Browsing by discipline "Genetics"
Now showing items 41-43 of 43
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(2019)Epithelial cells form a barrier between the tissue and the external environment. Epithelial morphogenesis refers to the shaping of epithelial layers and is a key step in the development of organisms. The actin cytoskeleton provides the cell its form and during epithelial morphogenesis, produces force to shape the cells. To achieve this, the actin cytoskeleton is organized into protrusive and contractile networks. In a living cell, these actin networks are dynamic, as the filaments are constantly undergoing assembly and disassembly. Actin-binding proteins regulate the turnover of actin filaments, but in epithelial morphogenesis, the regulatory role of most of these proteins is still relatively unknown. In all multicellular organisms, actin disassembly is controlled by ADF/cofilin. ADF/cofilin activity is furthermore enhanced by other actin-binding proteins, one of which is cyclase-associated protein (CAP). CAP promotes actin turnover by accelerating ADF/cofilin mediated actin disassembly and in recycling actin monomers to sites of actin polymerization. Unlike ADF/cofilin that regulates actin disassembly throughout the whole cell, CAP could be subject to more specific spatial regulation, as loss of CAP leads to F-actin accumulation on the apical side of epithelial cells. However, the role of CAP in morphogenetic cell rearrangements remains poorly known. In addition, the in vivo role of the biochemical functions of CAP has not been elucidated. The aim of this master’s thesis is to describe the role of CAP in regulating the actin cytoskeleton in the follicular epithelium of the fruit fly Drosophila melanogaster. For this purpose, chimeric mutant flies with homozygous CAP loss of function mutation were generated. Subsequently, the effect of the CAP loss of function was observed in follicle cell populations undergoing morphogenetic changes. In addition, CAP loss of function was rescued with different transgenes producing mutant CAP proteins to identify the protein domains of CAP with in vivo significance. In addition, a Drosophila CAP specific antibody was purified to be used in immunostaining. The ovaries were imaged using confocal microscopy. In this thesis, it is shown that CAP loss of function caused accumulation of filamentous actin in all observed follicular cell populations. Surprisingly, the actin turnover was rescued by all of the used CAP rescue transgenes, but the mutant transgenes exhibited phenotypes resembling the CAP loss of function in other epithelial tissues. Moreover, CAP loss of function caused defects in the follicle cell movement and cell spreading. The loss of function also caused expression changes in other actin-binding proteins. The findings of these thesis support the current knowledge of CAP importance for functional actin turnover in the follicle cells, even though the protein domain necessary for in vivo function could not be deciphered. Moreover, this project provides indication that CAP has an indispensable role in dynamic morphogenetic processes in the epithelium. Together with other actin-binding proteins, CAP could regulate epithelial actin turnover in spatially directed manner, providing force for epithelial cell adhesions or protrusions.
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(2015)Huntington's disease (HD) is a progressive neurodegenerative disorder that causes involuntary muscle movements, deteriorates muscle coordination and cognitive decline. Typical onset age of the disease is in mid age, although a juvenile form of HD is also known. The disease is inherited in an autosomal dominant manner via a mutation in the huntingtin gene (HTT). The characteristic mutation in HTT is an expansion of the glutamine stretch at the 5 end of the gene. Excessive amounts of glutamine residues alters the conformation and chemical features of the huntingtin protein (HTT) leading to accumulation of cellular aggregates. Although to date there are several known alterations in the cell that contribute to the disease, the pathogenesis of HD is not fully understood. Ubiquitin proteasome system (UPS) dismantles proteolytically unneeded or damaged proteins, which are targeted to proteolysis when ubiquitin tags are added to them. Deubiquitinating enzymes (DUB) recycle ubiquitin molecules by releasing them from proteasome substrates. Recycling of ubiquitin is critical to a cell as it maintains the free pool of the targeting molecule. Ubiquitin-specific protease 14 (USP14) is one of the DUB family enzymes and its distinctive function is to remove ubiquitin molecules from the tip of the ubiquitin chain and thus antagonize protein degradation. Although the specific function of the protein is unclear, it seems that USP14 operates as a fine regulator of protein turnover rate and in ER stress both in catalytic and non catalytic manner. The role of USP14 is especially emphasized in the nervous system, as it regulates synaptic transmission and neuronal development. Although it is suggested that dysfunction of UPS is involved in the pathogenesis of HD, the role of USP14 in the disease remains to be unknown. IU1 is a novel inhibitor of the catalytic domain of USP14. Studies with IU1 indicate that inhibition of USP14 enhances the clearance of aggregate prone proteins. The approach of this thesis was aimed to elucidate the routes of HD pathogenesis from diverse approaches. The general aim of the thesis was to investigate the role of USP14 in the wild-type PC6.3 cell model, and in the pathogenesis of HD by expressing HTT proteins with different lengths of glutamine stretches in PC6.3 cells. The specific aim of the study was to examine by western blot and microscopy analysis the pathogenic routes of HD that involve ER stress, oxidative stress, autophagy and mutant HTT aggregate dynamics. The function of USP14 was studied with overexpression of USP14, or by inhibiting its catalytic activity by IU1. The findings of this thesis show that overexpression of USP14 enhances the clearance of mutant HTT aggregates, and this effect is obtained in catalytic activity dependent manner. I show that upregulated USP14 is connected to improved clearance of mutant HTT and inhibition of autophagy, suggesting that the degradation is mediated via UPS. The catalytic activity of USP14 might also be important in ER stress regulation, as the results indicate that IU1 activates phosphorylation of both JNK and eIF2α. I was also able to establish a connection between USP14 and GADD34, as I show that GADD34 upregulates USP14. Finally, I show that catalytic inhibition of USP14 decreases the expression of antioxidant SOD2. The data in this thesis is lacking statistical significance, and it can be considered solely as a guideline. However, together these results indicate that the deubiquitinating activity of USP14 increases survival in PC6.3 cells in both a healthy and a HD model.
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(2020)Since the establishment of pathologic and cytogenetic laboratories, left-over material in the form of G-banded slides and cytogenetic fixed cells, as well as formalin-fixed, paraffin-embedded (FFPE) material, tissue samples, blood and bone marrow have been stored in archives for possible later reference. This material, which potentially contains rare and special cases, has been a welcome source for retrospective studies or e.g. for trying out new methods of analysis. Molecular genetic and molecular cytogenetic techniques such as Southern hybridization, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) have been successfully applied on different kinds of archival specimens. With this study we wanted to explore, both through a literature review and through a practical experiment, the history, present day and future use of such archival material in the light of molecular cytogenetics, including the challenges of DNA extraction, sample degradation, data analysis and interpretation as well as ethical issues. The experimental part had two main objectives, (1) to investigate the use of archived cytogenetic material in the form of G-banded slides and cytogenetic fixed cells for array-based CGH (aCGH), and (2) to explore abnormalities on chromosome 1q in hematologic malignancies. Extra material on the long arm of chromosome 1 is a common recurrent chromosomal abnormality that is present in many classes of hematologic cancers as either primary or secondary aberration. It is the most common structural aberration in multiple myeloma (MM), myeloproliferative disease (MPD) and myelodysplastic syndrome (MDS) and is also a frequent aberration in pediatric acute lymphoblastic leukemia (ALL). It has been associated with increased cell proliferation, disease progression and poor outcome, the mechanisms of which are not fully understood yet. To combine these two aims, we screened the patient database for relevant cases and searched the archive for corresponding samples. The idea was to find cases of hematologic malignancies with extra material on chromosome 1q that were available as cytogenetic slides, fixed cells and frozen bone marrow, find an ideal method of DNA extraction from slides (for other material ready protocols were available), analyze the samples with aCGH and compare the results. We wanted to prove the eligibility of archived cytogenetic material for aCGH analysis and at the same time study rearrangements of 1q in our samples. Starting with 38 patient cases, DNA extraction was performed with 2 different protocols, the latter of which, using a modified version of the Puregene® DNA Purification Kit Protocol for Blood Smears, turned out to be more successful. After having obtained sufficient DNA from several slide samples, we assessed DNA quality with agarose gel electrophoresis. Because slide DNA was too fragmented to be used for aCGH and whole genome amplification (WGA) was not a choice, the experiment was continued with archived fixed cells, bone marrow and archived DNA only. Using a high resolution 60-mer oligonucleotide 44K human CGH microarray platform, we analyzed 15 patient cases that were available both as fixed cell and DNA samples (2 cases), both as fixed cell and bone marrow samples (1 case), both as fixed cell and CPT™ cell samples (1 case), frozen bone marrow (7 cases) and DNA samples (4 cases). The malignancies were pediatric ALL (6 cases), adult ALL (1 case), chronic myeloid leukemia (CML, 2 cases), non-Hodgkin lymphoma (NHL, 1 case), Burkitt lymphoma (BL, 3 cases), Hodgkin's disease (HD, 1 case) and one undefined malignancy. After analysis with CGH Analytics software, we saw that aCGH results of fixed cells compared to results from bone marrow or DNA were almost identical, which confirmed that cytogenetic fixed cells were a reliable source for aCGH analysis. Challenges of working with archived material were witnessed in the form of CG-waves and centralization errors and deviations of the hybridization ratio diagram caused by partially degraded DNA. Concerning aberrations calls, 12 of the 15 cases had detectable aberrations on 1q, which included amplification of the whole 1q arm (1 pediatric ALL, 3 BL cases) and duplications or amplifications of parts of 1q (5 pediatric ALL, 2 CML and 1 undefined malignancy cases) in addition to other aberrations. Common break points were 1q21.2 (2 CML cases), 1q23.2 (1 pediatric ALL and 1 CML case) and 1q32.3 (2 pediatric ALL cases), and we detected 2 large common overlapping areas, at 1q21.2q23.2 and 1q25.3q32.2. The areas were, however, too vast for disease gene screening, spanning several Mb each. We could thus prove and confirm the value of cytogenetic archives for scientific studies. Array CGH with fixed cells was confirmed to work well, also frozen bone marrow and archived DNA were valuable sources. We would suggest further aCGH experiments with cytogenetic slides by applying WGA but would also suggest slide and fixed cell material to be used for Next Generation Sequencing, which has not been reported yet. Regarding aberrations on 1q, further studies to more accurately delimit relevant break points and common overlapping areas are recommended.
Now showing items 41-43 of 43