Faculty Bibliography

T cells play a critical role in host defense against viruses, intra- and extracellular microbes, and tumors. Because foreign antigen is presented amongst a vast majority of self-antigens, T cells have evolved the unique ability to discriminate "self" from "non-self" with high sensitivity and selectivity, enabling the elimination of foreign pathogens while largely avoiding self-reactivity. However, tissue-specific autoimmunity and tolerance to or eradication of cancer does not fit neatly into the self/non-self paradigm because the T cell responses in these situations are not directed to an exogenous pathogen, but rather most often to non-mutated self-proteins. Therefore, an important question is how the immune system establishes suitable thresholds that allow positively selected T cells to interact with selfligands in the periphery without causing overt activation. One hypothesis to explain how a T cell distinguishes among different types of self-ligands is the kinetic proof-reading theory, which relates signaling efficacy to the lifetime of the TCR (T cell receptor)-pMHC (peptide-major histocompatibility complex) interaction. More recently, T cell maturation associated signaling feedback pathways have also been hypothesized to play a role in T cell discrimination of between self-ligands. We are taking a variety of biophysical and cellular imaging approaches to determine how specific thresholds for T cell recognition of self-antigens are set. Our recent results [1] indicate that antitumor activity and autoimmunity are coupled and have a similar kinetic threshold; reducing autoimmunity cannot be accomplished without sacrificing efficacy of tumor killing. Therefore, an "optimal TCR affinity range" that leads to optimal tumor regression and minimal autoimmunity is elusive and treatment strategies focusing on increasing TCR affinities to a supraphysiological level has most likely little therapeutic benefit. Therefore, other approaches are needed to improve the balance between anti-tumor responses and autoimmunity. Our strategy to overcome this issue includes novel methods for careful biophysical engineering of tumor-specific TCRs to carefully balance tumorreactivity and autoimmunity. Furthermore, our recent preliminary data show that TCR-proximal signaling differs significantly between effector memory and central memory T cells due to differential constitutive activity and localization of signaling molecules. Understanding how activation signaling contributes to differences in memory T cell subset sensitivity may provide insight into how T cells can be manipulated to achieve optimal anti-tumor sensitivity. This could lead to adjuvants that target and enhance antigenspecific T cell anti-tumor efficacy. Together may lead to development of cancer immunotherapy approaches with improved outcomes

A systems-biology approach to complex disease (such as cancer) is now complementing traditional experience-based approaches, which have typically been invasive and expensive. The rapid progress in biomedical knowledge is enabling the targeting of disease with therapies that are precise, proactive, preventive, and personalized. In this paper, we summarize and classify models of systems biology and model checking tools, which have been used to great success in computational biology and related fields. We demonstrate how these models and tools have been used to study some of the twelve biochemical pathways implicated in but not unique to pancreatic cancer, and conclude that the resulting mechanistic models will need to be further enhanced by various abstraction techniques to interpret phenomenological models of cancer progression.

Wounds, both chronic and acute, continue to be a tremendous socioeconomic burden. As such, technologies drawn from many disciplines within science and engineering are constantly being incorporated into innovative wound healing therapies. While many of these therapies are experimental, they have resulted in new insights into the pathophysiology of wound healing, and in turn the development of more specialized treatments for both normal and abnormal wound healing states. Herein, we review some of the emerging technologies that are currently being developed to aid and improve wound healing after cutaneous injury.

It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level T-cell receptor stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level T-cell receptor stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4(+) T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types.

The formation and maintenance of neuromuscular synapses requires a complex exchange of signals between motor neurons and skeletal muscle fibers leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. As a consequence, acetylcholine receptors (AChRs) become highly concentrated in the postsynaptic membrane and arranged in perfect register with active zones in the presynaptic nerve terminal, insuring for rapid, robust and reliable synaptic transmission. During development, motor axons approach and recognize muscle that is primed, or prepatterned in the prospective synaptic region. Muscle prepatterning is established by MuSK, a receptor tyrosine kinase, and Lrp4, a member of the LDLR family. Lrp4 associates with MuSK and stimulates MuSK kinase activity, increasing Lrp4 and MuSK expression and causing the clustering of Lrp4 and MuSK. Once clustered, Lrp4 functions as a direct retrograde signal for presynaptic differentiation, causing motor axons to stop growing and develop specializations required for neurotransmitter release. Nascent synapses are stabilized by neuronal Agrin, which is released by motor nerve terminals and binds to Lrp4, stimulating further association between Lrp4 and MuSK and increasing MuSK kinase activity. Lrp4 thus has a central role in coordinating synaptic differentiation, as Lrp4 not only binds Agrin and stimulates postsynaptic differentiation but also acts in turn as a direct retrograde signal for presynaptic differentiation. Mutations in Agrin, Lrp4 and MuSK, as well as additional genes that function in this signaling pathway, cause congenital myasthenia, and auto-antibodies to Lrp4, MuSK, or AChRs are responsible for myasthenia gravis. I will summarize experiments that have contributed to this model of neuromuscular synapse formation, indicate how this knowledge has provided insight into causes for neuromuscular disease, and describe a therapeutic approach for preserving synapses and treating neuromuscular diseases

All vertebrate eggs are surrounded by an extracellular coat that supports growth of oocytes, protects oocytes, eggs, and early embryos, and participates in the process of fertilization. In mammals (platypus to human beings) the coat is called a zona pellucida (ZP) and in non-mammals (molluscs to birds), a vitelline envelope (VE). The ZP and VE are composed of just a few proteins that are related to one another and possess a common motif, called the zona pellucida domain (ZPD). The ZPD arose more than ~600 million years ago, consists of ~260 amino acids, and has 8 conserved Cys residues that participate in 4 intramolecular disulfides. It is likely that egg-coat proteins are derived from a common ancestral gene. This gene duplicated several times during evolution and gave rise to 3-4 genes in fish, 5 genes in amphibians, 6 genes in birds, and 3-4 genes in mammals. Some highly divergent sequences, N- and C-terminal to the ZPD, have been identified in egg-coat proteins and some of these sequences may be under positive Darwinian selection that drives evolution of the proteins. These and other aspects of egg-coat proteins, including their structure and synthesis, are addressed in this review.

Glioblastoma multiforme (GBM) is a deadly primary brain malignancy. Glioblastoma stem cells (GSC), which have the ability to self-renew and differentiate into tumor lineages, are believed to cause tumor recurrence due to their resistance to current therapies. A subset of GSCs is marked by cell surface expression of CD133, a glycosylated pentaspan transmembrane protein. The study of CD133-expressing GSCs has been limited by the relative paucity of genetic tools that specifically target them. Here, we present CD133-LV, a lentiviral vector presenting a single chain antibody against CD133 on its envelope, as a vehicle for the selective transduction of CD133-expressing GSCs. We show that CD133-LV selectively transduces CD133+ human GSCs in dose-dependent manner and that transduced cells maintain their stem-like properties. The transduction efficiency of CD133-LV is reduced by an antibody that recognizes the same epitope on CD133 as the viral envelope and by shRNA-mediated knockdown of CD133. Conversely, the rate of transduction by CD133-LV is augmented by overexpression of CD133 in primary human GBM cultures. CD133-LV selectively transduces CD133-expressing cells in intracranial human GBM xenografts in NOD.SCID mice, but spares normal mouse brain tissue, neurons derived from human embryonic stem cells and primary human astrocytes. Our findings indicate that CD133-LV represents a novel tool for the selective genetic manipulation of CD133-expressing GSCs, and can be used to answer important questions about how these cells contribute to tumor biology and therapy resistance.

Sleep and wake are fundamental behavioral states whose molecular regulation remains mysterious. Brain states and body functions change dramatically between sleep and wake, are regulated by circadian and homeostatic processes, and depend on the nutritional and emotional condition of the animal. Sleep-wake transitions require the coordination of several brain regions and engage multiple neurochemical systems, including neuropeptides. Neuropeptides serve two main functions in sleep-wake regulation. First, they represent physiological states such as energy level or stress in response to environmental and internal stimuli. Second, neuropeptides excite or inhibit their target neurons to induce, stabilize, or switch between sleep-wake states. Thus, neuropeptides integrate physiological subsystems such as circadian time, previous neuron usage, energy homeostasis, and stress and growth status to generate appropriate sleep-wake behaviors. We review the roles of more than 20 neuropeptides in sleep and wake to lay the foundation for future studies uncovering the mechanisms that underlie the initiation, maintenance, and exit of sleep and wake states.