<p>There has been a rapid shift in food environment of Western cultures that has increased consumption of diets high in fat and sugar, which have imparted negative effects on metabolic and neurocognitive health. There is also building evidence that the adverse effects of Western diet</p> <p>(WD) are different in males and females, such that males are impacted more at an earlier age and females are impacted later in life. The underlying biological mechanisms linking WD and neurocognitive health are often associated with energy dysregulation or neuroinflammation. WD</p> <p>disrupts glucose homeostasis and causes low grade inflammation in the body, and these can impact</p> <p>the brain by disrupting the blood-brain barrier (BBB). The BBB is the microvasculature found throughout the entire brain that tightly regulates what compounds get into the brain to ensure optimal neuronal function. WD disrupts the BBB, however, the effects of WD on BBB integrity</p> <p>in females and younger individuals remain largely unknown. Based on the metabolic and behavioral effects of WD, we hypothesized that the effects are age- and sex- specific. To test this, we gave male and female rats access to a WD for 8-10 weeks starting in juvenile period (post-natal</p> <p>day 21) or in adulthood (post-natal day 75), then measured body weight, behavior, glucose tolerance, the density of two different markers of BBB integrity. We also measured density of resident immune cells (microglia) to assess the relationship between inflammation and BBB integrity. First, we focused on the impact of hyperglycemia on the BBB since elevated glucose alters glucose transporter 1 (GLUT1). We found sex- and age- specific decreases in GLUT1 density in the prefrontal cortex and hippocampus—two brain regions commonly associated with neurocognitive impairments associated with WD. Correlational comparisons between WD and chow (CH) animals also found that the typically relationship between glucose tolerance and</p> <p>GLUT1 in the PFC and hippocampus were overall disrupted in WD animals. Second, we measured the leakage of albumin, a blood protein, since WD depletes the tight junctions that would typically prevent albumin from entering the brain and triggering a neuroinflammatory response. We did not find an increase in albumin density in WD animals, however, we found a main effect of age which</p> <p>offers insight to differential susceptibilities to BBB leakage. Third, we focused on inflammation and found that WD did not impact microglia density in our experiments, nor did it correlate with GLUT1, albumin, or behavior. Collectively, our findings support the hypothesis that the impact of</p> <p>WD on the BBB is sex- and age- specific, suggest that WD does not increase leakage of compounds such as albumin, and highlights the nuanced relationships between WD, metabolic disruption, behavioral deficits, and neuroinflammation.  </p>

Small extracellular vesicles (small EVs) are endogenous membrane-enclosed nanocarriers released from essentially all cells. They have been shown to carry proteins, lipids, nucleic acids to transmit biological signals throughout the body, including to the brain. Some evidence has suggested that small EVs can cross the blood-brain barrier (BBB), moving from the peripheral circulation to the central nervous system (CNS). The BBB is a dynamic barrier that regulates molecular trafficking between the peripheral circulation and the CNS. As a result, small EVs have attracted attention for their potential as a novel delivery platform for nucleic acid-based therapeutics across the BBB. Silencing RNAs (siRNAs) are a potent drug class but using “naked” siRNA is not feasible due to their short half-life, vulnerability to degradation and low penetration in cells. Despite the excitement for the development of small EV-based therapeutics, their clinical development is hampered by the lack of reliable methods for packing therapeutics into them. Reshke et al. has shown that cells can be genetically engineered to produce customizable small EVs packaged with siRNA against any protein by integrating the siRNA sequence into the pre- miR-451 structure. Mounting evidence has established that in a misfolded state, α-synuclein becomes insoluble and phosphorylated to form intracellular inclusions in neurons (known as Lewy bodies) which leads to Parkinson’s disease (PD) pathogenesis. Given that increased α-synuclein expression causes familial and idiopathic PD, decreasing its synthesis by using siRNA is an attractive therapeutic strategy. Here, we genetically engineered cells to produce small EVs packaged with siRNA against α-synuclein integrated in the pre-miR451 backbone, tested their ability to cross an in vitro BBB, and deliver its cargo to silence endogenous α-synuclein in neuron- like cells. The therapeutic potential of α-synuclein siRNA delivery by these small EVs was demonstrated by the strong mRNA (60-70%) and protein knockdown (43%) of α-synuclein in neuron-like cells. We also demonstrated that approximately at 4% and 2%, respectively of small EVs-derived from human brain endothelial cells (hCMEC/D3) and human embryonic kidney (HEK293T) were transported cross the in vitro BBB model. Interestingly, we observed that small EVs-derived from HEK293T deliver their cargo to induced brain endothelial cells (iBECs) (~74% α-synuclein mRNA reduction) but their rate of transport across BBB was lower and did not reduce α-synuclein mRNA expression in neuron-like cells, seeded on the far side of the BBB. Small EVs- derived from hCMEC/D3 reduced α-synuclein mRNA (40%) in neuron-like cells across the BBB model. This finding suggests that small EVs derived from different cell sources can undergo different intracellular trafficking routes, providing various opportunities to influence the efficiency of delivery and fate of intracellular cargo. Using small EVs-derived from hCMEC/D3, two different routes of administration, a single bolus intravenous (IV) or intra-carotid (ICD) injection, showed small EVs largely accumulated in the liver, spleen, small intestines and kidneys; and only a small amount of small EVs were detected in the brain. These results indicate that human brain endothelial cells may serve as a promising cell source for CNS treatments based on small EVs.

This thesis project serves to fill experimental gaps needed to advance the goal of performing pre-clinical trials using an orthotopic rat glioblastoma model to evaluate the efficacy of high-frequency electroporation (H-FIRE) and QUAD-CTX tumor receptor-targeted cytotoxic conjugate therapies, individually and in combination, in selectively and thoroughly treating glioblastoma multiforme. In order to achieve this, an appropriate model must be developed and characterized. I have transduced F98 rat glioma cells to express red-shifted firefly luciferase, which will facilitate longitudinal tumor monitoring in vivo through bioluminescent imaging. I have characterized their response to H-FIRE relative to DI TNC1 rat astrocytes. I have demonstrated the presence of the molecular targets of QUAD in F98 cells. The in vitro characterization of this model has enabled preclinical studies of this promising glioblastoma therapy in an immunocompetent rat model, an important step before advancing ultimately to clinical human trials. / Master of Science / Treating glioblastoma multiforme (GBM), a form of cancer found in the brain, has not been very successful; patients rarely live two years following diagnosis, and there have been no major breakthrough advances in treatment to improve this outlook for decades. We have been working on two treatments which we hope to combine. The first is high-frequency electroporation (H-FIRE), which uses electrical pulses to kill GBM cells while leaving healthy cells alive and blood vessels intact. The second is QUAD-CTX, which combines a toxin with two types of protein that attach to other proteins that are more common on the surface of GBM cells than healthy cells. We have shown these to be effective at disproportionately killing human GBM cells growing in a lab setting. Before H-FIRE and QUAD-CTX may be tested on humans, we need to show them to be effective in an animal model, specifically rats. I have chosen rat glioma cells that will behave similarly to human GBM and a rat species that will not have an immune response to them. I have made these cells bioluminescent so that we may monitor the tumors as they grow and respond to our treatments. I have also shown that QUAD-CTX kills these rat glioma cells, as does H-FIRE. Because of this work, we are ready to begin testing these two treatments in rats.

<p>Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some <em>in vitro</em> approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different <em>in vitro</em> systems that could help better mimic certain physiological systems that tend to not be mimicked by <em>in vivo</em> models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction.  Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in <em>in vivo</em> models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using <em>in vivo</em> models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.  </p>

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[ES] El tratamiento de enfermedades neurológicas está muy limitado por la ineficiente penetración de los fármacos en el tejido cerebral dañado debido a la barrera hematoencefálica (BHE), lo que imposibilita mejorar la salud del paciente. La BHE es un mecanismo de protección natural para evitar la difusión de agentes potencialmente peligrosas para el sistema nervioso central. No obstante, la BHE se puede inhibir mediante ultrasonidos focalizados e inyección de microburbujas de forma segura, localizada y transitoria, una tecnología empleada mundialmente. La principal ventaja es su carácter no invasivo, siendo así muy atractiva y cómoda para el paciente. Normalmente, la zona cerebral enferma se trata en su parte central empleando un único foco. Sin embargo, enfermedades como el Alzheimer o el Parkinson requieren un tratamiento sobre estructuras de geometría compleja y tamaño elevado, situadas en ambos hemisferios cerebrales. Por tanto, la tecnología actual está muy limitada al no cumplir dichos requisitos. Esta tesis doctoral tiene como objetivo el desarrollo de una técnica novedosa, basada en hologramas acústicos, para resolver las limitaciones presentes en los tratamientos neurológicos empleando ultrasonidos. Se estudian las lentes acústicas holográficas impresas en 3D, que acopladas a un transductor mono-elemento, permiten el control preciso del frente de onda ultrasónico tanto para (1) compensar las distorsiones que sufre el haz hasta alcanzar el cerebro, como (2) focalizarlo simultáneamente en regiones múltiples y de geometría compleja o formando de vórtices acústicos, proporcionando así efectividad en tiempo y coste. Por ello, la investigación desarrollada en esta tesis abre un camino prometedor en el campo de la biomedicina que permitirá mejorar los tratamientos neurológicos, además de aplicaciones en neuroestimulación o ablación térmica del tejido. / [CA] El tractament de malalties neurològiques està molt limitat per la ineficient penetració del fàrmac en el teixit cerebral danyat a causa de la barrera hematoencefàlica (BHE), i així no és possible una millora de salut del pacient. La BHE és un mecanisme de protecció natural per a evitar la difusió d'agents potencialment perillosos per al Sistema Nervios Central. No obstant això, aquesta barrera es pot inhibir mitjancant una tecnologia emprada mundialment basada en ultrasons focalitzats i injeccio de microbombolles. El principal avantatge és el seu caràcter no invasiu, sent així molt atractiva i còmoda per al pacient, i permet obrir la BHE de manera segura, localitzada i transitòria. Normalment, la zona cerebral malalta es tracta en la seua part central, emprant un unic focus. No obstant això, malalties com l'Alzheimer o el Parkinson requereixen un tractament al llarg d'estructures de geometria complexa i grandària elevada, situades en tots dos hemisferis cerebrals. Per tant, la tecnologia actual està fortament limitada al no complir amb aquests requeriments. Aquesta tesi doctoral està enfocada a investigar i desenvolupar una tècnica nova, basada en hologrames acústics, per a solucionar les limitacions presents en els tractaments neurològics. Una lent acústica holograca de baix cost impresa en 3D acoblada a un transductor d'element simple permet el control precs del front d'ona ultrasònic punt per a (1) compensar les distorsions que pateix el feix en el seu camí cap al cervell, i (2) focalització simultània del feix en regions multiples i de geometria complexa, proporcionant aix un tractament efectiu en temps i cost. Per això, la investigació desenvolupada en aquesta tesi demostra la possibilitat de realitzar qualsevol tractament neurològic, a més d'aplicacions en la neuroestimulació o l'ablació tèrmica dins del camp biomèdic. / [EN] Treatments for neurological diseases are strongly limited by the inefficient penetration of therapeutic drugs into the diseased brain due to the blood-brain barrier (BBB), and therefore no health improvement can be achieved. In fact, the BBB is a protection mechanism of the human body to avoid the diffusion of potentially dangerous agents into the central nervous system. Nevertheless, this barrier can be successfully inhibited by using a worldwide spread technology based on microbubble-enhanced focused ultrasound. Its main advantage is its non-invasive nature, thus defining a patient-friendly clinical procedure that allows to disrupt the BBB in a safe, local and transient manner. Conventionally, the diseased brain structure has been targeted in its center, with a single focus. However, Alzheimer's or Parkinson's Diseases do require that ultrasound is delivered to entire, complex-geometry and large-volume structures located at both hemispheres of the brain. Therefore, current technology presents several limitations as it does not fulfill these requirements. This doctoral thesis aims to develop a novel technique based on using focused ultrasound acoustic holograms to solve the existing limitations to treat neurological diseases. In this dissertation, we study 3D-printed holographic acoustic lenses coupled to a single-element transducer that allow to accurately control the acoustic wavefront to both (1) compensate distortions suffered by the beam in its path to the brain, and (2) simultaneous focusing in multiple and complex-geometry structures or acoustic vortex generation, providing a time- and cost- efficient procedure. Therefore, the research carried out throughout this thesis opens a promising path in the biomedical field to improve the treatment for neurological diseases, neurostimulation or tissue ablation applications. / Acknowledgments to the Spanish institution Generalitat Valenciana, which funding grant allowed me to develop this doctoral thesis, and as well funded my research stay at Columbia University. The development of the entire thesis was supported through grant Nª. ACIF/2017/045. Particularly, the research carried out in Chapter 3 and Chapter 4 was possible thanks to and supported through grant BEFPI/2019/075. Action co-financied by the Agència Valenciana de la Innovació through grant INNVAL10/19/016 and by the European Union through the Programa Operativo del Fondo Europeo de Desarrollo Regional (FEDER) of the Comunitat Valenciana 2014-2020 (IDIFEDER/2018/022). / Jiménez Gambín, S. (2021). Transcranial Ultrasound Holograms for the Blood-Brain Barrier Opening [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171373

Glioblastoma (GBM) is the most common and deadliest of the malignant primary brain tumors in humans, with a reported 5-year survival rate of only 6.8% despite years of extensive research. Failure to improve local tumor control rates and overall patient outcome is attributed to GBM's inherent therapeutic resistance. Marked heterogeneity, extensive local invasion within the brain parenchyma, and profound immunosuppression within the tumor microenvironment (TME) are some of the unique features that drive GBM therapeutic resistance. Furthermore, tumor cells are sequestered behind the blood-brain barrier (BBB), limiting delivery of effective therapeutics and immune cell infiltration into the local tumor. Electroporation-based therapies, such as irreversible electroporation (IRE) and second generation, high-frequency IRE (H-FIRE) represent attractive alternative approaches to standard GBM therapy given their ability to induce transient BBB disruption (BBBD), achieve non-thermal tumor cell ablation and stimulate local and systemic anti-tumor immune responses without significant morbidity. The following work explores the use of H-FIRE to overcome GBM-induced therapeutic resistance and improve treatment success. Chapter 1 opens with an overview of GBM and known barriers to treatment success. Here, we emphasize the utility of spontaneous canine gliomas as an ideal translational model for investigations into novel treatment approaches. Chapter 2 introduces novel ablation methods (i.e. IRE/H-FIRE) capable of targeting treatment-resistant cancer stem cells. The focus of Chapter 3 is to highlight IRE applications in a variety of spontaneous tumor types. In Chapter 4, we investigate the feasibility and local immunologic response of percutaneous H-FIRE for treatment of primary liver tumors using a spontaneous canine hepatocellular carcinoma (HCC) model. In chapter 5, we characterize the mechanisms of H-FIRE-mediated BBBD in an in vivo healthy rodent model. In Chapter 6, we characterize the local and systemic immune responses to intracranial H-FIRE in rodent and canine glioma models to enhance the translational value of our work. Collectively, our work demonstrates the potential for H-FIRE to overcome therapeutic resistance in GBM, thereby supporting its use as a novel, alternative treatment approach to standard therapy. / Doctor of Philosophy / Glioblastoma (GBM) is the most common and deadliest form of primary brain cancer in humans, with only 6.8% of people surviving 5-years after their diagnosis. GBM is characterized by a number of unique features that make it resistant to standard treatments, such as surgery, radiation and chemotherapy. Examples include: (1) extensive invasion of tumor cells into the brain, making complete removal via surgery very difficult; (2) tumor cells are protected by a structure called the blood-brain barrier (BBB), which restricts the entry of most drugs (i.e. chemotherapy) and many immune cells, into the brain, thereby preventing them from reaching tumor cells; (3) tumor cells produce substances that block the immune system from being able to detect the tumor itself, which allows it to continue to grow undetected. High-frequency irreversible electroporation (H-FIRE) represents a new approach for the treatment of GBM. H-FIRE uses electric pulses to temporarily or permanently injure cell membranes without the use of heat, which allows for very precise treatment. The following work explores the ways in which H-FIRE can interfere with specific GBM features that drive its resistance to treatment. Here, we demonstrate that H-FIRE is capable of temporarily disrupting the BBB and characterize the mechanisms by which this occurs. This allows for drugs and immune cells within the blood to enter the brain and access the tumor cells, particularly those extending beyond the visible tumor mass and invading the brain. We also illustrate the potential for H-FIRE treatment within the brain to stimulate local and systemic immune responses by causing the release of proteins from injured cells. Similar to a vaccine, these proteins are recognized by the immune system, which becomes primed to help fight off cancer cells within the body. The end result is an anti-tumor immune response. Collectively, this work supports the use of H-FIRE as an alternative treatment approach to standard therapy for GBM given its potential to overcome certain causes of treatment resistance.