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Engineered Exosomes For Delivery Of Therapeutic Sirnas To Neurons, Reka A. Haraszti May 2018

Engineered Exosomes For Delivery Of Therapeutic Sirnas To Neurons, Reka A. Haraszti

GSBS Dissertations and Theses

Extracellular vesicles (EVs), exosomes and microvesicles, transfer endogenous RNAs between neurons over short and long distances. We have explored EVs for siRNA delivery to brain. (1) We optimized siRNA chemical modifications and siRNA conjugation to lipids for EV-mediated delivery. (2) We developed a GMP-compatible, scalable method to manufacture active EVs in bulk. (3) We characterized lipid and protein content of EVs in detail. (4) We established how protein and lipid composition relates to siRNA delivering activity of EVs, and we reverse engineered natural exosomes (small EVs) into artificial exosomes based on these data.

We established that cholesterol-conjugated siRNAs passively associate ...


Dynamic Regulation At The Neuronal Plasma Membrane: Novel Endocytic Mechanisms Control Anesthetic-Activated Potassium Channels And Amphetamine-Sensitive Dopamine Transporters: A Dissertation, Luke R. Gabriel Jun 2013

Dynamic Regulation At The Neuronal Plasma Membrane: Novel Endocytic Mechanisms Control Anesthetic-Activated Potassium Channels And Amphetamine-Sensitive Dopamine Transporters: A Dissertation, Luke R. Gabriel

GSBS Dissertations and Theses

Endocytic trafficking dynamically regulates neuronal plasma membrane protein presentation and activity, and plays a central role in excitability and plasticity. Over the course of my dissertation research I investigated endocytic mechanisms regulating two neuronal membrane proteins: the anesthetic-activated potassium leak channel, KCNK3, as well as the psychostimulant-sensitive dopamine transporter (DAT). My results indicate that KCNK3 internalizes in response to Protein Kinase C (PKC) activation, using a novel pathway that requires the phosphoserine binding protein, 14-3-3β, and demonstrates for the first time regulated KCNK3 channel trafficking in neurons. Additionally, PKC-mediated KCNK3 trafficking requires a non-canonical endocytic motif, which is shared exclusively ...


Transposition Driven Genomic Heterogeneity In The Drosophila Brain: A Dissertation, Paola N. Perrat Jun 2012

Transposition Driven Genomic Heterogeneity In The Drosophila Brain: A Dissertation, Paola N. Perrat

GSBS Dissertations and Theses

In the Drosophila brain, memories are processed and stored in two mirrorsymmetrical structures composed of approximately 5,000 neurons called Mushroom Bodies (MB). Depending on their axonal extensions, neurons in the MB can be further classified into three different subgroups: αβ, α’β’ and γ. In addition to the morphological differences between these groups of neurons, there is evidence of functional differences too. For example, it has been previously shown that while neurotransmission from α’β’ neurons is required for consolidation of olfactory memory, output from αβ neurons is required for its later retrieval. To gain insight into the functional ...


Cellular And Molecular Mechanisms Driving Glial Engulfment Of Degenerating Axons: A Dissertation, Johnna E. Doherty Nov 2011

Cellular And Molecular Mechanisms Driving Glial Engulfment Of Degenerating Axons: A Dissertation, Johnna E. Doherty

GSBS Dissertations and Theses

The nervous system is made up of two major cell types, neurons and glia. The major distinguishing feature between neuronal cells and glial cells is that neurons are capable of transmitting action potentials while glial cells are electrically incompetent. For over a century glial cells were neglected and it was thought they existed merely to provide trophic and structural support to neurons. However, in the past few decades it has become increasingly clear that glial cell functions underlie almost all aspects of nervous system development, maintenance, and health. During development, glia act as permissive substrates for axons, provide guidance cues ...


Neuronal Diversification In The Postembryonic Drosophila Brain: A Dissertation, Suewei Lin Aug 2011

Neuronal Diversification In The Postembryonic Drosophila Brain: A Dissertation, Suewei Lin

GSBS Dissertations and Theses

A functional central nervous system (CNS) is composed of numerous types of neurons. Neurons are derived from a limited number of multipotent neural stem cells. Previous studies have suggested three major strategies nature uses to diversify neurons: lineage identity specification that gives an individual neural stem cell distinct identity based on its position in the developing CNS; temporal identity specification that gives neurons derived from a neural stem cell distinct identities based on their birth-order within the lineage; and binary cell fate specification that gives different identities to the two sister postmitotic neurons derived from the terminal division of a ...


Identification Of A Command Neuron Directing The Expression Of Feeding Behavior In Drosophila Melanogaster: A Dissertation, Thomas F. Flood May 2011

Identification Of A Command Neuron Directing The Expression Of Feeding Behavior In Drosophila Melanogaster: A Dissertation, Thomas F. Flood

GSBS Dissertations and Theses

Feeding is one of the most important behaviors for an animal’s survival. At a gross level, it is known that the nervous system plays a major role in the expression of this complex behavior, yet a detailed understanding of the neural circuits directing feeding behavior remains unknown. Here we identify a command neuron in Drosophila melanogaster whose artificial activation, using dTrpA1, a heat-activated cation channel, induces the appearance of complete feeding behavior. We use behavioral, genetic, cellular and optical imaging techniques to show that the induced behavior is composed of multiple motor programs and can function to uptake exogenous ...


Systems Level Processing Of Memory In The Fly Brain: A Dissertation, Michael Jonathan Krashes May 2009

Systems Level Processing Of Memory In The Fly Brain: A Dissertation, Michael Jonathan Krashes

GSBS Dissertations and Theses

Understanding the mechanisms of memory is vital in making sense of the continuity of the self, our experience of time and of the relation between mind and body. The invertebrate Drosophila melanogaster offers us an opportunity to study and comprehend the overwhelming complexity of memory on a smaller scale. The work presented here investigates the neural circuitry in the fly brain required for olfactory memory processing. Our observation that Dorsal Paired Medial (DPM) neurons, which project only to mushroom body (MB) neurons, are required during memory storage but not for acquisition or retrieval, led us to revisit the role of ...


Ethanol Sensitivity And Tolerance Of Rat Neuronal Bk Channels: A Dissertation, Patricia M. Wynne Dec 2008

Ethanol Sensitivity And Tolerance Of Rat Neuronal Bk Channels: A Dissertation, Patricia M. Wynne

GSBS Dissertations and Theses

BK channels are well studied targets of acute ethanol action. They play a prominent role in neuronal excitability and have been shown to play a significant role in behavioral ethanol tolerance in invertebrates. The focus of my work centers on the effects of alcohol on the BK channel and comprises studies that examine how subcellular location affects acute ethanol sensitivity and how duration of acute alcohol exposure impacts the development of rapid tolerance. My results also provide potential mechanisms which underlie acute sensitivity and rapid tolerance.

I first explore BK channel sensitivity to ethanol in the three compartments (dendrite, cell ...


Vps45p As A Model System For Elucidation Of Sec1/Munc18 Protein Function: A Dissertation, Melonnie Lynn Marie Furgason Dec 2008

Vps45p As A Model System For Elucidation Of Sec1/Munc18 Protein Function: A Dissertation, Melonnie Lynn Marie Furgason

GSBS Dissertations and Theses

Vesicular trafficking, the movement of vesicles between organelles and the plasma membrane for secretion, consists of multiple highly regulated processes. Many protein families function as specificity and regulatory determinants to ensure correct vesicle targeting and timing of trafficking events. The SNARE proteins dock and fuse vesicles to their target membranes. Sec1/Munc18 (SM) proteins regulate membrane fusion through interactions with the SNAREs—SM proteins have been shown to act as both inhibitors and stimulators of SNARE assembly and membrane fusion. However, the details of these SM protein functions are not understood.

Constructing a model of SM protein function has been ...


The Molecular Mechanisms Underlying The Polarized Distribution Of Drosophila Dscam In Neurons: A Dissertation, Shun-Jen Yang Oct 2008

The Molecular Mechanisms Underlying The Polarized Distribution Of Drosophila Dscam In Neurons: A Dissertation, Shun-Jen Yang

GSBS Dissertations and Theses

Neurons exhibit highly polarized structures, including two morphologically and functionally distinct domains, axons and dendrites. Dendrites and axons receive versus send information, and proper execution of each requires different sets of molecules. Differential distribution of membrane proteins in distinct neuronal compartments plays essential roles in neuronal functions. The major goal of my doctoral thesis was to study the molecular mechanisms that govern the differential distribution of membrane proteins in neurons, using the Drosophilalarval mushroom body (MB) as a model system.

My work was initiated by an observation of differential distribution of distinct Dscam isoforms in neurons. Dscam stands for ...


Analysis Of Cpeb Family Protein Member Cpeb4 Function In Mammalian Neurons: A Dissertation, Ming-Chung Kan Jun 2008

Analysis Of Cpeb Family Protein Member Cpeb4 Function In Mammalian Neurons: A Dissertation, Ming-Chung Kan

GSBS Dissertations and Theses

Local protein synthesis is required for long-term memory formation in the brain. One protein family, Cytoplasmic Polyadenylation Element binding Protein (CPEB) that regulates protein synthesis is found to be important for long-term memory formation possibly through regulating local protein synthesis in neurons. The well-studied member of this family, CPEB1, mediates both translational repression and activation of its target mRNAs by regulating mRNA polyadenylation. Mouse with CPEB1 KO shows defect in memory extinction but not long-term memory formation. Three more CPEB1 homologs (CPEB2-4) are identified in mammalian system. To test if CPEB2-4 may have redundant role in replacing CPEB1 in mediating ...


Modulation Of Neuropeptide Release Via Voltage-Dependent And -Independent Signaling In Isolated Neurohypophysial Terminals: A Dissertation, Cristina M. Velazquez-Marrero Apr 2008

Modulation Of Neuropeptide Release Via Voltage-Dependent And -Independent Signaling In Isolated Neurohypophysial Terminals: A Dissertation, Cristina M. Velazquez-Marrero

GSBS Dissertations and Theses

This thesis details my examination of several mechanisms for modulation of neuropeptide release via voltage-dependent and voltage-independent intraterminal signaling in isolated neurohypophysial terminals. The first part of this work characterizes depolarization-induced neuropeptide release in the absence of extracellular calcium. The goal of this project was to examine the relationship between depolarization-induced release of intracellular calcium stores and depolarization-secretion coupling of neuropeptides. We demonstrate that depolarization in the absence of extracellular calcium induced by either High K+ or electrical stimulation induces a rise in [Ca2+]i and subsequent neuropeptide release from Hypothalamic Neurohypophysial System (HNS) terminals. A portion of extracellular ...


Molecular And Neuronal Analysis Of Circadian Photoresponses In Drosophila: A Dissertation, Alejandro D. Murad Oct 2007

Molecular And Neuronal Analysis Of Circadian Photoresponses In Drosophila: A Dissertation, Alejandro D. Murad

GSBS Dissertations and Theses

Most organisms, from cyanobacteria to humans are equipped with circadian clocks. These endogenous and self-sustained pacemakers allow organisms to adapt their physiology and behavior to daily environmental variations, and to anticipate them. The circadian clock is synchronized by environmental cues (i.e. light and temperature fluctuations).

The fruit fly, Drosophila melanogaster, is well established as a model for the study of circadian rhythms. Molecular mechanisms of the Drosophilacircadian clock are conserved in mammals. Using genetic screens, several essential clock proteins (PER, TIM, CLK, CYC, DBT, SGG and CK-II) were identified in flies. Homologs of most of these proteins are ...


Neural Diversity In The Drosophila Olfactory Circuitry: A Dissertation, Sen-Lin Lai Jul 2007

Neural Diversity In The Drosophila Olfactory Circuitry: A Dissertation, Sen-Lin Lai

GSBS Dissertations and Theses

Different neurons and glial cells in the Drosophila olfactory circuitry have distinct functions in olfaction. The mechanisms to generate most of diverse neurons and glial cells in the olfactory circuitry remain unclear due to the incomprehensive study of cell lineages. To facilitate the analyses of cell lineages and neural diversity, two independent binary transcription systems were introduced into Drosophila to drive two different transgenes in different cells. A technique called ‘dual-expression-control MARCM’ (mosaic analysis with a repressible cell marker) was created by incorporating a GAL80-suppresible transcription factor LexA::GAD (GAL4 activation domain) into the MARCM. This technique allows the induction ...


Genetic Dissection Of The Neural Circuitry Underlying Memory Stability In Drosophila: A Dissertation, Alex Carl Keene Aug 2006

Genetic Dissection Of The Neural Circuitry Underlying Memory Stability In Drosophila: A Dissertation, Alex Carl Keene

GSBS Dissertations and Theses

Understanding how memory is formed requires looking beyond the genes involved to the neural circuitry and temporal aspects of memory. In this dissertation I have focused my investigation on Dorsal Paired Medial (DPM) neurons, two modulatory neurons essential for memory in Drosophila. DPM neurons highly express the amnesiac (amn) gene, which encodes for a putative pre-pro-neuropeptide. amn function in DPM neurons is required for memory. Here I provide evidence that DPM neurons are cholinergic and that acetylcholine (ACh) and AMN act as co-transmitters essential for DPM function. In order to investigate the temporal requirements of DPM output I blocked transmitter ...


The Regulation Of Nnos During Neuronal Differentiation And The Effect Of Nitric Oxide On Hdm2-P53 Binding: A Dissertation, Christopher M. Schonhoff Dec 2000

The Regulation Of Nnos During Neuronal Differentiation And The Effect Of Nitric Oxide On Hdm2-P53 Binding: A Dissertation, Christopher M. Schonhoff

GSBS Dissertations and Theses

Nitric oxide is a ubiquitous signaling molecule with both physiological and pathological functions in biological systems. Formed by the enzymatic conversion of arginine to citrulline, NO, has known roles in circulatory, immune and nervous tissues. In the nervous system nitric oxide has been implicated in long-term potentiation, neurotransmitter release, channel function, neuronal protection and neuronal degeneration. Much of our work has focused on yet another role for nitric oxide in cells, namely, neuronal differentiation.

During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model ...


Neuroprotection And Neurotransplantation Strategies In Models Of Parkinson’S Disease, Wendy R. Galpern May 1996

Neuroprotection And Neurotransplantation Strategies In Models Of Parkinson’S Disease, Wendy R. Galpern

GSBS Dissertations and Theses

Parkinson's disease (PD) is a neurodegenerative disorder characterized by dopaminergic cell death in the substantia nigra pars compacta (SNc) and dopamine (DA) depletion in the striatum. Current pharmacological treatments are aimed at the replacement of striatal DA via the administration of levodopa. While this therapy is beneficial initially, long-term treatment is associated with significant side effects, and disease progression continues. The present experiments investigate neuroprotective and neurotransplantation strategies as alternatives to palliative pharmacologic treatments.

The optimal therapeutic approach to neurodegenerative diseases would be to protect against cell death and prevent disease progression. PD is well-suited for such neuroprotective strategies ...


Myc And E1a Oncogenes Alter The Response Of Pc12 Cells To Nerve Growth Factor And Block Differentiation: A Thesis, Susan C. Schiavi Aug 1988

Myc And E1a Oncogenes Alter The Response Of Pc12 Cells To Nerve Growth Factor And Block Differentiation: A Thesis, Susan C. Schiavi

GSBS Dissertations and Theses

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc and E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors, activation of ribosomal S6 kinase, and ornithine decarboxylase induction were similar in myc and E1A expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. The ...