Matching Items (2)
Description
Traumatic brain injury (TBI) is a serious health problem around the world with few available treatments. TBI pathology can be divided into two phases: the primary insult and the secondary injury. The primary insult results from the bump or blow to the head that causes the initial injury. Secondary injury lasts from hours to months after the initial injury and worsens the primary insult, creating a greater area of tissue damage and cell death. Many current treatments focus on lessening the severity of secondary injury. Secondary injury results from the cyclical nature of tissue damage. Inflammatory pathways cause damage to tissue, which in turn reinforces inflammation. Since many inflammatory pathways are interconnected, targeting individual products within these pathways is impractical. A target at the beginning of the pathway, such as a receptor, must be chosen to break the cycle. This project aims to identify novel nanobodies that could temporarily inactivate the CD36 receptor, which is a receptor found on many immune and endothelial cells. CD36 initiates and perpetuates the immune system's inflammatory responses. By inactivating this receptor temporarily, inflammation and immune cell entry could be lessened, and therefore secondary injury could be attenuated. This project utilized phage display as a method of nanobody selection. The specific phage library utilized in this experiment consists of human heavy chain (V_H) segments, also known as domain antibodies (dAbs), displayed on M13 filamentous bacteriophage. Phage display mimics the process of immune selection. The target is bound to a well as a means of displaying it to the phage. The phage library is then incubated with the target to allow antibodies to bind. After, the well is washed thoroughly to detach any phage that are not strongly bound. The remaining phage are then amplified in bacteria and run again through the same assay to select for mutations that resulted in higher affinity binding. This process, called biopanning, was performed three times for this project. After biopanning, the library was sequenced using Next Generation sequencing (NGS). This platform enables the entire library to be sequenced, as opposed to traditional Sanger sequencing, which can only sequence single select clones at a time thereby limiting population sampling. This type of genetic sequencing allows trends in the complementarity determining regions (CDRs) of the domain antibody library to be analyzed, using bioinformatics programs such as RStudio, FastAptamer, and Swiss Model. Ultimately, two nanobody candidates were identified for the CD36 receptor.
ContributorsLundgreen, Kendall (Author) / Stabenfeldt, Sarah (Thesis director) / Ugarova, Tatiana (Committee member) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The WNT signaling pathway plays numerous roles in development and maintenance of adult homeostasis. In concordance with it’s numerous roles, dysfunction of WNT signaling leads to a variety of human diseases ranging from developmental disorders to cancer. WNT signaling is composed of a family of 19 WNT soluble secreted glycoproteins, which are evolutionarily conserved across all phyla of the animal kingdom. WNT ligands interact most commonly with a family of receptors known as frizzled (FZ) receptors, composed of 10 independent genes. Specific interactions between WNT proteins and FZ receptors are not well characterized and are known to be promiscuous, Traditionally canonical WNT signaling is described as a binary system in which WNT signaling is either off or on. In the ‘off’ state, in the absence of a WNT ligand, cytoplasmic β-catenin is continuously degraded by the action of the APC/Axin/GSK-3β destruction complex. In the ‘on’ state, when WNT binds to its Frizzled (Fz) receptor and LRP coreceptor, this protein destruction complex is disrupted, allowing β-catenin to translocate into the nucleus where it interacts with the DNA-bound T cell factor/lymphoid factor (TCF/LEF) family of proteins to regulate target gene expression. However in a variety of systems in development and disease canonical WNT signaling acts in a gradient fashion, suggesting more complex regulation of β-catenin transcriptional activity. As such, the traditional ‘binary’ view of WNT signaling does not clearly explain how this graded signal is transmitted intracellularly to control concentration-dependent changes in gene expression and cell identity. I have developed an in vitro human pluripotent stem cell (hPSC)-based model that recapitulates the same in vivo developmental effects of the WNT signaling gradient on the anterior-posterior (A/P) patterning of the neural tube observed during early development. Using RNA-seq and ChIP-seq I have characterized β-catenin binding at different levels of WNT signaling and identified different classes of β-catenin peaks that bind cis-regulatory elements to influence neural cell fate. This work expands the traditional binary view of canonical WNT signaling and illuminates WNT/β-catenin activity in other developmental and diseased contexts.
ContributorsCutts, Joshua Patrick (Author) / Brafman, David A (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Nikkhah, Mehdi (Committee member) / Wang, Xiao (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2019