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Undergraduate Theses, Department of Biology, 2013-2014
Adult stem cells are an important class of cells responsible for the maintenance and regeneration of the body’s many tissues. These cells are under heavy investigation for potential therapeutic roles. However, one of the rate limiting steps is an understanding of the exact mechanisms and genes that regulate this class of cells. To better understand the genetic program regulating adult stem cells, a forward genetic screen was utilized involving a two-stage screening process comprising a Flp/FRT primary screen and an EGUF/hid secondary screen . Out of 3,118 mutant males produced, 1,041 were recovered, of which 412 showed loss of germ line clones and one showed overproliferation of germline clones in the primary screen. Of the 412 germ cell loss mutations, 59 passed through a secondary screen for cell lethality (EGUF/hid). 6 mutant strains have been partially or completely mapped, uncovering germ cell loss mutations in DNA Replication-Related Element Factor (DREF), Apoptosis Inducing Factor (AIF), Guanylyl Cyclase 32E (Gyc32E) and two other loci, as well as a germ cell overproliferation mutation in Star. Further research on DREF has shown that the allele we uncovered genetically separates DREF’s role in cell division and DNA-replication from its role in adult stem cell maintenance. Furthermore, I have uncovered a novel antagonistic interaction between DREF and members of the NuRD complex that is essential for the regulation of germline stem cell maintenance. These results suggest that our understanding of the genetic program of adult stem cells can still be enriched by well-designed, classic genetic screens.
Adult stem cells are an important class of cells responsible for the maintenance and regeneration of the body’s many tissues. These cells are under heavy investigation for potential therapeutic roles. However, one of the rate limiting steps is an understanding of the exact mechanisms and genes that regulate this class of cells. To better understand the genetic program regulating adult stem cells, a forward genetic screen was utilized involving a two-stage screening process comprising a Flp/FRT primary screen and an EGUF/hid secondary screen . Out of 3,118 mutant males produced, 1,041 were recovered, of which 412 showed loss of germ line clones and one showed overproliferation of germline clones in the primary screen. Of the 412 germ cell loss mutations, 59 passed through a secondary screen for cell lethality (EGUF/hid). 6 mutant strains have been partially or completely mapped, uncovering germ cell loss mutations in DNA Replication-Related Element Factor (DREF), Apoptosis Inducing Factor (AIF), Guanylyl Cyclase 32E (Gyc32E) and two other loci, as well as a germ cell overproliferation mutation in Star. Further research on DREF has shown that the allele we uncovered genetically separates DREF’s role in cell division and DNA-replication from its role in adult stem cell maintenance. Furthermore, I have uncovered a novel antagonistic interaction between DREF and members of the NuRD complex that is essential for the regulation of germline stem cell maintenance. These results suggest that our understanding of the genetic program of adult stem cells can still be enriched by well-designed, classic genetic screens.
Collection
Undergraduate Theses, Department of Biology, 2013-2014
DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here I present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. In tests I directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. I applied cycled ligation assembly to construct chimeric proteins, revealing functional roles for individual domains of the Hedgehog signaling pathway protein PTCH1. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.
DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here I present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. In tests I directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. I applied cycled ligation assembly to construct chimeric proteins, revealing functional roles for individual domains of the Hedgehog signaling pathway protein PTCH1. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.
Collection
Undergraduate Theses, Department of Biology, 2013-2014
Recent work has established disruption of neurogenesis as a key cause of cognitive decline after use of brain irradiation to treat primary and metastatic tumors in children. Yet though this is widely accepted, little is known about the possibilities of restoration of normal neural stem cell (NSC) function after such a treatment, either through stem cell transplant or by promoting endogenous recovery by enhancing trophic effects. It is yet to be discovered whether endogenous quiescent neural stem cells (qNSCs) that are present in the irradiated brain have the potential to repopulate an injured neurogenic niche or whether resident stem cells are themselves damaged and are unable to repopulate the niche. It is also possible that a niche occupied by defective stem cells may simply block undamaged cells from occupying the niche. In this case, it may be necessary to ablate cells to create space for engraftment. Whereas most prior research has focused on solely anti-mitotic methods that spare rarely dividing quiescent NSCs, this research analyzes effective ablation of neural stem cells, including quiescent NSCs, in the dentate gyrus of the subgranular zone through the use of diphtheria toxin receptor-mediated cell death. My hypothesis was that partial ablation would show that qNSCs could repopulate the affected niche over time. After ablation treatment, it was observed that the number of nestin+ cells in the dentate gyrus (DG) was drastically reduced, and remained that way following a two-month recovery period. Along with this lack of renewal was partial ablation of neurogenesis in the olfactory bulb, evaluated by quantification of IdU+/CldU+ cells two months after ablation treatment. The data shown here provide new insight into the response of the neurogenic niche and surviving cells to effective ablation of local NSCs. This will potentially have a great impact on further research to be done regarding recovery procedures for irradiation-treated children.
Recent work has established disruption of neurogenesis as a key cause of cognitive decline after use of brain irradiation to treat primary and metastatic tumors in children. Yet though this is widely accepted, little is known about the possibilities of restoration of normal neural stem cell (NSC) function after such a treatment, either through stem cell transplant or by promoting endogenous recovery by enhancing trophic effects. It is yet to be discovered whether endogenous quiescent neural stem cells (qNSCs) that are present in the irradiated brain have the potential to repopulate an injured neurogenic niche or whether resident stem cells are themselves damaged and are unable to repopulate the niche. It is also possible that a niche occupied by defective stem cells may simply block undamaged cells from occupying the niche. In this case, it may be necessary to ablate cells to create space for engraftment. Whereas most prior research has focused on solely anti-mitotic methods that spare rarely dividing quiescent NSCs, this research analyzes effective ablation of neural stem cells, including quiescent NSCs, in the dentate gyrus of the subgranular zone through the use of diphtheria toxin receptor-mediated cell death. My hypothesis was that partial ablation would show that qNSCs could repopulate the affected niche over time. After ablation treatment, it was observed that the number of nestin+ cells in the dentate gyrus (DG) was drastically reduced, and remained that way following a two-month recovery period. Along with this lack of renewal was partial ablation of neurogenesis in the olfactory bulb, evaluated by quantification of IdU+/CldU+ cells two months after ablation treatment. The data shown here provide new insight into the response of the neurogenic niche and surviving cells to effective ablation of local NSCs. This will potentially have a great impact on further research to be done regarding recovery procedures for irradiation-treated children.
Book
1 online resource.
Temperature plays a vital role in shaping species' biology and biogeography, but the response of marine species to changes in temperature is still poorly understood over time scales relevant to climate change. In this thesis, I use a single species as a case study to explore acclimation and adaptation to temperature at the levels of gene sequence, gene expression, and whole-animal physiology. My study species is the European green crab, Carcinus maenas, a globally invasive temperate species that thrives across a wide range of environmental temperatures. By comparing an invasive species across seven populations in its native and invasive range, I was able to explore both short-term (invasive range) and long-term (native range) impacts of environmental temperature. To lay the groundwork for this project, I first review the literature on adaptation in marine invasive species. While quantitative research strongly suggests a role for adaptation, the dearth of integrated genetic-quantitative work severely limits our understanding of this process. The rest of this thesis attempts to fill this gap with empirical research. First, I describe the thermal physiology of green crabs in detail, and find that green crabs have high inherent eurythermality and acclimatory plasticity. Despite this thermal flexibility, I also observed potentially adaptive differentiation among populations, particularly within the native range. Population genetics supported a role for significant local adaptation between populations in the species' native range. I identified a number of specific genes likely involved in long-term adaptation between northern and southern native range populations, suggesting that innate immunity and muscle function may be under selection. These data also suggested a more limited role for ongoing, rapid adaptation in the species' invasive range. Patterns of gene expression integrate neatly with the genetic and physiological data, and I identified two groups of co-expressed genes whose expression appears related to adaptive differences in intraspecific physiology. Taken together, this project provides detailed, integrative evidence for the importance of acclimatory plasticity and both short- and long-term adaptation in success across a wide range of thermal environments in a high gene flow species. Finally, I discuss the broader implications of this work to species persistence in a rapidly changing ocean.
Temperature plays a vital role in shaping species' biology and biogeography, but the response of marine species to changes in temperature is still poorly understood over time scales relevant to climate change. In this thesis, I use a single species as a case study to explore acclimation and adaptation to temperature at the levels of gene sequence, gene expression, and whole-animal physiology. My study species is the European green crab, Carcinus maenas, a globally invasive temperate species that thrives across a wide range of environmental temperatures. By comparing an invasive species across seven populations in its native and invasive range, I was able to explore both short-term (invasive range) and long-term (native range) impacts of environmental temperature. To lay the groundwork for this project, I first review the literature on adaptation in marine invasive species. While quantitative research strongly suggests a role for adaptation, the dearth of integrated genetic-quantitative work severely limits our understanding of this process. The rest of this thesis attempts to fill this gap with empirical research. First, I describe the thermal physiology of green crabs in detail, and find that green crabs have high inherent eurythermality and acclimatory plasticity. Despite this thermal flexibility, I also observed potentially adaptive differentiation among populations, particularly within the native range. Population genetics supported a role for significant local adaptation between populations in the species' native range. I identified a number of specific genes likely involved in long-term adaptation between northern and southern native range populations, suggesting that innate immunity and muscle function may be under selection. These data also suggested a more limited role for ongoing, rapid adaptation in the species' invasive range. Patterns of gene expression integrate neatly with the genetic and physiological data, and I identified two groups of co-expressed genes whose expression appears related to adaptive differences in intraspecific physiology. Taken together, this project provides detailed, integrative evidence for the importance of acclimatory plasticity and both short- and long-term adaptation in success across a wide range of thermal environments in a high gene flow species. Finally, I discuss the broader implications of this work to species persistence in a rapidly changing ocean.
Collection
Undergraduate Theses, Department of Biology, 2013-2014
HIV usually makes use of either the CCR5 or CXCR4 co-receptors to gain entry into cells. Which coreceptor the virus uses (the tropism) determines the efficacy of different cell entry-inhibiting antiretroviral drugs, and is therefore important medical information. HIV tropism may be accurately assessed with sensitive recombinant virus phenotyping tests such as the Trofile assay, but these tests are often prohibitively expensive. Viral genome sequencing and interpretation of genotype using free online software programs such as Web PSSM and Geno2Pheno offer a low-cost alternative by predicting tropism from the V3 loop sequence in the HIV env gene. However, these programs' predictions are insensitive to minority viral variants and may be less accurate than the commercial recombinant virus phenotypic assays. We sequenced proviral subtype B HIV-1 DNA from peripheral blood mononuclear cell (PBMC) samples obtained from patients under drug-induced viral suppression and viral RNA from plasma samples obtained from the same patients after viral re-emergence. Predictions of re-emergent viral tropism were made by Geno2Pheno and Web PSSM assessment of the proviral DNA V3 loop sequences. Trofile tests were performed on plasma samples to provide a gold-standard re-emergent virus phenotype. Through statistical and qualitative analysis of these paired prediction and tropism data, we tested the hypothesis that the tropism of virus re-emerging into the blood following drug interruption can be accurately predicted by Geno2Pheno and Web PSSM analysis of proviral DNA. We show here that proviral DNA sequence analysis was able to predict re-emergent plasma virus tropism with 83.3% success, and exclusive R5 tropism with 87.0% success. Generalized linear modeling showed a highly significant relationship between prediction-tropism discordance and lower DNA-RNA sequence similarity. Qualitative analysis of clone env sequences demonstrated sequence interpretation accuracy by both Geno2Pheno and Web PSSM. Together, these results suggest that proviral PBMC DNA consensus sequences may fail to predict the re-emergent plasma virus tropism in more than 10% of cases. Differences between proviral DNA-based tropism predictions and re-emergent plasma virus phenotype may result from the selection of minority variants and the evolution of plasma virus.
HIV usually makes use of either the CCR5 or CXCR4 co-receptors to gain entry into cells. Which coreceptor the virus uses (the tropism) determines the efficacy of different cell entry-inhibiting antiretroviral drugs, and is therefore important medical information. HIV tropism may be accurately assessed with sensitive recombinant virus phenotyping tests such as the Trofile assay, but these tests are often prohibitively expensive. Viral genome sequencing and interpretation of genotype using free online software programs such as Web PSSM and Geno2Pheno offer a low-cost alternative by predicting tropism from the V3 loop sequence in the HIV env gene. However, these programs' predictions are insensitive to minority viral variants and may be less accurate than the commercial recombinant virus phenotypic assays. We sequenced proviral subtype B HIV-1 DNA from peripheral blood mononuclear cell (PBMC) samples obtained from patients under drug-induced viral suppression and viral RNA from plasma samples obtained from the same patients after viral re-emergence. Predictions of re-emergent viral tropism were made by Geno2Pheno and Web PSSM assessment of the proviral DNA V3 loop sequences. Trofile tests were performed on plasma samples to provide a gold-standard re-emergent virus phenotype. Through statistical and qualitative analysis of these paired prediction and tropism data, we tested the hypothesis that the tropism of virus re-emerging into the blood following drug interruption can be accurately predicted by Geno2Pheno and Web PSSM analysis of proviral DNA. We show here that proviral DNA sequence analysis was able to predict re-emergent plasma virus tropism with 83.3% success, and exclusive R5 tropism with 87.0% success. Generalized linear modeling showed a highly significant relationship between prediction-tropism discordance and lower DNA-RNA sequence similarity. Qualitative analysis of clone env sequences demonstrated sequence interpretation accuracy by both Geno2Pheno and Web PSSM. Together, these results suggest that proviral PBMC DNA consensus sequences may fail to predict the re-emergent plasma virus tropism in more than 10% of cases. Differences between proviral DNA-based tropism predictions and re-emergent plasma virus phenotype may result from the selection of minority variants and the evolution of plasma virus.
Book
1 online resource.
Global analysis of post-translational modifications (PTMs) by mass spectrometry facilitates interrogation of complex signaling networks. We have utilized this technology and developed analytical tools to combine peptide level data into high confidence PTM site identifications and to assess the quality of quantification measurements. We have applied this workflow in order to study three systems: 1) mitochondrial acetylation and phosphorylation during cardiac ischemia, 2) phosphorylation regulation Toxoplasma gondii egress from host cells, and 3) early phosphorylation events during hedgehog signaling. 1. Acetylation is a highly abundant modification in the mitochondrion, but we lack a general understanding of both its regulation and functional relevance, especially with regard to the acetyltransfer mechanism. To explore the nature of mitochondrial acetylation, we compared several features of mitochondrial phosphorylation and acetylation sites, showcasing stark differences between these two PTMs. We found that mitochondrial acetylation is biased towards ordered secondary protein structures, and that acetylation and succinylation modify the same residues in an unbiased, unenriched sample. We suggest that these results are consistent with low levels of background, non-functional acetylation. In order to understand acetylation regulation, we must know the mechanism of acetyl transfer. Our findings indicate that either a putative mitochondrial acetyltransferase or family of acyltransferases exist that operate non-specifically and recognize substrates in a matter entirely distinct from kinases, or else that a substantial fraction of observed mitochondrial lysine acylation results from non-enzymatic modification by acyl-CoAs. 2. We report the first phosphoproteomes of T. gondii and P. falciparum. These datasets demonstrated for the first time tyrosine phosphorylation in P. falciparum, and unusual phosphorylation-site motifs for P. falciparum. We observed extensive phosphorylation beyond the intercellular parasites' boundaries. In a follow-up study in T. gondii, we focused on the role of calcium-dependent protein kinase 3 (TgCDPK3), which regulates parasite egress from the host cell in the presence of a calciumionophore. We measured relative phosphorylation site abundance in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling by amino acids in cell culture (SILAC). Our results demonstrate that TgCDPK3 regulates several functions in addition to egress. We also observed novel phosphorylation events on proteins predicted to play a role in egress, and which implicate TgCDPK3 in regulating other calcium-dependent signaling pathways, including TgCDPK1. 3. Finally, we have initiated a study of the early phosphorylation signaling events in response to hedgehog signaling. Here, we report preliminary results that implicate a kinase, casein kinase 2 (CK2), in hedgehog signaling. Work is currently being undertaken in the Scott Lab to further characterize the role of CK2 in hedgehog signaling while we analyze biological replicates to expand proteome coverage and validate our initial hits.
Global analysis of post-translational modifications (PTMs) by mass spectrometry facilitates interrogation of complex signaling networks. We have utilized this technology and developed analytical tools to combine peptide level data into high confidence PTM site identifications and to assess the quality of quantification measurements. We have applied this workflow in order to study three systems: 1) mitochondrial acetylation and phosphorylation during cardiac ischemia, 2) phosphorylation regulation Toxoplasma gondii egress from host cells, and 3) early phosphorylation events during hedgehog signaling. 1. Acetylation is a highly abundant modification in the mitochondrion, but we lack a general understanding of both its regulation and functional relevance, especially with regard to the acetyltransfer mechanism. To explore the nature of mitochondrial acetylation, we compared several features of mitochondrial phosphorylation and acetylation sites, showcasing stark differences between these two PTMs. We found that mitochondrial acetylation is biased towards ordered secondary protein structures, and that acetylation and succinylation modify the same residues in an unbiased, unenriched sample. We suggest that these results are consistent with low levels of background, non-functional acetylation. In order to understand acetylation regulation, we must know the mechanism of acetyl transfer. Our findings indicate that either a putative mitochondrial acetyltransferase or family of acyltransferases exist that operate non-specifically and recognize substrates in a matter entirely distinct from kinases, or else that a substantial fraction of observed mitochondrial lysine acylation results from non-enzymatic modification by acyl-CoAs. 2. We report the first phosphoproteomes of T. gondii and P. falciparum. These datasets demonstrated for the first time tyrosine phosphorylation in P. falciparum, and unusual phosphorylation-site motifs for P. falciparum. We observed extensive phosphorylation beyond the intercellular parasites' boundaries. In a follow-up study in T. gondii, we focused on the role of calcium-dependent protein kinase 3 (TgCDPK3), which regulates parasite egress from the host cell in the presence of a calciumionophore. We measured relative phosphorylation site abundance in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling by amino acids in cell culture (SILAC). Our results demonstrate that TgCDPK3 regulates several functions in addition to egress. We also observed novel phosphorylation events on proteins predicted to play a role in egress, and which implicate TgCDPK3 in regulating other calcium-dependent signaling pathways, including TgCDPK1. 3. Finally, we have initiated a study of the early phosphorylation signaling events in response to hedgehog signaling. Here, we report preliminary results that implicate a kinase, casein kinase 2 (CK2), in hedgehog signaling. Work is currently being undertaken in the Scott Lab to further characterize the role of CK2 in hedgehog signaling while we analyze biological replicates to expand proteome coverage and validate our initial hits.
Collection
Undergraduate Theses, Department of Biology, 2013-2014
Identifying natural reservoirs and vectors for pathogens is central to understanding how animal diseases spill over into human populations. The human and animal disease-causing bacteria of the genus Bartonella often rely on a mammalian host and arthropod vector, and it has been suggested that bats and bat flies could fulfill these roles. Comparing the prevalence and genetic similarity of Bartonella spp. in Costa Rican bats and the bat flies parasitizing them, I found that Bartonella spp. were more prevalent in bat flies than in bats, 52.7% and 31.8% respectively, and that these Bartonella were diverse and sometimes shared between bats and bat flies. Phylogenetic analysis further revealed that some Bartonella identified in bats and bat flies were similar to strains known to infect humans and other mammals. The high prevalence and sharing of Bartonella in bat flies and bats supports a role for bat flies as a potential vector for Bartonella and other pathogens, while the genetic diversity and similarity to known species suggests that Bartonella could spill over from bats and bat flies into humans and animals sharing the landscape.
Identifying natural reservoirs and vectors for pathogens is central to understanding how animal diseases spill over into human populations. The human and animal disease-causing bacteria of the genus Bartonella often rely on a mammalian host and arthropod vector, and it has been suggested that bats and bat flies could fulfill these roles. Comparing the prevalence and genetic similarity of Bartonella spp. in Costa Rican bats and the bat flies parasitizing them, I found that Bartonella spp. were more prevalent in bat flies than in bats, 52.7% and 31.8% respectively, and that these Bartonella were diverse and sometimes shared between bats and bat flies. Phylogenetic analysis further revealed that some Bartonella identified in bats and bat flies were similar to strains known to infect humans and other mammals. The high prevalence and sharing of Bartonella in bat flies and bats supports a role for bat flies as a potential vector for Bartonella and other pathogens, while the genetic diversity and similarity to known species suggests that Bartonella could spill over from bats and bat flies into humans and animals sharing the landscape.
Book
1 online resource.
Epithelial cells sometimes display polarity along the axis orthogonal to the apicobasal axis -- a phenomenon referred to as planar cell polarity (PCP). This polarization biases organization of the cytoskeleton, and is required for a range of fundamental processes including, but not limited to, oriented cell division, polarized function of cilia, and the control of collective cell migration events during development. Consequently, deregulation or dysfunction of PCP genes has been linked to disorders including deafness as a result of misorientation of inner ear cells, neural tube closure defects because of a failure to execute convergent & extension, and tumor invasiveness. Despite having identified some of the key proteins required to achieve PCP, much is still unknown about their molecular functions. Evolutionarily conserved proteins, known as the core PCP proteins, are distributed asymmetrically within wing epithelial cells and form complexes within the proximal or distal cortical domains. Here, we demonstrate that these diametrically opposed complexes participate in a bi-directional negative feedback loop that simultaneously produces their asymmetric distribution within cells and reinforces the orientation of polarity to neighbors by transmitting polarity information intercellularly. We found that one of the core PCP proteins, Prickle (Pk), is important for negative feedback as it is required to mediate the exclusion of distal and proximal complexes from the plasma membrane during acquisition of asymmetry. Moreover, we found that the level of Pk is regulated by the Cullin1 (Cul1)/SkpA/Supernumerary limbs (Slimb) E3 ubiquitin ligase complex. Our observations lead us to believe that loss of, or excess, Pk perturbs PCP signaling through the disruption of feedback. Ultimately, these results highlight an underlying molecular mechanism for the generation of planar polarity. Finally, the approaches presented in this monograph can further help elucidate the molecular functions of the other core PCP proteins.
Epithelial cells sometimes display polarity along the axis orthogonal to the apicobasal axis -- a phenomenon referred to as planar cell polarity (PCP). This polarization biases organization of the cytoskeleton, and is required for a range of fundamental processes including, but not limited to, oriented cell division, polarized function of cilia, and the control of collective cell migration events during development. Consequently, deregulation or dysfunction of PCP genes has been linked to disorders including deafness as a result of misorientation of inner ear cells, neural tube closure defects because of a failure to execute convergent & extension, and tumor invasiveness. Despite having identified some of the key proteins required to achieve PCP, much is still unknown about their molecular functions. Evolutionarily conserved proteins, known as the core PCP proteins, are distributed asymmetrically within wing epithelial cells and form complexes within the proximal or distal cortical domains. Here, we demonstrate that these diametrically opposed complexes participate in a bi-directional negative feedback loop that simultaneously produces their asymmetric distribution within cells and reinforces the orientation of polarity to neighbors by transmitting polarity information intercellularly. We found that one of the core PCP proteins, Prickle (Pk), is important for negative feedback as it is required to mediate the exclusion of distal and proximal complexes from the plasma membrane during acquisition of asymmetry. Moreover, we found that the level of Pk is regulated by the Cullin1 (Cul1)/SkpA/Supernumerary limbs (Slimb) E3 ubiquitin ligase complex. Our observations lead us to believe that loss of, or excess, Pk perturbs PCP signaling through the disruption of feedback. Ultimately, these results highlight an underlying molecular mechanism for the generation of planar polarity. Finally, the approaches presented in this monograph can further help elucidate the molecular functions of the other core PCP proteins.
Collection
Undergraduate Theses, Department of Biology, 2013-2014
Pancreatic ductal adrenocarcinoma (PDAC) is one of the most aggressive cancers with poor prognosis at time of diagnosis and high mortality due to few effective treatment options. This work seeks to show the power of bioinformatics to quickly and efficiently repurpose FDA-approved drugs for PDAC. We used a systematic drug repositioning bioinformatics approach that compared a concise and comprehensive array of gene expression profiles to identify potential FDA approved drugs. Initial screening of the top candidates by MTT survival assay narrowed the search to Guanabenz, an alpha-2 receptor (ADRA2) agonist and antihypertensive drug. MTT survival assays showed that Guanabenz significantly lowered survival of various human and mouse PDAC cell lines. Immunoblot analysis showed Guanabenz attenuating pCREB (involved in GPCR signaling) and ERK ½ kinase in the Ras signaling pathway. Mouse models treated with Guanabenz showed the drug’s tumor suppressive properties with the prevention of preneoplastic legion (PanIN) formation, a landmark precursor in tumorigenesis of pancreatitis induced PDAC. These experiments confirm the identity of Guanabenz as a novel treatment for PDAC as well as the potential in targeting the ADRA2 pathway in PDAC drug development. Further, this work confirms drug repositioning as an effective way of identifying novel treatments in PDAC.
Pancreatic ductal adrenocarcinoma (PDAC) is one of the most aggressive cancers with poor prognosis at time of diagnosis and high mortality due to few effective treatment options. This work seeks to show the power of bioinformatics to quickly and efficiently repurpose FDA-approved drugs for PDAC. We used a systematic drug repositioning bioinformatics approach that compared a concise and comprehensive array of gene expression profiles to identify potential FDA approved drugs. Initial screening of the top candidates by MTT survival assay narrowed the search to Guanabenz, an alpha-2 receptor (ADRA2) agonist and antihypertensive drug. MTT survival assays showed that Guanabenz significantly lowered survival of various human and mouse PDAC cell lines. Immunoblot analysis showed Guanabenz attenuating pCREB (involved in GPCR signaling) and ERK ½ kinase in the Ras signaling pathway. Mouse models treated with Guanabenz showed the drug’s tumor suppressive properties with the prevention of preneoplastic legion (PanIN) formation, a landmark precursor in tumorigenesis of pancreatitis induced PDAC. These experiments confirm the identity of Guanabenz as a novel treatment for PDAC as well as the potential in targeting the ADRA2 pathway in PDAC drug development. Further, this work confirms drug repositioning as an effective way of identifying novel treatments in PDAC.
Book
1 online resource.
Lysine methylation is a chemical modification that occurs naturally in proteins. This post-translational modification is small and preserves the charge on the lysine sidechain, making relatively little chemical difference. However, misregulation of lysine methylation can cause a host of human cancers, developmental disorders, and other diseases. The most prominent example of lysine methylation occurs within histone proteins, but it can also occur on non-histone proteins. The proteomic extent of methylation and the diversity of processes it might regulate were previously unclear. The work presented here focuses on identifying the cellular "methylome" and understanding methylation signaling pathways within cells. I also examine the substrate repertoire of the methyltransferase SETMAR and identify the spliceosome component snRNP70 as a novel SETMAR substrate.
Lysine methylation is a chemical modification that occurs naturally in proteins. This post-translational modification is small and preserves the charge on the lysine sidechain, making relatively little chemical difference. However, misregulation of lysine methylation can cause a host of human cancers, developmental disorders, and other diseases. The most prominent example of lysine methylation occurs within histone proteins, but it can also occur on non-histone proteins. The proteomic extent of methylation and the diversity of processes it might regulate were previously unclear. The work presented here focuses on identifying the cellular "methylome" and understanding methylation signaling pathways within cells. I also examine the substrate repertoire of the methyltransferase SETMAR and identify the spliceosome component snRNP70 as a novel SETMAR substrate.
Collection
Undergraduate Theses, Department of Biology, 2013-2014
High levels of invasion and disturbance in the lowland wet forests of Hawaii have led to a need for novel management strategies for the conservation of biodiversity, carbon (C) storage, and other ecosystem services. The hybrid ecosystems approach represents a method by which species traits can be used in the creation of ecosystems which optimize desirable characteristics. In the lowland wet forests of Hawaii, C storage is one of these important traits, given the continual increase in atmospheric carbon and global environmental change. In this study we examined soil humus decomposition and leaf litter decomposition. We asked whether patterns seen in rates of litter turnover across nine species currently present in the lowland wet forests of the big island of Hawaii would be present in the turnover of humus originating from single species stands. We used a soda-lime absorption measurement assay to quantify soil decomposition over a period of four weeks and performed a four-month common site litter decomposition experiment. Total C efflux values for the full four week period ranged from 13.4 – 16.9 g C/kg soil, with little difference among species. Litter decomposition had greater variability. The fastest decomposition occurred in Thespesia populnea (milo) litter, with 17.6% of mass left at the end of the decomposition period, while Persea americana (avocado) litter was the slowest to decompose and maintained 78.6% of its mass. Our results suggest a decoupling of litter and humus decomposition rates. These findings provide additional understanding on the extent to which hybrid ecosystems may be able to influence C storage and turnover through species treatments.
High levels of invasion and disturbance in the lowland wet forests of Hawaii have led to a need for novel management strategies for the conservation of biodiversity, carbon (C) storage, and other ecosystem services. The hybrid ecosystems approach represents a method by which species traits can be used in the creation of ecosystems which optimize desirable characteristics. In the lowland wet forests of Hawaii, C storage is one of these important traits, given the continual increase in atmospheric carbon and global environmental change. In this study we examined soil humus decomposition and leaf litter decomposition. We asked whether patterns seen in rates of litter turnover across nine species currently present in the lowland wet forests of the big island of Hawaii would be present in the turnover of humus originating from single species stands. We used a soda-lime absorption measurement assay to quantify soil decomposition over a period of four weeks and performed a four-month common site litter decomposition experiment. Total C efflux values for the full four week period ranged from 13.4 – 16.9 g C/kg soil, with little difference among species. Litter decomposition had greater variability. The fastest decomposition occurred in Thespesia populnea (milo) litter, with 17.6% of mass left at the end of the decomposition period, while Persea americana (avocado) litter was the slowest to decompose and maintained 78.6% of its mass. Our results suggest a decoupling of litter and humus decomposition rates. These findings provide additional understanding on the extent to which hybrid ecosystems may be able to influence C storage and turnover through species treatments.
Collection
Undergraduate Theses, Department of Biology, 2013-2014
Gene targeting through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated homologous recombination is a novel and powerful technique with promising applications in human disease modeling and therapeutic development. However, one major factor limiting genome editing efficiency is the time and difficulty of obtaining a transgenic animal, which may require several generations of breeding and expensive maintenance. While the establishment of an animal line with endogenous Cas9 nuclease expression could potentially accelerate the study of both gene knock-out and overexpression models, particularly in primary cells, previous work has yet to test the feasibility of such a system. This project provided the first in vitro proof of concept for a Cas9 transgenic mouse line as a research tool through the generation and characterization of Cas9-expressing mouse cells. In NIH3T3 cells, constitutive expression of either the Cas9 nuclease or nickase resulted in toxicity, with high levels of wild-type Cas9 expression appearing to be lethal over time. Following delivery of a CRISPR guide RNA sequence into cultured Cas9-expressing mouse cells via lentivirus, efficient genome editing was also observed. These findings demonstrate the prospect of high-speed multiplex genome engineering in mouse primary cells with future clinical implications such as cancer modeling and gene therapy. Moving forward, the generation of an inducible Cas9 transgenic animal should be pursued.
Gene targeting through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated homologous recombination is a novel and powerful technique with promising applications in human disease modeling and therapeutic development. However, one major factor limiting genome editing efficiency is the time and difficulty of obtaining a transgenic animal, which may require several generations of breeding and expensive maintenance. While the establishment of an animal line with endogenous Cas9 nuclease expression could potentially accelerate the study of both gene knock-out and overexpression models, particularly in primary cells, previous work has yet to test the feasibility of such a system. This project provided the first in vitro proof of concept for a Cas9 transgenic mouse line as a research tool through the generation and characterization of Cas9-expressing mouse cells. In NIH3T3 cells, constitutive expression of either the Cas9 nuclease or nickase resulted in toxicity, with high levels of wild-type Cas9 expression appearing to be lethal over time. Following delivery of a CRISPR guide RNA sequence into cultured Cas9-expressing mouse cells via lentivirus, efficient genome editing was also observed. These findings demonstrate the prospect of high-speed multiplex genome engineering in mouse primary cells with future clinical implications such as cancer modeling and gene therapy. Moving forward, the generation of an inducible Cas9 transgenic animal should be pursued.
Collection
Undergraduate Theses, Department of Biology, 2013-2014
Cerebral arteriovenous malformations (AVMs), although rare, are a leading cause of intracerebral hemorrhage and stroke in young adults and children. Due to a poor understanding of the formation and cellular makeup of AVMs, available treatment options are complicated and invasive, and AVMs can regrow in some cases. Progenitor cells have been implicated in AVMs, possibly functioning in the abnormal growth characteristic of this lesion, but their physical presence has not been directly confirmed. By analyzing the protein expression in human AVM tissue in vivo and cells isolated from AVMs in vitro, this project works to clarify a potential progenitor presence and associated differentiation potential. First, Ki67 and CD133, accepted markers for cellular proliferation and stem cells respectively, were analyzed by immunohistochemistry in 29 human AVM tissue samples. In conjunction with previously identified expression patterns, the co-expression of progenitor-associated proteins in AVM tissue strongly indicates a progenitor presence. Second, two progenitor cell lines were successfully isolated from human cerebral AVMs by enrichment under progenitor conditions. The protein profile of these cell lines was then explored by immunocytochemistry to determine their in vitro differentiation potential. These cell lines maintain several characteristics of progenitor cells, including proliferation and expression of progenitor-associated proteins. Importantly, they have a unique protein signature, and the cells proliferate independently of growth factors. Additionally, human serum causes proliferation rather than differentiation. This project augments understanding of the fundamental nature of cerebral AVMs by identifying the presence of progenitor cells. Future research should extend these findings to determine the full characterization of these novel progenitors and their role in AVM formation and/or maintenance.
Cerebral arteriovenous malformations (AVMs), although rare, are a leading cause of intracerebral hemorrhage and stroke in young adults and children. Due to a poor understanding of the formation and cellular makeup of AVMs, available treatment options are complicated and invasive, and AVMs can regrow in some cases. Progenitor cells have been implicated in AVMs, possibly functioning in the abnormal growth characteristic of this lesion, but their physical presence has not been directly confirmed. By analyzing the protein expression in human AVM tissue in vivo and cells isolated from AVMs in vitro, this project works to clarify a potential progenitor presence and associated differentiation potential. First, Ki67 and CD133, accepted markers for cellular proliferation and stem cells respectively, were analyzed by immunohistochemistry in 29 human AVM tissue samples. In conjunction with previously identified expression patterns, the co-expression of progenitor-associated proteins in AVM tissue strongly indicates a progenitor presence. Second, two progenitor cell lines were successfully isolated from human cerebral AVMs by enrichment under progenitor conditions. The protein profile of these cell lines was then explored by immunocytochemistry to determine their in vitro differentiation potential. These cell lines maintain several characteristics of progenitor cells, including proliferation and expression of progenitor-associated proteins. Importantly, they have a unique protein signature, and the cells proliferate independently of growth factors. Additionally, human serum causes proliferation rather than differentiation. This project augments understanding of the fundamental nature of cerebral AVMs by identifying the presence of progenitor cells. Future research should extend these findings to determine the full characterization of these novel progenitors and their role in AVM formation and/or maintenance.
Book
1 online resource.
The hydrodynamic forces produced by breaking waves make the rocky intertidal zone one of the most physical stressful environments on Earth. Although the classical in-line hydrodynamic forces are well studied and characterized, an undescribed force -- known as the impingement force -- may be the largest hydrodynamic force in the intertidal zone. Impingement is characterized by a sharp, transient spike in force at the instant of wave arrival, and very few measurements of it exist. I measured impingement in the laboratory, using a gravity-driven water cannon to simulate waves and tested which variables affect impingement magnitude. I show that impingement is likely drag, rather than an undescribed hydrodynamic force; it likely occurs due to brief increases in flow velocity at the front of a wave. To assess the frequency and magnitudes of impingement events in the field, I built a force transducer to record high-frequency forces in the intertidal zone. Impingement events occur in 2-7% of waves in the field, but surprisingly, the average magnitude of impingement events is lower than the average maximum force produced by all recorded waves in this study. This suggests that impingement is not the largest hydrodynamic risk to organisms, and future work measuring flows in the intertidal zone should focus on identifying large transient forces regardless of where they occur in a wave. Using these measurements, I explore the likely effects of brief forces on intertidal organisms in two ways: first, I model a limpet as a mechanical spring-mass-damper system and show that its foot has the capacity to reduce the effective force on the animal. I also predict dislodgment rates of three species of gastropod using the maximum forces recorded in my field study. At my site, snails are very susceptible to wave-induced dislodgment. This dissertation illustrates the necessity of recording high-frequency flows when studying intertidal hydrodynamics.
The hydrodynamic forces produced by breaking waves make the rocky intertidal zone one of the most physical stressful environments on Earth. Although the classical in-line hydrodynamic forces are well studied and characterized, an undescribed force -- known as the impingement force -- may be the largest hydrodynamic force in the intertidal zone. Impingement is characterized by a sharp, transient spike in force at the instant of wave arrival, and very few measurements of it exist. I measured impingement in the laboratory, using a gravity-driven water cannon to simulate waves and tested which variables affect impingement magnitude. I show that impingement is likely drag, rather than an undescribed hydrodynamic force; it likely occurs due to brief increases in flow velocity at the front of a wave. To assess the frequency and magnitudes of impingement events in the field, I built a force transducer to record high-frequency forces in the intertidal zone. Impingement events occur in 2-7% of waves in the field, but surprisingly, the average magnitude of impingement events is lower than the average maximum force produced by all recorded waves in this study. This suggests that impingement is not the largest hydrodynamic risk to organisms, and future work measuring flows in the intertidal zone should focus on identifying large transient forces regardless of where they occur in a wave. Using these measurements, I explore the likely effects of brief forces on intertidal organisms in two ways: first, I model a limpet as a mechanical spring-mass-damper system and show that its foot has the capacity to reduce the effective force on the animal. I also predict dislodgment rates of three species of gastropod using the maximum forces recorded in my field study. At my site, snails are very susceptible to wave-induced dislodgment. This dissertation illustrates the necessity of recording high-frequency flows when studying intertidal hydrodynamics.
Book
1 online resource.
The goal of my thesis is to further understanding of the highly sophisticated yet relatively uncharacterized visual system of the octopus. To examine the functional organization of this system, I used electrophysiological recordings to document the response of groups of photoreceptors in the retina to polarized light stimulation. To examine in more detail the organization of the neurocircuitry that underlies visual processing in higher levels of the octopus visual system, I documented the expression patterns of a host of transcripts of key enzymes and receptors in the glutamatergic, cholinergic, and to a lesser degree in the dopaminergic, GABAergic, and octopaminergic neurotransmission pathways within the optic lobe. ADAPTATION AND FACILITATION IN THE RESPONSE TO POLARIZED LIGHT IN THE VISUAL SYSTEM OF OCTOPUS BIMACULOIDES. In an oceanic world of diminished color and contrast, polarized light detection is a powerful tool for overcoming visual challenges. Studies are making it increasingly clear that many animals use polarization information from light, for purposes ranging from detecting migration cues, to detecting prey and predators, to interspecies communication. Behavioral studies have documented the ability of cephalopods to not only detect differences in polarization of light, but in addition to alter the polarization of light incident on their iridophores, hinting at the possibility of a concealed communication that could exist with such intentional patterning. Electrophysiological recordings have indicated that polarization angle is indeed represented in the output signal of individual rhabdomeric photoreceptors within the cephalopod retina. However, the connectivity of those photoreceptors, or how they may process polarization information on the level of the retina, remains poorly understood. I tested both the adaptation and facilitation effects of stimulation with polarized light on groups of photoreceptors. I report that facilitation, consistent with a residual calcium hypothesis, occurs in short-term responses to stimulation. Over longer time courses of stimulation, adaptation effects on photoreceptor output occur independently of the polarization orientation of the adapting light, indicating that photoreceptors are connected to and communicating with each other in the retina to a much greater degree than previously postulated. The results I report here are the first indication that even on the level of retinal output, octopuses potentially possess not only polarization sensitivity, but also the capacity for true polarization vision. CHARACTERIZATION OF COMPONENTS OF THE GLUTAMATERGIC SIGNALING PATHWAY IN THE OPTIC LOBE OF OCTOPUS BIMACULOIDES: Glutamate is a simple molecule, which over the course of evolution has been recruited widely across the animal kingdom for use as a neurotransmitter. A diversity of expression of glutamate receptors allows for a wide variety of effects of glutamate on various cell populations, and underlies much of the subtle sorting of information that takes place in our own retinas. NMDA-type glutamate receptors have evolved to play a key role in memory and learning circuits. Glutamate has been long known to be present in the cephalopod visual system, but its specific roles and paths within the neurocircuitry therein remain poorly understood. Here I identify and document the expression pattern of the synthetic enzyme of glutamate, glutamine synthetase, as well as a host of receptors subunits from AMPA, kainate, and NMDA-type glutamate receptor subunit families. These data allow for more precise identification of glutamatergic cells in this system than was previously possible. CHARACTERIZATION OF COMPONENTS OF THE CHOLINERGIC SIGNALING PATHWAY IN THE OPTIC LOBE OF OCTOPUS BIMACULOIDES: Acetylcholine is also used across the animal kingdom as a neurotransmitter. The evolution of fast acting nicotinic acetylcholine receptors has allowed for this molecule to play a key role in rapid signal transduction in a large variety of neural pathways. In particular, acetylcholine is present in high concentration in the cephalopod optic lobes, and is has long been thought that acetylcholine plays a critical part as a neurotransmitter in cephalopod visual processing. Here I identified and documented the expression pattern of mRNA for the synthetic and packaging enzymes necessary for presynaptic cells to produce acetylcholine, as well as a host of acetylcholine receptor subunits present in the optic lobe. Of particular interest is the documentation of members of a novel class of inhibitory acetylcholine receptor subunits that has arisen independently in the molluscs. These data further the hypothesis that acetylcholine is indeed used prevalently in this system. THE IDENTIFICATION AND EXPRESSION PATTERNS OF COMPONENTS OF THE DOPAMINERGIC, GABAERGIC, AND OCTOPAMINERGIC NEUROTRANSMISSION PATHWAYS, AND A NOTE ON INHIBITORY GLUTAMATE RECEPTORS IN THE OPTIC LOBE OF OCTOPUS BIMACULOIDES: Many other classical neurotransmitters have been implicated in cephalopod visual processing, and here I present further data elucidating the cellular populations that participate in signaling using dopamine, GABA, and octopamine. Identification of the expression pattern of mRNA for both the synthetic enzyme and the signal-degrading transporter for dopamine implicates a small population of cells in the optic lobe as dopaminergic. Similarly, expression of mRNA for the degradative enzyme of GABA highlights a small but widespread population of cells that act to remove GABA from their synapses, presumably after receiving its signal. Though named for the octopus, octopamine presence and utility is poorly understood in the central brains of these animals; here I show the first documentation of the expression of the synthetic enzyme for octopamine within the optic lobe. The additional identification of inhibitory glutamate receptor subunit sequences in the transcriptome is also discussed.
The goal of my thesis is to further understanding of the highly sophisticated yet relatively uncharacterized visual system of the octopus. To examine the functional organization of this system, I used electrophysiological recordings to document the response of groups of photoreceptors in the retina to polarized light stimulation. To examine in more detail the organization of the neurocircuitry that underlies visual processing in higher levels of the octopus visual system, I documented the expression patterns of a host of transcripts of key enzymes and receptors in the glutamatergic, cholinergic, and to a lesser degree in the dopaminergic, GABAergic, and octopaminergic neurotransmission pathways within the optic lobe. ADAPTATION AND FACILITATION IN THE RESPONSE TO POLARIZED LIGHT IN THE VISUAL SYSTEM OF OCTOPUS BIMACULOIDES. In an oceanic world of diminished color and contrast, polarized light detection is a powerful tool for overcoming visual challenges. Studies are making it increasingly clear that many animals use polarization information from light, for purposes ranging from detecting migration cues, to detecting prey and predators, to interspecies communication. Behavioral studies have documented the ability of cephalopods to not only detect differences in polarization of light, but in addition to alter the polarization of light incident on their iridophores, hinting at the possibility of a concealed communication that could exist with such intentional patterning. Electrophysiological recordings have indicated that polarization angle is indeed represented in the output signal of individual rhabdomeric photoreceptors within the cephalopod retina. However, the connectivity of those photoreceptors, or how they may process polarization information on the level of the retina, remains poorly understood. I tested both the adaptation and facilitation effects of stimulation with polarized light on groups of photoreceptors. I report that facilitation, consistent with a residual calcium hypothesis, occurs in short-term responses to stimulation. Over longer time courses of stimulation, adaptation effects on photoreceptor output occur independently of the polarization orientation of the adapting light, indicating that photoreceptors are connected to and communicating with each other in the retina to a much greater degree than previously postulated. The results I report here are the first indication that even on the level of retinal output, octopuses potentially possess not only polarization sensitivity, but also the capacity for true polarization vision. CHARACTERIZATION OF COMPONENTS OF THE GLUTAMATERGIC SIGNALING PATHWAY IN THE OPTIC LOBE OF OCTOPUS BIMACULOIDES: Glutamate is a simple molecule, which over the course of evolution has been recruited widely across the animal kingdom for use as a neurotransmitter. A diversity of expression of glutamate receptors allows for a wide variety of effects of glutamate on various cell populations, and underlies much of the subtle sorting of information that takes place in our own retinas. NMDA-type glutamate receptors have evolved to play a key role in memory and learning circuits. Glutamate has been long known to be present in the cephalopod visual system, but its specific roles and paths within the neurocircuitry therein remain poorly understood. Here I identify and document the expression pattern of the synthetic enzyme of glutamate, glutamine synthetase, as well as a host of receptors subunits from AMPA, kainate, and NMDA-type glutamate receptor subunit families. These data allow for more precise identification of glutamatergic cells in this system than was previously possible. CHARACTERIZATION OF COMPONENTS OF THE CHOLINERGIC SIGNALING PATHWAY IN THE OPTIC LOBE OF OCTOPUS BIMACULOIDES: Acetylcholine is also used across the animal kingdom as a neurotransmitter. The evolution of fast acting nicotinic acetylcholine receptors has allowed for this molecule to play a key role in rapid signal transduction in a large variety of neural pathways. In particular, acetylcholine is present in high concentration in the cephalopod optic lobes, and is has long been thought that acetylcholine plays a critical part as a neurotransmitter in cephalopod visual processing. Here I identified and documented the expression pattern of mRNA for the synthetic and packaging enzymes necessary for presynaptic cells to produce acetylcholine, as well as a host of acetylcholine receptor subunits present in the optic lobe. Of particular interest is the documentation of members of a novel class of inhibitory acetylcholine receptor subunits that has arisen independently in the molluscs. These data further the hypothesis that acetylcholine is indeed used prevalently in this system. THE IDENTIFICATION AND EXPRESSION PATTERNS OF COMPONENTS OF THE DOPAMINERGIC, GABAERGIC, AND OCTOPAMINERGIC NEUROTRANSMISSION PATHWAYS, AND A NOTE ON INHIBITORY GLUTAMATE RECEPTORS IN THE OPTIC LOBE OF OCTOPUS BIMACULOIDES: Many other classical neurotransmitters have been implicated in cephalopod visual processing, and here I present further data elucidating the cellular populations that participate in signaling using dopamine, GABA, and octopamine. Identification of the expression pattern of mRNA for both the synthetic enzyme and the signal-degrading transporter for dopamine implicates a small population of cells in the optic lobe as dopaminergic. Similarly, expression of mRNA for the degradative enzyme of GABA highlights a small but widespread population of cells that act to remove GABA from their synapses, presumably after receiving its signal. Though named for the octopus, octopamine presence and utility is poorly understood in the central brains of these animals; here I show the first documentation of the expression of the synthetic enzyme for octopamine within the optic lobe. The additional identification of inhibitory glutamate receptor subunit sequences in the transcriptome is also discussed.
Collection
Undergraduate Theses, Department of Biology, 2013-2014
Recruitment of endogenous progenitors is critical during tissue repair. In the mammalian cochlea, damaged hair cells are not regenerated. As a result, hearing loss is permanent and negatively impacts the quality of life for over 48 million Americans. Adjacent to the cochlea in the inner ear are the vestibular organs responsible for our balance functions. Of the vestibular organs, the utricle requires mechanosensory hair cells to detect linear acceleration. After damage, non-mammalian utricles regenerate hair cells mitotically and non-mitotically. By contrast, mammalian utricles exhibit limited non-mitotic regeneration, with the source of regenerated hair cells currently unknown. Here, we used Lgr5-EGFP reporter mice to show that hair cell damage in neonatal mouse utricles activates the Wnt target gene, Lgr5, in supporting cells, which normally do not express Lgr5. Lineage tracing and time-lapse microscopy in utricles from Lgr5-EGFP-CreERT2; Rosa26R-tdTomato mice reveal that Lgr5+ cells non-mitotically become hair cell-like cells in vitro, a process termed “direct transdifferentiation.” Thus, Lgr5 marks damage-activated HC progenitors and may help reveal factors necessary for mammalian hair cell regeneration.
Recruitment of endogenous progenitors is critical during tissue repair. In the mammalian cochlea, damaged hair cells are not regenerated. As a result, hearing loss is permanent and negatively impacts the quality of life for over 48 million Americans. Adjacent to the cochlea in the inner ear are the vestibular organs responsible for our balance functions. Of the vestibular organs, the utricle requires mechanosensory hair cells to detect linear acceleration. After damage, non-mammalian utricles regenerate hair cells mitotically and non-mitotically. By contrast, mammalian utricles exhibit limited non-mitotic regeneration, with the source of regenerated hair cells currently unknown. Here, we used Lgr5-EGFP reporter mice to show that hair cell damage in neonatal mouse utricles activates the Wnt target gene, Lgr5, in supporting cells, which normally do not express Lgr5. Lineage tracing and time-lapse microscopy in utricles from Lgr5-EGFP-CreERT2; Rosa26R-tdTomato mice reveal that Lgr5+ cells non-mitotically become hair cell-like cells in vitro, a process termed “direct transdifferentiation.” Thus, Lgr5 marks damage-activated HC progenitors and may help reveal factors necessary for mammalian hair cell regeneration.
Book
1 online resource.
Stomata, epidermal valves made up of paired guard cells flanking a central pore, are found in most aerial tissues of plants, where they function to regulate gas exchange. Production of mature stomata involves a series of asymmetric cell divisions (ACDs) and cell fate transitions, and the molecular genetic regulation of this process has been extensively studied in the dicot Arabidopsis thaliana (thale cress). Yet, it remains largely unknown whether the same genetic regulators are used across a range of taxa, and how genes and networks might differ between species to produce (or accommodate) distinct developmental patterns. Indeed, stomatal ontogeny is remarkably diverse among plant species, with particularly intriguing differences between dicots and grasses. Whereas Arabidopsis stomata are sprinkled across the leaf surface seemingly at random, grasses produce stomata oriented along the leaf axis in specific linear cell files. In my thesis work, I have examined the genetic networks controlling stomatal development in grasses, focusing particularly on how these networks resemble or differ from those of Arabidopsis. My studies are conducted using the forage grass Brachypodium distachyon, a recently developed, genetically tractable model species related to wheat and barley. First, I report the basic characterization of Brachypodium stomatal development, as well as the identification of stomatal mutants via a forward genetic screen. Subsequently, I describe mutations in two orthologues of known Arabidopsis stomatal regulators: the bHLH gene BdICE1, which is required for production of mature stomata, and the MAPKKK gene BdYDA1, which enforces correct stomatal patterning. Although both mutants resemble their Arabidopsis counterparts, their phenotypes suggest subtly but intriguingly different roles in Brachypodium. BdICE1, unlike Arabidopsis ICE1, is not functionally redundant with its paralogue BdSCRM2, and the two Brachypodium genes have roles in promoting GMC and guard cell identity, respectively. Further, preliminary data suggest that the ICEs of Brachypodium may differ from those of Arabidopsis in their interactions with SPCH family bHLHs. Similarly, although BdYDA1, like Arabidopsis YDA, enforces stomatal patterning, it also regulates a number of other asymmetrically dividing epidermal cell lineages (e.g., hair and silica cell lineages), suggesting a role as a general regulator of fate asymmetry. Finally, I discuss prospects for using timelapse imaging and computational modeling to gain insight into stomatal lineage polarity, division, and fate specification processes in both dicots and grasses.
Stomata, epidermal valves made up of paired guard cells flanking a central pore, are found in most aerial tissues of plants, where they function to regulate gas exchange. Production of mature stomata involves a series of asymmetric cell divisions (ACDs) and cell fate transitions, and the molecular genetic regulation of this process has been extensively studied in the dicot Arabidopsis thaliana (thale cress). Yet, it remains largely unknown whether the same genetic regulators are used across a range of taxa, and how genes and networks might differ between species to produce (or accommodate) distinct developmental patterns. Indeed, stomatal ontogeny is remarkably diverse among plant species, with particularly intriguing differences between dicots and grasses. Whereas Arabidopsis stomata are sprinkled across the leaf surface seemingly at random, grasses produce stomata oriented along the leaf axis in specific linear cell files. In my thesis work, I have examined the genetic networks controlling stomatal development in grasses, focusing particularly on how these networks resemble or differ from those of Arabidopsis. My studies are conducted using the forage grass Brachypodium distachyon, a recently developed, genetically tractable model species related to wheat and barley. First, I report the basic characterization of Brachypodium stomatal development, as well as the identification of stomatal mutants via a forward genetic screen. Subsequently, I describe mutations in two orthologues of known Arabidopsis stomatal regulators: the bHLH gene BdICE1, which is required for production of mature stomata, and the MAPKKK gene BdYDA1, which enforces correct stomatal patterning. Although both mutants resemble their Arabidopsis counterparts, their phenotypes suggest subtly but intriguingly different roles in Brachypodium. BdICE1, unlike Arabidopsis ICE1, is not functionally redundant with its paralogue BdSCRM2, and the two Brachypodium genes have roles in promoting GMC and guard cell identity, respectively. Further, preliminary data suggest that the ICEs of Brachypodium may differ from those of Arabidopsis in their interactions with SPCH family bHLHs. Similarly, although BdYDA1, like Arabidopsis YDA, enforces stomatal patterning, it also regulates a number of other asymmetrically dividing epidermal cell lineages (e.g., hair and silica cell lineages), suggesting a role as a general regulator of fate asymmetry. Finally, I discuss prospects for using timelapse imaging and computational modeling to gain insight into stomatal lineage polarity, division, and fate specification processes in both dicots and grasses.
Book
1 online resource.
One of the central aspects of the biological program that guide the development of an organism is embedded in the regulated and sequential expression of genes as development progresses. A large part of this regulation is achieved through the temporal activation and repression of transcriptional initiation by the selective binding of regulatory proteins, such as transcription factors, to promoters during specific stages of development. Thus, being able to globally and precisely identify these processes are important steps in gaining a systems-level insight and understanding of the developmental program. The cell cycle of Caulobacter crescentus, an [alpha]-proteobacteria that undergoes cell differentiation and asymmetric cell division, has been used extensively as a model organism to study bacterial development. A cyclical and integrated genetic circuit involving five master regulatory proteins, including DnaA, GcrA, CtrA, and SciP, and the DNA methyl-transferase CcrM, whose presence and activities oscillate in space and time, orchestrate the many facets of the Caulobacter cell cycle including DNA replication, DNA methylation, organelle biogenesis, and cytokinesis. This genetic circuit is at the core of the Caulobacter developmental program. While microarrays have shown 19% of mRNAs undergo changes in RNA level during the cell cycle and development, it is unclear exactly which regulatory factors of the core circuit drive the changes in transcription at each specific locus, and how these regulatory factors act combinatorially to effect transcriptional outcomes has not been systematically dissected. In order to achieve these goals and to further define the transcriptional regulatory landscape that guides the cell cycle, a thorough and global analysis of Caulobacter transcription as a function of the cell cycle and developmental progression is needed. In this thesis, I devised a novel protocol combining 5' rapid amplification of cDNA ends (5' RACE) and high-throughput sequencing to globally map the precise locations of transcriptional start sites (TSSs) in the Caulobacter genome, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Using the TSSs identified and a RNA sequencing dataset, I made a functional annotation of operons and other transcriptional units in the genome. A large number of antisense transcripts were identified, and many of them are within essential cell cycle-regulated genes, including two master regulators, a previous unknown feature of the core cell cycle control circuit. Many critical genes and operons have multiple promoters, and these promoters are often independently regulated. Furthermore, approximately 25% of the cell cycle-regulated promoters are co-regulated by two or more master regulatory proteins of the core genetic circuit. These results revealed surprising transcriptional complexity and uncovered multiple new layers of transcriptional control mediating the bacterial cell cycle and development and represent the first in-depth analysis of TSS control in as a function bacterial cell cycle and developmental progression.
One of the central aspects of the biological program that guide the development of an organism is embedded in the regulated and sequential expression of genes as development progresses. A large part of this regulation is achieved through the temporal activation and repression of transcriptional initiation by the selective binding of regulatory proteins, such as transcription factors, to promoters during specific stages of development. Thus, being able to globally and precisely identify these processes are important steps in gaining a systems-level insight and understanding of the developmental program. The cell cycle of Caulobacter crescentus, an [alpha]-proteobacteria that undergoes cell differentiation and asymmetric cell division, has been used extensively as a model organism to study bacterial development. A cyclical and integrated genetic circuit involving five master regulatory proteins, including DnaA, GcrA, CtrA, and SciP, and the DNA methyl-transferase CcrM, whose presence and activities oscillate in space and time, orchestrate the many facets of the Caulobacter cell cycle including DNA replication, DNA methylation, organelle biogenesis, and cytokinesis. This genetic circuit is at the core of the Caulobacter developmental program. While microarrays have shown 19% of mRNAs undergo changes in RNA level during the cell cycle and development, it is unclear exactly which regulatory factors of the core circuit drive the changes in transcription at each specific locus, and how these regulatory factors act combinatorially to effect transcriptional outcomes has not been systematically dissected. In order to achieve these goals and to further define the transcriptional regulatory landscape that guides the cell cycle, a thorough and global analysis of Caulobacter transcription as a function of the cell cycle and developmental progression is needed. In this thesis, I devised a novel protocol combining 5' rapid amplification of cDNA ends (5' RACE) and high-throughput sequencing to globally map the precise locations of transcriptional start sites (TSSs) in the Caulobacter genome, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Using the TSSs identified and a RNA sequencing dataset, I made a functional annotation of operons and other transcriptional units in the genome. A large number of antisense transcripts were identified, and many of them are within essential cell cycle-regulated genes, including two master regulators, a previous unknown feature of the core cell cycle control circuit. Many critical genes and operons have multiple promoters, and these promoters are often independently regulated. Furthermore, approximately 25% of the cell cycle-regulated promoters are co-regulated by two or more master regulatory proteins of the core genetic circuit. These results revealed surprising transcriptional complexity and uncovered multiple new layers of transcriptional control mediating the bacterial cell cycle and development and represent the first in-depth analysis of TSS control in as a function bacterial cell cycle and developmental progression.
Book
1 online resource.
The Hedgehog signaling pathway performs essential and diverse roles in embryonic development and adult tissue homeostasis throughout the animal kingdom. Hedgehog proteins are secreted morphogens that emanate from localized pools of cells, generating a concentration gradient that communicates positional information to the cells within the tissue. A distinctive feature of Hedgehog proteins is covalent modification with a N-terminal palmitate and a C-terminal cholesterol, which strongly tether Hedgehog to cell membranes and modulate its distribution in tissues. Despite this hydrophobic character, Hedgehog proteins mobilize from expressing cells and travel far from their source to act directly upon distant responding cells. Although evidence exists for multiple mechanisms of Hedgehog mobilization, the actual physical form of the Hedgehog morphogen, as it is packaged and deployed in tissues, remains unknown. Studies in zebrafish have revealed a secreted factor, Scube2, that functions non-cell-autonomously to enable Hedgehog signaling. This led to the discovery that the mouse Scube2 protein can drive release of fully-lipidated Sonic Hedgehog from cell membranes in a soluble form that is potent in signaling assays. In this study, we have taken advantage of Scube2 activity to investigate the physical form of the active Shh morphogen. The signaling activity of this Scube2-released ShhNp is associated predominantly with a large, but discrete, protein complex approximately 250-300 kilodaltons in size. Further analysis suggests that this consists largely of a Scube2:Shh complex, although fully-lipidated Shh could also be present in other forms. Exracellular matrix glycans, such as heparan sulfate, contribute to the assembly and release of soluble, high molecular weight form of Shh by Scube2, and a heparin chain of 16 units suffices to potentiate Scube2-mediated release of ShhNp. This defined system has allowed us to produce large quantities of active morphogen for analysis, thus enabling us to begin addressing questions about the composition, stoichiomentry, and molecular architecture of the active morphogen.
The Hedgehog signaling pathway performs essential and diverse roles in embryonic development and adult tissue homeostasis throughout the animal kingdom. Hedgehog proteins are secreted morphogens that emanate from localized pools of cells, generating a concentration gradient that communicates positional information to the cells within the tissue. A distinctive feature of Hedgehog proteins is covalent modification with a N-terminal palmitate and a C-terminal cholesterol, which strongly tether Hedgehog to cell membranes and modulate its distribution in tissues. Despite this hydrophobic character, Hedgehog proteins mobilize from expressing cells and travel far from their source to act directly upon distant responding cells. Although evidence exists for multiple mechanisms of Hedgehog mobilization, the actual physical form of the Hedgehog morphogen, as it is packaged and deployed in tissues, remains unknown. Studies in zebrafish have revealed a secreted factor, Scube2, that functions non-cell-autonomously to enable Hedgehog signaling. This led to the discovery that the mouse Scube2 protein can drive release of fully-lipidated Sonic Hedgehog from cell membranes in a soluble form that is potent in signaling assays. In this study, we have taken advantage of Scube2 activity to investigate the physical form of the active Shh morphogen. The signaling activity of this Scube2-released ShhNp is associated predominantly with a large, but discrete, protein complex approximately 250-300 kilodaltons in size. Further analysis suggests that this consists largely of a Scube2:Shh complex, although fully-lipidated Shh could also be present in other forms. Exracellular matrix glycans, such as heparan sulfate, contribute to the assembly and release of soluble, high molecular weight form of Shh by Scube2, and a heparin chain of 16 units suffices to potentiate Scube2-mediated release of ShhNp. This defined system has allowed us to produce large quantities of active morphogen for analysis, thus enabling us to begin addressing questions about the composition, stoichiomentry, and molecular architecture of the active morphogen.
Collection
Undergraduate Theses, Department of Biology, 2013-2014
The insulating myelin sheath formed by oligodendrocytes is crucial for rapid conduction of action potentials. The understanding of how oligodendrocytes wrap this specialized membrane is limited. Actin, a key regulator of cell structure and movement, is hypothesized to play an important role (Bauer et al., 2009). Further investigation of the role of actin in myelination necessitates a cell-specific tool with fine temporal and dose- dependent control. We developed genetically encodable actin-modifying tools (gACTs). Using DNA sequences from endogenous actin-modifying proteins or bacterial toxins allowed us to harness their actin modifying power. At this stage, the constructs allow for temporal control and either dose-dependent or cell-specific control, but ultimately the tool will have all three features. To validate the constructs, we transfected HeLa cells and visualized actin morphology. We found clear actin morphology changes upon transfection for all five constructs with variable effectiveness and cytotoxicity. To provide temporal and dose-dependent control, we fused the destabilization domain (DD) to the constructs (Banaszynski et al., 2006). To provide temporal control and cell specificity, we replaced the strong cytomegalovirus (CMV) promoter with a myelin basic protein (MBP) promoter, conferring expression only in mature stages of oligodendrocytes. In addition to elucidating the role of actin in myelination, gACTs are a useful general cell biology tool to study actin in multicellular processes such as development or diseases such as multiple sclerosis.
The insulating myelin sheath formed by oligodendrocytes is crucial for rapid conduction of action potentials. The understanding of how oligodendrocytes wrap this specialized membrane is limited. Actin, a key regulator of cell structure and movement, is hypothesized to play an important role (Bauer et al., 2009). Further investigation of the role of actin in myelination necessitates a cell-specific tool with fine temporal and dose- dependent control. We developed genetically encodable actin-modifying tools (gACTs). Using DNA sequences from endogenous actin-modifying proteins or bacterial toxins allowed us to harness their actin modifying power. At this stage, the constructs allow for temporal control and either dose-dependent or cell-specific control, but ultimately the tool will have all three features. To validate the constructs, we transfected HeLa cells and visualized actin morphology. We found clear actin morphology changes upon transfection for all five constructs with variable effectiveness and cytotoxicity. To provide temporal and dose-dependent control, we fused the destabilization domain (DD) to the constructs (Banaszynski et al., 2006). To provide temporal control and cell specificity, we replaced the strong cytomegalovirus (CMV) promoter with a myelin basic protein (MBP) promoter, conferring expression only in mature stages of oligodendrocytes. In addition to elucidating the role of actin in myelination, gACTs are a useful general cell biology tool to study actin in multicellular processes such as development or diseases such as multiple sclerosis.