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Undergraduate Theses, Department of Biology, 2014-2015
β-catenin functions in the contexts of (1) cell development as a transcription factor for Wnt target genes and (2) cell-cell adhesion as an adaptor in cadherin-based adherens junctions. Mutations in β-catenin or in molecules regulating its localization and stability are associated with inappropriately triggered cell growth, weakened cell adhesion, and oncogenesis. Such correlation underscores the need to understand the molecular mechanisms by which β-catenin’s localization, and thereby its function, is regulated. An increasing body of evidence has suggested that the extent to which β-catenin partakes in either function may be influenced by phosphorylation. In particular, tyrosine phosphorylation of β-catenin has been qualitatively shown to modulate the affinities of two key binding partners at adherens junctions: cadherin and α-catenin. This study seeks to quantitatively assess the impact of tyrosine phosphorylation at specific sites on β-catenin’s affinity for cadherin and α-catenin. Through in vitro thermodynamic measurements by isothermal titration calorimetry (ITC) with β-catenin Y to E phosphomimics we show that tyrosine phosphorylation at Y142 and Y654 weakens β-catenin’s affinity for α-catenin and cadherin respectively. These results help construct a model of β-catenin regulation and may offer future therapeutic approaches for cancer.
β-catenin functions in the contexts of (1) cell development as a transcription factor for Wnt target genes and (2) cell-cell adhesion as an adaptor in cadherin-based adherens junctions. Mutations in β-catenin or in molecules regulating its localization and stability are associated with inappropriately triggered cell growth, weakened cell adhesion, and oncogenesis. Such correlation underscores the need to understand the molecular mechanisms by which β-catenin’s localization, and thereby its function, is regulated. An increasing body of evidence has suggested that the extent to which β-catenin partakes in either function may be influenced by phosphorylation. In particular, tyrosine phosphorylation of β-catenin has been qualitatively shown to modulate the affinities of two key binding partners at adherens junctions: cadherin and α-catenin. This study seeks to quantitatively assess the impact of tyrosine phosphorylation at specific sites on β-catenin’s affinity for cadherin and α-catenin. Through in vitro thermodynamic measurements by isothermal titration calorimetry (ITC) with β-catenin Y to E phosphomimics we show that tyrosine phosphorylation at Y142 and Y654 weakens β-catenin’s affinity for α-catenin and cadherin respectively. These results help construct a model of β-catenin regulation and may offer future therapeutic approaches for cancer.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
Introduction: The GABAA receptor (GABAAR) is an attractive, tractable drug discovery target. It remains unclear how native neural circuits of the hippocampus respond to drugs in this highly clinically relevant class. The CA1 region of the hippocampus is crucial for learning, memory and cognition, thus a key brain region to screen GABAergic compounds that may influence these processes. We developed a novel screen for GABAAR ligands, including general anesthetics, by measuring field inhibitory postsynaptic potentials (fIPSPs) in the CA1 area. While allowing many of the advantages of an in vitro preparation, field recordings mirror their in vivo counterparts, and unlike intracellular recordings, are minimally invasive to the neuron, typically remaining stable for many hours. Methods: 24-28 day old Sprague Dawley rats were anesthetized and decapitated. Brains were dissected and submerged in chilled artificial cerebrospinal fluid (ACSF). 400 μm thick coronal slices were cut and maintained in ACSF bubbled with 95% O2 and 5% CO2. fIPSPs were evoked through a bipolar tungsten stimulating electrode placed in the stratum pyramidale (SP) of the CA1 region and recorded by microelectrode 300-400 μm away in the SP of CA1. GABAergic fIPSPs were isolated with NMDA and AMPA receptor antagonists (d-APV, NBQX, kynurenic acid). fIPSP dependence on GABAAR was confirmed by blockade in high dose GABAAR antagonist, picrotoxin (PTX). GABAergic ligands were applied to slices, and their effects on magnitude and decay kinetics of the fIPSP were measured. Ligands tested include: propofol, isoflurane, midazolam, diazepam, flumazenil and furosemide (FUR) and PTX. Results: Hippocampal GABAergic inhibition can be classified by its duration and sensitivity to allosteric modulators like benzodiazepines (BZPs). We characterized the CA1 fIPSP with compounds known to affect these parameters; a subset of our data is summarized here. FUR, a selective antagonist of GABAA-fast, dose-dependently reduces fIPSP amplitude and prolongs its decay, suggesting that the fIPSP is largely mediated by GABAA-fast synapses. In comparison, PTX, a non-selective GABAAR antagonist, depressed evoked fIPSP amplitude without modifying the fIPSP decay. fIPSPs are also sensitive to BZPs, including midazolam and diazepam, both of which enhanced fIPSP amplitude, and prolonged decay time. Flumazenil, a BZP antagonist, blocked these effects. Conclusions: This method for studying synaptic inhibition has major advantages over conventional electrophysiological techniques: 1) it is extracellular, so key intracellular signaling molecules remain intact, 2) it detects changes in both tonic and phasic GABAAR mediated signaling, and finally 3) it is more stable and technically easier than whole-cell recording. Combining this fast, minimally cell invasive, neural population based approach affords a unique opportunity to assay multiple lead compounds for anesthetic efficacy in an intact, well characterized neural circuit with clear relevance to learning, memory and cognition.
Introduction: The GABAA receptor (GABAAR) is an attractive, tractable drug discovery target. It remains unclear how native neural circuits of the hippocampus respond to drugs in this highly clinically relevant class. The CA1 region of the hippocampus is crucial for learning, memory and cognition, thus a key brain region to screen GABAergic compounds that may influence these processes. We developed a novel screen for GABAAR ligands, including general anesthetics, by measuring field inhibitory postsynaptic potentials (fIPSPs) in the CA1 area. While allowing many of the advantages of an in vitro preparation, field recordings mirror their in vivo counterparts, and unlike intracellular recordings, are minimally invasive to the neuron, typically remaining stable for many hours. Methods: 24-28 day old Sprague Dawley rats were anesthetized and decapitated. Brains were dissected and submerged in chilled artificial cerebrospinal fluid (ACSF). 400 μm thick coronal slices were cut and maintained in ACSF bubbled with 95% O2 and 5% CO2. fIPSPs were evoked through a bipolar tungsten stimulating electrode placed in the stratum pyramidale (SP) of the CA1 region and recorded by microelectrode 300-400 μm away in the SP of CA1. GABAergic fIPSPs were isolated with NMDA and AMPA receptor antagonists (d-APV, NBQX, kynurenic acid). fIPSP dependence on GABAAR was confirmed by blockade in high dose GABAAR antagonist, picrotoxin (PTX). GABAergic ligands were applied to slices, and their effects on magnitude and decay kinetics of the fIPSP were measured. Ligands tested include: propofol, isoflurane, midazolam, diazepam, flumazenil and furosemide (FUR) and PTX. Results: Hippocampal GABAergic inhibition can be classified by its duration and sensitivity to allosteric modulators like benzodiazepines (BZPs). We characterized the CA1 fIPSP with compounds known to affect these parameters; a subset of our data is summarized here. FUR, a selective antagonist of GABAA-fast, dose-dependently reduces fIPSP amplitude and prolongs its decay, suggesting that the fIPSP is largely mediated by GABAA-fast synapses. In comparison, PTX, a non-selective GABAAR antagonist, depressed evoked fIPSP amplitude without modifying the fIPSP decay. fIPSPs are also sensitive to BZPs, including midazolam and diazepam, both of which enhanced fIPSP amplitude, and prolonged decay time. Flumazenil, a BZP antagonist, blocked these effects. Conclusions: This method for studying synaptic inhibition has major advantages over conventional electrophysiological techniques: 1) it is extracellular, so key intracellular signaling molecules remain intact, 2) it detects changes in both tonic and phasic GABAAR mediated signaling, and finally 3) it is more stable and technically easier than whole-cell recording. Combining this fast, minimally cell invasive, neural population based approach affords a unique opportunity to assay multiple lead compounds for anesthetic efficacy in an intact, well characterized neural circuit with clear relevance to learning, memory and cognition.
Book
1 online resource.
Technological developments in genomics over the past two decades have allowed biologists to address long-standing questions in ecology, evolutionary biology, and medicine, but also pose a substantial challenge for analysis and interpretation. This thesis addresses these challenges by developing and testing novel genomic tools and resources as well as leveraging them to address basic biological questions. The thesis chapters are united in their focus on the origin and maintenance of natural genetic variation as well as its influence on phenotypic variation. The first introductory chapter expands upon the unifying themes and briefly outlines the history of the study of genetic variation, with a specific focus on aneuploidy. The second chapter focuses on reference genome assembly by identifying advantages and limitations of a new synthetic long-read technology for de novo genome reconstruction. The third chapter introduces a low-cost method to survey genetic variation in non-model species by simultaneously building a transcriptome reference and discovering expressed single nucleotide polymorphisms from a population sample. These genomic data are then used to infer the demographic history of an introduced checkerspot butterfly, achieving parameter estimates that are consistent with the known population history. The final two chapters focus on application of high-throughput genomic technologies in a clinical setting, specifically in the context of preimplantation genetic screening (PGS) during in vitro fertilization. PGS data are then analyzed to study variation in chromosome copy number (i.e. aneuploidy) which is prevalent in early human development, but rarely survives to live birth. The fourth chapter contrasts the incidences of various forms of aneuploidy at different stages of preimplantation development, demonstrating that mitotic-origin aneuploidies are strongly selected against at the onset of zygotic genome activation. The final chapter provides evidence that a maternal genetic variant influences aneuploidy risk, identifying a promising candidate gene in the region. Together, these dissertation chapters develop and apply novel genomic approaches to achieve new biological insights and pave the way for future genomic studies.
Technological developments in genomics over the past two decades have allowed biologists to address long-standing questions in ecology, evolutionary biology, and medicine, but also pose a substantial challenge for analysis and interpretation. This thesis addresses these challenges by developing and testing novel genomic tools and resources as well as leveraging them to address basic biological questions. The thesis chapters are united in their focus on the origin and maintenance of natural genetic variation as well as its influence on phenotypic variation. The first introductory chapter expands upon the unifying themes and briefly outlines the history of the study of genetic variation, with a specific focus on aneuploidy. The second chapter focuses on reference genome assembly by identifying advantages and limitations of a new synthetic long-read technology for de novo genome reconstruction. The third chapter introduces a low-cost method to survey genetic variation in non-model species by simultaneously building a transcriptome reference and discovering expressed single nucleotide polymorphisms from a population sample. These genomic data are then used to infer the demographic history of an introduced checkerspot butterfly, achieving parameter estimates that are consistent with the known population history. The final two chapters focus on application of high-throughput genomic technologies in a clinical setting, specifically in the context of preimplantation genetic screening (PGS) during in vitro fertilization. PGS data are then analyzed to study variation in chromosome copy number (i.e. aneuploidy) which is prevalent in early human development, but rarely survives to live birth. The fourth chapter contrasts the incidences of various forms of aneuploidy at different stages of preimplantation development, demonstrating that mitotic-origin aneuploidies are strongly selected against at the onset of zygotic genome activation. The final chapter provides evidence that a maternal genetic variant influences aneuploidy risk, identifying a promising candidate gene in the region. Together, these dissertation chapters develop and apply novel genomic approaches to achieve new biological insights and pave the way for future genomic studies.
Book
1 online resource.
Protein sorting coordinates membrane dynamics with the clustering and isolation of proteins into discrete membrane domains. Proper protein sorting not only mediates intracellular homeostasis, for example, through the delivery of hydrolase enzymes to the lysosome or through degradation of long-lived proteins in autophagy. It also facilitates the communication between a cell and its environment (the extracellular milieu or neighboring cells) by regulating both ligand secretion as well as the availability of receptors at the plasma membrane by balancing receptor degradation with receptor recycling. Disruption of protein sorting pathways can therefore interfere with intracellular processes and prevent the interactions between cells necessary for the maintenance of tissue homeostasis. The nervous system, in particular, is sensitive to disruption of protein sorting pathways, as evidenced by the growing number of reports linking polymorphisms in protein sorting machinery to neurological disease, such as Alzheimer's Disease (AD). It is therefore necessary to elucidate the mechanisms controlling protein sorting so that we may better understand the normal biology underlying these processes and the consequences of their disruption in pathogenesis. Previous work has established that levels of beclin 1, a protein that functions in multiple membrane trafficking pathways, are decreased in the brains of Alzheimer's Disease patients. We show that, in addition to it's well-known function in regulating autophagy, beclin 1 regulates receptor recycling in two systems relevant to neurodegeneration: phagocytosis in microglia and TGF-[beta] signaling in neurons. We use immunocytochemistry, confocal microscopy, and live-cell imaging to demonstrate beclin 1 regulates receptor recycling through production of phosphatidylinositol-3-phosphate at vesicle membranes and recruitment of the retromer complex. We also use a combination of biochemical techniques, flow cytometry, and immunohistochemistry to show the functional consequence of disrupted receptor recycling in phagocytosis and the TGF-[beta] signaling pathway using both in vitro and in vivo models. In light of our findings, we discuss the implications of impaired beclin 1-mediated protein sorting in neurological disease.
Protein sorting coordinates membrane dynamics with the clustering and isolation of proteins into discrete membrane domains. Proper protein sorting not only mediates intracellular homeostasis, for example, through the delivery of hydrolase enzymes to the lysosome or through degradation of long-lived proteins in autophagy. It also facilitates the communication between a cell and its environment (the extracellular milieu or neighboring cells) by regulating both ligand secretion as well as the availability of receptors at the plasma membrane by balancing receptor degradation with receptor recycling. Disruption of protein sorting pathways can therefore interfere with intracellular processes and prevent the interactions between cells necessary for the maintenance of tissue homeostasis. The nervous system, in particular, is sensitive to disruption of protein sorting pathways, as evidenced by the growing number of reports linking polymorphisms in protein sorting machinery to neurological disease, such as Alzheimer's Disease (AD). It is therefore necessary to elucidate the mechanisms controlling protein sorting so that we may better understand the normal biology underlying these processes and the consequences of their disruption in pathogenesis. Previous work has established that levels of beclin 1, a protein that functions in multiple membrane trafficking pathways, are decreased in the brains of Alzheimer's Disease patients. We show that, in addition to it's well-known function in regulating autophagy, beclin 1 regulates receptor recycling in two systems relevant to neurodegeneration: phagocytosis in microglia and TGF-[beta] signaling in neurons. We use immunocytochemistry, confocal microscopy, and live-cell imaging to demonstrate beclin 1 regulates receptor recycling through production of phosphatidylinositol-3-phosphate at vesicle membranes and recruitment of the retromer complex. We also use a combination of biochemical techniques, flow cytometry, and immunohistochemistry to show the functional consequence of disrupted receptor recycling in phagocytosis and the TGF-[beta] signaling pathway using both in vitro and in vivo models. In light of our findings, we discuss the implications of impaired beclin 1-mediated protein sorting in neurological disease.
Book
1 online resource.
Genome editing is a process that entails precise manipulation of a DNA sequence. To achieve this, a define stretch of nucleotides is replaced by a new, exogenously provided, DNA template using the cellular DNA repair machinery as a mean to promote the exchange. By taking this approach, we not only can repair alterations that impede gene functions but we can also expand our knowledge of basic biological processes driven by genes. Different classes of site-specific hybrid nucleases have been engineered precisely for the purpose of deleting a DNA sequence in a targeted manner. There are three main supporting platforms, Zinc Finger Nucleases (ZFNs), Transcription Activator Like Effector Nucleases (TALENs) and Cluster Regulatory Interspaced Short Palindromic Repeats/Cas9 Nucleases (CRISPR/Cas9). Of these three, TALENs have the highest targeting range, the flexibility of modifying different genomes and cell types with the lowest off-target activity, features that promote TALENs as potential powerful therapeutic tools. To further characterize them, I have employed a biochemical approach that allowed me to determine their specificity, affinities and kinetics at both cognate and novel OFF target DNA binding sites. From this study it became apparent that TALENs' kinetic signature is the missing link that connects their in vitro behavior with in vivo activity.
Genome editing is a process that entails precise manipulation of a DNA sequence. To achieve this, a define stretch of nucleotides is replaced by a new, exogenously provided, DNA template using the cellular DNA repair machinery as a mean to promote the exchange. By taking this approach, we not only can repair alterations that impede gene functions but we can also expand our knowledge of basic biological processes driven by genes. Different classes of site-specific hybrid nucleases have been engineered precisely for the purpose of deleting a DNA sequence in a targeted manner. There are three main supporting platforms, Zinc Finger Nucleases (ZFNs), Transcription Activator Like Effector Nucleases (TALENs) and Cluster Regulatory Interspaced Short Palindromic Repeats/Cas9 Nucleases (CRISPR/Cas9). Of these three, TALENs have the highest targeting range, the flexibility of modifying different genomes and cell types with the lowest off-target activity, features that promote TALENs as potential powerful therapeutic tools. To further characterize them, I have employed a biochemical approach that allowed me to determine their specificity, affinities and kinetics at both cognate and novel OFF target DNA binding sites. From this study it became apparent that TALENs' kinetic signature is the missing link that connects their in vitro behavior with in vivo activity.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
Conservation and land management decisions are often based on an understanding of the distribution, abundance, and habitat requirements of wildlife. In this study, I examined the spatial distribution and abundance of the bobcat (Lynx rufus), a mid-sized predator, at Jasper Ridge Biological Preserve (JRBP) using camera traps. The initial camera trap survey was conducted from 2006 to 2008. I conducted another survey from 2014 to 2015. The comparison suggests that the relative abundance of bobcats has decreased substantially from the 2006-2008 survey to the 2014-2015 survey. This may be due to several factors, including the recent drought, increased human activity and construction, the incidence of notoedric mange, and increased puma abundance. The drought could indirectly affect bobcats by altering vegetation and reducing prey abundance. In both surveys, bobcats and their prey species exhibited crepuscular activity patterns, and bobcat occurrences did not vary by season. The distribution of bobcats was not predicted by canopy type or vegetation cover. As expected, bobcat abundance was positively correlated with prey abundance across both surveys. Human activity was measured only in the 2014 to 2015 survey and did not affect bobcat distribution, likely due to the temporal separation between the diurnal activity of humans and the crepuscular activity of bobcats. Unexpectedly, in the most recent survey, they were most abundant in locations with relatively high abundance of puma. In addition, bobcats and puma had a high degree of diel overlap. This could indicate that puma contribute to the decreased abundance of bobcats by competition or predation. Further research on the impacts of human activity, the recent drought, notoedric mange, and increased puma abundance are necessary, as well as continued camera trap monitoring of bobcats.
Conservation and land management decisions are often based on an understanding of the distribution, abundance, and habitat requirements of wildlife. In this study, I examined the spatial distribution and abundance of the bobcat (Lynx rufus), a mid-sized predator, at Jasper Ridge Biological Preserve (JRBP) using camera traps. The initial camera trap survey was conducted from 2006 to 2008. I conducted another survey from 2014 to 2015. The comparison suggests that the relative abundance of bobcats has decreased substantially from the 2006-2008 survey to the 2014-2015 survey. This may be due to several factors, including the recent drought, increased human activity and construction, the incidence of notoedric mange, and increased puma abundance. The drought could indirectly affect bobcats by altering vegetation and reducing prey abundance. In both surveys, bobcats and their prey species exhibited crepuscular activity patterns, and bobcat occurrences did not vary by season. The distribution of bobcats was not predicted by canopy type or vegetation cover. As expected, bobcat abundance was positively correlated with prey abundance across both surveys. Human activity was measured only in the 2014 to 2015 survey and did not affect bobcat distribution, likely due to the temporal separation between the diurnal activity of humans and the crepuscular activity of bobcats. Unexpectedly, in the most recent survey, they were most abundant in locations with relatively high abundance of puma. In addition, bobcats and puma had a high degree of diel overlap. This could indicate that puma contribute to the decreased abundance of bobcats by competition or predation. Further research on the impacts of human activity, the recent drought, notoedric mange, and increased puma abundance are necessary, as well as continued camera trap monitoring of bobcats.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
Despite synapse formation’s central role in neurological development, its underlying mechanisms still remain to be fully characterized. Until recent years, molecular studies of this process have largely ignored the neuronal extracellular matrix (ECM), as well as the basement membrane proteins that define the extent and functionality of the region. However, due to their localization at neuronal cell surfaces, basement membrane proteins are now increasingly being identified as prospective mediators of the guidance mechanisms necessary for proper circuit assembly. This investigation further develops an emerging role for the neuronal ECM in synapse formation. First, we describe a novel model of defective synapse formation in C. elegans, characterized by a loss-of-function allele for GON-1, a basement membrane protein (of the ADAMTS metalloprotease family) with putative ECM remodeling functions. Unbiased genetic screens carried out on this gon-1-/- background revealed neuronal ECM proteins with both enhancing and suppressing effects on the synapse-formation-defective phenotype. These modifier screen hits were then assayed for reproducible exacerbation or amelioration of this phenotype via both genetic knockout and translational inhibition experiments, implicating several in a cohesive, ECM-specific synapse development pathway centered on the GON-1 protein. Ultimately, this pathway must be characterized if neurobiologists are to understand nervous system development on a cellular level. In the meantime, these mechanisms are expected to provide vital insight into neurodevelopmental disorders (with particular optimism regarding autism spectrum disorders) and the demands of viable nerve tissue repair strategies, which will have enormous clinical significance in the coming decades.
Despite synapse formation’s central role in neurological development, its underlying mechanisms still remain to be fully characterized. Until recent years, molecular studies of this process have largely ignored the neuronal extracellular matrix (ECM), as well as the basement membrane proteins that define the extent and functionality of the region. However, due to their localization at neuronal cell surfaces, basement membrane proteins are now increasingly being identified as prospective mediators of the guidance mechanisms necessary for proper circuit assembly. This investigation further develops an emerging role for the neuronal ECM in synapse formation. First, we describe a novel model of defective synapse formation in C. elegans, characterized by a loss-of-function allele for GON-1, a basement membrane protein (of the ADAMTS metalloprotease family) with putative ECM remodeling functions. Unbiased genetic screens carried out on this gon-1-/- background revealed neuronal ECM proteins with both enhancing and suppressing effects on the synapse-formation-defective phenotype. These modifier screen hits were then assayed for reproducible exacerbation or amelioration of this phenotype via both genetic knockout and translational inhibition experiments, implicating several in a cohesive, ECM-specific synapse development pathway centered on the GON-1 protein. Ultimately, this pathway must be characterized if neurobiologists are to understand nervous system development on a cellular level. In the meantime, these mechanisms are expected to provide vital insight into neurodevelopmental disorders (with particular optimism regarding autism spectrum disorders) and the demands of viable nerve tissue repair strategies, which will have enormous clinical significance in the coming decades.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
The INO80 subfamily of chromatin remodelers, which includes the complexes INO80 and SWR1, have been known to play an important role in rearranging chromatin, hydrolyzing ATP to change the structure of nucleosomes. However, recent research has suggested that these complexes may also play a role during mitosis, specifically in microtubule dynamics. Preliminary studies from our lab have shown that ino80Δ shows ploidy defects and abnormal microtubule dynamics. Furthermore, high-throughput genetic analyses have shown negative interactions for both INO80 and SWR1 with various mitotic genes of interest, further suggesting that these complexes are involved in mitosis. However, the exact mechanisms underlying INO80 and SWR1’s mitotic functions remain unknown, particularly for the latter. This study sought to elucidate the roles of both complexes during mitosis, using an integrative approach of genetics, molecular biology, and microscopy techniques to answer this question. We hypothesized that INO80 and SWR1 affect chromosome segregation by affecting microtubule dynamics. Our genetic analyses show that INO80 and SWR1 function in parallel pathways to BIM1 and BIK1, two genes critical for microtubule dynamics in S. cerevisiae. Our in vivo live-cell imaging reveals that ino80Δ mutants have short spindles and elongated cell morphology. These results suggest that INO80 is involved in stabilizing microtubules and potentially affects other components of the cytoskeleton, including actin. On the other hand, although swr1Δ mutants also show shortened spindles, they also tend to have exaggerated astral microtubules (aMTs) as opposed to ino80Δ mutants, which show fewer aMTs. This finding opens the possibility that SWR1 and INO80 have complementary but opposite roles during mitosis. Further studies conducted on domain and module mutants of INO80 revealed that the HSA and spacer domains are specifically involved in the complex’s microtubule-stabilizing activity, which also apparently requires the ATPase function of the catalytic subunit. Genetic and fitness assays of SWR1 in conjunction with various kinetochore genes (CBF1, BUB1, MCM21, and CHL4) show negative interactions between the two groups. These results suggest that SWR1 may be acting at the microtubule-kinetochore interface, helping to define a mechanism for its effect on microtubule dynamics as observed through live-cell imaging. The findings of this study provide a novel view of a subfamily of chromatin remodelers, defining a mechanistic level of understanding for their function in mitosis. Chromosome missegregation is associated with many diseases such as cancer and congenital disorders. Because proper chromosome segregation is largely dependent on microtubule dynamics, determining the INO80 subfamily’s role in this process has meaningful implications for disease treatment in the future.
The INO80 subfamily of chromatin remodelers, which includes the complexes INO80 and SWR1, have been known to play an important role in rearranging chromatin, hydrolyzing ATP to change the structure of nucleosomes. However, recent research has suggested that these complexes may also play a role during mitosis, specifically in microtubule dynamics. Preliminary studies from our lab have shown that ino80Δ shows ploidy defects and abnormal microtubule dynamics. Furthermore, high-throughput genetic analyses have shown negative interactions for both INO80 and SWR1 with various mitotic genes of interest, further suggesting that these complexes are involved in mitosis. However, the exact mechanisms underlying INO80 and SWR1’s mitotic functions remain unknown, particularly for the latter. This study sought to elucidate the roles of both complexes during mitosis, using an integrative approach of genetics, molecular biology, and microscopy techniques to answer this question. We hypothesized that INO80 and SWR1 affect chromosome segregation by affecting microtubule dynamics. Our genetic analyses show that INO80 and SWR1 function in parallel pathways to BIM1 and BIK1, two genes critical for microtubule dynamics in S. cerevisiae. Our in vivo live-cell imaging reveals that ino80Δ mutants have short spindles and elongated cell morphology. These results suggest that INO80 is involved in stabilizing microtubules and potentially affects other components of the cytoskeleton, including actin. On the other hand, although swr1Δ mutants also show shortened spindles, they also tend to have exaggerated astral microtubules (aMTs) as opposed to ino80Δ mutants, which show fewer aMTs. This finding opens the possibility that SWR1 and INO80 have complementary but opposite roles during mitosis. Further studies conducted on domain and module mutants of INO80 revealed that the HSA and spacer domains are specifically involved in the complex’s microtubule-stabilizing activity, which also apparently requires the ATPase function of the catalytic subunit. Genetic and fitness assays of SWR1 in conjunction with various kinetochore genes (CBF1, BUB1, MCM21, and CHL4) show negative interactions between the two groups. These results suggest that SWR1 may be acting at the microtubule-kinetochore interface, helping to define a mechanism for its effect on microtubule dynamics as observed through live-cell imaging. The findings of this study provide a novel view of a subfamily of chromatin remodelers, defining a mechanistic level of understanding for their function in mitosis. Chromosome missegregation is associated with many diseases such as cancer and congenital disorders. Because proper chromosome segregation is largely dependent on microtubule dynamics, determining the INO80 subfamily’s role in this process has meaningful implications for disease treatment in the future.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
With today’s advances in technology, whole genomes can be sequenced in a matter of hours and the genetic basis of diseases can now be studied more closely. Even so, our understanding of disease is still incomplete without knowledge about the dynamic regulation of DNA and the consequent changes in the transcriptional profile of the cell. In the context of chromatin, the highly organized and packaged form of DNA, there are large multi-subunit protein complexes called chromatin remodelers that modulate the accessibility of DNA elements and the transcription of genes. In particular, the INO80 (inositol requiring 80) subfamily of ATP-dependent chromatin remodelers has been identified as a transcriptional regulator, and has been implicated in the regulation of cell proliferation. To further investigate the role of the INO80 chromatin remodeler in cell proliferation, RNA-sequencing was performed on INO80 yeast mutants and it was found that mitochondria related transcripts were up-regulated. Knowing that mitochondria play a significant role in metabolism and that the INO80 chromatin remodeler is also present in mammals, an in vitro mammalian model system with Ino80 knockouts was developed, and proliferation rates were studied. It was determined that there was no statistically significant difference in cellular proliferation rate between wild type and knockout cells. However, trends suggest Ino80 knockout MEFs rely more on mitochondrial function compared to wild type. Ultimately, understanding the INO80 complex’s role in cellular metabolism and cell proliferation is important to our understanding of what conditions a cell prefers to continue growth and to possibly even differentiate. This has some implications for the development of drugs that exploit the metabolic dependencies of cancer cells as a treatment option.
With today’s advances in technology, whole genomes can be sequenced in a matter of hours and the genetic basis of diseases can now be studied more closely. Even so, our understanding of disease is still incomplete without knowledge about the dynamic regulation of DNA and the consequent changes in the transcriptional profile of the cell. In the context of chromatin, the highly organized and packaged form of DNA, there are large multi-subunit protein complexes called chromatin remodelers that modulate the accessibility of DNA elements and the transcription of genes. In particular, the INO80 (inositol requiring 80) subfamily of ATP-dependent chromatin remodelers has been identified as a transcriptional regulator, and has been implicated in the regulation of cell proliferation. To further investigate the role of the INO80 chromatin remodeler in cell proliferation, RNA-sequencing was performed on INO80 yeast mutants and it was found that mitochondria related transcripts were up-regulated. Knowing that mitochondria play a significant role in metabolism and that the INO80 chromatin remodeler is also present in mammals, an in vitro mammalian model system with Ino80 knockouts was developed, and proliferation rates were studied. It was determined that there was no statistically significant difference in cellular proliferation rate between wild type and knockout cells. However, trends suggest Ino80 knockout MEFs rely more on mitochondrial function compared to wild type. Ultimately, understanding the INO80 complex’s role in cellular metabolism and cell proliferation is important to our understanding of what conditions a cell prefers to continue growth and to possibly even differentiate. This has some implications for the development of drugs that exploit the metabolic dependencies of cancer cells as a treatment option.
Book
1 online resource.
Global Change is complex and its effects on ecology and plant-insect interactions are specific. Thus, it is vital to examine the nuances in plant behavior to better understand how to mitigate anthropogenic change. The more data points we can accumulate on the line of plant communication, the better we are able to assess the effects of change in natural systems. The invasion of Centaurea solstitialis (Yellow starthistle) is both a local and national crisis. Current and future increases in CO2 will lead to further aggressive expansion of this weed. In my work I have found that Yellow starthistle may be dependent on indirect defense prior to flower-head formation, within a tri-trophic system that may have co-evolved prior to introduction of Yellow starthistle to North America, and identified the volatiles likely acting as indirect defense cues. In this first known report on the influence of climate change on volatile emission of Yellow starthistle in the field, global changes such as elevated temperature and CO2 were not found to influence the volatile profile of the invasive. However, its induced emission as an anti-herbivory strategy may be more effective under elevated CO2, hence explaining its increased growth under such conditions. Thus, for natural control purposes, attention should be given to monitoring both herbivore and predator populations during early growth of this weed. This work offers insight into the evolutionary purpose of volatile emission and will help document the ways in which ecosystem responses to global change may be facilitated by networks of chemical communication.
Global Change is complex and its effects on ecology and plant-insect interactions are specific. Thus, it is vital to examine the nuances in plant behavior to better understand how to mitigate anthropogenic change. The more data points we can accumulate on the line of plant communication, the better we are able to assess the effects of change in natural systems. The invasion of Centaurea solstitialis (Yellow starthistle) is both a local and national crisis. Current and future increases in CO2 will lead to further aggressive expansion of this weed. In my work I have found that Yellow starthistle may be dependent on indirect defense prior to flower-head formation, within a tri-trophic system that may have co-evolved prior to introduction of Yellow starthistle to North America, and identified the volatiles likely acting as indirect defense cues. In this first known report on the influence of climate change on volatile emission of Yellow starthistle in the field, global changes such as elevated temperature and CO2 were not found to influence the volatile profile of the invasive. However, its induced emission as an anti-herbivory strategy may be more effective under elevated CO2, hence explaining its increased growth under such conditions. Thus, for natural control purposes, attention should be given to monitoring both herbivore and predator populations during early growth of this weed. This work offers insight into the evolutionary purpose of volatile emission and will help document the ways in which ecosystem responses to global change may be facilitated by networks of chemical communication.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
Chromatin remodelers are protein complexes characterized by their role in manipulating and modifying chromatin and histones. They are known to regulate transcription and DNA damage response, but recent discoveries suggest they also regulate chromosome segregation and other aspects of mitosis, proposing a novel and undefined role for chromatin remodelers. Such is the case for INO80, a well studied 15 subunit protein complex. Preliminary data show a likely interaction between INO80 and microtubules, both of which are essential for proper chromosome segregation and correctly positioning components of the dividing cell. Thus, to maintain proper chromosome segregation and cell division, we hypothesize that the INO80 complex plays a vital role in mitosis by binding to and stabilizing microtubules. Using yeast genetics in S. cerevisiae, we found a strong association between the INO80 complex and tubulin after immunoprecipitation and separation by molecular density on a sucrose gradient. We next investigated this interaction in mutants of the various INO80 modules further to characterize each mutant’s relationship with microtubules. It was found that the N-terminal region of Ino80 was necessary for its association with tubulin. Furthermore, we investigated the DNA content of each mutant using FACS as well as its response to microtubule destabilizing agents in a fitness assay. Overall, investigating the role of chromatin remodeler INO80 in mitosis may elucidate a mechanism for developing chromosomal and other mitotic defects dependent on interactions between INO80’s subunits and microtubules. This mechanism of action may help characterize and explain the presence of mitotic defects in cancer cells.
Chromatin remodelers are protein complexes characterized by their role in manipulating and modifying chromatin and histones. They are known to regulate transcription and DNA damage response, but recent discoveries suggest they also regulate chromosome segregation and other aspects of mitosis, proposing a novel and undefined role for chromatin remodelers. Such is the case for INO80, a well studied 15 subunit protein complex. Preliminary data show a likely interaction between INO80 and microtubules, both of which are essential for proper chromosome segregation and correctly positioning components of the dividing cell. Thus, to maintain proper chromosome segregation and cell division, we hypothesize that the INO80 complex plays a vital role in mitosis by binding to and stabilizing microtubules. Using yeast genetics in S. cerevisiae, we found a strong association between the INO80 complex and tubulin after immunoprecipitation and separation by molecular density on a sucrose gradient. We next investigated this interaction in mutants of the various INO80 modules further to characterize each mutant’s relationship with microtubules. It was found that the N-terminal region of Ino80 was necessary for its association with tubulin. Furthermore, we investigated the DNA content of each mutant using FACS as well as its response to microtubule destabilizing agents in a fitness assay. Overall, investigating the role of chromatin remodeler INO80 in mitosis may elucidate a mechanism for developing chromosomal and other mitotic defects dependent on interactions between INO80’s subunits and microtubules. This mechanism of action may help characterize and explain the presence of mitotic defects in cancer cells.
Book
1 online resource.
Proteins are involved in every aspect of life owing to their highly diverse enzymatic and structural properties. Despite their importance in the regulation of life, proteins are vulnerable in living cells. Since this trait can easily lead to cellular defects, the stability of the protein folding process must be monitored. Failure to clear aberrant proteins results in the loss as well as gain of protein function, which may disrupt various cellular functions and lead to diseases in humans such as Alzheimer's, Parkinson's, and Huntington's diseases. Furthermore, cancer, one of the biggest causes of death, and aging, an inevitable defect for every human being, have been reported to be related to unfolded proteins. Thus, the maintenance of protein folding homeostasis is indispensable to all organisms, and the cell develops and contributes a significant amount of energy to ensuring that nascent proteins become their native state upon translation, to preventing the accumulation of aberrant proteins, and to maintaining the proper folding state of proteins. In chapter one I have briefly summarized the current understandings about the stress response related to unfolded proteins. However, we do not fully comprehend the cellular response to unfolded proteins. One reason why the response has not been well elucidated is because of lack of specific perturbants that induced the specific stresses in an acute manner. To overcome the barrier in elucidating the mechanisms inderlying the cellular response to unfolded proteins, we implemented a unique chemical biology approach developed by our group, known as destabilizing domains (DDs). Because the folding state of DDs can be conditionally controlled by a cell-permeable small molecule, we were able to create specific amounts of unfolded proteins inside cells in a simple and sudden manner by removing the ligand. Chapter two reports that acute unfolded protein stress from unfolded DDs elicit a coordinated transcriptional response from mammalian cells. The response is distinct from heat stress and the conventional unfolded protein response, and cells that trigger this response are more resistant than unstressed cells when challenged with other stressors. In addition, by changing the location of DDs, we demonstrated for the first time that the nucleus and cytoplasm have compartment specific responding elements to the stress from unfolded proteins. In the third chapter, we describe a series of experiments designed to elucidate another aspect of unfolded proteins, aggregation, by developing new method based on DD. We report a chemically controllable fluorescent aggregating protein that allows us to monitor the entire life of aggregates in living cells. This technology allows us to rapidly produce many small aggregates in seconds while monitoring the movement and coalescence of the aggregates in cells. We found that Hsc70 captures the aggregates extremely fast and prevents aggregation. This method is applicable for various experimental systems including living organisms, and further collaborations are currently taking place. The fourth and final chapter focus on our development of the new and third DD system to regulate a specific protein by a cell-permeable small molecule. The system based on the ligand binding domain of the estrogen receptor that can be regulated by one of the two synthetic ligands, CMP8 or 4-hydroxytamoxifen. It is orthogonal to other FKBP- and DHFR-based DD systems and will enable us to simultaneously and independently regulate three proteins in biological studies. We believe that this new technology to control protein activities will contribute to the advancement of science.
Proteins are involved in every aspect of life owing to their highly diverse enzymatic and structural properties. Despite their importance in the regulation of life, proteins are vulnerable in living cells. Since this trait can easily lead to cellular defects, the stability of the protein folding process must be monitored. Failure to clear aberrant proteins results in the loss as well as gain of protein function, which may disrupt various cellular functions and lead to diseases in humans such as Alzheimer's, Parkinson's, and Huntington's diseases. Furthermore, cancer, one of the biggest causes of death, and aging, an inevitable defect for every human being, have been reported to be related to unfolded proteins. Thus, the maintenance of protein folding homeostasis is indispensable to all organisms, and the cell develops and contributes a significant amount of energy to ensuring that nascent proteins become their native state upon translation, to preventing the accumulation of aberrant proteins, and to maintaining the proper folding state of proteins. In chapter one I have briefly summarized the current understandings about the stress response related to unfolded proteins. However, we do not fully comprehend the cellular response to unfolded proteins. One reason why the response has not been well elucidated is because of lack of specific perturbants that induced the specific stresses in an acute manner. To overcome the barrier in elucidating the mechanisms inderlying the cellular response to unfolded proteins, we implemented a unique chemical biology approach developed by our group, known as destabilizing domains (DDs). Because the folding state of DDs can be conditionally controlled by a cell-permeable small molecule, we were able to create specific amounts of unfolded proteins inside cells in a simple and sudden manner by removing the ligand. Chapter two reports that acute unfolded protein stress from unfolded DDs elicit a coordinated transcriptional response from mammalian cells. The response is distinct from heat stress and the conventional unfolded protein response, and cells that trigger this response are more resistant than unstressed cells when challenged with other stressors. In addition, by changing the location of DDs, we demonstrated for the first time that the nucleus and cytoplasm have compartment specific responding elements to the stress from unfolded proteins. In the third chapter, we describe a series of experiments designed to elucidate another aspect of unfolded proteins, aggregation, by developing new method based on DD. We report a chemically controllable fluorescent aggregating protein that allows us to monitor the entire life of aggregates in living cells. This technology allows us to rapidly produce many small aggregates in seconds while monitoring the movement and coalescence of the aggregates in cells. We found that Hsc70 captures the aggregates extremely fast and prevents aggregation. This method is applicable for various experimental systems including living organisms, and further collaborations are currently taking place. The fourth and final chapter focus on our development of the new and third DD system to regulate a specific protein by a cell-permeable small molecule. The system based on the ligand binding domain of the estrogen receptor that can be regulated by one of the two synthetic ligands, CMP8 or 4-hydroxytamoxifen. It is orthogonal to other FKBP- and DHFR-based DD systems and will enable us to simultaneously and independently regulate three proteins in biological studies. We believe that this new technology to control protein activities will contribute to the advancement of science.
Book
1 online resource.
Phosphorus (P) limits agricultural productivity because most soil P is found in pools of low plant-availability and external inputs that are used to increase plant-available P are only partially recovered in crops, resulting in low P use efficiency (PUE). Cover crops could reduce external P input requirements, increase PUE and stimulate soil P cycling by mobilizing soil P and by retaining soil P via plant uptake, especially in low-input agricultural systems. This dissertation seeks to determine if cover crops have similar effects on soil P cycling in intensive agricultural systems with relatively high soil P, using two long-term experiments in California, greenhouse experiments and nutrient budgets. In both field and laboratory conditions, legume cover crops had a greater potential to mobilize soil P than other cover crops, although in practice they did not mobilize soil P. In contrast, cereals had the strongest effect on soil P availability and P cycling by taking up more soil P than other cover crops. Regardless of cover crop type, P taken up in cover crop biomass was recycled rapidly in these systems: cover crop residues and mineral fertilizer contributed similarly to soil pools and wheat P uptake, with a greater contribution at lower soil P availability. However, cover crops had relatively small effects on long-term soil P dynamics relative to compost addition that was the main factor driving differences in P budgets computed at the farm-scale. Overall, cover crops have the potential to affect soil P cycling in these systems with relatively high soil P, although to a lesser degree than do composts.
Phosphorus (P) limits agricultural productivity because most soil P is found in pools of low plant-availability and external inputs that are used to increase plant-available P are only partially recovered in crops, resulting in low P use efficiency (PUE). Cover crops could reduce external P input requirements, increase PUE and stimulate soil P cycling by mobilizing soil P and by retaining soil P via plant uptake, especially in low-input agricultural systems. This dissertation seeks to determine if cover crops have similar effects on soil P cycling in intensive agricultural systems with relatively high soil P, using two long-term experiments in California, greenhouse experiments and nutrient budgets. In both field and laboratory conditions, legume cover crops had a greater potential to mobilize soil P than other cover crops, although in practice they did not mobilize soil P. In contrast, cereals had the strongest effect on soil P availability and P cycling by taking up more soil P than other cover crops. Regardless of cover crop type, P taken up in cover crop biomass was recycled rapidly in these systems: cover crop residues and mineral fertilizer contributed similarly to soil pools and wheat P uptake, with a greater contribution at lower soil P availability. However, cover crops had relatively small effects on long-term soil P dynamics relative to compost addition that was the main factor driving differences in P budgets computed at the farm-scale. Overall, cover crops have the potential to affect soil P cycling in these systems with relatively high soil P, although to a lesser degree than do composts.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
Stroke is a leading cause of human disability and death, yet continues to lack an effective treatment, as well as a thorough understanding of its pathological mechanisms. Currently, the post-stroke immune response is believed to be a pathological mechanism that exacerbates tissue damage and inhibits recovery. Within the immune system, a relatively rare leukocyte known as the Plasmacytoid Dendritic Cell (pDC) has been demonstrated to produce a disproportionately large amount of type 1 IFNs and pro-inflammatory cytokines through a TLR9-dependent pathway. Consequently, it is thought to serve as an orchestrator of the immune response. Therefore, this project sought to answer the question: How does pDC activity affect recovery after stroke? To answer this question, the effect of pDC activation via the TLR9-dependent pathway on post-stroke recovery was examined through usage of the widely accepted Middle Cerebral Artery Occlusion (MCAO) mouse model for stroke induction, the rotating beam test to assess recovery of motor function, neurological scoring, and confocal microscopy to visualize pDCs within the brain after stroke. It was found that activation of pDCs via the TLR9-dependent pathway leads to increased (statistically insignificant) infarct size. As well, pDC depletion facilitates short-term motor function recovery (partially significant), and a decrease in long-term development of infarct (statistically insignificant). This research is novel because it places pDC activity within the context of the post-stroke immune response, and has translational potential through elucidating a neuropathological mechanism of stroke.
Stroke is a leading cause of human disability and death, yet continues to lack an effective treatment, as well as a thorough understanding of its pathological mechanisms. Currently, the post-stroke immune response is believed to be a pathological mechanism that exacerbates tissue damage and inhibits recovery. Within the immune system, a relatively rare leukocyte known as the Plasmacytoid Dendritic Cell (pDC) has been demonstrated to produce a disproportionately large amount of type 1 IFNs and pro-inflammatory cytokines through a TLR9-dependent pathway. Consequently, it is thought to serve as an orchestrator of the immune response. Therefore, this project sought to answer the question: How does pDC activity affect recovery after stroke? To answer this question, the effect of pDC activation via the TLR9-dependent pathway on post-stroke recovery was examined through usage of the widely accepted Middle Cerebral Artery Occlusion (MCAO) mouse model for stroke induction, the rotating beam test to assess recovery of motor function, neurological scoring, and confocal microscopy to visualize pDCs within the brain after stroke. It was found that activation of pDCs via the TLR9-dependent pathway leads to increased (statistically insignificant) infarct size. As well, pDC depletion facilitates short-term motor function recovery (partially significant), and a decrease in long-term development of infarct (statistically insignificant). This research is novel because it places pDC activity within the context of the post-stroke immune response, and has translational potential through elucidating a neuropathological mechanism of stroke.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
Cilia are highly-conserved structures found in all major branches of eukaryotic tree that function in cellular motility, directional movement of extracellular fluid, and sensing of chemical and mechanical stimuli. Many developmental transitions are marked by cilium-dependent signaling events, and there is much interest in determining the mechanisms by which cells extend and retract cilia, given the relevance to human diseases caused by ciliary dysfunction (ciliopathies). Here, we present a chytrid fungus, Rhizoclosmatium globosum, as a possible model system for the study of the motile cilium, and characterize the process of cilium retraction in this organism. Chytrid fungi, unlike higher fungi, have zoospores with a single posterior cilium and a pair of centrioles at its base. We found cilium retraction in these organisms to be highly reproducible, occurring at the developmental shift between the motile, ciliated zoospore life-stage and reproductive, non-ciliated sporangium stage. Cilium retraction was accompanied by a simultaneous cytoplasmic rotation, suggesting that the cilium is “reeled” into the cell body. Sodium azide does not inhibit this process, indicating retraction may not require energy produced by the electron transport chain. The retracted cilium was visualized within the zoospore by immunofluorescence; it is initially coiled around the diameter of the cell, but is disassembled or degraded within 30 min. We showed chytrid cilium-related proteins to be more related to their human orthologs than those of C. reinhardtii and T. thermophila, the most commonly used single-cell, ciliated model systems. Due to the easily observable, reproducible nature of cilium retraction in chytrid fungi, and the relatively high homology between chytrid and human cilium-related proteins, we conclude that Rhizoclosmatium is an excellent candidate for a model system for the study of cilium retraction.
Cilia are highly-conserved structures found in all major branches of eukaryotic tree that function in cellular motility, directional movement of extracellular fluid, and sensing of chemical and mechanical stimuli. Many developmental transitions are marked by cilium-dependent signaling events, and there is much interest in determining the mechanisms by which cells extend and retract cilia, given the relevance to human diseases caused by ciliary dysfunction (ciliopathies). Here, we present a chytrid fungus, Rhizoclosmatium globosum, as a possible model system for the study of the motile cilium, and characterize the process of cilium retraction in this organism. Chytrid fungi, unlike higher fungi, have zoospores with a single posterior cilium and a pair of centrioles at its base. We found cilium retraction in these organisms to be highly reproducible, occurring at the developmental shift between the motile, ciliated zoospore life-stage and reproductive, non-ciliated sporangium stage. Cilium retraction was accompanied by a simultaneous cytoplasmic rotation, suggesting that the cilium is “reeled” into the cell body. Sodium azide does not inhibit this process, indicating retraction may not require energy produced by the electron transport chain. The retracted cilium was visualized within the zoospore by immunofluorescence; it is initially coiled around the diameter of the cell, but is disassembled or degraded within 30 min. We showed chytrid cilium-related proteins to be more related to their human orthologs than those of C. reinhardtii and T. thermophila, the most commonly used single-cell, ciliated model systems. Due to the easily observable, reproducible nature of cilium retraction in chytrid fungi, and the relatively high homology between chytrid and human cilium-related proteins, we conclude that Rhizoclosmatium is an excellent candidate for a model system for the study of cilium retraction.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
Inducing stable vascularization in engineered graft tissue is a major hurdle to the success of graft technology. Within the body, the vascular system frequently runs in parallel with the nervous system, and studies have shown that these cells may act synergistically. In order to better understand the benefits that a co-transplant of endothelial cells and neural-type cells might have, human umbilical vein endothelial cells (HUVECs) and myelinating Schwann cells (SCs) were grown in co-culture conditions and monitored. Vascular Endothelial Growth Factor (VEGF-A) and its receptors Vascular Endothelial Growth Factor Receptor 1 and 2 (VEGFR-1 and 2) are known to be highly influential in regulating the angiogenic process. To gauge whether co-culture of SCs has a beneficial impact on the activation of angiogenic sprouting, the expression of VEGFR-1 and VEGFR-2 as well as their activation in HUVECs was evaluated under varying co-culture conditions using GFP visualization of HUVECs and real time PCR analysis. While lower expression levels of VEGRF-2 were seen in all instances, defined expression patterns of VEGFR-1 and VEGF-A did emerge. Interestingly, visualization of the co-culture groups showed the formation of network-like structures not seen in the mono-culture groups in the absence of the usually required hydrogel matrix. Thus, the study may suggest the ability of SCs to facilitate HUVECs toward organization and an angiogenic, proliferative fate. With further research, this relationship may assist in vasculature growth and possible stabilization of vessels within large grafts.
Inducing stable vascularization in engineered graft tissue is a major hurdle to the success of graft technology. Within the body, the vascular system frequently runs in parallel with the nervous system, and studies have shown that these cells may act synergistically. In order to better understand the benefits that a co-transplant of endothelial cells and neural-type cells might have, human umbilical vein endothelial cells (HUVECs) and myelinating Schwann cells (SCs) were grown in co-culture conditions and monitored. Vascular Endothelial Growth Factor (VEGF-A) and its receptors Vascular Endothelial Growth Factor Receptor 1 and 2 (VEGFR-1 and 2) are known to be highly influential in regulating the angiogenic process. To gauge whether co-culture of SCs has a beneficial impact on the activation of angiogenic sprouting, the expression of VEGFR-1 and VEGFR-2 as well as their activation in HUVECs was evaluated under varying co-culture conditions using GFP visualization of HUVECs and real time PCR analysis. While lower expression levels of VEGRF-2 were seen in all instances, defined expression patterns of VEGFR-1 and VEGF-A did emerge. Interestingly, visualization of the co-culture groups showed the formation of network-like structures not seen in the mono-culture groups in the absence of the usually required hydrogel matrix. Thus, the study may suggest the ability of SCs to facilitate HUVECs toward organization and an angiogenic, proliferative fate. With further research, this relationship may assist in vasculature growth and possible stabilization of vessels within large grafts.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
Organisms’ responses to changes in external temperatures can dictate their ability or failure to survive and reproduce. Thus, the mechanisms that control these responses are thought to be highly conserved. However, the nature of these mechanisms and their interactions with sensory neural networks is not well understood on a very basic level. In particular, though it is known that many organisms’ internal processes are necessarily temperature-dependent due to the involvement of proteins with narrow optimum temperature ranges, a comparison of the function of these processes with and without the input of the relevant temperature-sensing neurons has not been performed. My research explores the relationship between external temperature changes and the corresponding changes in the locomotion of the model organism C. elegans, a nematode species, using MATLAB trackers to analyze spatial coordinates of the organisms in video clips captured under a microscope. Results indicate that locomotory speeds measured at discrete temperatures are more markedly different with the involvement of an intact thermoreceptor sensory neural network, whereas null mutant organisms with lack of function in their thermoreceptor neurons appeared to maintain a much more steady crawling speed across the same range of temperatures. With this information, I hope to facilitate continued exploration of the nature of temperature-dependent behaviors such as locomotion, and provide a foundation for acquiring related knowledge that may be applied to humans and other species.
Organisms’ responses to changes in external temperatures can dictate their ability or failure to survive and reproduce. Thus, the mechanisms that control these responses are thought to be highly conserved. However, the nature of these mechanisms and their interactions with sensory neural networks is not well understood on a very basic level. In particular, though it is known that many organisms’ internal processes are necessarily temperature-dependent due to the involvement of proteins with narrow optimum temperature ranges, a comparison of the function of these processes with and without the input of the relevant temperature-sensing neurons has not been performed. My research explores the relationship between external temperature changes and the corresponding changes in the locomotion of the model organism C. elegans, a nematode species, using MATLAB trackers to analyze spatial coordinates of the organisms in video clips captured under a microscope. Results indicate that locomotory speeds measured at discrete temperatures are more markedly different with the involvement of an intact thermoreceptor sensory neural network, whereas null mutant organisms with lack of function in their thermoreceptor neurons appeared to maintain a much more steady crawling speed across the same range of temperatures. With this information, I hope to facilitate continued exploration of the nature of temperature-dependent behaviors such as locomotion, and provide a foundation for acquiring related knowledge that may be applied to humans and other species.
Book
1 online resource.
Determining the factors that shape diversity and the persistence of species is a major aim of ecology and evolutionary biology, with direct conservation implications. Empirical data from present-day ecosystems have proven critical in characterizing how species interact with one another and their environment, but many of these studies lack a crucial element that would make them more applicable to projecting future dynamics: temporal resolution. I use Quaternary Caribbean lizards to investigate ecological theory about the repercussions of colonization and extinction on community structure. At a local scale, I find that the extinction of a large-bodied, predatory lizard, Leiocephalus, leads to ecological release in Anolis, a widespread Neotropical genus. This extinction is just one manifestation of a Caribbean-wide trend of size-biased and lineage-specific extinction, which results most dramatically in the extirpation of Leiocephalus from the Lesser Antilles, but also a loss of large-bodied lizards in other families. I then evaluate colonization events subsequent to extinction events in the Lesser Antilles. While I find that there are a few focal taxa that successfully colonize islands or are vulnerable to extirpation and extinction, the resulting communities are more heterogeneous than previous communities were. This contrasts with global trends of biotic homogenization and may reflect the realization of species richness-island area relationships in the Lesser Antilles. My results recapitulate empirical evidence from ongoing studies operating at ecological scales while also providing a glimpse into what the potential outcomes of continued colonization and extinctions will be during the Anthropocene.
Determining the factors that shape diversity and the persistence of species is a major aim of ecology and evolutionary biology, with direct conservation implications. Empirical data from present-day ecosystems have proven critical in characterizing how species interact with one another and their environment, but many of these studies lack a crucial element that would make them more applicable to projecting future dynamics: temporal resolution. I use Quaternary Caribbean lizards to investigate ecological theory about the repercussions of colonization and extinction on community structure. At a local scale, I find that the extinction of a large-bodied, predatory lizard, Leiocephalus, leads to ecological release in Anolis, a widespread Neotropical genus. This extinction is just one manifestation of a Caribbean-wide trend of size-biased and lineage-specific extinction, which results most dramatically in the extirpation of Leiocephalus from the Lesser Antilles, but also a loss of large-bodied lizards in other families. I then evaluate colonization events subsequent to extinction events in the Lesser Antilles. While I find that there are a few focal taxa that successfully colonize islands or are vulnerable to extirpation and extinction, the resulting communities are more heterogeneous than previous communities were. This contrasts with global trends of biotic homogenization and may reflect the realization of species richness-island area relationships in the Lesser Antilles. My results recapitulate empirical evidence from ongoing studies operating at ecological scales while also providing a glimpse into what the potential outcomes of continued colonization and extinctions will be during the Anthropocene.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
Circadian rhythms comprise a recurring pattern of behavioral and biological processes organized by a central pacemaker, the suprachiasmatic nucleus (SCN). Abnormal activity of this brain structure is associated with impaired hippocampal-dependent memory in both humans and animal models, but the process by which this occurs is unknown. Therefore, we used extracellular field potential recording techniques to assess the functional properties of the isolated hippocampus in circadian entrained and arrhythmic Siberian hamsters. This study finds evidence for plastic change in the hippocampus as a result of chronic non-circadian SCN activity. Input/output curves, paired-pulse tests, and induction of long-term potentiation were used to determine that intrinsic hippocampal circuitry and capacity for plasticity are conserved. Further examination in the presence of carbachol establishes that arrhythmia induces a change in cholinergic modulation of dentate gyrus function. For granule cells activated by perforant path stimulation, carbachol effected less depression of the population spike, and less paired-pulse facilitation, in arrhythmic animals compared to controls. Our findings are consistent with the hypothesis that a non-circadian firing pattern of SCN neurons could over-inhibit the medial septum, with learning deficits arising from attenuated septohippocampal signaling. Mitigating the cognitive detriment associated with circadian disruption is enormously relevant to shift workers, transmeridian flight crews, dementia patients, and the aging. Our model has high translational value and the present findings point to areas for pharmacological intervention as a therapeutic strategy. The relationships established here bring us closer to resolving learning deficits associated with circadian decline, and to understanding memory processes more broadly.
Circadian rhythms comprise a recurring pattern of behavioral and biological processes organized by a central pacemaker, the suprachiasmatic nucleus (SCN). Abnormal activity of this brain structure is associated with impaired hippocampal-dependent memory in both humans and animal models, but the process by which this occurs is unknown. Therefore, we used extracellular field potential recording techniques to assess the functional properties of the isolated hippocampus in circadian entrained and arrhythmic Siberian hamsters. This study finds evidence for plastic change in the hippocampus as a result of chronic non-circadian SCN activity. Input/output curves, paired-pulse tests, and induction of long-term potentiation were used to determine that intrinsic hippocampal circuitry and capacity for plasticity are conserved. Further examination in the presence of carbachol establishes that arrhythmia induces a change in cholinergic modulation of dentate gyrus function. For granule cells activated by perforant path stimulation, carbachol effected less depression of the population spike, and less paired-pulse facilitation, in arrhythmic animals compared to controls. Our findings are consistent with the hypothesis that a non-circadian firing pattern of SCN neurons could over-inhibit the medial septum, with learning deficits arising from attenuated septohippocampal signaling. Mitigating the cognitive detriment associated with circadian disruption is enormously relevant to shift workers, transmeridian flight crews, dementia patients, and the aging. Our model has high translational value and the present findings point to areas for pharmacological intervention as a therapeutic strategy. The relationships established here bring us closer to resolving learning deficits associated with circadian decline, and to understanding memory processes more broadly.
Book
1 online resource.
In neuroscience, methodological advancements bring about new discoveries, while unanswered questions prompt technical innovations. My thesis involves both aspects, contributing to the genetic toolkit in fruit flies as well as our understanding of olfactory behavior. Innate olfactory attraction and aversion are observed throughout the animal kingdom, but it is not well understood how such valences are encoded by the sensory circuits, how the relevant behaviors are implemented, or, more fundementally, to what extent attraction and aversion share principles of information processing. Using state-of-the-art genetic tools, I demonstrate that aversion is much more robust than attraction against blockade of the sensory circuits (Chapter 2), and that aversion engages specific kinematic and motor-related neurons (Chapter 3). Aversion and attraction are thus likely processed by distinct circuits and principles throughout the sensory-motor transformation. In addition, Chapter 4 not only provides another case where attraction but not averson was affected by a genetic perturbation, but may also link a circuit for specific behavior to a gene necessary for the function of the circuit. To further our ability to explore neural circuits, I developed a transcriptional reporter of intracellular calcium (TRIC, Chapter 5). TRIC signals in the sensory systems depend on neuronal activity, and it sucessfully quantified neuronal responses that change slowly, such as those of neuropeptide F-expressing neurons to sexual deprivation and neuroendocrine pars intercerebralis cells to food and arousal. In the last case, I also demonstrate that TRIC can be used for circuit manipulation. TRIC can thus monitor neuromodulatory circuits whose activity varies slowly with the physiological states of the animal, and its modular design will facilitate future optimizations for even broader applications.
In neuroscience, methodological advancements bring about new discoveries, while unanswered questions prompt technical innovations. My thesis involves both aspects, contributing to the genetic toolkit in fruit flies as well as our understanding of olfactory behavior. Innate olfactory attraction and aversion are observed throughout the animal kingdom, but it is not well understood how such valences are encoded by the sensory circuits, how the relevant behaviors are implemented, or, more fundementally, to what extent attraction and aversion share principles of information processing. Using state-of-the-art genetic tools, I demonstrate that aversion is much more robust than attraction against blockade of the sensory circuits (Chapter 2), and that aversion engages specific kinematic and motor-related neurons (Chapter 3). Aversion and attraction are thus likely processed by distinct circuits and principles throughout the sensory-motor transformation. In addition, Chapter 4 not only provides another case where attraction but not averson was affected by a genetic perturbation, but may also link a circuit for specific behavior to a gene necessary for the function of the circuit. To further our ability to explore neural circuits, I developed a transcriptional reporter of intracellular calcium (TRIC, Chapter 5). TRIC signals in the sensory systems depend on neuronal activity, and it sucessfully quantified neuronal responses that change slowly, such as those of neuropeptide F-expressing neurons to sexual deprivation and neuroendocrine pars intercerebralis cells to food and arousal. In the last case, I also demonstrate that TRIC can be used for circuit manipulation. TRIC can thus monitor neuromodulatory circuits whose activity varies slowly with the physiological states of the animal, and its modular design will facilitate future optimizations for even broader applications.