In the presented research we propose to improve on historically accepted radiobiological models via Monte Carlo simulation of radiation tracks passing through a cell nucleus modeled with up-to-date subnuclear structures. This is performed by generating a radiation track database using the Monte Carlo code, Geant4-DNA, that simulates radiation interactions at the nanometer scale of DNA. These tracks are called upon from the database and intersected with a cell nucleus model that incorporates DNA-containing structures. This allows for a Monte Carlo simulation of how DNA double strand breaks are produced by radiation. The results can be used to correlate to many experimentally observed biological endpoints, e.g. chromosome aberrations as well as cell death.

In the cell we can find a lot of small noncoding RNAs, which are important for many processes. Among those RNAs are small nuclear RNA uridin rich, which with proteins create U snRNP.These particles play important role in pre-mRNA splicing. In this process are noncoding sequences (introns) removed and coding sequences (exons) are joined. It is catalyzed by spliceosome. The core of this spliceosome is created by U1, U2, U4, U5 and U6 snRNP. They are essential for this process. Some steps of U snRNP biogenesis proceed in nuclear structures called Cajal bodies (CB). In my thesis I focused on factors, which are important for targeting U snRNA into CB. I used U2 snRNA like a model. With the aid of microinjection of fluorescently labeled U2 snRNA mutants I found, that the Sm binding site on U2 snRNA is essential for targeting to CB. Knock down of Sm B/B'showed us, that Sm proteins are necessary for transport U2 snRNA to CB. Sm proteins are formed on U2 snRNA by SMN complex. Deletion of SMN binding site on U2 snRNA had the same inhibition effect. From these results we can see, that Sm proteins and SMN complex are important for U2 snRNA biogenesis espacially for targeting into CB. Key words: U snRNP, Cajal body, U snRNA, cell nucleus

(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids. Their inositol head can be phosphorylated at three positions generating seven differently phosphorylated species. Cytoplasmic phosphoinositides regulate membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generate second messengers. In the nucleus, PIs are implicated in pre-mRNA processing, DNA transcription and chromatin remodelling. However, their nuclear functions are still poorly understood. Here we focus on nuclear phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). We describe their localization and interaction with proteins involved in regulation of DNA transcription. PI(4)P localizes to nuclear membrane, nuclear speckles and nucleoplasm. The majority of nuclear PI(4)P is associated with chromatin and colocalizes with H3K4me2. PI(4,5)P2 localizes to nucleoli and nuclear speckles. Besides, 30 % of nuclear PI(4,5)P2 forms small nucleoplasmic PI(4,5)P2 islets. They have carbon rich core, which is probably formed by lipids, and are surrounded by proteins and nucleic acids. The active form of RNA polymerase II associates with PI(4,5)P2 islets and DNA is actively transcribed in the vicinity of PI(4,5)P2 islets. Moreover,...

Polyglutamine (polyQ) diseases are a group of progressive neurodegenerative disorders, which are caused by the expansion of an existing glutamine-coding CAG repeat in the coding region of disease genes. The cell nucleus is a major site of polyQ toxicity, and gene transcription is compromised in polyQ-induced neurodegeneration. Understanding the nuclear translocation of mutant polyQ proteins is therefore crucial to unfold the complex pathogenic mechanisms that underlie the neuronal toxicity of polyQ disease. The polyQ domain is the only common sequence found among different mutant disease proteins. Nuclear transport signals have been identified in some, but not all, polyQ disease proteins. The detection of those mutant polyQ proteins that carry no classical nuclear transport signal, but not their normal counterparts, in the cell nucleus suggests the existence of uncharacterized nuclear transport signals in mutant polyQ proteins. Thus, the objective of the present study is to elucidate the nuclear transport pathway(s) adopted by an expanded polyQ domain and determine its correlation with polyQ toxicity. / Through a series of genetic and biochemical studies in cell culture, mouse and transgenic Drosophila models, exportin-1 was found to modulate the nucleocytoplasmic localization of mutant polyQ protein and its toxicity. Further, mutant polyQ protein was also demonstrated to be a novel transport substrate of exportin-1. By promoting the nuclear export of mutant polyQ protein, exportin-1 suppressed polyQ toxicity by reducing the interference of mutant polyQ protein on gene transcription. It was found that the protein level of exportin-1 diminished in the normal ageing process, which would result in an exaggeration of nuclear mutant polyQ toxicity. Thus, the age-dependent decline of exportin-1 level, at least in part, accounts for the progressive degeneration observed in polyQ patients. Results obtained from this project first demonstrated that expanded polyQ domain is a nuclear export signal, and further provided mechanistic explanation of how protein nuclear transport receptors modulate polyQ toxicity. / Chan, Wing Man. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0113. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 189-203). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-115).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 132-143).

(English) Phosphoinositides (PIs) are glycerophospholipids with a negative charge. As components of cell membranes, PIs are involved in membrane and cytoskeletal dynamics, cell movement and signalling, and the modulation of ion channels and transporters. Apart from the cytoplasm, phosphoinositides also localise to the cell nucleus. PIs play a role in crucial nuclear processes, such as DNA transcription, pre-rRNA and pre-mRNA processing, cell differentiation, DNA damage response, or apoptosis. Phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are the most abundant phosphoinositides in the cell. However, their exact localisation and function in the nucleus are largely unknown. Here, we describe their localisation at super-resolution level and their involvement in some nuclear processes. PI(4)P is present in nuclear lamina, nuclear speckles and nucleoli, and it forms small foci in nucleoplasm. The majority of nuclear PI(4)P localises to the nucleoplasm, whereas almost 16 % is present in nuclear speckles. On the other hand, the majority of nuclear PI(4,5)P2 localises to nuclear speckles, almost 30 % localises to nucleoplasm and the lesser portion to nucleoli. In the nucleoplasm, PI(4,5)P2 forms small foci called nuclear lipid islets (NLIs). Their core is...

(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids with inositol head (ring) which can be phosphorylated. Inositol ring phosphorylation yields in seven different PIs species which can be mono-, bis,- or tris-phosphorylated. Roles of cytoplasmic PIs have been extensively studied in for membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generating of second messengers. Nuclear PIs have been implicated in posttranscriptional processing of pre-mRNA, DNA transcription and chromatin remodelling. While cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. In this study we focus on description of localization of nuclear PIs in particular functional nuclear compartments, which enable us to reveal PIs involvement in nuclear processes. We also focused on identification of nuclear PIs involved in the regulation of genes transcription and revealed detailed mechanism of PI(4,5)P2 a PHF8 interaction in the regulation of ribosomal genes transcription. By two independent approaches, we have described PIs localization to the nuclear membrane, nuclear speckles, small foci in the nucleoplasm, and the nucleolus. This spread nuclear localization suggests and confirms PI's...

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Osmotic stress is a potent regulator of the normal function of cells that are exposed to osmotically active environments under physiologic or pathologic conditions. The ability of cells to alter gene expression and metabolic activity in response to changes in the osmotic environment provides an additional regulatory mechanism for a diverse array of tissues and organs in the human body. In addition to the activation of various osmotically- or volume-activated ion channels, osmotic stress may also act on the genome via a direct biophysical pathway. Changes in extracellular osmolality alter cell volume, and therefore, the concentration of intracellular macromolecules. In turn, intracellular macromolecule concentration is a key physical parameter affecting the spatial organization and pressurization of the nucleus. Hyper-osmotic stress shrinks the nucleus and causes it to assume a convoluted shape, whereas hypo-osmotic stress swells the nucleus to a size that is limited by stretch of the nuclear lamina and induces a smooth, round shape of the nucleus. These behaviors are consistent with a model of the nucleus as a charged core/shell structure pressurized by uneven partition of macromolecules between the nucleoplasm and the cytoplasm. These osmotically-induced alterations in the internal structure and arrangement of chromatin, as well as potential changes in the nuclear membrane and pores are hypothesized to influence gene transcription and/or nucleocytoplasmic transport. A further understanding of the biophysical and biochemical mechanisms involved in these processes would have important ramifications for a range of fields including differentiation, migration, mechanotransduction, DNA repair, and tumorigenesis. / Dissertation