O glioblastoma multiforme (GBM) pode desenvolver-se rapidamente sem evidências clínicas, radiológicas ou morfológicas de um tumor precursor menos maligno. Entretanto, um novo tumor pode desenvolver-se a partir de células gliais normais ou de suas precursoras, sendo chamado de GBM primário. Já meduloblastoma é um tumor embrionário maligno do cerebelo, cuja incidência ocorre preferencialmente em crianças de até 7 anos. Os tumores de cérebro se diferem entre si em nível molecular. A amplificação do gene EGFR (Receptor do Fator de Crescimento Epidermal) com consequente elevação da expressão do receptor de EGF (Fator de Crescimento Epidermal) é mais proeminente em GBM primário quando comparado com GBM secundário e está presente também em meduloblastoma. O presente trabalho consiste em investigar o perfil de expressão gênica da via EGF e o perfil proteico de genes supressores de tumor e oncogenes de linhagens de células tumorais de glioblastoma e meduloblastoma após o tratamento com terapia fotodinâmica (TFD). O conhecimento da ação da TFD em tumores cerebrais em nível molecular permitiu a detecção de genes que participam da regulação da expressão gênica de outras vias de sinalização como RTK/Ras/PI3-K e AKT/MAPK que são responsáveis pela proliferação celular aumentada, sobrevivência ou resistência a apoptos e perda de aderência e migração, podendo revelar alto grau de invasividade. Portanto, o tratamento com terapia fotodinâmica em células tumorais do cérebro acrescenta informações relevantes sobre o processo de proliferação celular e da biologia do câncer. / Glioblastoma multiforme (GBM) can develop quickly without clinical, radiological or morphological of a less malignant precursor tumor. However, a new tumor can grow from normal glial cells or their precursors, is called the primary GBM. Medulloblastoma is also a malignant embryonic tumor of the cerebellum, whose incidence occurs preferentially in children under 7 years. Brain tumors differ from between them at the molecular level. The amplification of the EGFR gene (Growth Factor Receptor Epidermal) with an increase in the expression of EGF receptor (Epidermal Growth Factor) is the main cause in primary GBM compared to secondary GBM and is also present in medulloblastoma. This work to investigate the gene expression profile of the EGF pathway and the protein profile of tumor suppressor genes and oncogenes tumor cell lines of glioblastoma and medulloblastoma after treatment with photodynamic therapy (PDT). PDT\'s share knowledge in brain tumors at the molecular level allowed to discovery of genes that participate in the regulation of gene expression of other RTK/Ras/PI3-K and AKT/MAPK signaling pathways such as that are responsible for the increased cell proliferation, survival or resistance to apoptosis and loss of adhesion and migration, and may reveal a high degree of invasiveness. For this reason, treatment with photodynamic therapy in brain tumor cells adds relevant information about the process of cellular proliferation and cancer biology.

Ng Yeung Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 180-209). / Abstracts in English and Chinese. / acknowledgements --- p.i / abstract --- p.ii / abstract in chinese --- p.vi / table of contents --- p.ix / list of tables --- p.xiii / list of figures --- p.xi v / list of abbreviations --- p.xvi / Chapter 1 --- chapter1 introduction and literature review --- p.1 / Chapter 1.1 --- Introduction of brain tumors --- p.1 / Chapter 1.2 --- Oligodendroglial tumors (OTs) --- p.3 / Chapter 1.2.1 --- Oligodendroglioma (OD) and anaplastic oligodendroglioma (AOD) --- p.3 / Chapter 1.2.1.1 --- WHO's definition and grading --- p.3 / Chapter 1.2.1.2 --- "Incidence, age, sex distribution, tumor location and survival rate" --- p.3 / Chapter 1.2.1.3 --- Clinical presentation --- p.4 / Chapter 1.2.1.4 --- Macroscopy and histopathology --- p.4 / Chapter 1.2.1.5 --- Immunohistochemistry --- p.5 / Chapter 1.2.1.6 --- Treatment --- p.6 / Chapter 1.2.2 --- Oligoastrocytoma (OA) and anaplastic oligoastrocytoma (AOA) --- p.11 / Chapter 1.2.2.1 --- WHO's definition and grading --- p.11 / Chapter 1.2.2.2 --- "Incidence, age, sex distribution, tumor location and survival rate" --- p.12 / Chapter 1.2.2.3 --- Clinical features --- p.12 / Chapter 1.2.2.4 --- Macroscopy and histopathology --- p.12 / Chapter 1.3 --- Overview of Genetic and Epigenetic Aberrations of OTs --- p.14 / Chapter 1.3.1 --- Chromosomal and genetic aberrations in OTs --- p.14 / Chapter 1.3.2 --- Candidate regions and genes on 1 p --- p.15 / Chapter 1.3.3 --- Candidate regions and genes on 19q --- p.20 / Chapter 1.3.4 --- Other aberrations in WHO grade II OTs --- p.24 / Chapter 1.3.5 --- Progression-associated aberrations in ODs --- p.25 / Chapter 1.3.6 --- Chromosomal and genetic aberrations in OAs --- p.29 / Chapter 1.4 --- Correlation of genetic alterations with response to therapy and survival --- p.31 / Chapter 1.4.1 --- Response to PCV chemotherapy correlates with lp and combined lp/19q status in patients with AODs --- p.31 / Chapter 1.4.2 --- Survival of patients with AODs correlates with lp/19q status --- p.32 / Chapter 1.4.3 --- WHO grade II ODs behavior and lp/19q status --- p.32 / Chapter 1.4.4 --- Response to other therapies (temozolomide and radiotherapy) and lp/19q status in patients with ODs --- p.33 / Chapter 1.4.5 --- lp and 19q loss in OAs and diffuse astrocytomas --- p.34 / Chapter 1.5 --- Microarray-based expression profiling of OTs --- p.35 / Chapter 1.6 --- Description of p73 protein --- p.37 / Chapter 1.6.1 --- Introduction of p73 --- p.37 / Chapter 1.6.2 --- p73: gene structure and splicing variants --- p.37 / Chapter 1.6.3 --- Signaling in p73 --- p.40 / Chapter 1.6.4 --- Regulation ofp73 protein stability and transcriptional activity --- p.43 / Chapter 1.6.4.1 --- Regulation by DNA damage --- p.43 / Chapter 1.6.4.2 --- Regulation by oncogenes --- p.44 / Chapter 1.6.4.3 --- Interaction with viral proteins --- p.44 / Chapter 1.6.5 --- Role of p73 in the nervous system --- p.45 / Chapter 1.6.6 --- p73 in cancer --- p.45 / Chapter 1.6.6.1 --- p73 knockout mice --- p.45 / Chapter 1.6.6.2 --- Alteration of p73 expression in human cancers --- p.46 / Chapter 1.6.7 --- p73 and chemosensitivity --- p.50 / Chapter CHAPTER2 --- AIMS OF STUDY --- p.51 / Chapter CHAPTER3 --- MATERIALS AND METHODS --- p.53 / Chapter 3.1 --- Tumor and blood samples --- p.53 / Chapter 3.2 --- Cell culture --- p.53 / Chapter 3.3 --- DNA extraction from frozen tissues and blood samples --- p.54 / Chapter 3.4 --- Detection of allelic loss of chromosome lp --- p.58 / Chapter 3.4.1 --- LOH analysis --- p.58 / Chapter 3.4.2 --- Fluorescence in situ Hybridization (FISH) analysis on Paraffin and Frozen Sections --- p.60 / Chapter 3.6 --- DNA sequencing analysis --- p.62 / Chapter 3.7 --- Analysis of Methylation --- p.63 / Chapter 3.7.1 --- Bisulfite sequencing --- p.63 / Chapter 3.7.2 --- Methylation-specific polymerase chain reaction (MSP) --- p.66 / Chapter 3.8 --- Northern Blot analysis --- p.68 / Chapter 3.9 --- RNA isolation and cDNA preparation --- p.70 / Chapter 3.10 --- Laser microdissection and RNA extraction from microdissected tumor cells --- p.71 / Chapter 3.10.1 --- Conventional RT-PCR --- p.71 / Chapter 3.11 --- Primer design for TP73 and its isoforms --- p.74 / Chapter 3.12 --- Real-time RT-PCR --- p.77 / Chapter 3.12.1 --- Real-time RT-PCR for TP73 and its isoforms --- p.78 / Chapter 3.12.2 --- Real-time RT-PCR for KIAA0495 --- p.79 / Chapter 3.13 --- Statistical analyses --- p.81 / Chapter CHAPTER4 --- RESULTS --- p.82 / Chapter 4.1 --- Genes annotated in the minimally deleted regions --- p.82 / Chapter 4.2 --- Expression analyses of TP73 and its isoforms in ODs by quantitative real-time RT-PCR --- p.85 / Chapter 4.3 --- Methylation analysis of TP73 in ODs by methylation sensitive PCR (MSP) --- p.97 / Chapter 4.4 --- A rapid screen of candidate genes for aberrant expression in microdissected tumors --- p.100 / Chapter 4.5 --- Quantitative real-time RT-PCR of KIAA0495 gene --- p.103 / Chapter 4.6 --- Mutation analysis of KIAA0495 gene --- p.110 / Chapter 4.7 --- Methylation analysis of KIAA0495 in ODs by bisulfite sequencing…… --- p.112 / Chapter 4.8 --- Detection of allelic loss of lp by LOH analysis and interphase FISH --- p.121 / Chapter 4.9 --- Two-hit inactivation of KIAA0495 gene in ODs --- p.126 / Chapter 4.10 --- Tissue distribution of KIAA0495 gene --- p.130 / Chapter 4.11 --- Bioinformatics of KIAA0495 --- p.133 / Chapter CHAPTER5 --- DISCUSSION --- p.146 / Chapter 5.1 --- Expression analysis of TP73 and its isoforms in ODs by isoform-specific RT-PCR --- p.148 / Chapter 5.2 --- Methylation status ofTP73 in ODs --- p.153 / Chapter 5.3 --- A rapid screening of candidate genes for aberrant expressionin microdissected tumors --- p.156 / Chapter 5.4 --- Expression pattern of KIAA0495 mRNA in a large cohort of ODs --- p.157 / Chapter 5.5 --- No somatic mutation in coding region of KIAA0495 --- p.158 / Chapter 5.6 --- Methylation status of putative promoter region of KIAA0495 in ODs --- p.159 / Chapter 5.7 --- Status of chromosome lp in ODs --- p.161 / Chapter 5.8 --- Two-hit inactivation of KIAA0495 gene in ODs by promoter hypermethylation and allelic loss of lp --- p.162 / Chapter 5.9 --- Evaluation of expression of KIAA0495 gene as a marker for the response to chemotherapy and prognostic marker in patients with OTs --- p.164 / Chapter 5.10 --- Tissue distribution of KIAA0495 --- p.166 / Chapter 5.11 --- "KIAA0495 cDNA sequence, protein sequence and potential functional features" --- p.167 / Chapter 5.12 --- Candidate tumor suppressor genes on lp in other type of tumors with loss of lp --- p.171 / Chapter CHAPTER6 --- CONCLUSIONS --- p.174 / Chapter CHAPTER7 --- FUTURE STUDIES --- p.177 / Chapter CHAPTER8 --- REFERENCES --- p.180

O glioblastoma multiforme (GBM) pode desenvolver-se rapidamente sem evidências clínicas, radiológicas ou morfológicas de um tumor precursor menos maligno. Entretanto, um novo tumor pode desenvolver-se a partir de células gliais normais ou de suas precursoras, sendo chamado de GBM primário. Já meduloblastoma é um tumor embrionário maligno do cerebelo, cuja incidência ocorre preferencialmente em crianças de até 7 anos. Os tumores de cérebro se diferem entre si em nível molecular. A amplificação do gene EGFR (Receptor do Fator de Crescimento Epidermal) com consequente elevação da expressão do receptor de EGF (Fator de Crescimento Epidermal) é mais proeminente em GBM primário quando comparado com GBM secundário e está presente também em meduloblastoma. O presente trabalho consiste em investigar o perfil de expressão gênica da via EGF e o perfil proteico de genes supressores de tumor e oncogenes de linhagens de células tumorais de glioblastoma e meduloblastoma após o tratamento com terapia fotodinâmica (TFD). O conhecimento da ação da TFD em tumores cerebrais em nível molecular permitiu a detecção de genes que participam da regulação da expressão gênica de outras vias de sinalização como RTK/Ras/PI3-K e AKT/MAPK que são responsáveis pela proliferação celular aumentada, sobrevivência ou resistência a apoptos e perda de aderência e migração, podendo revelar alto grau de invasividade. Portanto, o tratamento com terapia fotodinâmica em células tumorais do cérebro acrescenta informações relevantes sobre o processo de proliferação celular e da biologia do câncer. / Glioblastoma multiforme (GBM) can develop quickly without clinical, radiological or morphological of a less malignant precursor tumor. However, a new tumor can grow from normal glial cells or their precursors, is called the primary GBM. Medulloblastoma is also a malignant embryonic tumor of the cerebellum, whose incidence occurs preferentially in children under 7 years. Brain tumors differ from between them at the molecular level. The amplification of the EGFR gene (Growth Factor Receptor Epidermal) with an increase in the expression of EGF receptor (Epidermal Growth Factor) is the main cause in primary GBM compared to secondary GBM and is also present in medulloblastoma. This work to investigate the gene expression profile of the EGF pathway and the protein profile of tumor suppressor genes and oncogenes tumor cell lines of glioblastoma and medulloblastoma after treatment with photodynamic therapy (PDT). PDT\'s share knowledge in brain tumors at the molecular level allowed to discovery of genes that participate in the regulation of gene expression of other RTK/Ras/PI3-K and AKT/MAPK signaling pathways such as that are responsible for the increased cell proliferation, survival or resistance to apoptosis and loss of adhesion and migration, and may reveal a high degree of invasiveness. For this reason, treatment with photodynamic therapy in brain tumor cells adds relevant information about the process of cellular proliferation and cancer biology.

<p>While drug-loaded biodegradable polymer microparticles have found many therapeutic applications, bulk manufacturing methods produce heterogeneous populations of particles. A more highly controlled manufacturing method may provide the ability improve the microparticle characteristics such as the drug release profile. Microfluidic droplet-makers manipulate liquids on the scale of tens of microns and can produce highly regular and controlled emulsions. However, microfluidic droplet manufacturing is not typically designed for clinical translation and the chemicals used are often not biocompatible.</p><p>I developed a two-chip PDMS-based microfluidic device that can manufacture PLGA microparticle loaded with hydrophilic or hydrophobic drugs. I characterized protein-loaded microparticles made using this device and compared them with bulk-generated microparticles. The microfluidics-generated microparticles had similar release curves and encapsulation efficiencies as bulk-generated microparticles but a much narrower size distribution. I generated peanut protein-loaded microparticles with this device and tested them in a mouse model of peanut allergy, improving the particles as the project evolved to have a higher loading level and lower burst release. The microparticles improved the safety and efficacy of an immunotherapy protocol. I also encapsulated hydrophilic and hydrophobic chemotherapeutic drugs for a brain cancer model.</p> / Dissertation

Background Cancer immunotherapy has yielded many successes. Yet to some hard-to-treat brain tumors, such as glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), it still lacks substantial improvement. Neoantigens resulting from mutations in malignant cells are the key targets for employing adoptive cell therapies. A novel therapeutical strategy may be developed based on the identification of T cell receptors (TCRs) targeting specific neoantigens. Methods Previous work had been done to provide essential materials, including candidate neoantigen peptides, human leukocyte antigen (HLA) genotypes, and peripheral blood mononuclear cell (PBMCs) from patients and healthy donors (HDs). Autologous antigen-presenting cells (APCs) and T cells were isolated from PBMCs for in vitro assays. The activation of T cells against peptides was evaluated by the upregulation of 41BB utilizing flow cytometry (FACS). The cell populations with positive signals were sorted through FACS for TCR sequencing directly or after rapid cell expansion. Results T cells and APCs from 12 HDs were isolated. T cells from 10 HDs were analyzed after in vitro stimulation. T cells from HD30 showed reactions to several public neoantigens; while T cells from HD49 and HD53 showed reactions also to private neoantigens restricted in GBM patient C6. Conclusion The upregulation of 41BB indicated the activation of T cells and the existence of reactive TCRs against either public or private neoantigens in some HDs. Those reactive TCRs and their encoding sequences were the fundamentals of future works. Due to practical reasons, TCR sequencing cannot be done within this project. In future works, wildtype peptides will be included to further validate the results, ensuring identified TCRs recognize neoantigens specifically. Furthermore, the identified TCRs will be cloned and transferred to freshly isolated T cells to confirm their functionality. Keywords Cancer immunotherapy, brain cancer, neoantigen, MHC/HLA, TCR

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer with an average survival time of 15 months. GBM is considered incurable with even the most aggressive multimodal therapies and is characterized by near universal recurrence. Irreversible electroporation (IRE) is a cellular ablation method currently being investigated as a therapy for a variety of cancers. Application of IRE involves insertion of electrodes into tissue to deliver pulsed electric fields (PEFs), which destabilize the cell membrane past the point of recovery, thereby inducing cell death. While this treatment modality has numerous advantages, the lack of selectivity for malignant cells limits its application in the brain where damage to healthy tissue is especially deleterious. In this dissertation we hypothesize that a form of IRE therapy, high-frequency IRE (H-FIRE), may be able to act as a selective targeted therapy for GBM due to its ability to create an electric field inside a cell to interact with altered inner organelles. Through a comprehensive investigation involving experimental testing combined with numerical modeling, we have attained results in strong support of this hypothesis. Using tissue engineered hydrogels as our platform for therapy testing, we demonstrate selective ablation of GBM cells. We develop mathematical models that predict the majority of the electric field produced by H-FIRE pulses reach the inside of the cell. We demonstrate that the increased nuclear to cytoplasm ratio (NCR) of malignant GBM cells compared to healthy brain—evidenced in vivo and in in vitro tissue mimics—is correlated with greater ablation volumes and thus lower electric field thresholds for cell death when treated with H-FIRE. We enhance the selectivity achieved with H-FIRE using a molecularly targeted drug that induces an increase in NCR. We tune the treatment pulse parameters to increase selective malignant cell killing. Finally, we demonstrate the ability of H-FIRE to ablate therapy-resistant GBM cells which are a focus of many next-generation GBM therapies. We believe the evidence presented in this dissertation represents the beginning stages in the development of H-FIRE as a selective therapy to be used for treatment of human brain cancer. / Ph. D.

No description available.

Malignant gliomas (MG), most notably glioblastoma multiforme (GBM), are among the most aggressive of all malignancies. High-grade variants of this type of brain cancer are generally considered incurable with singular or multimodal therapies. Many patients with GBM die within one year of diagnosis, and the 5-year survival rate in people is approximately 10%. Despite extensive research in diagnostic and therapeutic technologies, very few developments have emerged that significantly improve survival over the last seven decades. Irreversible electroporation (IRE) is a new non-thermal focal tissue ablation technique that uses low-energy electric pulses to destabilize cell membranes, thus achieving tissue death. The procedure is minimally invasive and is performed through small electrodes inserted into the tissue with treatment duration of about one minute. The pulses create an electric field that induces an increase in the resting transmembrane potential (TMP) of the cells in the tissue. The induced increase in the TMP is dependent on the electric pulse parameters. Depending on the magnitude of the induced TMP the electric pulses can have no effect, transiently increase membrane permeability or cause spontaneous death. In this dissertation we hypothesize that irreversible electroporation is capable of ablating normal (gray and white matter) and pathological (MG and/or GBM) brain tissue in a highly focused non-thermal manner that is modulated through pulse parameters and electrode configuration. Through a comprehensive experimental and numerical investigation, we tested and attained results strongly supporting our hypothesis. Specifically, we developed numerical models that were capable of simulating an entire IRE treatment protocol and would take into account pulse parameters (e.g. duration, frequency, repetition rate and strength) in addition to the dynamic changes in tissue electrical conductivity due to electroporation and joule heating, as well as biologically relevant processes such as blood perfusion and metabolic heat. We also provided a method to isolate the IRE effects from undesired thermal damage in models that were validated with real-time temperature measurements during the delivery of the pulses. Finally we outlined a procedure to use 3D volumetric reconstructions of IRE lesions using patient specific MRI scans in conjunction with the models described for establishing field thresholds or performing treatment planning prior to the surgical procedure; thus supplying the readers with the tools and understanding necessary to design appropriate treatment protocols for their specific application. Experimentally we presented the first systematic in vivo study of IRE in normal canine brain and the multimodal treatment of a canine MG patient. We confirmed that the procedure can be applied safely in the brain and was well tolerated clinically. The lesions created with IRE were sub-millimeter in resolution and we achieved 75% tumor volume reduction within 3 days post-IRE in the patient. In addition to the sharp delineation between necrotic and normal brain, the treatments spared the major blood vessels, making it appropriate for treatment of tumors adjacent to, or enveloping critical vascular structures. We believe that irreversible electroporation will play a key role in the treatment of intracranial disorders including malignant brain cancer in which the intent is to focally kill undesired tissue while minimizing damage to surrounding healthy tissue. / Ph. D.

The purpose of this research is to validate various forms of mathematical modeling of glioblastoma multiforme (GBM) expressed as differential equations, numerically. The first work was involved in the numerical solution of the reaction-convection model, efficacy of which is expressed in terms of survival time. It was calculated using simple numerical scheme for the standard-of-care treatment in clinics which includes surgery followed by the radiation and chemotherapy. Survival time using all treatment options increased significantly to 57 weeks compared to that of surgery close to 14 weeks. It was also observed that survival time increased significantly to 90 weeks if tumor is totally resected. In reaction-diffusion model using simple numerical scheme, tumor cell density patterns due to variation in patient specific tumor parameters such as net proliferation rate and diffusion coefficient were computed. Significant differences were observed in the patterns while using dominant diffusion and proliferation rate separately. Numerical solution of the tumor growth model under the anti-angiogenic therapy revealed some impacts in optimum tumor growth control however it was not significant. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

This thesis investigates rest-activity and sleep profiles in neurodegeneration and brain cancer. Study 1 comprised longitudinal field assessments of rest-activity, sleep and memory in controls and memory-impairment individuals with: subjective memory complaint (SMC), amnestic mild cognitive impairment (aMCI), mild and moderate Alzheimer’s disease (AD). Four questions were addressed: (1) is SMC a prodromal stage of AD? (2) do characteristics of SMC predict future decline? (3) does cholinergic medication (ChEI) impact rest-activity and sleep of moderate AD patients? and (4) are there factors predicting response to ChEI? Study 2 assessed rest-activity and melatonin rhythms in a brain cancer patient (JJB), and post-mortem analysis of brain tissue assessed infiltration of cancer cells on the circadian clock (SCN). Both studies used questionnaires, cognitive tests, electroencephalography and actigraphy simultaneously at patients’ homes. In Study 1, the SMC group showed a reduced activity amplitude to be correlated with increasing memory impairment severity, lower sleep quality and efficiency. Increased sleep fragmentation was observed in all memory-impaired groups, although not correlated to impairment severity. Increased fragmentation of rest-activity rhythm correlated with increasing memory impairment severity in all groups except SMC. Following ChEI medication with donepezil, moderate AD patients showed increased sleep fragmentation, probably due to potentiation of available acetylcholine known to maintain arousal. Higher daytime-activity and lower activity in the rest-phase, when drug-naïve, predicted improved cognition following ChEIs. In Study 2, cancer cell infiltration of the patient’s SCN was confirmed. However, a robust circadian rest-activity period with a misaligned melatonin phase, was recorded, indicating that the effects of partial SCN lesions in humans are complex and this result was possibly in part are due to the masking effect of social behaviour.