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Description
The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial

The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial proteins expressed in human cell lines, yet they exhibit an organizing principle: that genes and proteins may be treated as modular units that can be moved from their native organism to a novel one. However, protein behavior is always unpredictable; drop-in functionality is not guaranteed.

My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.

1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.

2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
ContributorsDaer, René (Author) / Haynes, Karmella (Thesis advisor) / Brafman, David (Committee member) / Nielsen, David (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2017
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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,

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
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Description
Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome

Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin.

I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions.

SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug.

Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.
ContributorsBarrett, Cassandra M (Author) / Haynes, Karmella A (Thesis advisor) / Rege, Kaushal (Committee member) / Mills, Jeremy (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2019
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Description
Breast cancer is the second leading cause of disease related death in women, contributing over

40,000 fatalities annually. The severe impact of breast cancer can be attributed to a poor

understanding of the mechanisms underlying cancer metastasis. A primary aspect of cancer

metastasis includes the invasion and intravasation that results in cancer cells

Breast cancer is the second leading cause of disease related death in women, contributing over

40,000 fatalities annually. The severe impact of breast cancer can be attributed to a poor

understanding of the mechanisms underlying cancer metastasis. A primary aspect of cancer

metastasis includes the invasion and intravasation that results in cancer cells disseminating from

the primary tumor and colonizing distant organs. However, the integrated study of invasion and

intravasation has proven to be challenging due to the difficulties in establishing a combined tumor

and vascular microenvironments. Compared to traditional in vitro assays, microfluidic models

enable spatial organization of 3D cell-laden and/or acellular matrices to better mimic human

physiology. Thus, microfluidics can be leveraged to model complex steps of metastasis. The

fundamental aim of this thesis was to develop a three-dimensional microfluidic model to study the

mechanism through which breast cancer cells invade the surrounding stroma and intravasate into

outerlying blood vessels, with a primary focus on evaluating cancer cell motility and vascular

function in response to biochemical cues.

A novel concentric three-layer microfluidic device was developed, which allowed for

simultaneous observation of tumor formation, vascular network maturation, and cancer cell

invasion/intravasation. Initially, MDA-MB-231 disseminated from the primary tumor and invaded

the acellular collagen present in the adjacent second layer. The presence of an endothelial network

in the third layer of the device drastically increased cancer cell invasion. Furthermore, by day 6 of

culture, cancer cells could be visually observed intravasating into the vascular network.

Additionally, the effect of tumor cells on the formation of the surrounding microvascular network

within the vascular layer was evaluated. Results indicated that the presence of the tumor

significantly reduced vessel diameter and increased permeability, which correlates with prior in vivo

data. The novel three-layer platform mimicked the in vivo spatial organization of the tumor and its

surrounding vasculature, which enabled investigations of cell-cell interactions during cancer

invasion and intravasation. This approach will provide insight into the cascade of events leading up

to intravasation, which could provide a basis for developing more effective therapeutics.
ContributorsNagaraju, Supriya (Author) / Nikkhah, Mehdi (Thesis advisor) / Ebrahimkhani, Mohammad (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2017
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Description
The pathophysiology of Alzheimer’s disease (AD) remains difficult to precisely ascertain in part because animal models fail to fully recapitulate many aspects of the disease and postmortem studies do not allow for the study of the pathophysiology. In vitro models of AD generated with patient derived human induced pluripotent stem

The pathophysiology of Alzheimer’s disease (AD) remains difficult to precisely ascertain in part because animal models fail to fully recapitulate many aspects of the disease and postmortem studies do not allow for the study of the pathophysiology. In vitro models of AD generated with patient derived human induced pluripotent stem cells (hiPSCs) could provide new insight into disease mechanisms. Although many protocols exist to differentiate hiPSCs to neurons, standard practice relies on two-dimensional (2-D) systems, which do not accurately mimic the complexity and architecture of the in vivo brain microenvironment. This research aims to create three-dimensional (3-D) models of AD using hiPSCs, which would enhance the understanding of AD pathophysiology thereby, enabling the generation of effective therapeutics.
ContributorsLundeen, Rachel (Author) / Brafman, David (Thesis advisor) / Kiani, Samira (Committee member) / Ebrahimkhani, Mohammad (Committee member) / Arizona State University (Publisher)
Created2017
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Description
Ideas from coding theory are employed to theoretically demonstrate the engineering of mutation-tolerant genes, genes that can sustain up to some arbitrarily chosen number of mutations and still express the originally intended protein. Attention is restricted to tolerating substitution mutations. Future advances in genomic engineering will make possible the ability

Ideas from coding theory are employed to theoretically demonstrate the engineering of mutation-tolerant genes, genes that can sustain up to some arbitrarily chosen number of mutations and still express the originally intended protein. Attention is restricted to tolerating substitution mutations. Future advances in genomic engineering will make possible the ability to synthesize entire genomes from scratch. This presents an opportunity to embed desirable capabilities like mutation-tolerance, which will be useful in preventing cell deaths in organisms intended for research or industrial applications in highly mutagenic environments. In the extreme case, mutation-tolerant genes (mutols) can make organisms resistant to retroviral infections.

An algebraic representation of the nucleotide bases is developed. This algebraic representation makes it possible to convert nucleotide sequences into algebraic sequences, apply mathematical ideas and convert results back into nucleotide terms. Using the algebra developed, a mapping is found from the naturally-occurring codons to an alternative set of codons which makes genes constructed from them mutation-tolerant, provided no more than one substitution mutation occurs per codon. The ideas discussed naturally extend to finding codons that can tolerate t arbitrarily chosen number of mutations per codon. Finally, random substitution events are simulated in both a wild-type green fluorescent protein (GFP) gene and its mutol variant and the amino acid sequence expressed from each post-mutation is compared with the amino acid sequence pre-mutation.

This work assumes the existence of synthetic protein-assembling entities that function like tRNAs but can read k nucleotides at a time, with k greater than or equal to 5. The realization of this assumption is presented as a challenge to the research community.
ContributorsAmpofo, Prince Kwame (Author) / Tian, Xiaojun (Thesis advisor) / Kiani, Samira (Committee member) / Kuang, Yang (Committee member) / Arizona State University (Publisher)
Created2019
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Description
Extracellular Vesicles (EVs), particularly exosomes, are of considerable interest as tumor biomarkers since tumor-derived EVs contain a broad array of information about tumor pathophysiology including its metabolic and metastatic status. However, current EV based assays cannot distinguish between EV biomarker changes by altered secretion of EVs during diseased conditions like

Extracellular Vesicles (EVs), particularly exosomes, are of considerable interest as tumor biomarkers since tumor-derived EVs contain a broad array of information about tumor pathophysiology including its metabolic and metastatic status. However, current EV based assays cannot distinguish between EV biomarker changes by altered secretion of EVs during diseased conditions like cancer, inflammation, etc. that express a constant level of a given biomarker, stable secretion of EVs with altered biomarker expression, or a combination of these two factors. This issue was addressed by developing a nanoparticle and dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker level(s) to EV abundance, revealing average expression levels of EV biomarker under observation. In this approach, EVs are captured from complex samples (e.g. serum), stained with a lipophilic dye and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels. EVs from malignant (PANC-1) and nonmalignant pancreatic cell lines (HPNE) exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM, and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its flexible design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple swapping of the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker utilizing a workflow that is suitable for rapid clinical translation.
ContributorsRodrigues, Meryl (Author) / Hu, Tony (Thesis advisor) / Nikkhah, Mehdi (Committee member) / Kiani, Samira (Committee member) / Smith, Barbara (Committee member) / Han, Haiyong (Committee member) / Arizona State University (Publisher)
Created2019
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Description
The CRISPR/Cas9 gene-editing tool is currently in clinical trials as the excitement about its therapeutic potential is exponentially growing. However, many of the developed CRISPR based genome engineering methods cannot be broadly translated in clinical settings due to their unintended consequences. These consequences, such as immune reactions to CRISPR, immunogenic

The CRISPR/Cas9 gene-editing tool is currently in clinical trials as the excitement about its therapeutic potential is exponentially growing. However, many of the developed CRISPR based genome engineering methods cannot be broadly translated in clinical settings due to their unintended consequences. These consequences, such as immune reactions to CRISPR, immunogenic adverse events following receiving of adeno-associated virus (AAV) as one of the clinically relevant delivery agents, and CRISPR off-target activity in the genome, reinforces the necessity for improving the safety of CRISPR and the gene therapy vehicles. Research into designing more advanced CRISPR systems will allow for the increased ability of editing efficiency and safety for human applications. This work 1- develops strategies for decreasing the immunogenicity of CRISPR/Cas9 system components and improving the safety of CRISPR-based gene therapies for human subjects, 2- demonstrates the utility of this system in vivo for transient repression of components of innate and adaptive immunity, and 3- examines an inducible all-in-one CRISPR-based control switch to pave the way for controllable CRISPR-based therapies.
ContributorsMoghadam, Farzaneh (Author) / Kiani, Samira (Thesis advisor) / LaBaer, Josh (Committee member) / Ebrahimkhani, Mo (Committee member) / Arizona State University (Publisher)
Created2020
Description
A genetically engineered line of human induced pluripotent stem cells was used to study the effects of gene expression on cell fate. These cells were designed to activate expression of the gene GATA6 when exposed to the small molecule doxycycline. This gene was chosen because it plays an

A genetically engineered line of human induced pluripotent stem cells was used to study the effects of gene expression on cell fate. These cells were designed to activate expression of the gene GATA6 when exposed to the small molecule doxycycline. This gene was chosen because it plays an important role in the developmental biology stages of liver formation. Because of the way the cells were engineered, a given population would have a heterogeneous expression of GATA6 because each cell could have a different copy number of the exogenous gene. This variation allows for the differentiation of multiple cell types, and is used to grow liver organoids. The early liver organoid samples were studied via immunofluorescent staining, imaging, and quantitative image analysis. It was originally hypothesized that absolute gene expression was not the most important factor in determining cell fate, but relative gene expression was. This meant that the spatial location of the cells and their local environment were critical in determining cell fate. In other words, the level of GATA6 of a cell is important, but so is the level of GATA6 in the surrounding cells, or neighborhood, of that cell. This hypothesis was analyzed with the creation of various Neighborhood Impact Factor (NIF) methods. Multiple time points of growth were analyzed to study the temporal effect, in addition to the gene expression and NIF influence on a cell’s fate. Direct gene expression level showed correlation with certain cell fate markers. In addition to GATA6 expression levels, NIF results from early and late time point experiments show statistical significance with relatively small neighborhood radii. The NIF analysis was useful for examining the effect of neighboring cells and determining the size of the neighborhood – how far cells influence one another. While these systems are complex, the NIF analysis provides a way to look at gene expression and its influence in spatial context.
ContributorsCarter, Shaylina Rae (Author) / Ebrahimkhani, Mohammad R (Thesis advisor) / Kiani, Samira (Thesis advisor) / Nikkhah, Mehdi (Committee member) / Arizona State University (Publisher)
Created2017