Medulloblastoma is a devastating disease of the cerebellum, and the most common solid pediatric malignancy of the central nervous system. Recently, transcriptome-wide profiling has dissected medulloblastoma from one single disease into four disparate molecular subgroups – namely WNT, SHH, Group3 and Group4. Distinct genomic, cytogenetic, mutational and clinical spectra associated with these subgroups highlight the pressing need for targeted therapies, of which encouraging preliminary results have been generated. While the promise of personalized medicine is within our reach, improved understanding of the molecular mechanisms driving pathogenesis is critical to this process. The intent of my PhD thesis research was to characterize the molecular mechanisms contributing to medulloblastoma pathogenesis, and the clinical impact of these aberrations. Through a combinatorial use of genetic and epigenetic profiling, next-generation sequencing and bioinformatics analyses we have identified subsets of tumors with transcriptional signatures that influence their clinical properties. Furthermore, our results have shed light on the establishment of the normal cerebellar cytoarchitecture, identifying a physiological glutamate gradient with critical implications to both cerebellar development and disease. This thesis stresses the importance of interrogating medulloblastoma in a subgroup-specific manner. Our findings demonstrate the utility of pursuing an integrated (copy number, mutational, transcriptional and epigenetic) molecular approach, to further our understanding of the pathobiology of medulloblastoma. Finally, we propose rationale therapeutic targets that may improve the treatment of aggressive variants of this disease.

Medulloblastoma is a devastating disease of the cerebellum, and the most common solid pediatric malignancy of the central nervous system. Recently, transcriptome-wide profiling has dissected medulloblastoma from one single disease into four disparate molecular subgroups – namely WNT, SHH, Group3 and Group4. Distinct genomic, cytogenetic, mutational and clinical spectra associated with these subgroups highlight the pressing need for targeted therapies, of which encouraging preliminary results have been generated. While the promise of personalized medicine is within our reach, improved understanding of the molecular mechanisms driving pathogenesis is critical to this process. The intent of my PhD thesis research was to characterize the molecular mechanisms contributing to medulloblastoma pathogenesis, and the clinical impact of these aberrations. Through a combinatorial use of genetic and epigenetic profiling, next-generation sequencing and bioinformatics analyses we have identified subsets of tumors with transcriptional signatures that influence their clinical properties. Furthermore, our results have shed light on the establishment of the normal cerebellar cytoarchitecture, identifying a physiological glutamate gradient with critical implications to both cerebellar development and disease. This thesis stresses the importance of interrogating medulloblastoma in a subgroup-specific manner. Our findings demonstrate the utility of pursuing an integrated (copy number, mutational, transcriptional and epigenetic) molecular approach, to further our understanding of the pathobiology of medulloblastoma. Finally, we propose rationale therapeutic targets that may improve the treatment of aggressive variants of this disease.

The blood brain barrier (BBB) remains a major challenge to the advancement and application of systemic anti-cancer therapeutics into the central nervous system. The structural and physiological delivery constraints of the BBB significantly limit the effectiveness of conventional chemotherapy, thereby making systemic administration a non-viable option for the vast majority of chemotherapy agents. Furthermore, the lack of specificity of conventional systemic chemotherapy when applied towards malignant brain tumors remains a major shortcoming. Hence novel therapeutic strategies that focus both on targeted and enhanced delivery across the BBB are warranted. In recent years nanoparticles (NPs) have emerged as attractive vehicles for efficient delivery of targeted anti-cancer therapeutics. In particular, gold nanoparticles (AuNPs) have gained prominence in several targeting applications involving systemic cancers. Their enhanced permeation and retention within permissive tumor microvasculature provide a selective advantage for targeting. Malignant brain tumors also exhibit transport-permissive microvasculature secondary to blood brain barrier disruption. Hence AuNPs may have potential relevance for brain tumor targeting. However, the permeation of AuNPs across the BBB has not been well characterized, and hence is a potential limitation for successful application of AuNP-based therapeutics within the central nervous system (CNS). In this dissertation, we designed and characterized AuNPs and assessed the role of polyethylene glycol (PEG) on the physical and biological properties of AuNPs. We established a size-dependent permeation profile with respect to core size as well as PEG length when AuNPs were assessed through a transport-permissive in-vitro BBB. This study was the first of its kind to systematically examine the influence of design on permeation of AuNPs through transport-permissive BBB. Given the significant delivery limitations through the non-transport permissive and intact BBB, we also assessed the role of magnetic resonance imaging (MRI) guided focused ultrasound (MRgFUS) disruption of the BBB in enhancing permeation of AuNPs across the intact BBB and tumor BBB in vivo. MRgFUS is a novel technique that can transiently increase BBB permeability thereby allowing delivery of therapeutics into the CNS. We demonstrated enhanced delivery of AuNPs with therapeutic potential into the CNS via MRgFUS. Our study was the first to establish a definitive role for MRgFUS in delivering AuNPs into the CNS. In summary, this thesis describes results from a series of research projects that have contributed to our understanding of the influence of design features on AuNP permeation through the BBB and also the potential role of MRgFUS in AuNP permeation across the BBB.

The study was conducted to develop a technique that measures the amount of gadolinium based contrast agent accumulated in a head tumour by x-ray fluorescence, while a patient is exposed to neutrons or during external beam radiotherapy planning. In this research, measurements of the gadolinium concentration in a vessel simulating a brain tumour located inside a head phantom, by the x-ray fluorescence method were taken, where the Magnevist contrast medium which has gadolinium atom, in the tumour vessel, was excited by a 36 GBq (0.97 Ci) 241Am source that emits gamma rays of 59.54 keV, in 35.7 % of it’s decays, resulting the emission of characteristic fluorescence of gadolinium at 42.98 keV that appeared in the X-ray fluorescence spectrum. A Cadmium Telluride (CdTe) detector was used to evaluate and make an analysis of the gadolinium concentration. Determinations of the gadolinium content were obtained directly from the detector measurements of XRF from gadolinium in the exposed tumour vessel. The intensity measured by the detector was proportional to the gadolinium concentration in the tumour vessel. These concentrations of gadolinium were evaluated for dose assessment. The positioning of the head phantom was selected to be in the lateral and vertex positions for different sizes of tumour vessels. Spherical tumour vessels of 1.0, 2.0, 3.0 cm and an oval tumour vessel of 2.0 cm diameter and 4.0 cm length, containing the gadolinium agent, contained concentration between 5.62 to 78.63 mg/ml. They were placed at different depths inside a head phantom at different positions in front of the detector and the source for the measurements. These depths ranged from 0.5 cm to 5.5 cm between the center of the tumour and interior wall of the head phantom surface. The total number of measurements in all four sizes of the tumour vessel was 478; 78 examinations of a 1.0 cm spherical tumour vessel, 110 examinations of a 2.0 cm spherical tumour vessel, 150 examinations of a 3.0 cm spherical tumour vessel and 140 examinations of a 2.0 x 4.0 cm ellipsoid tumour vessel. To measure the size and the shape of the tumour by the alternative radiographic method, a general x-ray machine with radiograph film was used. Based on that, the appropriate shape of concentration could be selected for therapy. The differences of optical density in the x-ray films showed that the noise was increased with low concentration of the Gd. Because radiographic film may be subjected to different chemical processes where the darkness will be affected, these measurements would be very hard to be quantitative. Accordingly it is difficult to use the film for Gd concentrations. The obtained data show that the method works very well for such measurements.

The blood brain barrier (BBB) remains a major challenge to the advancement and application of systemic anti-cancer therapeutics into the central nervous system. The structural and physiological delivery constraints of the BBB significantly limit the effectiveness of conventional chemotherapy, thereby making systemic administration a non-viable option for the vast majority of chemotherapy agents. Furthermore, the lack of specificity of conventional systemic chemotherapy when applied towards malignant brain tumors remains a major shortcoming. Hence novel therapeutic strategies that focus both on targeted and enhanced delivery across the BBB are warranted. In recent years nanoparticles (NPs) have emerged as attractive vehicles for efficient delivery of targeted anti-cancer therapeutics. In particular, gold nanoparticles (AuNPs) have gained prominence in several targeting applications involving systemic cancers. Their enhanced permeation and retention within permissive tumor microvasculature provide a selective advantage for targeting. Malignant brain tumors also exhibit transport-permissive microvasculature secondary to blood brain barrier disruption. Hence AuNPs may have potential relevance for brain tumor targeting. However, the permeation of AuNPs across the BBB has not been well characterized, and hence is a potential limitation for successful application of AuNP-based therapeutics within the central nervous system (CNS). In this dissertation, we designed and characterized AuNPs and assessed the role of polyethylene glycol (PEG) on the physical and biological properties of AuNPs. We established a size-dependent permeation profile with respect to core size as well as PEG length when AuNPs were assessed through a transport-permissive in-vitro BBB. This study was the first of its kind to systematically examine the influence of design on permeation of AuNPs through transport-permissive BBB. Given the significant delivery limitations through the non-transport permissive and intact BBB, we also assessed the role of magnetic resonance imaging (MRI) guided focused ultrasound (MRgFUS) disruption of the BBB in enhancing permeation of AuNPs across the intact BBB and tumor BBB in vivo. MRgFUS is a novel technique that can transiently increase BBB permeability thereby allowing delivery of therapeutics into the CNS. We demonstrated enhanced delivery of AuNPs with therapeutic potential into the CNS via MRgFUS. Our study was the first to establish a definitive role for MRgFUS in delivering AuNPs into the CNS. In summary, this thesis describes results from a series of research projects that have contributed to our understanding of the influence of design features on AuNP permeation through the BBB and also the potential role of MRgFUS in AuNP permeation across the BBB.

No description available.

The study was conducted to develop a technique that measures the amount of gadolinium based contrast agent accumulated in a head tumour by x-ray fluorescence, while a patient is exposed to neutrons or during external beam radiotherapy planning. In this research, measurements of the gadolinium concentration in a vessel simulating a brain tumour located inside a head phantom, by the x-ray fluorescence method were taken, where the Magnevist contrast medium which has gadolinium atom, in the tumour vessel, was excited by a 36 GBq (0.97 Ci) 241Am source that emits gamma rays of 59.54 keV, in 35.7 % of it’s decays, resulting the emission of characteristic fluorescence of gadolinium at 42.98 keV that appeared in the X-ray fluorescence spectrum. A Cadmium Telluride (CdTe) detector was used to evaluate and make an analysis of the gadolinium concentration. Determinations of the gadolinium content were obtained directly from the detector measurements of XRF from gadolinium in the exposed tumour vessel. The intensity measured by the detector was proportional to the gadolinium concentration in the tumour vessel. These concentrations of gadolinium were evaluated for dose assessment. The positioning of the head phantom was selected to be in the lateral and vertex positions for different sizes of tumour vessels. Spherical tumour vessels of 1.0, 2.0, 3.0 cm and an oval tumour vessel of 2.0 cm diameter and 4.0 cm length, containing the gadolinium agent, contained concentration between 5.62 to 78.63 mg/ml. They were placed at different depths inside a head phantom at different positions in front of the detector and the source for the measurements. These depths ranged from 0.5 cm to 5.5 cm between the center of the tumour and interior wall of the head phantom surface. The total number of measurements in all four sizes of the tumour vessel was 478; 78 examinations of a 1.0 cm spherical tumour vessel, 110 examinations of a 2.0 cm spherical tumour vessel, 150 examinations of a 3.0 cm spherical tumour vessel and 140 examinations of a 2.0 x 4.0 cm ellipsoid tumour vessel. To measure the size and the shape of the tumour by the alternative radiographic method, a general x-ray machine with radiograph film was used. Based on that, the appropriate shape of concentration could be selected for therapy. The differences of optical density in the x-ray films showed that the noise was increased with low concentration of the Gd. Because radiographic film may be subjected to different chemical processes where the darkness will be affected, these measurements would be very hard to be quantitative. Accordingly it is difficult to use the film for Gd concentrations. The obtained data show that the method works very well for such measurements.

Background: Brain tumors are the most common solid tumors found in children. Current research is determining whether diagnosing brain tumors earlier will help improve prognosis and reduce long-term deficits; however, childhood brain tumors are often diagnosed late with a median time of 1-4 months from onset of symptoms. Prolonged symptom intervals before diagnosis have been associated with life-threatening risks, neuro-cognitive disabilities, and detrimental professional relationships between healthcare providers and families. Pediatric brain tumor clinical presentations are often non-specific and resemble less serious illnesses; therefore, healthcare providers are failing to include this in their differential diagnoses list. Purpose: To assess healthcare provider knowledge of signs and symptoms of pediatric brain tumors using The Brain Pathways Guideline. Methods: A one group pre-test and post-test e-mailed separately to nurse practitioners that have active membership in National Association of Pediatric Nurse Practitioners (NAPNAP) Arizona Chapter. Results: The Wilcoxon Signed Rank Test revealed that the matched test scores were not statistically significant (p=0.157) after viewing The Brain Pathways Guideline educational materials. Conclusion: The results of this study did not show a statistically significant difference in the test scores and therefore it cannot be concluded that presenting an evidence-based guideline to assist healthcare providers to assess and diagnose patients with brain tumors will be helpful to improve pre-diagnostic symptom intervals.

abstract: Patients with malignant brain tumors have a median survival of approximately 15 months following diagnosis, regardless of currently available treatments which include surgery followed by radiation and chemotherapy. Improvement in the survival of brain cancer patients requires the design of new therapeutic modalities that take advantage of common phenotypes. One such phenotype is the metabolic dysregulation that is a hallmark of cancer cells. It has therefore been postulated that one approach to treating brain tumors may be by metabolic alteration such as that which occurs through the use of the ketogenic diet (KD). The KD is high-fat, low-carbohydrate diet that induces ketosis and has been utilized for the non-pharmacologic treatment of refractory epilepsy. It has been shown that this metabolic therapy enhances survival and potentiates standard therapy in mouse models of malignant gliomas, yet the anti-tumor mechanisms are not fully understood. The current study reports that KetoCal® (KC; 4:1 fat:protein/carbohydrates), fed ad libitum, alters hypoxia, angiogenic, and inflammatory pathways in a mouse model of glioma. Tumors from animals maintained on KC showed reduced expression of the hypoxia marker carbonic anhydrase 9 (CA IX), a reduction in hypoxia inducible factor 1-alpha (HIF-1α) and decreased activation of nuclear factor kappa B (NF-κB). Animals maintained on KC also showed a reduction in expression of vascular endothelial growth factor receptor 2 (VEGFR2) and decreased microvasculature in their tumors. Further, peritumoral edema was significantly reduced in animals fed the KC and protein analysis showed significantly altered expression of the tight junction protein zona occludens-1 (ZO-1) and the water channeling protein aquaporin-4 (AQP4), both of which have been implicated in malignant processes in glioma, including the formation of peritumoral edema in patients. Taken together the data suggests that KC alters multiple processes involved in malignant progression of gliomas. A greater understanding of the effects of the ketogenic diet as an adjuvant therapy will allow for a more rational approach to its clinical use. / Dissertation/Thesis / Masters Thesis Biology 2014

The primary objective of this proposal was to determine whether mitochondrial oxidative stress and variation in a particular mtDNA lineage contribute to the risk of developing cortical dysplasia and are potential contributing factors in epileptogenesis in children. The occurrence of epilepsy in children is highly associated with malformations of cortical development (MCD). It appears that MCD might arise from developmental errors due to environmental exposures in combination with inherited variation in response to environmental exposures and mitochondrial function. Therefore, it is postulated that variation in a particular mtDNA lineage of children contributes to the effects of mitochondrial DNA damage on MCD phenotype. Quantitative PCR and dot blot were used to examine mitochondrial oxidative damage and single nucleotide polymorphism (SNP) in the mitochondrial genome in brain tissue from 48 pediatric intractable epilepsy patients from Miami Children’s Hospital and 11 control samples from NICHD Brain and Tissue Bank for Developmental Disorders. Epilepsy patients showed higher mtDNA copy number compared to normal health subjects (controls). Oxidative mtDNA damage was lower in non-neoplastic but higher in neoplastic epilepsy patients compared to controls. There was a trend of lower mtDNA oxidative damage in the non-neoplastic (MCD) patients compared to controls, yet, the reverse was observed in neoplastic (MCD and Non-MCD) epilepsy patients. The presence of mtDNA SNP and haplogroups did not show any statistically significant relationships with epilepsy phenotypes. However, SNPs G9804A and G9952A were found in higher frequencies in epilepsy samples. Logistic regression analysis showed no relationship between mtDNA oxidative stress, mtDNA copy number, mitochondrial haplogroups and SNP variations with epilepsy in pediatric patients. The levels of mtDNA copy number and oxidative mtDNA damage and the SNPs G9952A and T10010C predicted neoplastic epilepsy, however, this was not significant due to a small sample size of pediatric subjects. Findings of this study indicate that an increase in mtDNA content may be compensatory mechanisms for defective mitochondria in intractable epilepsy and brain tumor. Further validation of these findings related to mitochondrial genotypes and mitochondrial dysfunction in pediatric epilepsy and MCD may lay the ground for the development of new therapies and prevention strategies during embryogenesis.