreased the level of autophagy, however in cell line T47-D led to its activation. We detected no difference between basal levels of autophagy in sensitive subline SK-BR-3 compared to resistant subline SK-BR-3-PAC/REZ, but we observed increased basal level of autophagy in sensitive subline MCF-7 compared to the resistant subline. Increase or decrease level of autophagy did not affect taxane resistance, except activation of autophagy in resistant subline SK-BR-3-PAC/REZ, that further increased the resistance to paclitaxel. Taxane application in cell line T47-D increased the levels of βII- and βIII-tubuline expression, however we did not find any similar effect in other tested...

The exocyst, an octameric protein complex conserved among all eukaryotes, mediates tethering of the vesicle prior to its fusion with the target membrane. Apart from the function of exocyst in exocytosis, new studies from both mammalian and plant fields report its involvement in the cellular self-eating process called autophagy. In land plants the number of paralogs of some exocyst subunits is extraordinarily large. There are 23 paralogs of Exo70 subunit in Arabidopsis thaliana. It is supposed that these paralogs have acquired functional specialization during the evolution - including involvement in autophagy. Using yeast two- hybrid assay it is shown here that Exo70B1 and Exo70B2, but not other Arabidopsis Exo70 paralogs interact with Atg8, an autophagosomal marker. The proximity of these two paralogs and Atg8 in vivo was confirmed by independent Förster resonance energy transfer (FRET) method. Interestingly, interaction of Atg8f with Exo70B2 paralog appears to be stronger than with Exo70B1. Exo70B1-mRUBY expressed under the natural promoter shows punctate membrane structures that are mostly static. That changes after the tunicamycin treatment - movement of some of these dots was induced. Homology modeling of Exo70B1 and Exo70B2 proteins tertiary structure in combination with bioinformatic prediction based...

Exocyst is a binding protein complex, which is evolutionary conserved in yeast, animal and in plant cells. It has crucial role in regulation of cell morfogenesis and cell polarity. The function of the exocyst complex is binding of secretoric vesicle to the proper side on plasma membrane in penultimate step of exocytosis. This process is essecial for function and survival of cell. Another process crucial for the cell is autophagy. In plants autophagy plays important role in the responses to nutrient starvation, senescence, abiotic and biotic stress. RabG3b are small GTPases, which have positive role in autophagy. In this work I described the interaction between RabG3b and some of subunits of exocyst complex: Exo70B1, Exo70B2 and Exo84b. I also studied changes in morfogenesis of tonoplast by induction and inhibition of authophagy and induction of anthocyans synthesis in Arabidopsis thaliana.

Francisella tularensis is a facultative intracellular pathogenic bacterium, which causes disease named tularemia. For the entrance to the host cells Francisella uses host's cell mechanisms by which it is incorporated into cell phagosome. Subsequently, it escapes from phagosome to cytosole where bacterial growth takes place. Some of bacteria are cleared from cytosol by autophagy, from another ones dsDNA is released. This DNA is recognized by cytosolic receptors, which form inflammasome complex. Inflammasome sets off pathway leading to the death of infected cell. Since the penetration to the cell Francisella modulates cell signallization in its own benefit to ensure enough time and nutrients for its growth. Francisella do not act only in the infected cells, where it reduces recognition of itself and clearance from cytosol, but it also induces secretion of factors, which moderate activation of adaptive immunity of the host. Key words: Francisella, tularemia, fagosome, inflammasome, autophagy, adaptive immunity

Toll like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that play a central role in host cell recognition and responses to pathogens. Primarily they are responsible for induction and regulation of the innate and adaptive immune responses whereby the effector function is executed chiefly by differentiated myeloid cells. Somewhat unexpectedly, TLRs have been also shown to be involved in direct pathogen sensing by bone marrow-derived hematopoietic stem cells (HSCs) and hematopoietic progenitors when, under inflammatory conditions, the rapid generation of innate immune effector cells that effectively combat the infection is of utmost priority. While it has been recognized that the release of inflammatory cytokines from inflamed tissues along with the changes in proportions of differentiating cells in the bone marrow (BM) as well as the BM niche can nudge the differentiation of adult BM-derived cells towards myeloid cells and granulocytes, a direct role of TLRs expressed by HSCs in this process has been demonstrated only recently. However, whether a similar mechanism operates also during embryonic hematopoiesis is unknown. Here we show that TLRs and their adaptor proteins are functionally expressed during early stages of embryogenesis by short-lived maternally-transferred...

Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...

Charles University in Prague Pharmaceutical Faculty in Hradec Králové Department of Pharmacology and Toxicology Student: Anastasiya Shkut Supervisors: Mgr. Jana Mandíková, Virginia Motilva Ph.D. Title of diploma thesis: The role of melatonin in SIRT1 and p-AMPK regulation in HT-29 cell line. Sirituin 1 (SIRT1) is NAD+ dependent deacetylase present in wide range of organisms including mammals. It was found to extend life span in yeasts during calorie restriction (CR) conditions. SIRT1 deacetylates many regulator proteins and thus control metabolic status of cell as well as AMP-activated kinase (AMPK), which is also energy regulator enzyme depending on NAD+ levels in cell. Both of them play roles in cancer and could influence autophagy, although the exact mechanism remains unclear. We focused our study on hormone melatonin, which has anti-inflammatory and anti-cancer effects, to study its influence on human colon cancer cell line HT-29. If it has impact on SIRT1 and AMPK and what is hierarchic relationship between SIRT1 and AMPK. Also we observed its possible influence on autophagy. We used Western blotting (WB) technique and from our results it seems that melatonin has significant effect on SIRT1, which might activate AMPK as well as autophagy. Nevertheless some of results did not have sufficient...

Vacuole is very important plant cell organelle which can occupy 90% of cell volume. It provides wide range of functions. Considering enormous size of the vacuole, vesicle trafficking into the plant vacuole is major vesicle movement inside the cell. Transport pathway into the vacuole is very dynamic field of plant cell biology. This sorting machinery shares similarities within all eukaryotes, but plants also have their own specificities. Soluble cargo is targeted through secretory pathways by vacuolar sorting receptors (VSRs). These proteins due to its transmembrane localization can interact with sorted cargo and take it to the right organelle within the cell. Membrane fusion is facilitated with Rab-GTPase and SNARE protein complexes. A special kind of vesicle traffic is autophagy, the self consuming process that protects the cell from various type of stress or enables apoptosis.

Autophagy is a cellular proces, taht allows degradation of a portion of cytoplasm, protein aggregates or entire organelles. Major function of autophagy is the maintainance of cellular homeostasis, the protection against stress and mobilization of internal resources. However, autophagy also has a role in imunity, development and differentiation. Autophagic signaling can interact with apoptotic machinery at several levels via regulatory proteins of both pathways or via mutual degradation or cleavage of the components of both pathways. Autophagy can communicate with both extrinsic and intrinsic pathways of apoptosis. Under certain circumstances can autophagy by itself also induce cell death. Autophagic cell death called also programmed cell death of type II is accompanied by massive vacuolization and lysosomal autodestruction of the affected cell. Autophagic cell death was documented during Drosophila development but also in mammalian cells. Autophagy also play importamt role in tumorogenesis, where it can either protect tumor cells against various stresses or it can contribute to their death. Further research of autophagic signaling and mechanisms of communication between autophagy and apoptosis may ont only extend our knowledge on these essential processes but can also contribute to cancer therapy. Powered...

Autophagy is a degradation pathway, conserved from yeast to mammals. The uniqueness of this pathway lies in its function, it is applied in the cell especially under the adverse conditions. It helps the cell to deliver essential nutrients for life, it removes the damaged or superfluous organelles, protein aggregates and helps with recycling and maintains a constant inner environment. These functions can prolong cell life and the cells survive the adverse conditions. Autophagy may induce the programmed cell death type II. This paper describes the basic of autophagy machinery, regulation and influence of yeast autophagy to adapt to the stressful conditions. Understanding the mechanism and regulation of autophagy in yeast may help with the study of autophagy in mammals. In mammals, this degradation pathway disorders cause many diseases (especially neurodegenerative), autophagy also effects the formation of tumors. Powered by TCPDF (www.tcpdf.org)

Exocyst is an octameric protein complex, conserved across all Eukaryotes. Its role, originally described in yeast, resides in a tethering of the secretory vesicles to the plasma membrane prior to the membrane fusion of the two membranes. Subunits SEC3 and EXO70 are believed to be spatial landmarks for the vesicles delivery. While yeast genome encodes single EXO70, we find dozens of them in land plants (23 in Arabidopsis). This work is focused at a role of the exocyst complex in plant cells. Its first part documents, that exocyst is essential for delivery of the cell wall components, namely pectins, but also for pathogen induced secondary cell wall thickening. Second part reveals an unconventional role of EXO70B1 subunit harboring exocyst subcomplex at an autophagic pathway to the vacuole and raises many questions about plant secretory pathway.

Cellen maken niet alleen voortdurend nieuwe eiwitten en bouwen steeds nieuwe structuren, maar moeten ook steeds delen kunnen afbreken, recyclen en afvoeren. Autofagie is een mechanisme dat cellen in staat stelt om onbruikbare of beschadigde cellulaire componenten af te breken. Wanneer dit mechanisme ontregeld raakt, kan dit onder meer kanker, neurodegeneratie en veroudering veroorzaken. UMCG-promovenda Jing Yang bracht dit proces nader in kaart. Uit het onderzoek blijkt dat FOXO1, een transcriptiefactor geassocieerd met cellulaire veroudering, autofagie kan stimuleren. Intrigerend was dat dit geheel onafhankelijk blijkt van de transcriptionele activiteit van FOXO1 en dat het de ontwikkeling van tumoren in een diermodel verminderde. Dit suggereert dat activering van autofagie mogelijk kan helpen kanker te bestrijden. Ook ontdekteYang dat FOXO1 ervoor kan zorgen dat verkeerd gevouwen eiwitten worden afgebroken, net voordat ze zich in de hersenen ophopen. Daarmee lijkt het bruikbaar in de strijd tegen neurodegeneratieve ziekten, zoals de ziekte van Parkinson, de ziekte van Huntington ziekten en ALS, die worden veroorzaakt door ophoping van verkeerd gevouwen eiwitten. Tenslotte vonden we dat een verhoogde autofagie-activiteit werd geassocieerd met mutaties in een gen coderend voor het eiwit hepatocystin, hetgeen een zeldzame erfelijke vorm van leverziekte (PCLD) veroorzaakt. Anders gezegd, bij PCLD is remming van autofagie (in plaats van activering zoals bij kanker en neurodegeneratie) een optie als therapeutische behandeling.

Cellen maken niet alleen voortdurend nieuwe eiwitten en bouwen steeds nieuwe structuren, maar moeten ook steeds delen kunnen afbreken, recyclen en afvoeren. Autofagie is een mechanisme dat cellen in staat stelt om onbruikbare of beschadigde cellulaire componenten af te breken. Wanneer dit mechanisme ontregeld raakt, kan dit onder meer kanker, neurodegeneratie en veroudering veroorzaken. UMCG-promovenda Jing Yang bracht dit proces nader in kaart. Uit het onderzoek blijkt dat FOXO1, een transcriptiefactor geassocieerd met cellulaire veroudering, autofagie kan stimuleren. Intrigerend was dat dit geheel onafhankelijk blijkt van de transcriptionele activiteit van FOXO1 en dat het de ontwikkeling van tumoren in een diermodel verminderde. Dit suggereert dat activering van autofagie mogelijk kan helpen kanker te bestrijden. Ook ontdekteYang dat FOXO1 ervoor kan zorgen dat verkeerd gevouwen eiwitten worden afgebroken, net voordat ze zich in de hersenen ophopen. Daarmee lijkt het bruikbaar in de strijd tegen neurodegeneratieve ziekten, zoals de ziekte van Parkinson, de ziekte van Huntington ziekten en ALS, die worden veroorzaakt door ophoping van verkeerd gevouwen eiwitten. Tenslotte vonden we dat een verhoogde autofagie-activiteit werd geassocieerd met mutaties in een gen coderend voor het eiwit hepatocystin, hetgeen een zeldzame erfelijke vorm van leverziekte (PCLD) veroorzaakt. Anders gezegd, bij PCLD is remming van autofagie (in plaats van activering zoals bij kanker en neurodegeneratie) een optie als therapeutische behandeling.

Cellen maken niet alleen voortdurend nieuwe eiwitten en bouwen steeds nieuwe structuren, maar moeten ook steeds delen kunnen afbreken, recyclen en afvoeren. Autofagie is een mechanisme dat cellen in staat stelt om onbruikbare of beschadigde cellulaire componenten af te breken. Wanneer dit mechanisme ontregeld raakt, kan dit onder meer kanker, neurodegeneratie en veroudering veroorzaken. UMCG-promovenda Jing Yang bracht dit proces nader in kaart. Uit het onderzoek blijkt dat FOXO1, een transcriptiefactor geassocieerd met cellulaire veroudering, autofagie kan stimuleren. Intrigerend was dat dit geheel onafhankelijk blijkt van de transcriptionele activiteit van FOXO1 en dat het de ontwikkeling van tumoren in een diermodel verminderde. Dit suggereert dat activering van autofagie mogelijk kan helpen kanker te bestrijden. Ook ontdekteYang dat FOXO1 ervoor kan zorgen dat verkeerd gevouwen eiwitten worden afgebroken, net voordat ze zich in de hersenen ophopen. Daarmee lijkt het bruikbaar in de strijd tegen neurodegeneratieve ziekten, zoals de ziekte van Parkinson, de ziekte van Huntington ziekten en ALS, die worden veroorzaakt door ophoping van verkeerd gevouwen eiwitten. Tenslotte vonden we dat een verhoogde autofagie-activiteit werd geassocieerd met mutaties in een gen coderend voor het eiwit hepatocystin, hetgeen een zeldzame erfelijke vorm van leverziekte (PCLD) veroorzaakt. Anders gezegd, bij PCLD is remming van autofagie (in plaats van activering zoals bij kanker en neurodegeneratie) een optie als therapeutische behandeling.

The production of mitochondrial reactive oxygen species and the resulting oxidative stress is an important phenomenon driving long-lasting research and intense discussions. Knowledge of exact mechanisms of reactive oxygen species production and pathways leading to their formation could help us to directly affect their production, a task with potential terapeutic implications. The molecular nature of the production of reactive oxygen species by some enzymes has already been well documented, but others still remain controversial and current theories are obviously far from the truth. Much more interesting is the question of physiological importace of this production. The reactive oxygen species were considered harmful factors clearly distorting the integrity of the organism for a long time. However, recent research suggest that their existence can also be beneficial and effective. Evidently they can serve as a signaling molecules in several metabolic and regulatory pathways occurring in the organism. This bachelor thesis offers insight into the current state of knowledge. It focuses on the most detailed description of the reactive oxygen species production by mitochondrial respiratory chain enzymes. Furthermore, it deals with some signaling cascades, where involvement of mitochondrially generated...

Autophagy is an essential, homeostatic process - survival mechanism that protects cells by various ways: cells break down their own components to recycle nutrients, remodel and dispose unwanted cytoplasmic constituents. Autophagy is involved in the degradation of long-lived proteins and entire organelles, but paradoxically, considering important prosurvival functions, autophagy may be deleterious. It plays an important role during development, tumor suppression, immunity and is required for the adaptation to environmental stresses such as starvation. Recent studies indicate, that autophagy is a central player in the immunological control of bacterial, parasitic and viral infections. The process of autophagy may degrade intracellulal pathogens. This work describes the mechanism of autophagy and highlights the role of autophagy in innate and adaptive imunity, summarizes some advances in understanding the functions of autophagy and its possible roles in the causation and prevention of human deseases.

Currently, it is growing evidence that autophagy is involved in the prevention of various diseases, which of course also includes heart diseases. This thesis is therefore aimed at clarifying the role of autophagy in the heart, especially during ischemia and subsequent reperfusion. Autophagy is a physiological cellular process by which the cell maintains homeostasis by eliminating long-lived proteins and damaged organelles. The role of autophagy during ischemia/reperfusion in the heart is complex. Predominantly it functions as a pro-survival pathway, because it protects the heart from ischemia or hypoxia. However, when triggered over, which happens during reperfusion, it may lead to cell death. In the heart autophagy is activated in response to various stimuli, such as decrease in ATP and subsequent activation of AMPK, protein Bnip3, reactive oxygen and nitrogen species, the opening of mitochondrial permeability transition pore, endoplasmic reticulum stress or unfolded protein response.