BIOMED PhD Thesis Defense

Title:
CYCLOPS 2.0: Improving and Applying Circadian Phase Reconstruction in Confounded Datasets

Speaker:
Jan Alexander Hammarlund, PhD Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University

Advisors:
Ron C. Anafi, MD, PhD
Assistant Professor
Perelman School of Medicine
University of Pennsylvania

Andres Kriete, PhD
Associate Dean for Academic Affairs
Teaching Professor
School of Biomedical Engineering, Science and Health Systems
Drexel University

Details:
Circadian rhythms regulate the timing of thousands of genes across human tissues, aligning physiology with the 24-hour day. However, most transcriptomic datasets lack time-of-day annotations, limiting our ability to study these rhythms and their disruption in disease. We improve CYCLOPS, an unsupervised algorithm for inferring internal circadian time from gene expression data, by introducing CYCLOPS 2.0—a covariate-aware model that adjusts for batch effects and non-circadian confounders. Benchmarking confirms its improved accuracy in recovering latent circadian structure under noisy conditions.

We apply CYCLOPS 2.0 to breast cancer and GTEx datasets to reconstruct circadian transcriptional order. In breast tumors, especially luminal A, we observe persistent but reprogrammed rhythms, with enhanced cycling in EMT and immune pathways. A novel metric, CYCLOPS magnitude (CMag), quantifies global rhythm strength and correlates with reduced five-year survival. Functional assays confirm that circadian disruption reduces invasiveness, linking molecular rhythms to metastatic behavior.

We identify circadian eQTLs (cQTLs)—variants that affect rhythmic parameters of gene expression—in adipose, muscle, and skin. Many cQTLs are not detected by traditional methods and frequently colocalize with GWAS loci and circadian transcription factor motifs. These findings reveal a temporally dynamic layer of gene regulation with clinical and functional relevance.

BIOMED PhD Research Proposal

Title:
Roles of Decorin in the Assembly and Retention of Nascent Cartilage ECM Constituents in Native and Regenerative Matrices

Speaker:
Thomas Li, PhD Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University

Advisor:
Lin Han, PhD
Professor
School of Biomedical Engineering, Science and Health Systems
Drexel University

Details:
Articular cartilage is a specialized load-bearing tissue whose mechanical resilience depends on the structural integration of its two primary extracellular matrix (ECM) constituents: collagen II fibrils and the aggrecan supramolecular network. Disruption of this integration, particularly through aggrecan loss, is a hallmark of early osteoarthritis (OA) and is not adequately addressed by current regenerative strategies. Emerging evidence highlights the role of decorin, a small leucine-rich proteoglycan, in coordinating ECM architecture, yet its precise function in regulating nascent matrix assembly and retention remains poorly understood. This thesis elucidates how decorin modulates the formation and stability of newly synthesized cartilage ECM components under both normal and degenerative conditions.

Using bio-orthogonal click-labeling, we visualized the temporal and spatial deposition of nascent glycosaminoglycans and proteins in both explant and 3D methacrylated hyaluronan (MeHA) hydrogel models. We found that decorin loss accelerates the release of nascent aggrecan without affecting collagen II synthesis, suggesting a role in proteoglycan retention. Exogenous decorin treatments mitigated matrix loss only when administered before catabolic stimulation, indicating protective effects are contingent on early matrix incorporation. Surface plasmon resonance further revealed high-affinity binding between decorin and aggrecan and enhanced ternary interactions between decorin, collagen II, and aggrecan, supporting its role as a “physical linker."

In MeHA hydrogels, decorin overexpression via AAV2 gene therapy is hypothesized to improve matrix retention, collagen network formation, and mechanotransduction under dynamic loading. Together, these findings establish decorin as a key mediator of cartilage ECM integration and resilience, with translational implications for regenerative medicine. By advancing our understanding of how nascent matrix structure is regulated, this work sets the stage for decorin-based therapeutic strategies aimed at enhancing tissue repair and delaying OA progression.

BIOMED Master's Thesis Defense

Title:
Optimizing Stability of Engineered Anti-RAS bioPROTAC for Rapid Degradation of Undruggable Proteins

Speaker:
Shaila Rao, Master's Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University
 
Advisors:
Andrew Tsourkas, PhD
Professor
Department of Bioengineering
University of Pennsylvania

Kara Spiller, PhD
URBN Professor of Biomedical Innovation
School of Biomedical Engineering, Science and Health Systems
Drexel University

Details:
Proteins that are commonly mutated and overexpressed in cancers are called oncoproteins and are typical targets for cancer therapies. Cancer treatment faces significant challenges due to the presence of "undruggable" proteins—key drivers of tumor growth and survival that lack suitable binding sites for conventional therapeutics. Undruggable proteins constitute a great percentage of all proteins and have thus been a focus of drug and therapeutic delivery. Due to their structure lacking deep drug-binding sites or having a large smooth surface, traditional inhibitors cannot successfully bind to and block or degrade the protein. Many off-target effects result from this, which can cause unwanted physiological responses while leaving the target protein relatively uninhibited. A key example of undruggable oncoproteins is the Ras family of proteins, a key player in many cancer pathways.

Small-molecule inhibitors, degraders, and other biotherapeutics have been developed to target oncoproteins. A powerful small-molecule drug, known as a Proteolysis Targeting Chimera (PROTAC), has been developed. A PROTAC is made of two functional parts: a target protein binding domain and an E3 ubiquitin ligase recruiting domain. PROTACs have superior degradation properties compared to traditional small-molecule degraders due to their structure, which allows them to bring the target protein and an E3 ligase in proximity so that the target protein can be tagged with ubiquitin for Ubiquitin-proteasome degradation. While PROTACs can be designed to degrade undruggable proteins, their application becomes limited due to their size and inability to enter cells.

These small-molecule drugs allow for the degradation of druggable proteins. Using a protein-based biologic method, on the other hand, can more effectively target “undruggable” proteins. One of these protein-based degraders, known as a bioPROTAC, was developed. bioPROTACs are heterobifunctional degrader fusion proteins consisting of a target-binding domain, a linker, and an E3 ligase complex. Compared to the PROTAC, bioPROTACs have higher success rates, fewer design constraints, are biodegradable, and can clear up to 95% of the target protein within hours of treatment. An anti-RAS bioPROTAC was developed by the Tsourkas Lab at the University of Pennsylvania. Points of improvement were identified, one of which was to improve the stability of these anti-RAS bioPROTACs. It was found that the bioPROTACS were subject to autoubiquitination, which means the bioPROTAC was being artificially tagged for self-destruction, leading to reduced stability, potency, and specificity.

To stabilize the biodegrader, the lysine sites prone to auto-ubiquitination on the wild-type bioPROTAC were identified and substituted with an arginine, which is known as a K to R mutation. Lysine and arginine are both positively charged and have similar structures, but arginine is resistant to auto-ubiquitination. A Q5 Site Directed Mutagenesis, followed by Minipreps and sequencing, provided plasmid samples to be expressed into proteins in E. coli and purified via the lab’s sortase-tag expressed protein ligation (STEPL) protocol. These mutated bioPROTACs were tested for auto-ubiquitination and their ability to drive KRAS ubiquitination and degradation in vitro. Several protein engineering techniques and machinery were employed, including but not limited to Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE), protein expression and purification, lipid nanoparticle (LNP) formulation, and flow cytometry. The performance of the individual mutants informed which K to R mutations were combined into a single gene block plasmid. The same process for individual mutations was followed to develop a single engineered anti-RAS bioPROTAC molecule, likely with increased stability and potency.

BIOMED PhD Research Proposal

Title:
Optimization of Process Parameters for Filament Fabrication of PEKK and Si3N4-PEKK for Total Knee (TK) Arthroplasty

Speaker:
Tabitha Derr, PhD Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University

Advisor:
Steven Kurtz, PhD
Research Professor
School of Biomedical Engineering, Science and Health Systems
Drexel University

Details:
Total knee arthroplasty (TKA) is a procedure in which the knee joint is replaced by an artificial implant to reduce pain and restore mobility. With the aging population and increases in obesity and osteoarthritis, TKA is becoming increasingly frequent. Despite years of development, instability, loosening and infection remain the most common causes of implant failure leading to revision surgery. Therefore, it remains imperative to address these remaining concerns to ensure successful outcomes of TKA.

Traditionally, TKA is composed of cobalt-chromium and titanium femoral and tibial components that are fused to the bone with an articulating polyethylene insert positioned between them. With increasing concerns of stress shielding and wear of metallic implants, interest has shifted to PAEK materials that are bioinert and have mechanical properties more similar to bone. Additionally, these materials can be additively manufactured via fused filament fabrication (FFF), enabling faster and cheaper production as well as architecture that supports osseointegration to reduce risks of loosening and instability. An emerging PAEK in the medical field is polyetherketoneketone (PEKK). This polymer has lower processing temperatures than other widely used PAEKs, easing manufacturing pressures. Furthermore, PEKK’s antibacterial and osseointegrative properties can be enriched through the addition of a bioactive filler. Silicon nitride (Si3N4) has been shown to have both antibacterial and osseointegrative properties. Therefore, incorporating Si3N4 into a PEKK composite allows for the combination of PEKK’s superior mechanical properties with Si3N4’s antimicrobial and osseointegrative benefits.

To effectively use PEKK and Si3N4-PEKK implants created via FFF in TKA applications, they must have adequate mechanical properties while retaining accurate dimensions. Therefore, it is necessary to better understand their FFF processing requirements, specifically how their processing affects the resultant mechanical strengths and dimensional tolerances. This research aims to investigate and optimize the FFF processing and post-processing of PEKK and Si3N4-PEKK, in an effort to improve the understanding of potential new biomaterials for TKA applications.

The School of Biomedical Engineering, Science and Health Systems is pleased to announce its 7th Annual Immune Modulation & Engineering Symposium (IMES).