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Monogenic Defects of the Type I Interferons Signaling Pathway in Humans: Molecular and Clinical Implications
Get PDFThe germ theory of disease, which dictates that microorganisms colloquially referred to as germs can invade humans or other hosts and cause disease, has remained the dominant conceptualization of infectious disease since the late nineteenth century. Since that time, growing appreciation for the extent of subclinical infections, that is, infections where the germ is detectable in its host without causing overt disease, and the Mendelian inheritance patterns of some infectious diseases have reinvigorated the study of the human genetic basis of infectious disease. This theory, so far demonstrated for a number of bacterial, fungal, and viral infections, holds that germs are a necessary but not sufficient condition for infectious disease with susceptibility determined by the genetics of individual members of the host species. In the first part of this thesis, I describe a child with inherited, complete interferon regulatory factor-9 (IRF9) deficiency who suffered from a life-threatening pulmonary influenza infection. This discovery adds to the growing body of evidence which indicates that susceptibility to severe primary infections, even from common pathogens such as influenza virus, is genetically determined. IRF9 is a critical regulator of innate anti-viral immunity in the type I and III interferon (IFN) signaling pathways, which share many signaling proteins. Consistent with this role, I show that the patient\u27s cells had drastically reduced responses to type I IFN which led to increased influenza virus replication. Profiling the transcriptome of the patient\u27s cells following stimulation with type I IFN revealed that the transcriptional response to IFN is not completely abolished in the patient\u27s cells. Indeed, the patient\u27s cells induced a small but partially overlapping set of antiviral mediators when compared to transcriptional responses from healthy control cells. Unfortunately, type III IFN signaling could not be directly assessed as fibroblasts, the cell line used throughout for viral and biochemical analyses, do not express the type III IFN receptor. These data demonstrate that IRF9 deficiency disturbs type I, and likely type III, IFN signaling, and that this deficiency underlies the patient\u27s severe influenza infection. In the second part of this thesis, I describe the first cases of inherited IFN IFNα/b receptor-1 (IFNAR1) deficiency in children who suffered severe complications following vaccination with attenuated viral vaccines but who were otherwise healthy. IFNAR1 is one of the two subunits that form the receptor for type I IFNs, and I show that type I IFN signaling is abolished in the patients\u27 cells. Consistent with a defect in type I IFN signaling, the patients\u27 cells exhibit a profound susceptibility to all viruses tested in vitro, in contrast with the patients\u27 specific and, compared to other primary immunodeficiencies, relatively mild phenotype. This susceptibility was dependent on the absence of a functional IFNAR1 receptor, which we established through complementation experiments. Taken together, these two studies interrogate the functions of the type I and III IFN pathways that are critical for human immunity to viral diseases and provide evidence for an underappreciated non-redundancy within these pathways
Pfaff, D. Origins of Human Socialization
No full textDonald W. Pfaff. Origins of human socialization
Origins of Human Socialization introduces a new concept on the origins of basic human instinct. The book combines the three disciplinary approaches, including neuroscience, paleoanthropology, and developmental psychology as an intertwined foundation for prosocial behavior. It argues that humans have the basic brain mechanisms for prosocial activity, offering new insights into more sophisticated social behavior. It also examines both visual and auditory systems in both humans and animals to explain the evolution of social interactions. Written by world-renowned researcher Dr. Donald Pfaff, this book is the first to explore why we have basic social instinct and how it works.
For centuries, researchers have argued over the foundations of human behavior in society. Anthropologists point to transitions from hunter/gathers to urban dwellers leading to human domestication. Developmental psychologists highlight social competencies in babies. Neuroscientists focus on specific genetic and neurochemical mechanisms that attribute to social behavior. This book brings all of these important areas together in an interdisciplinary approach that helps readers understand how they are linked.https://digitalcommons.rockefeller.edu/ru-authors/1177/thumbnail.jp
DEC-205 as a Unique Receptor for Adsorptive Endocytosis
No full textSlide 4-5: DEC-205 as a unique receptor for adsorptive endocytosishttps://digitalcommons.rockefeller.edu/endocytosis/1004/thumbnail.jp
The Efficacy of Dendritic Cells in Cross Presentation
No full textSlide 4-20: The efficacy of dendritic cells in cross presentation (internalized nonreplicating proteins on MHC class I)https://digitalcommons.rockefeller.edu/endocytosis/1018/thumbnail.jp
Proliferation of OVA - Specific CD8+ T Cells
No full textSlide 4-13: Proliferation of OVA-specific CD8+ T cells when dendritic cells are targeted with small amounts of α-DEC: OVA conjugates in the steady statehttps://digitalcommons.rockefeller.edu/endocytosis/1012/thumbnail.jp
Innate Resistance
No full textInnate resistance to several transplantable tumors loaded with α-Galactosyl ceramidehttps://digitalcommons.rockefeller.edu/nkt-cells/1009/thumbnail.jp
Release of Cytokines into the Serum
No full textRelease of cytokines into the serum after injection of B16 melanoma loaded with α-Galactosyl ceramidehttps://digitalcommons.rockefeller.edu/nkt-cells/1007/thumbnail.jp
Visualizing Protease Activation, Retrochmp3 Activity, and VPR Recruitment During HIV-1 Assembly
Get PDFThis thesis covers my contributions to the field of HIV-1 assembly and the field of science communication. Over the course of my studies, I have determined when the HIV-1 protease becomes active during the assembly of new virions, elucidated the mechanism by which the protein retroCHMP3 has an antiviral function, and examined the kinetics of recruitment of the HIV-1 accessory protein Vpr. I have also added significantly to science communication research through a study which evaluated the effectiveness of science summary methods. HIV-1 virions assemble at the plasma membrane of infected cells. The assembly of new viruses is driven by the HIV-1 Gag structural polyprotein and involves a number of viral and host proteins to produce infectious virions. Although many of these factors have been studied extensively, there are still steps during viral assembly that have yet to be fully characterized. In Chapter 2 of this thesis, I will discuss my study of the activation of the HIV-1 protease which is necessary for infectious particle production. Previous research has suggested that protease becomes active prior to scission of the particle from the cell, but there was no study which specifically measured protease activation and its cleavage of Gag during the assembly process. The timing of protease activation and Gag cleavage directly affects particle morphology and infectivity. Using polarization TIRF microscopy, I have directly measured protease activation and Gag cleavage. My results suggest that protease becomes active prior to recruitment of the host ESCRT proteins and scission from the cell. Chapter 3 of this thesis describes my work on the protein retroCHMP3, which was performed in collaboration with Sundquist lab at the University of Utah. The retroCHMP3 protein was originally found in squirrel monkeys and is a truncated and mutated version of the endogenous CHMP3 ESCRT protein. Data from the Sundquist lab shows that retroCHMP3 inhibits the budding of many ESCRT-dependent viruses, including HIV-1, while not preventing essential ESCRT-dependent cell functions such as cytokinesis. To determine the mechanism of retroCHMP3, I used TIRF imaging to show that retroCHMP3 alters ESCRT recruitment to sites of HIV-1 assembly. This altered recruitment suggests that a delay in scission occurs when retroCHMP3 is present in cells. My results also suggest that there is a loss of HIV-1 proteins back into the cell due to protease activation as assayed by loss of fluorescence. As a confirmation of these results, I show that this loss of fluorescence was rescued by genetic inactivation of the protease. We suspect that the retroCHMP3 alteration of ESCRT function affects viral budding more than cellular events like cytokinesis due to the need for viruses to assemble quickly to avoid cellular defense mechanisms and loss of viral proteins from protease activation. My final contribution to the HIV-1 field is a study of the recruitment kinetics of the HIV-1 accessory protein Vpr. My study of Vpr will also be presented in Chapter 3 of this thesis. Vpr is a protein which increases infectivity in infected animals and humans. It is packaged specifically into virions through interactions with the Gag protein. Through simultaneous TIRF imaging of Gag and Vpr, I was able to show that Vpr has a delayed accumulation compared to Gag, suggesting that Vpr is not bound to Gag before coming to the membrane. This work hints at possible interactions between Vpr and the host ESCRT proteins which have recruitment sites close to the Vpr recruitment site. Finally, Chapter 4 of this thesis will show my findings regarding the effectiveness of different science summaries. Science summaries like video abstracts, graphical abstracts, and plain language summaries all help with accessibility of research papers. To study the efficacy of each of those summaries, I created a survey which showed participants video abstracts, graphical abstracts, plain language summaries, and academic abstracts from two HIV-1 research papers. My findings suggested that video abstracts and plain language summaries are both effective ways to summarize scientific research while graphical abstracts and academic abstracts are not as effective. After presenting all of my work in Chapters 2-4, I will discuss the implications and immediate future directions of each contribution I have made. These discussions will be in Chapter 5 of this thesis
DDI1/2 and RTF2-Dependent Regulation of RNASE H2 at the Replisome Ensures Genome Stability
Get PDFProteasome shuttle proteins, DNA Damage Inducible 1 (DDI1) and DNA Damage Inducible 2 (DDI2), are active in the replication stress response (Kottemann et al., 2018). Under conditions of replication stress, DDI1/2 function to remove Replication Termination Factor 2 (RTF2) from the replisome. Persistence of RTF2 at the replisome compromises the replication stress response, resulting in accumulation of single-stranded DNA, inefficient replication fork restart, genome instability, and cell death. During unperturbed replication, RTF2 travels with the replication fork (Kottemann et al., 2018). Here we examine the function of RTF2 and address why RTF2\u27s removal from the replisome is necessary for recovery from replication stress. We find that loss of RTF2 results in murine embryonic lethality and that RTF2 is required for normal DNA replication elongation speeds. By isolation of proteins on nascent DNA (iPOND), replisomes deficient for RTF2 concurrently lack RNase H2, a heterotrimeric enzyme responsible for removing RNA in the context of RNA-DNA heteroduplexes. Like RTF2, RNase H2 is necessary for normal replication speed. RTF2- deficient cells exhibit phenotypes consistent with loss of RNase H2 and an increase in genome-embedded ribonucleotides, including the accumulation of DNA damage. In the absence of DDI1/2, the persistence of RNase H2 at a stalled replication fork also compromises the replication stress response. We show that direct replication restart is dependent on PRIM1, the primase component of DNA polymerase α. The dependence on PRIM1 for replication restart is greater than its requirement during processive DNA replication. Our data suggest there might be a competition between PRIM1 and RNase H2, which regulates the synthesis and degradation of the RNA primer. Our data support a model whereby the DDI-RTF2 axis regulates RNase H2 levels at the replisome during stress. RNase H2 travels with the replication fork to remove genome-embedded ribonucleotides resulting from random polymerase incorporation or inefficient Okazaki fragment maturation. However, upon DNA damage and replication stalling, RNase H2 must be removed from the replication fork to allow for proper restart using PRIM1-synthesized RNA primers. The work presented here provides a new perspective on the reorganization of the replisome that is required upon replication stress and addresses a fundamental need for regulation of replication-coupled ribonucleotide incorporation during DNA replication and repair
Small-Molecule Activation of Yap for Inner-Ear Regeneration – and Beyond
Get PDFHippo signaling is an evolutionarily conserved pathway that restricts organ growth during development and suppresses regeneration in mature organs. Using a highthroughput phenotypic screen, we have identified a potent, non-toxic, and reversible inhibitor of Hippo signaling. An ATP-competitive inhibitor of Lats kinases, the compound causes Yap-dependent proliferation of murine supporting cells in the inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. The compound promotes the initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear. In conjunction with the Tri- Institutional Therapeutics Discovery Institute, we have thoroughly characterized the compound and generated a suite of over 60 derivatives with improved characteristics such as potency, pharmacokinetics, and specificity. Together, these compounds offer powerful tools for molecular investigations of development, stem cell biology, and regeneration; it is even plausible that drugs related to this novel thiazolimine class will prove useful in therapeutic contexts