abstract: Cellular assays are the backbone of biological studies - be it for tissue modeling, drug discovery, therapeutics, or diagnostics. Two-dimensional (2D) cell culture has been deployed for several decades to garner physiologically relevant information and predict data before the cost-intensive animal testing. Although 2D techniques have been valuable for cellular assays, they have a colossal limitation - they do not adequately consider the natural three-dimensional (3D) microenvironment of the cells. As a result, they sometimes provide misleading statistics. Therefore, it is important to develop a 3D model that predicts cellular behaviors and their interaction with neighboring cells and extracellular matrix (ECM) in a more realistic manner. In recent biomedical research, various platforms have been modeled to generate 3D prototypes of tissues, spheroids, in vitro that could allow the study of cellular responses resembling in vivo environments, such as matrices, scaffolds, and devices. But most of these platforms have drawbacks such as lack of spheroid size control, low yield, or high cost associated with them. On the other hand, Amikagel is a low cost, high-fidelity platform that can facilitate the convenient generation of tumor and stem cell spheroids. Furthermore, Amikabeads are aminoglycoside-derived hydrogel microbeads derived from the same monomers as Amikagel. They are a versatile platform with several chemical groups that can be exploited for encapsulating the spheroids and investigating the delivery of bioactive compounds to the cells. This thesis is focused on engineering novel 3D tumor and stem cell models generated on Amikagel and encapsulated in Amikabeads for proximal delivery of bioactive compounds and applications in regenerative medicine. / Dissertation/Thesis / Z-stacks of confocal images of spheroids encapsulated in Amikabeads (compilations of sections) / Masters Thesis Bioengineering 2020

The heat shock response (HSR) is a robust cellular reaction to mitigate protein damage from heat and other challenges to the proteome. This protective molecular program in humans is controlled by heat shock transcription factor 1 (HSF1). Activation of HSF1 leads to the induction of an array of cytoprotective genes, many of which code for chaperones. These chaperones, known as heat shock proteins (HSPs), are responsible for maintaining the functional integrity of the proteome. HSPs achieve this by promoting proper folding and assembly of nascent proteins, refolding denatured proteins, and processing for degradation proteins and aggregates which cannot be returned to a functional conformation. The powerful ability of the heat shock response to promote cell survival makes its master regulator, HSF1, an important point of research. To garner a better understanding of HSF1, we reviewed the role of the highly dynamic HSF1 protein structure and investigated how HSF1 affects cancer cell behavior and drug response. Cancers can be characterized in part by abhorrent replication, self-sufficient growth signaling, invasion, and evasion of apoptosis. HSF1 has been found to promote proliferation, invasion, and drug resistance in several types of cancer; including lung and ovarian cancer. Ovarian cancer has elevated levels of HSF1, but the role of HSF1 in ovarian cancer behavior had not been previously examined. Researching the role of HSF1 in ovarian cancer is merited, because treatment outcomes are poor due to the high frequency of late stage detection and drug resistance. We hypothesized that HSF1 is important in the malignant growth and drug resistance of ovarian cancer. We have created ovarian cancer cell lines with inducible knockdown of HSF1 to investigate how HSF1 contributes to the behavior of ovarian cancer. This allowed us to examine the behavior of cells in the absence HSF1. Both 2D and 3D spheroid tissue culture models were used to study how HSF1 contributes to the growth and invasion of ovarian cancer cells after treatment with the transforming growth factor β (TGFβ) cytokine. Additionally, we studied how HSF1 reduction modulates the response to multiple therapeutic drugs. Our research shows that HSF1 induces epithelial-mesenchymal transition (EMT) in a 3D growth model. Our work also demonstrates that reduction of HSF1 sensitizes ovarian cancer cells to multiple drugs.

<p dir="ltr">Tuberculosis (TB) remains a global public health crisis, causing over 10 million new infections and 1.3 million deaths in 2022 alone. TB is caused by <i>Mycobacterium tuberculosis </i>(<i>Mtb</i>), which initiates heterogeneous pathology in the lungs, including granulomas and cavities. Granulomas are organized structures of immune cells, traditionally thought to contain bacteria. Cavities are pathological spaces caused by the destruction of extracellular matrix (ECM), which can worsen disease outcomes and cause long-lasting pulmonary impairment.<i> In vitro </i>methods are commonly used to study host-pathogen interactions in <i>Mtb</i> infection, and recent developments have led to models that represent the TB granuloma environment more closely than traditional cell culture. These advances include the development of 3D models and the inclusion of physiological ECM components like collagen. Increasing complexity has been accomplished in a piece-wise manner – minimally necessary components are included to minimize cost while maintaining throughput and tractability. This creates a need for tools to analyze these systems and, more importantly, integrate the independent data created. We developed an agent-based model to characterize multiple <i>in vitro</i> models of TB and apply it to 1) separate the contributions of dimension and structure to bacterial control in granuloma-like spheroids and 2) explore how the interactions of collagen and matrix metalloproteinases (MMP) contribute to clinically relevant outputs such as bacterial load and ECM destruction. The model provides insights into the role of granuloma structure and the conflicting results of MMP inhibition, generating new hypotheses to be tested in tandem with <i>in vitro</i> models.</p>