Glioblastoma Multiforme (GBM) is the most common and lethal of human primary central nervous system tumours, with a median survival of 14-16 months despite surgery, radiation and chemotherapy. A reason for this dismal prognosis is insufficient understanding of the ontogeny of GBMs, which are highly heterogeneous at a pathological level. This pathological diversity, between and within GBMs as well as varying grades of gliomas, is not fully explained on the grounds of an oncogenic stimulus. Interaction with the tumour microenvironment, as well as inherent characteristics of the tumour cell of origin are likely a source of this heterogeneity. In this thesis we describe the use of a novel mouse model which integrates Cre-Lox mediated and Tet-regulated gene expression. This system in combination with germline and somatic strategies has enabled us to interrogate how the state in glial development and the region in the brain where transformation occurs influence the process of gliomagenesis. The findings of this thesis suggest that the state of glial development at which a mutation is introduced is an important determinant of gliomagenesis. In support of this, we showed that early progenitors in the radial glial lineage are more susceptible to transformation than those, which have committed to a gliogenic lineage and are presumably further along in the process of differentiation. Highlighting the interplay between genetic alterations and the molecular changes that accompany the process of differentiation. Despite findings that suggest that neurogenic regions of the adult brain are more susceptible to transformation, we show that this is not always the case and instead, transformation is dependent on an interaction between specific combinations of genetic mutations and susceptible cell types regardless of the region of origin. Results from this thesis highlight the need to view the tumourigenic process of gliomas in the context of normal brain development as the cell context of oncogene expression may determine the phenotype and biologic aggressiveness of the tumour. Thus, the results of genetic or epigenetic alterations leading to brain tumours may be quite different in different cells of the hierarchy, suggesting unique treatment targets and strategies depending on the cell of origin.

For the treatment of cancer, immunotherapy has some inherent advantages over other treatment modalities: disseminated disease can be eradicated due to the systemic nature of immunity, the immune system is effective against a wide range of targets, long-term memory can offer added protection against disease relapse, immunotherapy should be relatively non-toxic, and it can be synergistically combined with other treatment platforms such as radiation and chemotherapy. Type 1 immune responses are thought to be superior for the treatment of cancer and, as the quintessential Th1 polarizing cytokine, interleukin-12 (IL-12) holds much promise; however, optimal therapeutic protocols have yet to be developed and clinical results have fallen short of this promise. The in vivo IL-12 experiments described here highlight a characteristic of cellular therapy that has not previously been appreciated. That is, the effect of cell-mediated cytokine delivery on the immediate microenvironment and how that affects the immune response initiated. This observation has implications for the clinical application of IL-12 therapy but may also prove to be an important consideration when studying other immunostimulants. I have herein developed a novel in vitro assay system that I have used to dissect the cellular responses to IL-12 and to identify the signals that are required for activation of a cluster of differentiation 4 (CD4)+ effector population that affects leukaemia cell clearance both in vitro and in vivo. This work, and the future studies proposed, will expand our understanding of the potential of IL-12 immunotherapy and enhance our ability to manipulate therapeutic conditions to favour the desired response. Moreover, the in vitro assay system offers a method for further characterization of CD4+ effector cells and the development of protocols to initiate their potent anticancer activity.

Iroquois (Irx) homeobox genes have important and redundant functions during embryogenesis. Irx3/5 double knock out (Irx3/5 KO) mouse embryos exhibit severe hindlimb phenotypes. In these mutant hindlimbs, digit 1 and tibia are absent, moreover femur and pelvis are hypoplastic. Here, we demonstrate that Irx3/5 are expressed in the hindlimb field prior to limb bud initiation, and are required at this early stage for the pattern formation along the anteroposterior axis. Their early function is involved in prepatterning and positioning the Shh expression domain. In addition, Irx3/5 KO mutant hindlimb buds have a mild outgrowth defect and increased cell death at early stages of limb development, which may explain the small hindlimb bud size in these mutant embryos. To examine whether Irx3/5-expressing cells are the origin of lost and affected structures in Irx3/5 KO mutant hindlimbs, targeting vectors with Cre genes inserted into the Irx5 locus have been generated.

Autism is a severe childhood neuropsychiatric condition characterized by impairments in socialization and communication, and by restricted and repetitive behaviours. Autism spectrum disorder (ASD) is a complex and largely unknown disease with a strong genetic basis, multiple genes involved and environmental factors determining its phenotype. Interestingly, the DLX1/DLX2 and DLX5/DLX6 bigene clusters are located in autism susceptibility loci and Dlx genes are involved in GABAergic interneurons differentiation and migration to the cortex during forebrain development. Dlx gene expression is controlled by different cis-regulatory elements. Of these, 4 are active in the forebrain, URE2, I12b, I56ii and I56i. In order to determine the role of the DLX genes in ASD, variants were found in gene exons and in cis-regulatory elements in autistic individuals. A single nucleotide polymorphism (SNP), a change of an adenine for a guanine, was identified in I56i enhancer. Finding a SNP in I56i was very surprising considering that it is located in a Dlx binding motif highly conserved among >40 species. We showed, using in vitro approaches, that the presence of this SNP affects the affinity of Dlx for their binding site and reduces the transcriptional activation of the enhancer. The SNP also affects activity of the I56i enhancer in transgenic mice. In order to determine the real impact of the SNP in vivo, mutant mice harboring the SNP in their I56i enhancer were produced. That involved the insertion of the I56i enhancer with the SNP, using homologous recombination in mouse embryonic stem cells to replace the wild type version of the enhancer. With these mutant mice, we demonstrated that, in vivo, this SNP reduces Dlx5 and Dlx6 expression in the forebrain. Furthermore, this decrease in Dlx5/Dlx6 expression could affect the differentiation and/or migration of specific populations of inhibitory interneurons in the forebrain. No distinct iv behavioural phenotypes were observed between wild type mice and those carrying the SNP, during social interaction and anxiety tests. Therefore, these results suggest that even a subtle change in a regulatory element can have an impact in the development of the forebrain and may even contribute to disorders such as autism.

The goal of this study was to show that quantifiable metabolic changes may be used to screen for cancer. NIH III nude mice (n=22) were injected with human GBM cells, with daily urine samples collected pre and post-injection. 14 mice were injected with saline to serve as controls. The measurement of metabolite concentrations took place on an 800 MHz NMR spectrometer. 34 metabolites were identified and quantified, through targeted profiling, with Chenomx Suite 5.1. Univariate statistical analysis showed that 3 metabolites (2-oxoglutarate, glucose and trimethylamine n-oxide) were significantly altered in the presence of tumour, while PCA and PLS-DA models found the maximum variance between the healthy and tumour-bearing groups. Receiver operating characteristic (ROC) curve analysis was applied to the data set to provide a measure of clinical utility. ROC statistics were as high as 0.85 for the analysis of individual metabolites, 0.939 for the analysis of metabolite pairs and 0.996 for PLS-DA models. These results show that metabolomics has potential as a screening tool for cancer. / Medical Physics

国立情報学研究所で電子化したコンテンツを使用している。

国立情報学研究所で電子化したコンテンツを使用している。

国立情報学研究所で電子化したコンテンツを使用している。

The 2-50 transgenic mouse is a mutant containing the functional exons of human c-myc, an oncogene and cell cycle controller, under the control of the minimal sequence of the promoter of myelin basic protein, a component of the sheath surrounding axons of neurons. The transgene complex is expressed only in oligodendrocytes during a limited period of neonatal life, and is not detectable in large amounts. The animals suffer significant loss of oligodendrocyte precursor cells prior to myelination and onset of myelin formation is delayed. These animals elaborate only an incomplete myelin sheath in the central nervous system. Quantitative genetic analysis was used to characterize the transgene insertion and optic nerves from transgenic and non-transgenic animals were used for light and election microscopy, for electrophysiological testing and for immunohistochemical studies of glial cell subpopulations and axonal cytoskeletal components.

Doctor of Philosophy (PhD) / The field of epigenetics emerged primarily from studies in Drosophila, and is now being studied intensively by mammalian biologists. In order to increase our knowledge of epigenetic gene control in the mouse, I have studied modifiers of epigenetic gene silencing. My main method of investigation involved the characterisation of mutants from a sensitised ENU mutagenesis screen performed previously in our laboratory. The screen was carried out in an FVB/NJ strain carrying a variegating GFP transgene expressed in erythrocytes. To date we have recovered 12 dominant (D) and seven recessive (R) mutant mouse lines from this screen that display altered transgene expression. We have named these Mommes (Modifiers of murine metastable epialleles). I investigated the phenotype and attempted to identify the underlying causative mutation of two of these Momme mutants. MommeD6 is a semi-dominant, homozygous lethal mutation that acts as a suppressor of variegation with respect to the GFP transgene. This mutation has a large effect on the level of expression of the transgene in expressing cells, but little effect on the percentage of cells expressing the transgene. MommeD6 is linked to a 2.5 Mbp interval on chromosome 14. MommeD9 is a semi-dominant, homozygous lethal mutation that acts as an enhancer of variegation with respect to the GFP transgene. Mutants have a tendency to become obese as they age, show abnormal haematology profiles, and females develop infertility. MommeD9 is linked to a 17.4 Mbp region on chromosome 7. I produced and studied a strain carrying the same GFP transgene but in a new strain background, C57BL/6J. This strain provided an opportunity to look for strain-specific modifiers of expression of the GFP transgene. Several regions were mapped to chromosomal locations. Further work will be needed to identify the genes involved. This mouse will be useful in future mutagenesis screens of this type.