An integrated functional mapping and source localization platform for brain research

The premise of this dissertation is to create a highly integrated platform that combines the most current recording technologies for brain research through the development of new algorithms for three-dimensional (3D) functional mapping and 3D source localization. The recording modalities that were integrated include: Electroencephalography (EEG), Optical Topographic Maps (OTM), Magnetic Resonance Imaging (MRI), and Diffusion Tensor Imaging (DTI). This work can be divided into two parts: The first part involves the integration of OTM with MRI, where the topographic maps are mapped to both the skull and cortical surface of the brain. This integration process is made possible through the development of new algorithms that determine the probes location on the MRI head model and warping the 2D topographic maps onto the 3D MRI head/brain model. Dynamic changes of the brain activation can be visualized on the MRI head model through a graphical user interface. The second part of this research involves augmenting a fiber tracking system, by adding the ability to integrate the source localization results generated by commercial software named Curry. This task involved registering the EEG electrodes and the dipole results to the MRI data. Such Integration will allow the visualization of fiber tracts, along with the source of the EEG, in a 3D transparent brain structure. The research findings of this dissertation were tested and validated through the participation of patients from Miami Children Hospital (MCH). Such an integrated platform presented to the medical professionals in the form of a user-friendly graphical interface is viewed as a major contribution of this dissertation. It should be emphasized that there are two main aspects to this research endeavor: (1) if a dipole could be situated in time at its different positions, its trajectory may reveal additional information on the extent and nature of the brain malfunction; (2) situating such a dipole trajectory with respect to the fiber tracks could ensure the preservation of these fiber tracks (axons) during surgical interventions, preserving as a consequence these parts of the brain that are responsible for information transmission.

Blood-brain barrier in vitro model: A tissue engineering approach and validation

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

Prenatal environmental exposure and neurodevelopmentally important gene expression in malformed brain tissue from pediatric intractable epilepsy patients

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.

The potential role of environmental exposures and genomic signaling in development of central nervous system tumors

The etiology of central nervous system tumors (CNSTs) is mainly unknown. Aside from extremely rare genetic conditions, such as neurofibromatosis and tuberous sclerosis, the only unequivocally identified risk factor is exposure to ionizing radiation, and this explains only a very small fraction of cases. Using meta-analysis, gene networking and bioinformatics methods, this dissertation explored the hypothesis that environmental exposures produce genetic and epigenetic alterations that may be involved in the etiology of CNSTs. A meta-analysis of epidemiological studies of pesticides and pediatric brain tumors revealed a significantly increased risk of brain tumors among children whose mothers had farm-related exposures during pregnancy. A dose response was recognized when this risk estimate was compared to those for risk of brain tumors from maternal exposure to non-agricultural pesticides during pregnancy, and risk of brain tumors among children exposed to agricultural activities. Through meta-analysis of several microarray studies which compared normal tissue to astrocytomas, we were able to identify a list of 554 genes which were differentially expressed in the majority of astrocytomas. Many of these genes have in fact been implicated in development of astrocytoma, including EGFR, HIF-1α, c-Myc, WNT5A, and IDH3A. Reverse engineering of these 554 genes using Bayesian network analysis produced a gene network for each grade of astrocytoma (Grade I-IV), and ‘key genes’ within each grade were identified. Genes found to be most influential to development of the highest grade of astrocytoma, Glioblastoma multiforme (GBM) were: COL4A1, EGFR, BTF3, MPP2, RAB31, CDK4, CD99, ANXA2, TOP2A, and SERBP1. Lastly, bioinformatics analysis of environmental databases and curated published results on GBM was able to identify numerous potential pathways and geneenvironment interactions that may play key roles in astrocytoma development. Findings from this research have strong potential to advance our understanding of the etiology and susceptibility to CNSTs. Validation of our ‘key genes’ and pathways could potentially lead to useful tools for early detection and novel therapeutic options for these tumors.

Targeted Brain Derived Neurotropic Factors (BDNF) Delivery across the Blood-Brain Barrier for Neuro-Protection Using Magnetic Nano Carriers: An In-Vitro Study

Parenteral use of drugs; such as opiates exert immunomodulatory effects and serve as a cofactor in the progression of HIV-1 infection, thereby potentiating HIV related neurotoxicity ultimately leading to progression of NeuroAIDS. Morphine exposure is known to induce apoptosis, down regulate cAMP response element-binding (CREB) expression and decrease in dendritic branching and spine density in cultured cells. Use of neuroprotective agent; brain derived neurotropic factor (BDNF), which protects neurons against these effects, could be of therapeutic benefit in the treatment of opiate addiction. Previous studies have shown that BDNF was not transported through the blood brain barrier (BBB) in-vivo.; and hence it is not effectivein-vivo. Therefore development of a drug delivery system that can cross BBB may have significant therapeutic advantage. In the present study, we hypothesized that magnetically guided nanocarrier may provide a viable approach for targeting BDNF across the BBB. We developed a magnetic nanoparticle (MNP) based carrier bound to BDNF and evaluated its efficacy and ability to transmigrate across the BBB using an in-vitro BBB model. The end point determinations of BDNF that crossed BBB were apoptosis, CREB expression and dendritic spine density measurement. We found that transmigrated BDNF was effective in suppressing the morphine induced apoptosis, inducing CREB expression and restoring the spine density. Our results suggest that the developed nanocarrier will provide a potential therapeutic approach to treat opiate addiction, protect neurotoxicity and synaptic density degeneration.

Expression of the retinoic acid catabolic enzyme CYP26B1 in the human brain to maintain signaling homeostasis

Funding was provided by the Wellcome Trust grant WT081633MA-NCE and Biological Sciences Research Council Grant BB/K001043/1. Prof Fragoso is the recipient of a Post Doctoral Science without Borders grant from the Brazilian National Council for Scientific and Technological Development (CNPq, 237450/2012-7).

Phytochemical-loaded mesoporous silica nanoparticles for nose-to-brain olfactory drug delivery

Central nervous system (CNS) drug delivery is often hampered due to the insidious nature of the blood-brain barrier (BBB). Nose-to-brain delivery via olfactory pathways have become a target of attention for drug delivery due to bypassing of the BBB. The antioxidant properties of phytochemicals make them promising as CNS active agents but possess poor water solubility and limited BBB penetration. The primary aim of this study was the development of mesoporous silica nanoparticles (MSNs) loaded with the poorly water-soluble phytochemicals curcumin and chrysin which could be utilised for nose-to-brain delivery. We formulated spherical MSNP using a templating approach resulting in ∼220nm particles with a high surface porosity. Curcumin and chrysin were successfully loaded into MSNP and confirmed through Fourier transformation infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and HPLC approaches with a loading of 11-14% for curcumin and chrysin. Release was pH dependant with curcumin demonstrating increased chemical stability at a lower pH (5.5) with a release of 53.2%±2.2% over 24h and 9.4±0.6% for chrysin. MSNP were demonstrated to be non-toxic to olfactory neuroblastoma cells OBGF400, with chrysin (100μM) demonstrating a decrease in cell viability to 58.2±8.5% and curcumin an IC50 of 33±0.18μM. Furthermore confocal microscopy demonstrated nanoparticles of <500nm were able to accumulate within cells with FITC-loaded MSNP showing membrane localised and cytoplasmic accumulation following a 2h incubation. MSNP are useful carriers for poorly soluble phytochemicals and provide a novel vehicle to target and deliver drugs into the CNS and bypass the BBB through olfactory drug delivery.

Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Regulates Dendritic Cells and Myeloid Derived Suppressor Cells Development in the Lymphoma Microenvironment

Calcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies.

We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.

In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.

Dynamic classifiers for neonatal brain monitoring

Brain injury due to lack of oxygen or impaired blood flow around the time of birth, may cause long term neurological dysfunction or death in severe cases. The treatments need to be initiated as soon as possible and tailored according to the nature of the injury to achieve best outcomes. The Electroencephalogram (EEG) currently provides the best insight into neurological activities. However, its interpretation presents formidable challenge for the neurophsiologists. Moreover, such expertise is not widely available particularly around the clock in a typical busy Neonatal Intensive Care Unit (NICU). Therefore, an automated computerized system for detecting and grading the severity of brain injuries could be of great help for medical staff to diagnose and then initiate on-time treatments. In this study, automated systems for detection of neonatal seizures and grading the severity of Hypoxic-Ischemic Encephalopathy (HIE) using EEG and Heart Rate (HR) signals are presented. It is well known that there is a lot of contextual and temporal information present in the EEG and HR signals if examined at longer time scale. The systems developed in the past, exploited this information either at very early stage of the system without any intelligent block or at very later stage where presence of such information is much reduced. This work has particularly focused on the development of a system that can incorporate the contextual information at the middle (classifier) level. This is achieved by using dynamic classifiers that are able to process the sequences of feature vectors rather than only one feature vector at a time.

Market-oriented new product development of health promoting foods for the ageing population

Major factors influencing food development and food marketing strategies in global market places at present can be attributable to the changing age structure of the population. The significant shifts in global age structure will inevitably lead to the number of people aged 60 reaching an all-time high of one billion by the year 2020. The rapidly growing population of ageing people globally represents a large, neglected and very much under-developed category within the Food Industry. The primary focus of this study was the integration of knowledge creation techniques at early NPD stages, for the development of market-oriented new health promoting foods for the ageing population. The methodology of this study was centered on an exploratory sequential mixed methods strategy. Stage one of the study involved in-depth semi-structured interviews with 16 Stakeholders to facilitate the need identification stage of the NPD process. The main outputs identified were the need for: the fortification of foods for a preventative nutrition approach, the development of foods that targeted age-related conditions such as cognitive, heart, gut and bone health, the integration of ageing compensatory packaging adaptations and the creation of marketing messages with an active lifestyle message. Stage two consisted of a market-oriented computer assisted NPD technique, a user centered design interaction (UCD) to integrate consumers as co-creators throughout the idea generation stage of the NPD process. The most important product attributes identified in this stage included: products targeted at brain and cognitive health, liquid based beverages, easy to use packaging with environmentally friendly elements, simplistic marketing with a clear focus on health not age and realistic health claims constructed with consumer friendly terminology. Finally, Stage three used an abbreviated means-end chain (MEC) analysis to complete the concept development stage of the NPD process. This stage identified commercial information that could be used by food firms for the development of positioning and communication strategies. Equally, the information generated could be of high strategic importance to governments, policy makers, health professionals and medical professionals. The values and goals listed in this stage included: better overall health, active lifestyle, optimum nutrition and wellbeing feelings. Overall, this research illustrated that knowledge creation techniques can assist firms in the development of market-oriented health promoting foods for the ageing population.

Classifying Child Maltreatment by Brain Imaging Data

Thesis (Master's)--University of Washington, 2016-08

Synthetic Polymer and Microbubble Systems for Gene Delivery to the Brain

Thesis (Ph.D.)--University of Washington, 2016-08

New tools for cognitive and motor rehabilitation: development and clinical validation

Nervous system disorders are associated with cognitive and motor deficits, and are responsible for the highest disability rates and global burden of disease. Their recovery paths are vulnerable and dependent on the effective combination of plastic brain tissue properties, with complex, lengthy and expensive neurorehabilitation programs. This work explores two lines of research, envisioning sustainable solutions to improve treatment of cognitive and motor deficits. Both projects were developed in parallel and shared a new sensible approach, where low-cost technologies were integrated with common clinical operative procedures. The aim was to achieve more intensive treatments under specialized monitoring, improve clinical decision-making and increase access to healthcare. The first project (articles I – III) concerned the development and evaluation of a web-based cognitive training platform (COGWEB), suitable for intensive use, either at home or at institutions, and across a wide spectrum of ages and diseases that impair cognitive functioning. It was tested for usability in a memory clinic setting and implemented in a collaborative network, comprising 41 centers and 60 professionals. An adherence and intensity study revealed a compliance of 82.8% at six months and an average of six hours/week of continued online cognitive training activities. The second project (articles IV – VI) was designed to create and validate an intelligent rehabilitation device to administer proprioceptive stimuli on the hemiparetic side of stroke patients while performing ambulatory movement characterization (SWORD). Targeted vibratory stimulation was found to be well tolerated and an automatic motor characterization system retrieved results comparable to the first items of the Wolf Motor Function Test. The global system was tested in a randomized placebo controlled trial to assess its impact on a common motor rehabilitation task in a relevant clinical environment (early post-stroke). The number of correct movements on a hand-to-mouth task was increased by an average of 7.2/minute while the probability to perform an error decreased from 1:3 to 1:9. Neurorehabilitation and neuroplasticity are shifting to more neuroscience driven approaches. Simultaneously, their final utility for patients and society is largely dependent on the development of more effective technologies that facilitate the dissemination of knowledge produced during the process. The results attained through this work represent a step forward in that direction. Their impact on the quality of rehabilitation services and public health is discussed according to clinical, technological and organizational perspectives. Such a process of thinking and oriented speculation has led to the debate of subsequent hypotheses, already being explored in novel research paths.

Effects of unilateral visual deprivation on the developing avian brain

The effect of varying states of visual deprivation on the development of the optic lobes and cerebral hemispheres has been studied in the chick where the visual pathways are totally crossed over. Unilateral eye extirpation of the new hatched chick resulted in arrested development of the contralateral but not ipsilateral lobe, as measured by weight, protein content and acetylcholinesterase activity. Similar effects but of smaller magnitude were observed in the cerebral hemispheres. Histologic and enzymic evidence revealed the absence of significant degeneration in the optic lobe contralateral to an eye removed 17 days previously. These results were observed in the optic lobes of operated animals maintained either in light of in darkness between 3 and 17 days after hatch. However, in the inpaired cerebral hemispheres, differences could only be detected in birds kept the light. The effects of unilateral eyelid suturing on the development of chick brain regions were also examined. In this group, all asymmetrical differences observed within paired brain regions were totally light dependent and confined to the cerebral hemispheres. The hemisphere contralateral to the sutured eye weighed less and had less acetylcholinesterase activity than its paired hemisphere. The cerebral hemispheres of monocularly treated birds manifested effects of similar magnitude whether the treatment was enucleation or suturing. This suggests that the complete development of the associative centers in avian cerebral hemispheres is dependent on both intact innervation and on the information content of the visual input. © 1969.

Development of radiotracers for nuclear imaging of poly(ADP-ribose) polymerase-1 and the translocator protein in glioblastoma.

Glioblastoma (GBM) is a highly aggressive and fatal brain cancer that is associated with a number of diagnostic, therapeutic, and treatment monitoring challenges. At the time of writing, inhibition of a protein called poly (ADP-ribose) polymerase-1 (PARP-1) in combination with chemotherapy was being investigated as a novel approach for the treatment of these tumours. However, human studies have encountered toxicity problems due to sub-optimal PARP-1 inhibitor and chemotherapeutic dosing regiments. Nuclear imaging of PARP-1 could help to address these issues and provide additional insight into potential PARP-1 inhibitor resistance mechanisms. Furthermore, nuclear imaging of the translocator protein (TSPO) could be used to improve GBM diagnosis, pre-surgical planning, and treatment monitoring as TSPO is overexpressed by GBM lesions in good contrast to surrounding brain tissue. To date, relatively few nuclear imaging radiotracers have been discovered for PARP-1. On the other hand, numerous tracers exist for TSPO many of which have been investigated in humans. However, these TSPO radiotracers suffer from either poor pharmacokinetic properties or high sensitivity to human TSPO polymorphism that can affect their binding to TSPO. Bearing in mind the above and the high attrition rates associated with advancement of radiotracers to the clinic, there is a need for novel radiotracers that can be used to image PARP-1 and TSPO. This thesis reports the pre-clinical discovery programme that led to the identification of two potent PARP-1 inhibitors, 4 and 17, that were successfully radiolabelled to generate the potential SPECT and PET imaging agents [123I]-4 and [18F]-17 respectively. Evaluation of these radiotracers in mice bearing subcutaneous human GBM xenografts using ex vivo biodistribution techniques revealed that the agents were retained in tumour tissue due to specific PARP-1 binding. This thesis also describes the pre-clinical in vivo evaluation of [18F]-AB5186, which is a novel radiotracer discovered previously within the research group with potential for PET imaging of TSPO. Using ex vivo autoradiography and PET imaging the agent was revealed to accumulate in intracranial human GBM tumour xenografts in good contrast to surrounding brain tissue, which was due to specific binding to TSPO. The in vivo data for all three radiolabelled compounds warrants further pre-clinical investigations with potential for clinical advancement in mind.