Matching Items (2)
Filtering by
- All Subjects: Intravasation
- Creators: Kiani, Samira
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 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.
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
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 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.
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