While the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility, concerns have been raised regarding the risk of chromosomal abnormalities in pregnancies derived from ICSI. Studies on sperm from infertile men have suggested that this population may produce higher rates of aneuploid sperm. Thus, we hypothesized that defects in early meiotic events may contribute to both male infertility and the production of aneuploid sperm. We used immunofluorescent techniques to observe the synapsis and recombination of chromosomes during meiosis, and fluorescent in-situ hybridization (FISH) to assess sperm aneuploidy. We analyzed testicular tissue from thirty-one men (10 fertile and 21 infertile men). We observed that ~36% (5/14) of men with impaired spermatogenesis displayed reduced genome-wide recombination. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. We combined immunofluorescent and FISH techniques to study recombination patterns on chromosomes 13, 18 and 21 in fifteen men (5 fertile and 10 infertile men). Four of the infertile men displayed altered recombination distributions on at least one of the chromosome arms studied. Finally, we examined early meiotic events in two biopsies from an azoospermic t(8;13) carrier. While global recombination rates were not altered, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and γH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes, suggesting that regions of autosomal chromosomes that fail to synapse undergo transcriptional silencing in humans. In summary, we observed that a subset of infertile men display alterations in the number and position of meiotic crossovers, which may contribute to both infertility and an increased risk of sperm aneuploidy. The fidelity of synapsis is also a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility.

Homologous recombination (HR) is a faithful mechanism for the repair of double-stranded DNA breaks (DSBs) and plays a critical role in maintaining the integrity of genomic DNA. The product of the Breast Cancer Susceptibility 2 (BRCA2) gene functions as a recombination mediator in HR-directed repair of DSBs. BRCA2 interacts directly with RAD51, the central recombinase of HR, through highly conserved repetitive motifs of 30-40 amino acids, named BRC repeats, and regulates the formation of the RAD51-ssDNA nucleoprotein filament. There is significant variability in the number of BRC repeats among taxa. However, all mammalian BRCA2 orthologs have eight BRC repeats, which display different characteristics in in vitro studies of RAD51-ssDNA nucleoprotein filament. To test the importance of the number of BRC repeats and to evaluate the role of individual BRC repeats in HR, BRCA2 variants bearing different combinations of BRC repeats were generated using BAC-recombineering, expressed in murine hybridoma cells, and assayed for the ability to stimulate HR using a gene targeting assay. The BRCA2 variant bearing BRC repeats 1 to 4 decreased the efficiency of HR and increased the level of Rad51 protein, whereas the BRCA2 variant bearing BRC repeats 5 to 8 significantly stimulated HR, but had no effect on the level of Rad51. These results supported the hypothesis that BRC repeats are not functionally equivalent, but rather have different, perhaps reinforcing functions in HR. / Canadian Institutes of Health Research

Oxidative stress and histone deacetylase (HDAC) inhibition has been implicated as potential mechanisms in valproic acid (VPA) teratogenicity. Reactive oxygen species (ROS) can target DNA to cause oxidative DNA damage and DNA double strand breaks (DSBs) which can be repaired through homologous recombination (HR). HR is not an error free process and can result in detrimental genetic changes. In this present study we evaluated the role of HDAC inhibition in VPA-initiated HR. HDAC inhibition may indirectly alter repair activity as a result of increased expression of genes involved in HR or indirectly by causing DNA damage which initiates repair. The first objective was to investigate the ability of VPA to cause HDAC inhibition in the Chinese hamster ovary (CHO) 33 cell line. Using immunblotting, an increase in acetylated histone H3 and H4 protein levels was observed throughout 24 hr exposure to 5 mM VPA. Secondly, to investigate whether VPA affects the activity of DNA DSB repair, CHO 33 cells were transfected with either the endonuclease I-SceI plasmid to induce a site specific DSB or the empty plasmid, pGem. However, no increase in the difference in HR between VPA and media exposed I-Sce1 transfected cells compared to cells transfected with pGem was observed, which suggests that VPA does not affect DNA repair activity. Thirdly, to determine if VPA-induced HDAC inhibition increases susceptibility to DNA damage, immunocytochemistry revealed an increase in the number of γ-H2AX foci throughout 24 hr exposure to 5 mM VPA. To determine if oxidative stress may play a role in mediating VPA-induced DNA DSBs, another recombination study was carried out in which cells were pretreated with 400 U/ml of PEG-catalase prior to VPA treatment. The observed protective effect of PEG-catalase against VPA-induced HR and the generation of intracellular ROS by VPA suggest ROS may also play a role in VPA-initiated HR. However, in our DNA oxidation study, no increase in the oxidized nucleosides, 8-hydroxy-2'-deoxyguanosine and 5-hydroxycytosine was observed after VPA treatment. These studies suggest that HDAC inhibition and ROS signalling may play other roles in DNA maintenance and cell cycle arrest in initiating DNA DSBs and HR repair. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2009-08-12 14:27:16.327

Benzene is a ubiquitous environmental pollutant and a known human leukemogen. Early-life exposure to environmental carcinogens, including benzene, may lead to genomic instability in the fetus, ultimately leading to an increased risk for the development of childhood cancers including leukemia. It is possible that exposure to benzene results in DNA damage that may either be left unrepaired or be repaired erroneously causing genotoxicity. The first objective of this study was to determine if exposure of fetal hematopoietic cells to p-benzoquinone, a known toxic metabolite of benzene, increased DNA recombination in the pKZ1 model of mutagenesis. A significant increase in recombination was observed following exposure to 25 μM and 50 μM p-benzoquinone for 2, 4, 8, and 24 hours. A significant increase in recombination was also observed following exposure to 25 μM p-benzoquinone for 30 min, 45 min, and 1 hour, but not 15 min as compared to vehicle alone. Secondly, this study determined if exposure of fetal hematopoietic cells to p-benzoquinone resulted in DNA damage using γ-H2A.X as a marker for DNA double strand breaks and 8-hydroxy-2’-deoxyguanosine as a marker of oxidative DNA damage. A significant increase in γ-H2A.X foci formation was observed following exposure to 25 μM p-benzoquinone for 30 min, 45 min and one hour. Exposure of fetal hematopoietic cells to 25 μM p-benzoquinone did not significantly increase oxidative DNA damage at any of the examined time points. The third objective of this study was to determine whether or not reactive oxygen species were involved in the observed increase in DNA damage and recombination. Exposure to 25 μM p-benzoquinone for 15 min and 30 min, but not 45 min or one hour, led to an increase in reactive oxygen species production as measured by 5-(and-6)-chloromethyl-2-7-dichlorodihydrofluorescein diacetate fluorescence. Additionally, pretreatment with 400 U/mL PEG-catalase, an antioxidative enzyme, attenuated the increases in both DNA recombination and DNA double strand breaks as compared to treatment with p-benzoquinone alone. These studies indicate that p-benzoquinone is able to induce DNA damage and recombination in fetal hematopoieitic cells and that reactive oxygen species and oxidative stress may be important in the mechanism of toxicity. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2010-07-23 15:44:05.381

The gene that produces the precursor RNA transcript to the three largest ribosomal RNA molecules (rDNA) is present in multiple copies and organized into gene clusters. They represent 0.5% of the diploid human genome but are critical for cellular viability. The individual genes possess very high levels of sequence identity and are present in high local concentration, making them ideal substrates for genomic rearrangement driven by dysregulated homologous recombination. Our laboratory has developed a sensitive physical assay capable of detecting recombination-mediated genomic restructuring in the rDNA by monitoring changes in lengths of the individual clusters. In order to determine whether dysregulated recombination is a potential driving force of genomic instability in human cancer, adult patients with either lung or colorectal cancer, and pediatric patients with leukemia were prospectively recruited and assayed. Over half of the adult solid tumors show detectable rDNA rearrangements relative to either surrounding non-tumor tissue or normal peripheral blood. In contrast, there is a greatly reduced frequency of alteration in pediatric leukemia. This finding makes rDNA restructuring one of the most common chromosomal alterations in adult solid tumors, illustrates the dynamic plasticity of the human genome, and may have prognostic or predictive value in disease progression.

<p>Recombination is the process in which genetic material is exchanged between one's homologous chromosome pairs during egg or sperm development (meiosis). Recombination is necessary for proper segregation of chromosomes during meiosis, and also plays a role in purging deleterious mutations, accelerating adaptation, and influencing the distribution of genomic features over evolutionary time. While recombination is clearly an important process, recombination rate is known to vary within and between individuals, populations, and species. Furthermore, what causes this variation remains relatively unknown. Using empirical and sequenced based estimates of recombination rate for the closely related species <italic>Drosophila pseudoobscura</italic> and <italic>Drosophila miranda</italic>, I seek to understand where recombination happens across the genome, to what extent recombination changes between species, and what genomic features are responsible for these changes. These data will deepen our understanding of mechanisms determining the recombination landscape, and shed light on generalized patterns and exceptions of recombination rate variation across the tree of life.</p> / Dissertation

Multijunction solar cells made from a combination of CQDs of differing sizes and thus bandgaps are a promising means by which to increase the energy harvested from the Sun’s broad spectrum. In this dissertation, we first report the systematic engineering of 1.6 eV PbS CQD solar cells, optimal as the front cell responsible for visible wavelength harvesting in tandem photovoltaics. We rationally optimize each of the device’s collecting electrodes—the heterointerface with electron accepting TiO2 and the deep-work-function hole-collecting MoO3 for ohmic contact—for maximum efficiency. Room-temperature processing enables flexible substrates, and permits tandem solar cells that integrate a small-bandgap back cell atop a low thermal-budget larger-bandgap front cell. We report an electrode strategy that enables a depleted heterojunction CQD PV device to be fabricated entirely at room temperature. We develop a two-layer donor-supply electrode (DSE) in which a highly doped, shallow work function layer supplies a high density of free electrons to an ultrathin TiO2 layer via charge-transfer doping. Using the DSE we build all-room-temperature-processed small-bandgap (1 eV) colloidal quantum dot solar cells suitable for use as the back junction in tandem solar cells. We further report in this work the first efficient CQD tandem solar cells. We use a graded recombination layer (GRL) to provide a progression of work functions from the hole-accepting electrode in the bottom cell to the electron-accepting electrode in the top cell. The recombination layers must allow the hole current from one cell to recombine, with high efficiency and low voltage loss, with the electron current from the next cell. We conclude our dissertation by presenting the generalized conditions for design of efficient graded recombination layer solar devices. We demonstrate a family of new GRL designs experimentally and highlight the benefits of the progression of dopings and work functions in the interlayers.

While the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility, concerns have been raised regarding the risk of chromosomal abnormalities in pregnancies derived from ICSI. Studies on sperm from infertile men have suggested that this population may produce higher rates of aneuploid sperm. Thus, we hypothesized that defects in early meiotic events may contribute to both male infertility and the production of aneuploid sperm. We used immunofluorescent techniques to observe the synapsis and recombination of chromosomes during meiosis, and fluorescent in-situ hybridization (FISH) to assess sperm aneuploidy. We analyzed testicular tissue from thirty-one men (10 fertile and 21 infertile men). We observed that ~36% (5/14) of men with impaired spermatogenesis displayed reduced genome-wide recombination. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. We combined immunofluorescent and FISH techniques to study recombination patterns on chromosomes 13, 18 and 21 in fifteen men (5 fertile and 10 infertile men). Four of the infertile men displayed altered recombination distributions on at least one of the chromosome arms studied. Finally, we examined early meiotic events in two biopsies from an azoospermic t(8;13) carrier. While global recombination rates were not altered, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and γH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes, suggesting that regions of autosomal chromosomes that fail to synapse undergo transcriptional silencing in humans. In summary, we observed that a subset of infertile men display alterations in the number and position of meiotic crossovers, which may contribute to both infertility and an increased risk of sperm aneuploidy. The fidelity of synapsis is also a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility.

Barley (Hordeum vulgare L. subsp. vulgare) is an important crop and ranks fourth in overall production of the major cereal crops in the world. Like other cereal crops, barley suffers from a narrowing of its genetic base and susceptibility to diseases, pests and environmental stresses. H. bulbosum is a possible source of desirable genes for introgressing into barley to restore genetic diversity and improve current cultivars. Sexual hybridisation between barley and H. bulbosum is the main method for interspecific gene transfer in barley breeding but there are several barriers to overcome. Two of these are reduced recombination and the ability to identify recombinants quickly and efficiently. The aim in this thesis was to gain a better understanding of meiotic chromosomal behaviour in the two species and their hybrids and to improve the characterisation of recombinants from the hybrids. To study the events during meiosis, synaptonemal complex (SC) analysis was carried out on the two species and two H. vulgare - H. bulbosum hybrids. The results indicated that there were interspecific and intraspecific variations in SC length. Mean SC length was positively correlated with recombination frequency but not related to genome size. This suggests that the ratios of mean SC length to genome size (SC/DNA) show divergence among these Hordeum examples. An hypothesis based on the conformation of chromatin associated with axial element, which is dependent on SC/DNA ratio, was presented to explain the relationship between SC length and recombination frequency. Chromosome pairing in the two hybrids was determined by observation at pachytene and metaphase I (MI). Mean percentages of synapses were similar but there were different frequencies of MI pairing between these two hybrids, indicating that different mechanisms may regulate synapsis and MI pairing in the hybrids. To investigate meiotic recombination, genomic in situ hybridisation (GISH) was performed on the two hybrids at MI and anaphase I (AI). It was observed that intergenomic pairing and recombination events occur in distal chromosome segments. A great discrepancy between mean pairing and recombination frequencies was observed in both hybrids and several possible reasons for this discrepancy were discussed. Hybrid 102C2 with high MI pairing had a significantly higher recombination frequency than the low pairing 103K5, suggesting that high MI pairing appears to be associated with high recombination in the hybrids. An interesting finding is that the ratio of recombination to MI pairing in 103K5 (l:8.9) is twice as high compared with 102C2 (l:17). However, the mechanism for this difference in the ratio between the two hybrids remains unknown. Sequential fluorescence in situ hybridisation (FISH) and GISH were used successfully to localise the introgressions in selfed progeny from a tetraploid hybrid derived from chromosome-doubled 102C2 (102C2/colch). This procedure is fast, cheap and can efficiently detect and locate introgressions. Several disease-resistant recombinants were analysed in more details and leaf rust and powdery mildew resistance was associated with distal introgressions on chromosomes 2HS and 2HL (leaf rust) and 2HS (powdery mildew). It is possible that the leaf rust and powdery mildew resistances were closely linked in the distal region of 2HS. A considerable variation in introgression size was observed at similar chromosomal sites among the different recombinants, which will provide useful information for map-based cloning of genes.

Barley (Hordeum vulgare L. subsp. vulgare) is an important crop and ranks fourth in overall production of the major cereal crops in the world. Like other cereal crops, barley suffers from a narrowing of its genetic base and susceptibility to diseases, pests and environmental stresses. H. bulbosum is a possible source of desirable genes for introgressing into barley to restore genetic diversity and improve current cultivars. Sexual hybridisation between barley and H. bulbosum is the main method for interspecific gene transfer in barley breeding but there are several barriers to overcome. Two of these are reduced recombination and the ability to identify recombinants quickly and efficiently. The aim in this thesis was to gain a better understanding of meiotic chromosomal behaviour in the two species and their hybrids and to improve the characterisation of recombinants from the hybrids. To study the events during meiosis, synaptonemal complex (SC) analysis was carried out on the two species and two H. vulgare - H. bulbosum hybrids. The results indicated that there were interspecific and intraspecific variations in SC length. Mean SC length was positively correlated with recombination frequency but not related to genome size. This suggests that the ratios of mean SC length to genome size (SC/DNA) show divergence among these Hordeum examples. An hypothesis based on the conformation of chromatin associated with axial element, which is dependent on SC/DNA ratio, was presented to explain the relationship between SC length and recombination frequency. Chromosome pairing in the two hybrids was determined by observation at pachytene and metaphase I (MI). Mean percentages of synapses were similar but there were different frequencies of MI pairing between these two hybrids, indicating that different mechanisms may regulate synapsis and MI pairing in the hybrids. To investigate meiotic recombination, genomic in situ hybridisation (GISH) was performed on the two hybrids at MI and anaphase I (AI). It was observed that intergenomic pairing and recombination events occur in distal chromosome segments. A great discrepancy between mean pairing and recombination frequencies was observed in both hybrids and several possible reasons for this discrepancy were discussed. Hybrid 102C2 with high MI pairing had a significantly higher recombination frequency than the low pairing 103K5, suggesting that high MI pairing appears to be associated with high recombination in the hybrids. An interesting finding is that the ratio of recombination to MI pairing in 103K5 (l:8.9) is twice as high compared with 102C2 (l:17). However, the mechanism for this difference in the ratio between the two hybrids remains unknown. Sequential fluorescence in situ hybridisation (FISH) and GISH were used successfully to localise the introgressions in selfed progeny from a tetraploid hybrid derived from chromosome-doubled 102C2 (102C2/colch). This procedure is fast, cheap and can efficiently detect and locate introgressions. Several disease-resistant recombinants were analysed in more details and leaf rust and powdery mildew resistance was associated with distal introgressions on chromosomes 2HS and 2HL (leaf rust) and 2HS (powdery mildew). It is possible that the leaf rust and powdery mildew resistances were closely linked in the distal region of 2HS. A considerable variation in introgression size was observed at similar chromosomal sites among the different recombinants, which will provide useful information for map-based cloning of genes.