Plants that are heterozygous for genetic markers but do not produce segregating progeny may be suspected of carrying a mutation conditioning apomixis. Seed stocks in which heterozygous plants could be identified phenotypically were treated with a chemical mutagen. These seed stocks were heterozygous for recessive genetic markers, and/or heterozygous for a chromosome translocation. Spikes from heterozygous M1 plants were harvested and seeded in bulk. Spikes from heterozygous M2 plants were harvested and planted in M3 rows. The M3 rows were observed for the absence of segregating progeny and/or were observed cytologically for the presence of a heterozygous translocation. M3 rows not segregating for the genetic markers were crossed onto plants homozygous for the genetic markers. The F1 progenies were observed for an expected ratio of 1 normal: 1 recessive plant. All nonsegregating lines were found to be non-heterozygous. These lines most likely occurred due to seed and pollen contamination or were the result of crossing over between genetic markers. Fertile M2 plants were harvested from the treated heterozygous translocation seed stock. Normally, barley plants heterozygous for a translocation will produce semisterile spikes. Plants that would normally be semisterile but are fertile could be carrying a mutation conditioning apomixis. Progeny of the fertile M2 plants were examined cytologically for the presence of the heterozygous translocation. All selected lines contained the normal seven pairs of chromosomes and were the result of seed or pollen contamination. Seed stocks which could eliminate the problem of contamination in future experiments were developed and discussed. Haploviable mutants closely linked with the male sterile locus, msg2, were isolated in these seed stocks. Haploviable mutants are recognized by upset genetic ratios of alleles linked with the mutant. Selfed progenies of plants carrying a haploviable mutation contained fertile and male sterile plants in about a 1:1 ratio. Mostly male sterile progenies were obtained when plants heterozygous for the haploviable mutant and the male sterile allele were crossed onto male sterile plants. Four lines containing haploviable mutants were evaluated for their usefulness in producing all male sterile lines for hybrid barley production.

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Primary hyperparathyroidism (pHPT) is a pathology associated with one or multiple hyperfunctioning parathyroid glands.  The disease prevalence occurs in roughly 1-2% of the population primarily post-menopausal women.  The molecular pathology of the disease is poorly understood.  Elevated serum calcium levels in the setting of an inappropriately elevated parathyroid hormone level are indicative of the disease process.  The ultimate treatment of the disease is to remove the hyperfunctioning gland. The aim of this thesis was to examine potential genetic and epigenetic aberrations that are potentially disease causing. The methylation signature of normal and pathological parathyroid tissue has yet to be investigated.  DNA was bisulphite modified and analyzed using the Infinium HumanMethylation27 BeadChip. Distinct hierarchical clustering of genes with altered DNA methylation profiles in normal and pathologic parathyroid tissue was evident.  DNA hypermethylation of CDKN2B, CDKN2A, WT1, SFRP1, SFRP2, and SFRP4 known to be important in the development of parathyroid tumors were associated with reduced gene expression in both benign and malignant parathyroid tumors. Familial primary hyperparathyroidism (FPHPT) may occur due to an underlying germ-line mutation in the MEN1, CASR, or HRPT2/CDC73 genes.  Eighty-six young (≤45 years of age) patients with clinically non-syndromic PHPT underwent genetic analysis.  Eight of 86 (9.3%) young patients with clinically non-familial PHPT displayed deleterious germ-line mutations in the susceptibility genes (4 MEN1, 3 CASR, and 1 HRPT2/ CDC73). Accumulation of non-phosphorylated active β -catenin has been reported to commonly occur in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT).  We assessed possible β-catenin stabilizing mutations in a large series of parathyroid adenomas. A total of one hundred and eighty sporadic parathyroid adenomas were examined for mutations in exon 3 of the CTNNB1gene. The mutation S33C (TCT >TGT) was detected by direct-DNA sequencing of PCR fragments in 1 out of 180 sporadic parathyroid adenomas (0.68 %). Eight matched tumor-constitutional DNA pairs from patients with sporadic parathyroid adenomas underwent whole-exome capture and high-throughput sequencing.  Four of eight tumors displayed a frame shift deletion or nonsense mutations within the MEN1 gene, which was accompanied by loss of heterozygosity of the remaining wild-type allele.  One tumor harbored a Y641N mutation of the histone methyltransferase EZH2 gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in the additional 185 parathyroid adenomas revealed a high rate of MEN1 mutations (35%).

At least some areas of the embryonic blastoderm in Drosophila melanogaster appear to be determined by substances produced under control of the maternal genome without involvement of the embryonic genome. Study of mutant genes controlling the production of such morphogenic substances would provide valuable information about these substances and the nature of determination itself. In this thesis, the adult ovaries in offspring of females containing sex chromosomes originating from a wild-type strain of Drosophila melanogaster collected on Margarita Island, Venezuela have been examined. After selection on such chromosomes, over 50% of the offspring produced by homozygous females contained either one or two agametic gonads. The delayed expression is due to temperature-sensitive gene expression duringoogenesis. Two micron sections of normal and abnormal ovaries of this strain indicate the mesodermal components of the abnormal ovaries including the ovarian sheaths and the ovariole epithelial sheaths, were normal. In addition, attachment of the ovaries to the oviducts are normal. The abnormal ovarioles contain cells which appear to be mesodermally-derived follicle cells, but no normal egg chambers are seen. The germaria also contain follicle cells but no cells resembling stem cells or cystocytes are seen. It is concluded: (1) the development of ovarian germinal and mesodermal components are completely independent and (2) the mutant gene(s) prevent normal differentiation of pole cells into germ cells.

Biological and environmental factors contribute collectively to the development of human cancers. The primary focus of this research project was to investigate the impact of germline gene mutations, as a significant biological factor, on 29 major primary human cancers. For this I obtained data from multiple databases, including the Genetic Association Database (GAD), Sanger database (COSMIC), HGMD database, OMIM data and PubMed literature. Using the Extraction Transform and Load (ETL) process, 424 genes were obtained with 8,879 cancer mutation records. By integrating these gene mutation records a Human Cancer Map (HCM) was constructed, from which several sub-maps were derived based on particular mutation classes. Furthermore, a Protein-Protein Interaction Map (PPIM) was constructed based on the encoded proteins of the 424 gene set. Several key questions were addressed using the HCM and its sub-maps including the following: (i) Are individual groups of primary cancers associated with specific subset of genes (within the 424 full set)? (ii) Are groups of primary cancers associated with particular mutation classes? (iii) If both questions prove to be true, are groups of cancers associated with particular mutation class of target genes? This project also explored whether a corresponding Protein-Protein Interaction Map, derived from the Missense/Non-sense Mutation portion of the HCM gene set, would provide further information on gene associations between primary cancers in terms of the consequent identical amino acid changes involved. Results showed that: (1) closely-connected human cancers in the HCM exhibited a strong association with a particular mutation class; (2) Missense /Nonsense and Regulatory mutations played a central role in connecting cancers (i.e. via primary nodes) and so significantly influenced the construction of the HCM; (3) Genes with Missense/Nonsense and Regulatory mutations tended to be involved in cancer-associated pathways; (4) Using the kappa test to measure the extent of agreement between two connected primary cancers in the sub-HCMs, BRCA1, BRCA2, PALB2, MSH2, MSH6, MLH1, CDKN2A, and TP53 showed highest agreement for 5 of 10 mutation classes; (5) From the PIPM, it was evident that BRCA1, MSH6, BARD1, TP53, MSH2 and CHEK2 proteins best connected Breast, Ovarian, Prostate and Bowel primary cancers, and so the latter could represent 'driver proteins' for these cancers. In summary, this project has approached the analysis of gene involvement in human primary cancers from the starting position of the mutation class that harbours the specific gene mutation. Together with their downstream resultant alterations in the associated proteins, this analysis can provide insights into the relatedness of primary human cancers and their potential gene hierarchies. These data may therefore help us to understand more fully the etiology, diagnosis and potentially personalized treatments for cancer.

Assisted reproductive technologies (ART) emerged in the late 1970’s as a therapy for human infertility. Up till now more than 3 million babies have been conceived through ART, demonstrating the safety and efficiency of the technique. Published reports showed an increase in the rate of imprinting disorders (Beckwith Wiedemann Syndrome, Angelman Syndrome, etc.) in babies born after ART. What are the effects imposed through ART and should researchers reassess its safety and implications on the future offspring? Throughout this thesis, I analyzed the methylation patterns of germ cells and embryos to determine whether in vitro maturation and in vitro fertilization have a negative impact on the epigenetic patterns. Furthermore, DNA methylation was compared between sperm of infertile and presumably fertile controls in order to understand whether epigenetic disturbances lead to infertility at the first place. The occurrence of methylation aberrations in germ cells of infertile patients could be transmitted to new-borns and then cause epigenetic disorders. In order to elucidate the imprinting status within single cells, I developed a new technique based on limiting dilution where bisulfite treated DNA is distributed across several wells before amplification. This allowed methylation measurement at the single allele level as well parent of origin detection. In a total of 141 sperm samples from couples undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) including 106 with male factor or combined infertility and 28 with female infertility, I detected a significant correlation between lower quality of semen parameters (sperm count, percentage of abnormal sperm, and percentage of motile sperm) and the rate of imprinting errors. ALU repeats displayed a higher methylation in sperm DNA of patients leading to a pregnancy and live birth, compared to patients in which pregnancy was not achieved or a spontaneous abortion occurred. A discriminant analysis based on ALU methylation allowed correct classification of >70% of cases. Preliminary data from illumina methylation arrays where more than 27,000 CpGs were analyzed determined that only a single CpG site from the open reading frame C14orf93 was significantly different between the infertile and presumably fertile control group. However, further improvements on data normalization might permit detection of other differentially methylated regions. Comparison of embryos after natural conception, in vitro fertilized embryos from superovulated oocytes, and embryos achieved through fertilization of in vitro cultured oocytes revealed no dramatic effect on the imprinting patterns of Igf2r, H19, and Snrpn. Oocyte cryotop vitrification did not result in a dramatic increase of imprinting mutations in oocytes even though the rate of sporadic methylation errors in single Snrpn CpGs were higher within the in-vitrified group. Collectively, the results I will present within this thesis suggest an increase in the rate of imprinting errors within the germ cells of infertile patients, in addition to a decrease in genome wide methylation of ALU repetitive elements. I did not observe a detrimental effect on the methylation patterns of oocytes and the resulting embryos using in vitro maturation of oocytes and/or standard IVF with in vivo grown superovulated oocytes. / Assistierte Reproduktionstechniken (ART) wurden in den späten 1970er Jahren als Therapie für unfruchtbare Paare mit Kinderwunsch etabliert. Bis zum heutigen Tage wurden dank ART weltweit mehr als 3 Millionen Kinder geboren, ein eindrucksvoller Beweis für die Sicherheit und Effizienz dieser Methode. Dennoch zeigen veröffentlichte Studien einen Anstieg in der Rate von Imprinting-Erkrankungen (Beckwith Wiedemann-Syndrom, Angelman-Syndrom, etc.) bei Kindern, die nach assistierter Reproduktion geboren wurden. Es stellt sich die Frage, welche Effekte durch ART ausgelöst werden können und ob eine neue Einschätzung dieser Methode bezüglich ihrer gesundheitlichen Implikationen für künftige Generationen notwendig ist. In dieser Arbeit habe ich mögliche negative Effekte von in vitro-Maturation und -Fertili-sierung auf Methylierungsmuster humaner und muriner Keimzellen, sowie Maus-Embryonen untersucht. Aberrante DNA-Methylierungsmuster in Keimzellen von infertilen Patienten könnten auf die Neugeborenen übertragen werden und epigenetische Erkrankungen zur Folge haben. Ob epigenetische Störungen im Zusammenhang mit Infertilität stehen, wurde außerdem durch den Vergleich der DNA-Methylierung von Spermien infertiler und fertiler Männer untersucht. Um den Imprintigstatus auf Einzelzellebene zu bestimmen, habe ich basierend auf „Limiting Dilution“ eine neue Methode entwickelt. Bei diesem Verfahren wird Bisulfit-behandelte DNA vor der PCR-Amplifikation in mehrere Reaktionsgefässe verdünnt. Dies erlaubt die Methylierungsanalyse einzelner Allele und die Detektion elternspezifischer Methylierungsmuster. Mit insgesamt 141 Sperma-Proben von Paaren, die sich einer in vitro- Fertilisierung (IVF) oder einer Intrazytoplasmischen Spermieninjektion (ICSI) unterzogen hatten, davon 28 mit weiblicher und 106 mit männlicher oder kombinierter Unfruchtbarkeit, konnte ich einen positiven Zusammenhang zwischen der Rate an Imprinting-Fehlern und geringer Sperma-Qualität (gemessen an Standardparametern) ableiten. ALU-Sequenzen zeigten in Spermien-DNA von Patienten mit erfolgreicher Schwangerschaft und Geburt eine höhere Methylierung als von Patienten mit fehlgeschlagener Schwangerschaft oder Spontanabort. Eine auf der ALU-Methylierung basierende Diskriminanzanalyse konnte mehr als 70% aller Fälle korrekt klassifizieren. Vorläufige Daten aus Experimenten mit Illumina Methylierungs-Arrays mit einer Auflösung von mehr als 27.000 CpG-Positionen identifizierten einen signifikanten Gruppenunterschied zwischen Patienten- und Kontrollgruppe für eine CpG-Position innerhalb des offenen Leserahmens C14orf93. Verbesserungen der Datenauswertung (Normalisierung, Testung etc.) sollten die Entdeckung weiterer differenziell methylierter Regionen erlauben. Der Vergleich von Mausembryos aus natürlicher Konzeption, aus in vitro kultivierten und fertilisierten Oozyten und aus in vitro Fertilisation nach Superovulation zeigte keine dramatischen Effekte auf die Imprinting-Muster der geprägten Gene Igf2r, H19 und Snrpn. Das gilt auch für Cryo-Top vitrifizierte Oozyten, wenn auch die Rate sporadischer Methylierungsfehler einzelner CpG-Positionen in Snrpn etwas höher war als in den Kontrollgruppen. Zusammengenommen lassen die in dieser Arbeit präsentieren Resultate auf eine Zunahme an Imprinting-Fehlern und eine genomweite Abnahme der Methylierung repetitiver ALU-Sequenzen in den Keimzellen infertiler Patienten schließen.