2020 Research Projects

Research Project Descriptions

Rice grain sized ingestible, implantable, and biodegradable sensors

Imagine swallowing a pill that could diagnose a disease and then immediately disintegrate. In this project, we will explore new ways to create ingestible and implantable sensors that physically attach to and interact with the tissue. We will exploit the body's mechanical and electrical properties to develop smaller and safer sensors that could alter the standard of care for patients worldwide.

Applications of anti-CRISPRs toward control of Drosophila function

CRISPR-based technologies have been revolutionizing biotechnology by providing an easy way to target the genome. In our lab, we focus not only on using CRISPR for gene editing applications, but also gene regulation: turning the production of proteins up and down in a specific manner to control cellular function. We have recently reported on an extension of these techniques using anti-CRISPRs, proteins that can disable CRISPR systems, by using these anti-CRISPRs as a control module to tune the activity of CRISPR technologies. Some applications and future development of these techniques are as follows:

  1. We have integrated anti-CRISPRs stably into cells and shown that future genome editing via CRISPR is prevented (we term this genomic "write-protection"). We now seek to demonstrate this concept in whole organisms (fruit flies). We seek to use these write-protected flies to demonstrate their ability to combat the spread of CRISPR gene drives (a technology that allows for the inheritance of genetic elements at higher than normal Mendelian rates) as a potential safeguard.
  2. We have demonstrated that control elements can be adapted onto anti-CRISPRs to generate chemical control over gene regulation. We wish to build upon this and other work to investigate the utility of attaching pre-existing cellular control signals to anti-CRISPRs to create endogenously regulated gene expression. This may culminate in achieving control of gene expression in a development- or tissue-specific manner in fruit flies.
  3. We have also demonstrated that anti-CRISPRs can be used to create a pre-programmed gene expression circuit (synthetic pulse generator). We seek to expand this to other CRISPR systems, allowing for the development of more sophisticated genetic circuits.

The student will be able to work on a subset of one of these problems, depending on individual interest.

Light-Controllable Enzymes for Neurobiology

Modern synthetic biology tools allow us to investigate the dynamic cellular signaling pathways that control health and disease. In the Lin lab, one active line of research focuses on the development of light-controlled enzymes. The lab has developed a photodissociating green fluorescent protein called pdDronpa that can be reversibly switched between a dimeric and monomeric state using cyan and UV light, respectively. By genetically inserting two copies of pdDronpa into natural enzymes, this tool has been used to gain light-mediated control over a range of enzymes, including kinases and Cas9. In the dark, the photoswitchable enzymes (psEnzymes) are inactive due to shielding of the active site by dimeric pdDronpa, while illumination with cyan light causes pdDronpa dissociation and enzyme activation by allowing substrates to access the active site.

Currently, we are working on improving the properties of pdDronpa to enable more sensitive spatial and temporal control of psEnzymes in neurons. We are especially interested in utilizing these optogenetic tools to investigate enzymes involved in the dynamical and bidirectional remodeling of neuronal connections, synapses. Specifically, two forms of plasticity called long-term potentiation and long-term depression result in molecular reorganizations that, respectively, strengthen or weaken synapses. In the healthy brain, these processes are believed to drive learning and memory function. In contrast, dysregulation of synaptic plasticity has been associated with drug abuse and mental illness.

During the project, we will work on optimizing specific properties of pdDronpa. We are for example interested in testing circular permutation of pdDronpa (i.e. moving the termini of the protein to new positions in the amino acid sequence while maintaining the overall protein architecture) since this can be used to optimize the geometry of our psEnzyme designs. This part of the project involves protein engineering and biophysical characterization of pdDronpa variants. It will introduce you to protein structure inspection, DNA cloning and mutagenesis, protein expression and purification from E. coli, and biophysical characterization of the proteins. In this way, you will contribute to refining our understanding of the photodissociation mechanism of pdDronpa.

During the project, you will also have the opportunity to gain experience with cell culture technique, fluorescence microscopy, and screening via cell-based activity assays. One example involves the redesign of an existing psEnzyme (e.g. a kinase) by inserting a circularly permuted pdDronpa and screening the performance. Overall, this will help us establish more detailed guidelines for the design of better psEnzymes.

Role of cell membrane composition in regulating Wnt growth factor signaling

The processes of cell growth, division, and specialization must be carefully controlled during the development of an organism to ensure successful progression through each developmental stage. These processes must also remain highly coordinated in adulthood to maintain proper functioning of the body's tissues and promote healing following injury. One class of growth factors that control these processes are called Wnt proteins. As central mediators of cell growth and specialization, defects in Wnt signaling pathways can contribute to many diseases including bone disorders, diabetes, and cancer. Therefore, there is tremendous interest in therapeutically targeting Wnt signaling pathways in these diseases, as well as in stem cell therapy and regenerative medicine.

To transmit its signal from cell to cell, a Wnt protein must first bind its receptor Frizzled (Fzd) on the cell surface. Upon binding Wnt, Fzd recruits the protein Dishevelled (Dvl) to the cell membrane. Dvl then organizes the formation of a larger signaling complex, sending a message to the cell nucleus to activate genes controlling cell growth and proliferation. Despite the critical role of Fzd and Dvl in transmitting Wnt signals, little is known about how these proteins interact.

This project aims to determine what factors are important for a productive Fzd-Dvl interaction. We will purify Fzd and Dvl proteins and use these as tools to perform a range of biochemical and biophysical assays and structural studies. We will also mutate specific regions of both Fzd and Dvl and perform signaling assays in cells to investigate the effects of these mutations on Wnt signaling outputs. By completing this project, we will clarify the initial events that occur upon Wnt stimulation of cells and provide fundamental information useful for future therapeutic modulation of this crucial signaling pathway.The processes of cell growth, division, and specialization must be carefully controlled during the development of an organism to ensure successful progression through each developmental stage. These processes must also remain highly coordinated in adulthood to maintain proper functioning of the body's tissues and promote healing following injury. One class of growth factors that control these processes are called Wnt proteins. As central mediators of cell growth and specialization, defects in Wnt signaling pathways can contribute to many diseases including bone disorders, diabetes, and cancer. Therefore, there is tremendous interest in therapeutically targeting Wnt signaling pathways in these diseases, as well as in stem cell therapy and regenerative medicine.

To transmit its signal from cell to cell, a Wnt protein must first bind its receptor Frizzled (Fzd) on the cell surface. Upon binding Wnt, Fzd recruits the protein Dishevelled (Dvl) to the cell membrane. Dvl then organizes the formation of a larger signaling complex, sending a message to the cell nucleus to activate genes controlling cell growth and proliferation. Despite the critical role of Fzd and Dvl in transmitting Wnt signals, little is known about how these proteins interact.

This project aims to determine what factors are important for a productive Fzd-Dvl interaction. We will purify Fzd and Dvl proteins and use these as tools to perform a range of biochemical and biophysical assays and structural studies. We will also mutate specific regions of both Fzd and Dvl and perform signaling assays in cells to investigate the effects of these mutations on Wnt signaling outputs. By completing this project, we will clarify the initial events that occur upon Wnt stimulation of cells and provide fundamental information useful for future therapeutic modulation of this crucial signaling pathway.

Molecular investigations in the Alzheimer’s brain, in micro- and macro-scale

The project will be in the field of neuroscience and brain imaging using a variety of methods. The scholar will have the opportunity to study human and/or mouse brain tissue (in a clinical or preclinical/animal setting), and get involved in experiments, modeling or analysis to the desired extent.

About the methods:

In the past years, new imaging techniques based on X-rays and MRI have been developed that allow detailed and quantitative molecular investigations of samples without the need for histology sectioning. The scholar will be involved in imaging experiments and data analysis thereof, related to characterization of mouse or human brain microstructure, focusing on Alzheimer’s disease.

The main methods that will be used are:

  • X-ray scattering and X-ray fluorescence, which take place in synchrotron facilities of the SLAC National Accelerator Laboratory, adjacent to main Stanford campus
  • MRI scanning in Stanford’s 3T/7T clinical or 7T animal scanners
  • Electron microscopy, 2D or 3D histology (involving tissue clearing & accompanying confocal/two-photon microscopy techniques)

The scholar will use all or a subset of these techniques to conduct a scientific project tailored to her/his interests. This can be any of the following possibilities or combinations of thereof:

  1. X-ray scattering data provide a wealth of molecular and structural information on the tissue, (eg. myelin content, neuronal orientations, brain connectivity, elemental mapping) and the recently developed X-ray scattering tensor tomography (Liebi, Georgiadis et al., Nature, 2015) has opened new avenues towards providing this information non-destructively on bulk samples. The scholar can get involved in the synchrotron experiments throughout the year, and will learn how to retrieve quantitative information in Alzheimer’s human brain tissue.
  2. MRI is widely used to study brain in clinics and in research, but its information lacks specificity. New methods, such as diffusion MRI, enable a quantitative approach, and are rapidly expanding. The scholar will have the chance to get involved with related experiments and corresponding analysis of Alzheimer’s brain post-mortem tissue, to study the molecular and microstructural underpinnings of the disease.
  3. Method correlation: Given MRI’s lack of specificity, further validation of the sample’s molecular and microstructural properties is needed. After MRI scanning, the scholar will retrieve information on the same samples using specific methods, such X-ray scattering, light or electron microscopy.  Then the scholar will explore associations with the MRI methods in order to derive molecular markers related to Alzheimer’s disease.

The experiments as well as the associated teachings are expected to take place throughout the academic year, with most of data analysis, reaching results, drawing conclusions and putting them in form of a presentation taking place in the summer.

The Role of Astrocytes in Parkinson's Disease

Parkinson’s disease (PD) is a debilitating movement disorder that affects over 10 million people worldwide. Astrocytes, the most abundant cells in the brain, play a key role in PD progression. Under typical conditions, astrocytes support neuron survival. However PD alters astrocytes in a way that promotes neuron loss. 

Although the precise cause of PD is unclear, we know that rare forms of the disease are linked to family genetics. The major cause of genetic PD are mutations in an enzyme called leucine rich repeat kinase 2 (LRRK2). In general, these mutations increase LRRK2 activity and result in deleterious changes within cells. For instance, mutant LRRK2 activity triggers defective protein clearance, impairs trafficking of cargoes across the cell, and removes signaling organelles. Importantly however, the role of LRRK2 in astrocytes is unclear. 

Therefore, we are studying mutant LRRK2 activity in astrocytes. In other cell-types, we have identified a pathway by which mutant LRRK2 phosphorylates a subset of membrane trafficking proteins called Rab GTPases. In particular, when one of these Rab GTPases, Rab10, is phosphorylated by mutant LRRK2, it prevents the formation of key signaling organelles called primary cilia. Loss of primary cilia disrupts cell-cell communication and likely contributes to neuron loss in PD. As a starting point to this project, we will determine if the LRRK2/Rab10 pathway is conserved in astrocytes. We will use advanced imaging and biochemical tools to study astrocytes from mutant LRRK2 rodents and human induced pluripotent stem cells. Our goal is to provide critical and fundamental information related to the molecular basis of PD.

Engineering the mechanics of implant-tissue interface to reduce implant rejection

The Egyptians used gold wires to fix broken teeth in 2500BC. Today, we have sophisticated biomedical devices such as pacemakers and biosensors that help save millions of lives every day. Despite the prevalence of these devices, the fundamental nature of the interaction between living tissue and synthetic material, how the body recognizes the material, and why the foreign material is rejected, remains elusive.

In Dr. Gurtner's lab at Stanford Surgery, we use novel surgical animal models, patient-derived tissue specimen and advanced engineering techniques to define the role of mechanical stress in biomedical implant rejection. The undergraduate student will have the freedom to choose and design a project within the scope of the lab's focus. For example, the student can choose to focus on bioinformatics analyses, single cell sequencing techniques,  animal research or patient EMR analyses. 

I have mentored several successful high school, undergraduate and graduate students before. Prospective students require no previous experience. Enthusiasm for research and a willingness to learn new things is all I'm looking for. Interested students can contact me at jaganpa@stanford.edu.