Matching Items (25)
Description
Synthetic biology is an emerging field which melds genetics, molecular biology, network theory, and mathematical systems to understand, build, and predict gene network behavior. As an engineering discipline, developing a mathematical understanding of the genetic circuits being studied is of fundamental importance. In this dissertation, mathematical concepts for understanding, predicting, and controlling gene transcriptional networks are presented and applied to two synthetic gene network contexts. First, this engineering approach is used to improve the function of the guide ribonucleic acid (gRNA)-targeted, dCas9-regulated transcriptional cascades through analysis and targeted modification of the RNA transcript. In so doing, a fluorescent guide RNA (fgRNA) is developed to more clearly observe gRNA dynamics and aid design. It is shown that through careful optimization, RNA Polymerase II (Pol II) driven gRNA transcripts can be strong enough to exhibit measurable cascading behavior, previously only shown in RNA Polymerase III (Pol III) circuits. Second, inherent gene expression noise is used to achieve precise fractional differentiation of a population. Mathematical methods are employed to predict and understand the observed behavior, and metrics for analyzing and quantifying similar differentiation kinetics are presented. Through careful mathematical analysis and simulation, coupled with experimental data, two methods for achieving ratio control are presented, with the optimal schema for any application being dependent on the noisiness of the system under study. Together, these studies push the boundaries of gene network control, with potential applications in stem cell differentiation, therapeutics, and bio-production.
ContributorsMenn, David J (Author) / Wang, Xiao (Thesis advisor) / Kiani, Samira (Committee member) / Haynes, Karmella (Committee member) / Nielsen, David (Committee member) / Marshall, Pamela (Committee member) / Arizona State University (Publisher)
Created2018
Description
Calcium is the only ion capable of triggering electrical and chemical reactions in cells which are part of essential biomolecular processes, such as gene transcription and ion flux. Calcium homeostasis, the control of concentration levels, is therefore crucial for the proper functioning of cells. For example, cardiomyocytes, the cells that form cardiac muscle, rely on calcium transfer process to produce muscle contraction.
The purpose of this work is to study aspects of calcium homeostasis in the model organism Saccharomyces cerevisiae, common yeast. Using luminometric techniques, the response of the yeast was monitored against a set of changes in the environment calcium abundance. The results indicate a complex response as both increase and decreases of external calcium induce elevations in cytosolic calcium concentrations.
Calcium is transferred across compartments by means of channels. In Saccharomyces cerevisiae, many of them have been identified; Cch1p-Mid1p, Vcx1p, Pmc1p, Pmr1p, and Yvc1p. Their participation in calcium homeostasis is well established. Observations of cytosolic calcium increase after a hypertonic shock are mainly associated with influx of ions from the environment though the Cch1p-Mid1p. This process is generally considered as driven by calcium concentration gradients. However, recent studies have suggested that the plasma membrane channel, Cch1p-Mid1p, may possess more sophisticated regulation and sensory mechanisms. The results of our experiments support these ideas.
We carried out experiments that subjected yeast to multiple shocks: a hypertonic shock followed by either a second hypertonic shock, a hypotonic shock, or a yeast dilution pulse where the solution volume increases by the calcium concentration has only a small change. The cytosolic calcium concentration of a yeast population was monitored via luminometry.
The main result of this study is the observation of an unexpected response to the combination of hypertonic and hypotonic shocks. In this case it was observed that the cytosolic calcium concentration increased after both shocks. This indicates that cytosolic calcium increases are not solely driven by the presence of concentration gradients. The response after the hypotonic pulse arises from more complex mechanisms that may include sensor activity at the membrane channels and the release of calcium from internal storages.
The purpose of this work is to study aspects of calcium homeostasis in the model organism Saccharomyces cerevisiae, common yeast. Using luminometric techniques, the response of the yeast was monitored against a set of changes in the environment calcium abundance. The results indicate a complex response as both increase and decreases of external calcium induce elevations in cytosolic calcium concentrations.
Calcium is transferred across compartments by means of channels. In Saccharomyces cerevisiae, many of them have been identified; Cch1p-Mid1p, Vcx1p, Pmc1p, Pmr1p, and Yvc1p. Their participation in calcium homeostasis is well established. Observations of cytosolic calcium increase after a hypertonic shock are mainly associated with influx of ions from the environment though the Cch1p-Mid1p. This process is generally considered as driven by calcium concentration gradients. However, recent studies have suggested that the plasma membrane channel, Cch1p-Mid1p, may possess more sophisticated regulation and sensory mechanisms. The results of our experiments support these ideas.
We carried out experiments that subjected yeast to multiple shocks: a hypertonic shock followed by either a second hypertonic shock, a hypotonic shock, or a yeast dilution pulse where the solution volume increases by the calcium concentration has only a small change. The cytosolic calcium concentration of a yeast population was monitored via luminometry.
The main result of this study is the observation of an unexpected response to the combination of hypertonic and hypotonic shocks. In this case it was observed that the cytosolic calcium concentration increased after both shocks. This indicates that cytosolic calcium increases are not solely driven by the presence of concentration gradients. The response after the hypotonic pulse arises from more complex mechanisms that may include sensor activity at the membrane channels and the release of calcium from internal storages.
ContributorsMintz, David Anthony (Co-author) / Parker, Augustus (Co-author) / Solis, Francisco (Thesis director) / Marshall, Pamela (Committee member) / School of Mathematical and Natural Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Calcium is the only ion capable of triggering electrical and chemical reactions in cells which are part of essential biomolecular processes, such as gene transcription and ion flux. Calcium homeostasis, the control of concentration levels, is therefore crucial for the proper functioning of cells. For example, cardiomyocytes, the cells that form cardiac muscle, rely on calcium transfer process to produce muscle contraction.
The purpose of this work is to study aspects of calcium homeostasis in the model organism Saccharomyces cerevisiae, common yeast. Using luminometric techniques, the response of the yeast was monitored against a set of changes in the environment calcium abundance. The results indicate a complex response as both increase and decreases of external calcium induce elevations in cytosolic calcium concentrations.
Calcium is transferred across compartments by means of channels. In Saccharomyces cerevisiae, many of them have been identified; Cch1p-Mid1p, Vcx1p, Pmc1p, Pmr1p, and Yvc1p. Their participation in calcium homeostasis is well established. Observations of cytosolic calcium increase after a hypertonic shock are mainly associated with influx of ions from the environment though the Cch1p-Mid1p. This process is generally considered as driven by calcium concentration gradients. However, recent studies have suggested that the plasma membrane channel, Cch1p-Mid1p, may possess more sophisticated regulation and sensory mechanisms. The results of our experiments support these ideas.
We carried out experiments that subjected yeast to multiple shocks: a hypertonic shock followed by either a second hypertonic shock, a hypotonic shock, or a yeast dilution pulse where the solution volume increases by the calcium concentration has only a small change. The cytosolic calcium concentration of a yeast population was monitored via luminometry.
The main result of this study is the observation of an unexpected response to the combination of hypertonic and hypotonic shocks. In this case it was observed that the cytosolic calcium concentration increased after both shocks. This indicates that cytosolic calcium increases are not solely driven by the presence of concentration gradients. The response after the hypotonic pulse arises from more complex mechanisms that may include sensor activity at the membrane channels and the release of calcium from internal storages.
The purpose of this work is to study aspects of calcium homeostasis in the model organism Saccharomyces cerevisiae, common yeast. Using luminometric techniques, the response of the yeast was monitored against a set of changes in the environment calcium abundance. The results indicate a complex response as both increase and decreases of external calcium induce elevations in cytosolic calcium concentrations.
Calcium is transferred across compartments by means of channels. In Saccharomyces cerevisiae, many of them have been identified; Cch1p-Mid1p, Vcx1p, Pmc1p, Pmr1p, and Yvc1p. Their participation in calcium homeostasis is well established. Observations of cytosolic calcium increase after a hypertonic shock are mainly associated with influx of ions from the environment though the Cch1p-Mid1p. This process is generally considered as driven by calcium concentration gradients. However, recent studies have suggested that the plasma membrane channel, Cch1p-Mid1p, may possess more sophisticated regulation and sensory mechanisms. The results of our experiments support these ideas.
We carried out experiments that subjected yeast to multiple shocks: a hypertonic shock followed by either a second hypertonic shock, a hypotonic shock, or a yeast dilution pulse where the solution volume increases by the calcium concentration has only a small change. The cytosolic calcium concentration of a yeast population was monitored via luminometry.
The main result of this study is the observation of an unexpected response to the combination of hypertonic and hypotonic shocks. In this case it was observed that the cytosolic calcium concentration increased after both shocks. This indicates that cytosolic calcium increases are not solely driven by the presence of concentration gradients. The response after the hypotonic pulse arises from more complex mechanisms that may include sensor activity at the membrane channels and the release of calcium from internal storages.
ContributorsParker, Augustus Carrucciu (Co-author) / Mintz, David (Co-author) / Solis, Francisco (Thesis director) / Marshall, Pamela (Committee member) / School of Mathematical and Natural Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
This project details the synthesis and analysis of five analogs of model compound NEt-4IB (6-[ethyl(4-isobutoxy-3-isopropylphenyl)amino]nicotinic acid), that target the retinoid-X-receptor (RXR). These molecules were synthesized by substituting, adding, or removing substituents in the nitrogen-containing ring of NEt-4IB. The parent compound is a RXR partial agonist and has proven to be effective in the treatment of type II diabetes without the unwanted side effects seen with full agonists. Many of the current drugs used to treat type II diabetes are accompanied by adverse effects including increased triglyceride levels, weight gain, and hypoglycemia. Biological evaluation with KK-Ay (obese diabetic) model mice indicates that NEt-4IB may even be more effective than current drugs on the market, like pioglitazone. As a result, it is predicted that due to such structural similarity, the analogs synthesized for this work will perform equally, if not better than, NEt-4IB.
ContributorsMaiorella, Emma Lauren (Author) / Wagner, Carl (Thesis director) / Marshall, Pamela (Committee member) / School of Mathematical and Natural Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
In humans, infections, disease, inflammation, and other injuries to specific tissues have been shown to cause delays in the onset of puberty. It is known that steroid hormones and insulin play a role in these delays, yet it is not understood what is happening with the immune system during this response. Similar results have been found in the fruit fly, Drosophila melanogaster, in which damage to adult precursor tissues triggers developmental delays. This project addresses the immune component of the injury response in Drosophila. The goal is to identify which immune response genes, if any, show a significant change in expression after injury. The general methodologies used were first inducing injury via a temperature- sensitive expression of cell death genes in wing precursor tissues, then examining changes in gene expression of immune response genes before and after injury using real-time PCR. The results show that injury increases the expression of genes Drs, CecA1, and Def while decreasing expression of Rel, Dpt, PGRP-LE, and Tl. The changes in immune gene expression following injury suggest the possibility of an immune component to the systemic injury response. These results can further be explored by using mutations of the immune genes to examine their direct effects on the systemic injury response. This research can eventually lead to preventative measures to protect against developmental delays due to infections and diseases in humans.
ContributorsDuprey, Deanna Jeanette (Author) / Hackney, Jennifer (Thesis director) / Marshall, Pamela (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Natural Sciences (Contributor) / School of Social and Behavioral Sciences (Contributor)
Created2014-12
Description
Virtually all animals require relatively predictable developmental schedules in order to fulfill the cycle of life. Cell death and severe inflammation alter steroid hormone production and can disrupt the timing of developmental transitions such as puberty. In the fruit fly, Drosophila melanogaster, injury to wing precursor tissues has been shown to result in decreased steroid hormone levels and delay development. The effects of damage to other tissues have not yet been explored. Here, the larval salivary glands were damaged in order to observe how injuring these specific tissues affect the timing of developmental transitions. Damage was induced by tissue-specific, temperature sensitive activation of cell death genes. The results indicated that death to salivary gland cells accelerates the Drosophila time to adult eclosion and that the observed acceleration of development is age-dependent. Insight into the effects of injury on development in Drosophila can potentially lead to information about development in other organisms, including humans, following injury or chronic inflammation.
ContributorsRippere, Alicia Leann (Author) / Hackney, Jennifer (Thesis director) / Marshall, Pamela (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Natural Sciences (Contributor)
Created2015-05
Description
Bexarotene (Targretin®) is an FDA approved drug used to treat cutaneous T-cell lymphoma (CTCL), as well as off-label treatments for various cancers and neurodegenerative diseases. Previous research has indicated that bexarotene has a specific affinity for retinoid X receptors (RXR), which allows bexarotene to act as a ligand-activated-transcription factor and in return control cell differentiation and proliferation. Bexarotene targets RXR homodimerization to drive transcription of tumor suppressing genes; however, adverse reactions occur simultaneously when bound to other nuclear receptors. In this study, we used novel bexarotene analogs throughout 5 iterations synthesized in the laboratory of Dr. Wagner to test for their potency and ability to bind RXR. The aim of our study is to quantitatively measure RXR homodimerization driven by bexarotene analogs using a yeast two-hybrid system. Our results suggests there to be several compounds with higher protein activity than bexarotene, particularly in generations 3.0 and 5.0. This higher affinity for RXR homodimers may help scientists identify a compound that will minimize adverse effects and toxicity of bexarotene and serve as a better cancer treatment alternative.
ContributorsSeto, David Hua (Author) / Marshall, Pamela (Thesis director) / Wagner, Carl (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Natural Sciences (Contributor) / School of Social and Behavioral Sciences (Contributor)
Created2015-05
Description
Universal healthcare has become a regular feature of most developed nations around the world. This characteristic, however, does not extend to the United States, where some 28.2 million Americans remain uninsured or underinsured. In the past few years, the US has been on the precipice of major healthcare overhaul which has brought the debate on government-sponsored coverage to the forefront of political discourse. This thesis explores what it may mean to establish affordable access to healthcare as a right for all Americans. In doing so, it utilizes rule-utilitarian principles to define and assess the moral obligation of the United States' federal and state governments to provide sufficient coverage to all qualifying individuals within the country. This paper focuses on evaluating the current healthcare system in the United States while concentrating particularly on how its fragmented approach limits its success and longevity. It then offers a cross-comparison with the universal healthcare systems of Canada, France, and Japan, nations that outperform the United States in most healthcare measures such as life expectancy, infant and under-5 mortality, medical costs per capita, and disease prevalence. The free-market criticisms of government-provided coverage and its alternative private-insurance-based approach to healthcare in the US are also deliberated. In light of these considerations, this thesis concludes with a commentary on what healthcare reform could look like for the nation as well as examines how a utilitarian appeal to rights likely makes the best case for adopting universal government-sponsored healthcare coverage in the United States.
ContributorsKhan, Sameera (Author) / Manninen, Bertha (Thesis director) / Marshall, Pamela (Committee member) / School of Mathematical and Natural Sciences (Contributor) / School of Humanities, Arts, and Cultural Studies (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Cancer, a disease which affects many lives, has been the topic of interest for this research. Treatment options are often available to help lessen the effects of the disease and in regards to cutaneous T-cell lymphoma (CTCL), no cure currently exists. An FDA approved drug by the name of Bexarotene has been developed to provide chemotherapeutic effects within CTCL. Bexarotene has also been used in trials of breast cancer, lung cancer, glioblastoma multiforme and various neurodegenerative diseases. Yet the medication often causes serious side effects including hyperthyroidism, raised triglyceride levels and cutaneous toxicity. The focus of this research is to synthesize a hydroxylated analog compound of Bexarotene in efforts to produce a molecule that provides better chemotherapeutic effects while also lessening the various side effects caused. Synthesis of the molecule followed various organic chemistry techniques and reactions to create the final product. Melting point analysis, NMR and other various characterization data helped to confirm the synthesis of the intended molecule. Preliminary bioassay data results of the analog compound showed similar potency to that of Bexarotene. Further testing, however, will be required to determine the full pharmacokinetic profile of the molecule. Future direction of the research focuses on both further testing of the hydroxylated analog as well synthesizing newer analog compounds to find a molecule that can provide the best effects within cutaneous T-cell lymphoma and the various other diseases as well.
ContributorsMinasian, Ani Christina (Author) / Wagner, Carl (Thesis director) / Marshall, Pamela (Committee member) / School of Social and Behavioral Sciences (Contributor) / School of Mathematical and Natural Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Synthetic gene networks have evolved from simple proof-of-concept circuits to
complex therapy-oriented networks over the past fifteen years. This advancement has
greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a
mechanism that cells use to achieve a discrete number of mutually exclusive states in
response to environmental inputs. However, complex contextual connections of gene
regulatory networks in natural settings often impede the experimental establishment of
the function and dynamics of each specific gene network.
In this work, diverse synthetic gene networks are rationally designed and
constructed using well-characterized biological components to approach the cell fate
determination and state transition dynamics in multistable systems. Results show that
unimodality and bimodality and trimodality can be achieved through manipulation of the
signal and promoter crosstalk in quorum-sensing systems, which enables bacterial cells to
communicate with each other.
Moreover, a synthetic quadrastable circuit is also built and experimentally
demonstrated to have four stable steady states. Experiments, guided by mathematical
modeling predictions, reveal that sequential inductions generate distinct cell fates by
changing the landscape in sequence and hence navigating cells to different final states.
Circuit function depends on the specific protein expression levels in the circuit.
We then establish a protein expression predictor taking into account adjacent
transcriptional regions’ features through construction of ~120 synthetic gene circuits
(operons) in Escherichia coli. The predictor’s utility is further demonstrated in evaluating genes’ relative expression levels in construction of logic gates and tuning gene expressions and nonlinear dynamics of bistable gene networks.
These combined results illustrate applications of synthetic gene networks to
understand the cell fate determination and state transition dynamics in multistable
systems. A protein-expression predictor is also developed to evaluate and tune circuit
dynamics.
complex therapy-oriented networks over the past fifteen years. This advancement has
greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a
mechanism that cells use to achieve a discrete number of mutually exclusive states in
response to environmental inputs. However, complex contextual connections of gene
regulatory networks in natural settings often impede the experimental establishment of
the function and dynamics of each specific gene network.
In this work, diverse synthetic gene networks are rationally designed and
constructed using well-characterized biological components to approach the cell fate
determination and state transition dynamics in multistable systems. Results show that
unimodality and bimodality and trimodality can be achieved through manipulation of the
signal and promoter crosstalk in quorum-sensing systems, which enables bacterial cells to
communicate with each other.
Moreover, a synthetic quadrastable circuit is also built and experimentally
demonstrated to have four stable steady states. Experiments, guided by mathematical
modeling predictions, reveal that sequential inductions generate distinct cell fates by
changing the landscape in sequence and hence navigating cells to different final states.
Circuit function depends on the specific protein expression levels in the circuit.
We then establish a protein expression predictor taking into account adjacent
transcriptional regions’ features through construction of ~120 synthetic gene circuits
(operons) in Escherichia coli. The predictor’s utility is further demonstrated in evaluating genes’ relative expression levels in construction of logic gates and tuning gene expressions and nonlinear dynamics of bistable gene networks.
These combined results illustrate applications of synthetic gene networks to
understand the cell fate determination and state transition dynamics in multistable
systems. A protein-expression predictor is also developed to evaluate and tune circuit
dynamics.
ContributorsWu, Fuqing (Author) / Wang, Xiao (Thesis advisor) / Haynes, Karmella (Committee member) / Marshall, Pamela (Committee member) / Nielsen, David (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2017