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The aim of the study was to investigate whether non-thermal levels of radiofrequency (RF) fields, characteristic of some mobile phones, might be directly genotoxic when applied in vitro to unstimulated G0 or stimulated human lymphocytes. Also, the study aimed to investigate the possibility that RF fields might act epipigenetically when combined with x-rays, by modifying their effect when applied in vitro to G0 lymphocytes. In addition, the possibility of RF fields inducing apoptosis in murine neuroblastoma (N2a) cells was also examined. G0 lymphocytes from 4 donors were exposed for a total of 24 h to a continuous or an intermittent RF signal. The signals were 935 MHz GSM (Global System for Mobile Communication) Basic, 1800 MHz GSM Basic, 935 MHz continuous wave (CW) carrier frequency, and 935 MHz GSM Talk. Stimulated lymphocytes were exposed for a total of 48 h to intermittent 1800 MHz RF signals that were GSM Basic or the carrier frequency only. The RF fields used for the 24 h exposure of N2a cells were all at 935 MHz and consisted of GSM Basic, GSM Talk and a CW signal. The chosen Specific energy Absorption values of the signals were either 1 or 2 W/kg. These values are near the upper limit of actual energy absorbed in localised tissue by a person from some mobile phones. The field was applied to G0 human lymphocytes either alone or combined with an exposure to 1 Gy x-rays given immediately before or after the RF field. A dose of 4 Gy x-rays was used as a positive control for apoptosis induction in N2a cells and in the study with stimulated lymphocytes no x-rays were used. The lymphocytes were assayed by several standard methods to demonstrate genotoxicity. Unstable chromosome aberrations (stimulated lymphocytes and those exposed in G0), sister chromatid exchanges (SCE) and cytokinesis blocked micronuclei (MN) (lymphocytes exposed in G0). In addition the SCE and MN assays allowed nuclear division indices (NDI) to be calculated as NDI defines the cell cycle progression of lymphocytes after PHA stimulation and how this might be affected by RF exposure. N2a cells were assessed by fluorescence microscopy for levels of apoptosis at a number of time points post RF field or x-ray exposure, between 0 and 48 h. Three independent assays that detect different stages of the apoptotic pathway were used, the Annexin V binding, caspase activation and in situ end labelling. By comparison with appropriate sham exposed samples no effect of RF fields alone could be found in G0 or PHA stimulated lymphocytes exposed in vitro. Also, RF fields did not modify any measured effects of x-rays either given before or after RF exposure. No statistically significant difference in apoptosis levels were observed between RF exposed and sham exposed N2a cells in either a proliferating or differentiated state for any assay at any time point post exposure.

Errors in DNA replication can cause chromosome instability and gross chromosomal rearrangements (GCRs). For my thesis work I investigate how chromosome instability can originate in the telomere. Here I report how defects in Cdc13, a telomere specific protein, lead to chromosome instability and GCRs in Saccharomyces cerevisiae. Using a temperature sensitive mutant of Cdc13, I find that cdc13-induced instability can be induced in a single cell cycle and synergizes with replication stress (dNTP depletion via hydroxyurea). Additionally, I find that Cdc13 has to be functional during the cell’s S phase to suppress chromosome instability. Further genetic analysis suggests that that cdc13-induced chromosome instability depends on the generation of single stranded (ss)DNA, but not on the activity of canonical double strand break (DSB) repair pathways such as homologous recombination or non-homologous end joining. Finally, I demonstrate that telomeric unstable chromosomes can later progress and trigger rearrangements at centromeric loci. This system, using the conditional nature of the cdc13 mutation, promises a more complex analysis of the ontogeny of chromosome instability: in this case from errors semi-conservative DNA replication through the telomere to the formation and resolution of unstable chromosomes.

Orientador: Cesar Martins / Abstract: One of the biggest challenges in chromosome biology is to understand the occurrence and complex genetics of extra, non-essential karyotype elements, commonly known as supernumerary B chromosomes (Bs). Bs are present in diverse species of eukaryotes and their molecular characterization remains elusive for years. A distinguished feature that makes them different from the normal chromosomes (called A chromosomes) is their way of inheritance in irregular fashion. Over the last decades, their genetic composition, function and evolution have remained an unresolved query, although a few successful attempts have been made to address these phenomena. The non-Mendelian inheritance and unpairing/non-recombining abilities make the B chromosomes immensely interesting for genomics studies, thus arising different questions about their genetic composition, survival, maintenance and role inside the cell. This study aims to uncover these phenomena in different species. Here, we sequenced the genomes of three model organisms including fish species Astyanax mexicanus and Astyanax correntinus, and grasshopper Abracris flavolineata with (B+) and without Bs (B-) to identify the B-localized sequences, called B chromosome blocks (“B-blocks”). We established approaches for this analysis that comprised of steps such as comparative genomics analysis and annotation of B chromosomal genes and DNA repeat types. The next generation sequencing (NGS) analyses identified thousands of genes fragments as well as... (Complete abstract click electronic access below) / Doutor

by Jenney Yin-mei Tse. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 122-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Bik-Yu Hui. / "July 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 187-203). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thesis (M. Ed.)--Kutztown State College. / Source: Masters Abstracts International, Volume: 45-06, page: 3060. Typescript. Includes bibliographical references (leaves 32-34).

The ultimate purpose of this study was to investigate AHP2 protein function in Arabidopsis; AHP2 protein is known to form a heterodimer with MND1. But to do this an existing chromosome spreading protocol had to be modified to reproducibly provide large numbers of well preserved chromosomes and a multi-criteria meiotic staging method was developed to accurately identify chromosome spreads at specific prophase I substages. As well a technique for combined immuno-cytochemistry and fluorescent in situ hybridization (FISH) had to be developed. Coimmuno-localization of AHP2 and MND1 proteins, in wild type meiocytes, revealed synchronous temporal organization (signal initially peaks, for both, during zygotene) but spatially the AHP2 signal appeared exclusively as chromosome axis-associated foci whereas the MND1 signal was diffuse with some foci suggesting that MND1 may also be localizing to loop regions. This finding strongly suggests that MND1 also functions independently of AHP2 during meiosis. We coupled transmission electron microscopy (TEM) analysis of ultrathin sections of meiotic nuclei with light microscope (LM) analysis of chromosome spreads and demonstrated that ahp2 meiocytes fail to stabilize chromosome close alignment that normally occurs during zygotene. My method of combining 2-Bromo-5-deoxyUridine (BrdU) - determination of meiosis duration and the relative durations of each substage allowed us to calculate the absolute duration of each prophase I substage in wild type and revealed that early to mid-zygotene was prolonged in the ahp2 mutant. This finding was consistent with the ahp2 mutant’s overall lack of stabilized chromosome alignment. Scanning electron microscopy (SEM) showed that the Arabidopsis ahp2 short stamen filament was due to reduced cell elongation in filament parenchyma (epidermal) cells. Detection of illegitimate connections between chromosomes at anaphase I may trigger cell-to-cell signals that result in reduced epidermal cell elongation in the stamen filament. Finally, I report that the short arms of NOR-bearing chromosomes 2 and 4 homologously pair and synapse in the ahp2 mutant despite an overall lack of stabilized pairing. This NOR phenomenon has been observed in Drosophila but ours is the first report of the ability of NORs to locally induce pairing and synapsis in plants.

The ultimate purpose of this study was to investigate AHP2 protein function in Arabidopsis; AHP2 protein is known to form a heterodimer with MND1. But to do this an existing chromosome spreading protocol had to be modified to reproducibly provide large numbers of well preserved chromosomes and a multi-criteria meiotic staging method was developed to accurately identify chromosome spreads at specific prophase I substages. As well a technique for combined immuno-cytochemistry and fluorescent in situ hybridization (FISH) had to be developed. Coimmuno-localization of AHP2 and MND1 proteins, in wild type meiocytes, revealed synchronous temporal organization (signal initially peaks, for both, during zygotene) but spatially the AHP2 signal appeared exclusively as chromosome axis-associated foci whereas the MND1 signal was diffuse with some foci suggesting that MND1 may also be localizing to loop regions. This finding strongly suggests that MND1 also functions independently of AHP2 during meiosis. We coupled transmission electron microscopy (TEM) analysis of ultrathin sections of meiotic nuclei with light microscope (LM) analysis of chromosome spreads and demonstrated that ahp2 meiocytes fail to stabilize chromosome close alignment that normally occurs during zygotene. My method of combining 2-Bromo-5-deoxyUridine (BrdU) - determination of meiosis duration and the relative durations of each substage allowed us to calculate the absolute duration of each prophase I substage in wild type and revealed that early to mid-zygotene was prolonged in the ahp2 mutant. This finding was consistent with the ahp2 mutant’s overall lack of stabilized chromosome alignment. Scanning electron microscopy (SEM) showed that the Arabidopsis ahp2 short stamen filament was due to reduced cell elongation in filament parenchyma (epidermal) cells. Detection of illegitimate connections between chromosomes at anaphase I may trigger cell-to-cell signals that result in reduced epidermal cell elongation in the stamen filament. Finally, I report that the short arms of NOR-bearing chromosomes 2 and 4 homologously pair and synapse in the ahp2 mutant despite an overall lack of stabilized pairing. This NOR phenomenon has been observed in Drosophila but ours is the first report of the ability of NORs to locally induce pairing and synapsis in plants.