Germ cell tumours (GCTs) are clinically and pathologically heterogeneous neoplasms that arise at gonadal (testicular/ovarian) and extra-gonadal sites. The chemotherapy burden for patients with malignant germ cell tumours (mGCTs) that require treatment results in substantial longterm side-effects, and, furthermore, poor-risk patients have < 50% survival. Consequently, identifying common molecular changes and novel therapeutic targets in mGCTs is of major clinical importance. MicroRNAs are short, non-protein coding RNAs that regulate gene expression. We previously showed that miR-99a-5p/-100-5p and miR-125b-5p are among the most frequently underexpressed microRNAs in mGCTs, regardless of anatomical site, histological type or patient age. The present study investigates the upstream causes and downstream consequences of such under-expression. The mature form of miR-125b-5p is the product of two genomic loci, which form a cluster with either miR-99a-5p (on chromosome 21q) or miR-100-5p (on chromosome 11q). MiR-99a-5p/- 100-5p share identical 'seed' regions (at nucleotide positions 2-7), which determine their mRNA targets. Cross-reactivity experiment revealed that both miR-99a-5p and miR-100-5p probes were highly cross-reactive to each other's target (from 91% to 95%), indicating functional overlap. Linear regression analysis of qRT-PCR data reveals a strong positive correlation between miR-99a-5p/-100-5p and miR-125b-5p levels (R2 =0.989) in mGCTs, strongly suggesting co-regulation. Primary microRNA transcripts (pri-miR-99a/-100 and pri-miR-125b), and other genes that colocalise to these miRNA clusters (e.g. BLID on chromosome 11), were quantified by RT-qPCR in four representative cell lines - TCam2, 1411H, 2102Ep, and GCT44 - which were derived from a range of common histological types of mGCTs. A significant down-regulation (p < 0.0001) of all primary transcripts was observed, suggesting transcriptional repression of the entire cluster regions. Treatment of the cell lines with 5'-azacytidine resulted in significant upregulation of all three miRNAs (p < 0.002), as well as BLID (p < 0.02). The methylation status of potential CpG islands at the region of interest on chromosome 11 and chromosome 21 was therefore investigated by Pyrosequencing. Significant hyper methylation was found in 2102Ep, 1411H and GCT44 cell lines, suggesting that the miR-99a-5p/-100-5p and miR-125b-5p clusters are likely transcriptionally silenced by DNA methylation. To assess the functional relevance of these microRNAs in GCT progression, co-transfection of microRNA mimics (8.3 nM miR-99a-5p/-100-5p + 8.3 nM miR-125b-5p) was performed. A significant decrease in cell growth was seen in 1411H (p < 0.01) and TCam2 (p < 0.03) cells. To identify the mimics' downstream mRNA targets, HumanHT-12 v4 Expression Bead Chip (Illumina) mRNA arrays were used and data analysed using Sylamer. This analysis showed that mimic-treated cells were enriched in downregulated genes involved in pro-proliferative mechanisms. Among those, further functional characterisation focussed in particular on TRIM71, FGFR3, E2F7 and LIN28A. Moreover, restoring miR-99a-5p/-100-5p and miR-125b-5p in TCam2 cells also resulted in G0-G1 accumulation, consistent with a cell cycle effect. These data support a functionally important role for miR99a-5p/-100-5p and miR-125b-5p in GCT progression. They also raise the possibility of a therapeutic replenishment approach for treating these, and potentially other, tumours.

During tumor inception and progression, culprit gene variants confer selective advantage to progenitor cancer cells, allowing them to outcompete normal cells and proliferate uncontrollably. Both regions of somatic amplification as well as germline DNA sequence changes may be variants that are positively selected by the tumor. Traditionally, these two variant classes have been studied independently. While many discoveries have been made in such a manner, independent examination of these classes possesses certain limitations. Integrated examination of these two classes holds the potential to reveal specific nucleotide alleles that are amplified in the tumor, which in turn may reveal proximal genes. We present methods that focus on such integration. The first, the Amplification Distortion Test (ADT), aims to detect nucleotide alleles that are selectively amplified across tumor samples. Motivated to apply ADT on nascent next generation sequencing data, we developed a novel Hidden Markov Model-based method - Haplotype Amplification in Tumor Sequences (HATS) - that analyzes tumor and matched normal sequence data, along with training data for linkage information, to infer amplified alleles and haplotypes in regions of copy number gain. HATS is designed to handle biases in read data as well as accommodate rare variants. We demonstrate that HATS infers the amplified alleles more accurately on simulated and real tumor data than does an alternate naïve approach, especially at low to intermediate sequence coverage levels, and when allele-specific biases or stromal contamination is present. We present these methods with the motivation that they may aid the cancer community in identifying novel causal or associated putative variants.

Cancer often develops from specific DNA alterations, and these cancer-associated mutations influence precision cancer treatment. These alterations can be specific to the tumor DNA (somatic mutations) or they can be heritable and present in normal and tumor DNA (germline mutations). Germline variants can affect how patients respond to therapy and can influence clinical surveillance of patients and their families. While identifying cancer-associated germline variants traditionally required studying families with inherited cancer predispositions, large-scale cancer sequencing cohorts enable alternative analysis of germline variants. In this dissertation, we develop and apply multiple strategies for analyzing germline DNA from cancer sequencing cohorts. First, we develop the Tumor-Only Boosting Identification framework (TOBI) to learn biological features of true somatic mutations and generate a classification model that identifies DNA variants with somatic characteristics. TOBI has high sensitivity in identifying true somatic variants across several cancer types, particularly in known driver genes. After predicting somatic variants with TOBI, we assess the identified somatic-like germline variants for known oncogenic germline variants and enrichment in biological pathways. We find germline and somatic variants inactivating the Fanconi anemia pathway in 11% of patients with bladder cancer. Finally, we investigate germline, diagnosis, and relapse variants in a large cohort of patients with pediatric acute lymphoblastic leukemia (ALL). Our somatic analysis captures known ALL driver genes, and we describe the sequential order of diagnosis and relapse mutations, including late events in NT5C2. We apply both the TOBI framework and guidelines American College of Medical Genetics and Genomics to identify potentially cancer-associated germline variants, and nominate nonsynonymous variants in TERT and ATM.

Primordial germ cells (PGCs) are the precursors of the germ cell lineage that eventually differentiate into mature spermatozoa and oocytes. Although present throughout the animal kingdom, the specification and migration of PGCs differs widely between species. In vertebrates, avians are evolutionary divergent from mammals and therefore allow a comparative system in which to study germ cell development in higher organisms. Unlike mouse, PGCs can be isolated from the chicken embryo, expanded and cultured long term in vitro. Analysis of these cells showed that cultured chicken PGCs maintain the characteristics of their in vivo counterparts, including the expression of key germ cell specific markers and cell surface adhesion proteins, and thus, are an ideal system to study germ cell biology. Further characterisation revealed that an avian homologue of the zinc finger transcription factor PRDM14, essential for the specification of the mammalian germ cell lineage, was expressed in chicken PGCs. cPRDM14 was found to be expressed in PGCs in vitro and in vivo from early developmental stages until expression is lost by embryonic day 10 and subsequently re-expressed in the adult testis. The expression of cPRDM14 suggested that this gene may play a conserved role in the avian germ cell lineage. To investigate the function of cPRDM14, a novel single piggyBac transposon vector containing a reverse tetracycline activator protein and a tetracycline response element-regulated promoter was developed. Testing of the integrated transposon revealed that expression was tightly regulated and it was possible to conditionally express one gene product whilst simultaneously reducing the expression of a second gene, both in vitro and in vivo. This vector system was fully functional in PGCs, and was used to create transgenic founder chickens capable of having gene expression manipulated in germ cells at various developmental stages. Transgenic offspring were produced and the transgene was inducible at early developmental stages in the G1 animals. The un-induced transgene proved to be toxic to early embryos so a transgenic line of birds could not be produced. The inducible transposon was used to knockdown cPRDM14 expression in chicken PGCs. Knockdown of this gene led to reduced PGC numbers and increased cell death, both in vitro and in ovo. Expression of the pluripotency factor cNANOG was also significantly reduced which may explain the increased cell death. The knockdown of cPRDM14 also led to an increased susceptibility of PGCs to spontaneously de-differentiate to pluripotent embryonic germ cells (EGCs). cPRDM14 knockdown PGCs exhibited elevated levels of phosphorylated ERK, a target of the FGF signalling pathway. It was possible to prevent de-differentiation of the knockdown PGCs by removing ectopic FGF from the media. Furthermore, a sustained high level of FGF signalling in the media was sufficient to drive the de-differentiation of control PGCs to EGCs, suggesting that increased FGF signalling was key to the de-differentiation process. Extensive epigenetic remodelling of mouse PGCs occurs during embryonic development and PRDM14 was shown to be involved in this process. Chicken PGCs in vitro, contain several key histone modifications (H3K4me3, H3K9me2 and H3K27me3) and are 5-methyl cytosine (5-mC) positive. Immunohistochemical analysis of these markers in PGCs, at various stages during early embryonic development, suggests that these cells do not undergo the extensive epigenetic remodelling found in their mammalian counterparts. In contrast to the mouse germ cell lineage, knockdown of cPRDM14 in cultured PGCs had no noticeable effect on the epigenetic status of chicken PGCs. Together these results demonstrate that cPRDM14 is essential for the survival and maintenance of germ cell identity in chicken PGCs, but may not be critical for maintaining the epigenetic status of these cells.

Copies of author's previously published articles inserted. Bibliography: p. 199-254. In eutherian mammals, the size of the testes and number of spermatozoa produced and stored in the excurrent ducts vary widely between species, with the hydromyine rodents of Australia exhibiting a greater range of interspecific variation than any other closely related group of species. This study compared the efficiency of germ cell production and sperm storage capacity in the extra-testicular ducts of two arid zone species, the plains rat, Pseudomys australis, and the spinifex hopping mouse, Notomys alexis, that have vast differences in testes size and number of stored spermatozoa. Results are discussed.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Pennisetum Rich, is one of the most important genera in the family Poaceae because it includes forage and crop species such as Pennisetum purpureum Schumach. and Pennisetum glaucum (L.) R. Br. Both P. purpureum and P. glaucum have a number of cultivars and varieties arising due to natural crossing which are very difficult to distinguish morphologically. P. purpureum and P. glaucum also hybridize naturally because they are protogynous and cross pollinated. The resulting hybrids are highly sterile and resemble P. purpureum. Lepidopteran stem borers cause great yield loss in maize produced by resource-poor farmers in Africa and are managed by habitat management or push-pull strategies, in which P. purpureum cultivars and hybrids are used as a trap crop. The aims of this project were to genotype different P. purpureum cultivars and hybrids using Amplified Fragment Length Polymorphism (AFLP) as well as Random Amplified Polymorphic DNA (RAPD) in order to identify cultivars and hybrids and possible misidentifications, assess the congruency of results between AFLPs and RAPDs and to attempt to relate these results to the oviposition preference of Chilo partellus for different P. purpureum cultivars. The individuals to be fingerprinted were collected from several countries in sub-Saharan Africa, a few from the USA and one from China. The AFLP analysis of these individuals were done with primer combinations EcoRI/MseI and Mlul/Msel on polyacrylamide gels and an ABI 3130 xl Genetic Analyzer respectively. The automated sequencer visualized more bands than the polyacrylamide gels. The RAPD technology was not developed any further after 17 primers were tested and no polymorphic bands detected. Overall results indicated that cultivars did not cluster according to geographical origin, and cultivars known by popular names did not always cluster together, indicating diversity within the cultivar or misidentifications. An example of a misidentification is the cultivar Green Gold being no other than cultivar Harare, or cultivar Swaziland 3 being cultivar Sanitas. Proper management by nursery managers cannot be stressed enough, as this will prevent plants getting mixed up, causing confusion. There was no relationship between the relatedness of cultivars and moth oviposition preference. The AFLP technology could be a powerful tool for the DNA fingerprinting and molecular characterization of this grass species, but poor germ plasm management negates its application. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2008.

The formation of germ cells in Drosophila melanogaster is dependent on the presence of ribonucleoprotein complexes called polar granules. A key component of these complexes is Oskar, a novel protein which has been shown to nucleate the granules. To investigate whether Oskar plays a further role in polar granule formation, I cloned the oskar gene from D. immigrans flies (osk[superscript imm]) and introduced it into D. melanogaster flies using P-element transformation. I found that osk[superscript imm] was able to rescue both the posterior patterning and germ cell formation defects of embryos from oskar mutant mothers. In addition, I found that the polar granules of embryos containing only Osk[superscript imm] as a source of Oskar protein resemble those found in D. immigrans embryos, indicating a new role for Oskar in determining the morphology of the polar granules. Germ cell formation in Drosophila is succeeded by migration of the germ cells to the site of gonad formation. A second line of research presented in this dissertation describes analysis of a novel protein important for both germ cell formation and migration, Bluestreak (Blue). Embryos from either heterozygous or homozygous Blue-mothers display defects in germ cell number and shape. I found that the ovaries of Blue-females have defects in the localization of Staufen and Oskar, sufficient to cause a reduction in pole cell number in embryos. In addition, genetic analysis of the interaction between Bluestreak and mutants which affect pole cell migration implicates Bluestreak in this process. Finally, I found that Blue localizes to centrosomes along with [gamma]-tubulin throughout the embryo, and to the nuclear membrane in pole cells. My findings introduce the possibility that Bluestreak may act to regulate germ cell migration in Drosophila.

No description available.

In most animals, oogenesis involves a syncytial “cyst” stage. Cysts are produced by incomplete mitotic divisions of gonial precursor cells, leaving the resulting cystocytes interconnected by cytoplasmic bridges. The bridges subsequently break down, liberating the developing gametes. In some animals (e.g. meroistic insects) cysts are “polarized”, such that certain cystocytes differentiate as supportive nurse cells, rather than oocytes. The variability of cysts in animal oogenesis contrasts with the relative universality of spermatogenic cysts, making the functional importance of cysts in oogenesis unclear. I have studied oogenesis in a polychaete worm, Ophryotrocha labronica (Annelida: Dorvilleidae). These worms produce polarized, two-celled oogenic cysts with one nurse cell and one oocyte. Such cysts resemble their better-characterized counterparts in meroistic insects. However, using a variety of light- and electron-microscopic techniques, I show here that the resemblance between O. labronica and meroistic insects is largely superficial. Rather, the roles of nurse cells and the mechanisms underlying cystocyte differentiation are quite distinct in both groups. Therefore, similarities between these polychaetes and insects are probably examples of convergent evolution rather than homology. These observations underscore the plasticity of oogenesis among animals. Mechanisms by which germ cells become distinct from somatic cells in animals are also a subject of considerable research activity. Two general modes of germ-cell specification have been described in animals: deterministic specification, which is typical of established model species (e.g., Drosophila melanogaster and Caenorhabditis elegans) and inductive specification, which, though it is the more-common mode among animals, has not been well studied. As an annelid worm, O. labronica likely specifies its germ cells inductively, and therefore has potential to serve as a model species for studies of inductive germ cell specification. Realizing this potential, however, will require the development of genetic resources for this species. I describe the beginnings of such work here: the isolation and characterization of a vasa/PL10-like gene whose expression is largely restricted to germ cells, the construction of a cDNA library, and the refinement of methods for in situ hybridization and immunostaining to visualize gene expression in whole worms.