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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-β 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-β 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-β 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-β 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.
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.
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.
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.
Book
1 online resource.
The diverse community of microbes that inhabits the human bowel, known as the gut microbiota, is vitally important to human health. While the identity and abundance of the microbes has been widely studied, little is known about the ways in which they interact with their host. The breakdown of the host-microbiota symbiosis has been linked to numerous diseases, including inflammatory bowel diseases, autoimmune diseases and even autism. Host-expressed proteins are essential for maintaining this mutualistic relationship and serve as reporters on the status of host-microbiota interactions. Previously, a global means for non-invasively monitoring the expression of proteins by the host in the gastrointestinal tract has not been described. In this thesis, I elucidate the need for an unbiased method to monitor host responses to the microbiota and describe a mass spectrometry-based method we developed, called host-centric proteomics of stool, which can simultaneously measure hundreds of host secreted proteins. I then illustrate how this method can be used to study host protein secretions in various regions of the gastrointestinal tract and the host responses to antibiotic-associated pathogens.
The diverse community of microbes that inhabits the human bowel, known as the gut microbiota, is vitally important to human health. While the identity and abundance of the microbes has been widely studied, little is known about the ways in which they interact with their host. The breakdown of the host-microbiota symbiosis has been linked to numerous diseases, including inflammatory bowel diseases, autoimmune diseases and even autism. Host-expressed proteins are essential for maintaining this mutualistic relationship and serve as reporters on the status of host-microbiota interactions. Previously, a global means for non-invasively monitoring the expression of proteins by the host in the gastrointestinal tract has not been described. In this thesis, I elucidate the need for an unbiased method to monitor host responses to the microbiota and describe a mass spectrometry-based method we developed, called host-centric proteomics of stool, which can simultaneously measure hundreds of host secreted proteins. I then illustrate how this method can be used to study host protein secretions in various regions of the gastrointestinal tract and the host responses to antibiotic-associated pathogens.
Book
1 online resource.
Inclusion bodies (IB) containing aggregated forms of disease-associated proteins and polyubiquitin conjugates are universal histopathological features of neurodegenerative disease, but the mechanisms that govern recruitment of ubiquitinated proteins to IB are not well understood. Ubiquitin (Ub) has previously been proposed to target proteins to IB for degradation. In this study, we use conditionally destabilized reporters that undergo misfolding and ubiquitination upon removal of stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IB composed of an N-terminal fragment of mutant huntingtin (htt), the causative protein in Huntington's disease (HD). We show that reporters are excluded from IB in the presence of stabilizing ligand, but are recruited to IB following ligand washout. However, we find that Ub conjugation is not necessary or sufficient to target reporters to IB. Moreover, misfolded reporters and Ub conjugates are stable at IB. These data indicate that compromised folding states, not conjugation to Ub, specifies IB recruitment.
Inclusion bodies (IB) containing aggregated forms of disease-associated proteins and polyubiquitin conjugates are universal histopathological features of neurodegenerative disease, but the mechanisms that govern recruitment of ubiquitinated proteins to IB are not well understood. Ubiquitin (Ub) has previously been proposed to target proteins to IB for degradation. In this study, we use conditionally destabilized reporters that undergo misfolding and ubiquitination upon removal of stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IB composed of an N-terminal fragment of mutant huntingtin (htt), the causative protein in Huntington's disease (HD). We show that reporters are excluded from IB in the presence of stabilizing ligand, but are recruited to IB following ligand washout. However, we find that Ub conjugation is not necessary or sufficient to target reporters to IB. Moreover, misfolded reporters and Ub conjugates are stable at IB. These data indicate that compromised folding states, not conjugation to Ub, specifies IB recruitment.
Book
1 online resource.
Increasing temperatures associated with anthropogenic climate change are likely to surpass the thermal limits of many species in the coming decades. Predictions of species response to these changes are predicated on the assumption that heat tolerance is static through time. Countless studies, however, have demonstrated that individual organisms and populations can shift their upper thermal limits through either acclimation, in which individuals exposed to increased temperatures shift their physiology, or adaptation, in which whole populations shift their thermal limits through natural selection for the most heat tolerant genotypes. These processes are especially relevant to foundation species, such as reef-building corals, as these organisms are often sessile, and so cannot easily shift their range. In this dissertation, I examined patterns and processes involved in acclimation and adaptation in reef-building corals, including their impacts on upper thermal limits, the genes and cellular pathways involved, and the relative roles of the two mechanisms over small and large spatial scales. In Chapter 1, I conducted a time series analysis of acclimation. I found an increase in heat tolerance after one week at higher temperatures and a corresponding shift in gene expression, resulting in a dampened response to heat stress. In Chapter 2, I utilized a small-scale natural temperature gradient to search for genetic variants associated with corals adapted to different temperatures. I found 114 genetic variants that differed between warm and cool microclimates separated by less than 1km. In Chapter 3, I tested these variants using a reciprocal transplants between warm and cool microclimates. I found that corals from warm microclimates survived more often, but a subset of genetic variants associated with survival were also associated with lower growth, suggesting a tradeoff in overall fitness. In Chapters 4 and 5, I examined tradeoffs and mechanisms of adaptation and acclimation over a large spatial scale: across latitude. Using a combination of acclimation and heat stress experiments, I found evidence for a high degree of plasticity in upper thermal limit for corals at both low and high latitudes. Despite this plasticity, however, there were still significant fixed effects between locations, suggesting large roles for both acclimation and adaptation in determining heat tolerance across latitude. In Chapter 5, I examined the molecular basis of these shifts in thermal tolerance. I found divergence at both the sequence and gene expression levels for populations from low and high latitude locations, eventually leading to the discovery that the two populations were separate cryptic species: a warm adapted congener and a more broadly distributed congener. This allowed me to compare gene expression patterns between the two cryptic species with different evolutionary histories, finding transcriptional differences across a broad range of cellular functions that could be involved in adaptive thermal tolerance. I also found a set of genes whose expression was tightly linked with bleaching status; these genes are therefore good candidates 'biomarkers' that could be used for prediction of heat stress and tolerance. Together, these studies highlight important roles for both acclimation and adaptation in determining thermal tolerance in corals and provide insights useful for predicting the response of these ecologically and economically important taxa to future climate change.
Increasing temperatures associated with anthropogenic climate change are likely to surpass the thermal limits of many species in the coming decades. Predictions of species response to these changes are predicated on the assumption that heat tolerance is static through time. Countless studies, however, have demonstrated that individual organisms and populations can shift their upper thermal limits through either acclimation, in which individuals exposed to increased temperatures shift their physiology, or adaptation, in which whole populations shift their thermal limits through natural selection for the most heat tolerant genotypes. These processes are especially relevant to foundation species, such as reef-building corals, as these organisms are often sessile, and so cannot easily shift their range. In this dissertation, I examined patterns and processes involved in acclimation and adaptation in reef-building corals, including their impacts on upper thermal limits, the genes and cellular pathways involved, and the relative roles of the two mechanisms over small and large spatial scales. In Chapter 1, I conducted a time series analysis of acclimation. I found an increase in heat tolerance after one week at higher temperatures and a corresponding shift in gene expression, resulting in a dampened response to heat stress. In Chapter 2, I utilized a small-scale natural temperature gradient to search for genetic variants associated with corals adapted to different temperatures. I found 114 genetic variants that differed between warm and cool microclimates separated by less than 1km. In Chapter 3, I tested these variants using a reciprocal transplants between warm and cool microclimates. I found that corals from warm microclimates survived more often, but a subset of genetic variants associated with survival were also associated with lower growth, suggesting a tradeoff in overall fitness. In Chapters 4 and 5, I examined tradeoffs and mechanisms of adaptation and acclimation over a large spatial scale: across latitude. Using a combination of acclimation and heat stress experiments, I found evidence for a high degree of plasticity in upper thermal limit for corals at both low and high latitudes. Despite this plasticity, however, there were still significant fixed effects between locations, suggesting large roles for both acclimation and adaptation in determining heat tolerance across latitude. In Chapter 5, I examined the molecular basis of these shifts in thermal tolerance. I found divergence at both the sequence and gene expression levels for populations from low and high latitude locations, eventually leading to the discovery that the two populations were separate cryptic species: a warm adapted congener and a more broadly distributed congener. This allowed me to compare gene expression patterns between the two cryptic species with different evolutionary histories, finding transcriptional differences across a broad range of cellular functions that could be involved in adaptive thermal tolerance. I also found a set of genes whose expression was tightly linked with bleaching status; these genes are therefore good candidates 'biomarkers' that could be used for prediction of heat stress and tolerance. Together, these studies highlight important roles for both acclimation and adaptation in determining thermal tolerance in corals and provide insights useful for predicting the response of these ecologically and economically important taxa to future climate change.
Book
1 online resource.
A single fertilized embryo gives rise to more than 200 distinct cell types in the human body. Regulation of cell type specific gene expression is one of the most critical molecular processes in specifying and maintaining these diverse cell fates. Here we show that cell type specific transcriptional repressors, along with activators, play key roles in selective gene expression in cell fate determination. In the Drosophila male germ line stem cell lineage, when progenitor cells cease mitotic proliferation and initiate terminal differentiation, they turn on one of the most dramatic cell type specific transcriptional programs in Drosophila. To investigate how this dramatic cell type specific gene expression is set up, I developed an in vivo synchronous differentiation system to identify the first transcripts turned up, with the idea that these early expressed genes may include "first regulators" that help initiate the terminal differentiation program. Progenitor mitotic spermatogonia that accumulated in bam-/- mutant testes were triggered to differentiate by a single heat shock pulse of expression of Bam. Global gene expression profiling by microarray and RNA-seq across time as spermatogonia synchronously differentiate into spermatocytes identified three distinct steps - 'downregulation', 'initiation', and 'terminal differentiation' -- in the gene expression cascade in the switch. Functional knock down of the first 39 upregulated genes identified 16 new genes required for proper differentiation, 6 of which exhibited particularly strong and early defects. One of the newly identified early genes, tZnF, encodes a multiple zinc finger spermatocyte-specific nuclear protein, required for repressing gene expression of somatic transcripts in male germ cells. Results from double mutant analysis suggest that tZnF is required in spermatocytes to prevent promiscuous activation of somatic transcripts by the testis-specific Meiotic Arrest Complex (tMAC) when it activates testis specific transcripts required for spermatocyte differentiation My results demonstrate how a cell can prevent collateral activation by a promiscuous activator to ensure correct differentiation of a specific cell type. In chapter 4, I characterize five additional genes identified from the early regulator screen. The products of the five genes --testis-specific ATP synthase β chain (CG5389); B9 domain (CG14870); pipsqueak type homeodomain like domain (CG30401); smooth or hnRNP-L, RNA binding domain (CG9218); and tBRD-1, bromodomain (CG13597) -- have diverse molecular functions, ranging from signaling, transcription, and RNA-processing, suggesting that terminal differentiation into spermatocytes is a complicated process involving complex regulatory mechanisms.
A single fertilized embryo gives rise to more than 200 distinct cell types in the human body. Regulation of cell type specific gene expression is one of the most critical molecular processes in specifying and maintaining these diverse cell fates. Here we show that cell type specific transcriptional repressors, along with activators, play key roles in selective gene expression in cell fate determination. In the Drosophila male germ line stem cell lineage, when progenitor cells cease mitotic proliferation and initiate terminal differentiation, they turn on one of the most dramatic cell type specific transcriptional programs in Drosophila. To investigate how this dramatic cell type specific gene expression is set up, I developed an in vivo synchronous differentiation system to identify the first transcripts turned up, with the idea that these early expressed genes may include "first regulators" that help initiate the terminal differentiation program. Progenitor mitotic spermatogonia that accumulated in bam-/- mutant testes were triggered to differentiate by a single heat shock pulse of expression of Bam. Global gene expression profiling by microarray and RNA-seq across time as spermatogonia synchronously differentiate into spermatocytes identified three distinct steps - 'downregulation', 'initiation', and 'terminal differentiation' -- in the gene expression cascade in the switch. Functional knock down of the first 39 upregulated genes identified 16 new genes required for proper differentiation, 6 of which exhibited particularly strong and early defects. One of the newly identified early genes, tZnF, encodes a multiple zinc finger spermatocyte-specific nuclear protein, required for repressing gene expression of somatic transcripts in male germ cells. Results from double mutant analysis suggest that tZnF is required in spermatocytes to prevent promiscuous activation of somatic transcripts by the testis-specific Meiotic Arrest Complex (tMAC) when it activates testis specific transcripts required for spermatocyte differentiation My results demonstrate how a cell can prevent collateral activation by a promiscuous activator to ensure correct differentiation of a specific cell type. In chapter 4, I characterize five additional genes identified from the early regulator screen. The products of the five genes --testis-specific ATP synthase β chain (CG5389); B9 domain (CG14870); pipsqueak type homeodomain like domain (CG30401); smooth or hnRNP-L, RNA binding domain (CG9218); and tBRD-1, bromodomain (CG13597) -- have diverse molecular functions, ranging from signaling, transcription, and RNA-processing, suggesting that terminal differentiation into spermatocytes is a complicated process involving complex regulatory mechanisms.
Book
1 online resource.
This thesis contains studies on the G2 phase of the cell cycle, which is arguably the most mysterious aspect of how cells prepare for cell division. Chapter 1 gives a historical overview of research on G2 and introduces key concepts and molecular players. G2 phase differs across model systems. It is absent in budding yeast as well as the early embryos of Xenopus and Drosophila. It is particularly well-understood in later embryonic development of Drosophila and in fission yeast, where an important regulator is mitotic phosphatase Cdc25. In mammalian tissue culture cells, the model system that is closest to cells of the human body, both Cdc25 and RCC1, a protein that emerged out of the analysis of mammalian cell-cycle mutants, present tantalizing possibilities for how the G2 phase may be regulated, but many unanswered questions remain. Chapter 2 presents results on how the duration of G2 phase affects mitotic progression, in particular the timeliness of anaphase. This work uses MCF10A cells that express fluorescently tagged PCNA, a marker of DNA replication, and fluorescently tagged histone H2B, a marker of DNA morphology, in live-cell microscopy to directly measure durations of all cell-cycle phases. G2 phase is then shortened by PD0166285, a drug known to induce premature mitosis. Shortening G2 phase results in a prolonged interval from nuclear envelope breakdown to anaphase that is partially rescued by inhibiting the spindle assembly checkpoint and not affected by inhibiting protein synthesis. Chapter 3 presents possible future directions for these studies. It contains preliminary data on the effect of shortened G2 on microtubule-kinetochore attachment, repair of DNA damage, and the centrosome.
This thesis contains studies on the G2 phase of the cell cycle, which is arguably the most mysterious aspect of how cells prepare for cell division. Chapter 1 gives a historical overview of research on G2 and introduces key concepts and molecular players. G2 phase differs across model systems. It is absent in budding yeast as well as the early embryos of Xenopus and Drosophila. It is particularly well-understood in later embryonic development of Drosophila and in fission yeast, where an important regulator is mitotic phosphatase Cdc25. In mammalian tissue culture cells, the model system that is closest to cells of the human body, both Cdc25 and RCC1, a protein that emerged out of the analysis of mammalian cell-cycle mutants, present tantalizing possibilities for how the G2 phase may be regulated, but many unanswered questions remain. Chapter 2 presents results on how the duration of G2 phase affects mitotic progression, in particular the timeliness of anaphase. This work uses MCF10A cells that express fluorescently tagged PCNA, a marker of DNA replication, and fluorescently tagged histone H2B, a marker of DNA morphology, in live-cell microscopy to directly measure durations of all cell-cycle phases. G2 phase is then shortened by PD0166285, a drug known to induce premature mitosis. Shortening G2 phase results in a prolonged interval from nuclear envelope breakdown to anaphase that is partially rescued by inhibiting the spindle assembly checkpoint and not affected by inhibiting protein synthesis. Chapter 3 presents possible future directions for these studies. It contains preliminary data on the effect of shortened G2 on microtubule-kinetochore attachment, repair of DNA damage, and the centrosome.
Collection
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.