Doctoral Thesis Defenses
After completing their scientific training, doctoral candidates working at NYU Grossman School of Medicine discuss the significance of their original research at a thesis defense. Members of the public are welcome to attend. Below are some recent thesis defenses.
Doctoral Thesis Defense 2022
Congratulations to everyone who defended!
Mericien Venzon—Cadwell Lab
Title: The Role of Microbial Byproducts in the Reproductive Success of Free-Living and Parasitic Nematodes
Date: April 26, 2022
Abstract: Soil-transmitted helminths (STHs) are parasitic worms that typically inhabit mammalian intestines and infect one in four of the world’s population. Efficacies of treatment have been found to be largely species dependent, with cure rates for the whipworm, Trichuris trichiura, less than half that of the other two major STHs. A distinguishing feature of Trichuris nematodes is that these parasitic worms reproduce within the digestive tracts of humans and other mammalian hosts shedding thousands of eggs daily, facilitating their sustained presence in the environment and hampering eradication efforts. Although this aspect of the lifecycle places Trichuris in a microbiota-rich environment, the role of gut commensal bacteria or bacterial metabolic byproducts in successful infection and reproductive development within its host are unknown. In this thesis, we employ a novel pipeline using the well-characterized, free-living nematode C. elegans to identify microbial factors with conserved roles in the reproduction of nematodes.
A screen for E. coli mutants that impair C. elegans fertility identified genes in fatty acid biosynthesis and ethanolamine utilization pathways, including fabH and eutN. Trichuris muris eggs displayed defective hatching in the presence of E. coli deficient in fabH or eutN due to reduction in arginine or elevated levels of aldehydes, respectively. Remarkably, T. muris reared in gnotobiotic mice colonized with these E. coli mutants displayed profound abnormalities including morphological defects and a failure to lay viable eggs. We propose that C. elegans can be used as a system for investigating the effects of specific microbial genes and pathways in supporting the parasitic nematode life cycle and aid in further understanding the transkingdom interactions that sustain human disease.
Doctoral Thesis Defenses 2021
Below are abstracts defended during 2021.
Naoya Yamaguchi—Knaut Lab
Title: Generation of Traction Stress by a Migrating Tissue in the Zebrafish Embryo
Date: October 18, 2021
Abstract: Cell migration is fundamental to almost all biological processes. In animal development, homeostasis, and disease, tissues physically interact with their outside environment to move. How cells in tissues in vivo generate forces against the surrounding environment (referred to as traction stress) for motility is unclear. I addressed this question by studying the migration of zebrafish posterior lateral line primordium (primordium).
Using electron microscopy, a GFP-tagged Laminin reporter line, and genetic perturbations of laminin, I identified the basement membrane (BM) as a substrate for primordium migration. This predicted that the primordium interacts with the BM and transmits the forces from its internal cytoskeleton to the BM through cell-extracellular matrix (ECM) adhesions. Therefore, I asked whether the primordium cells form focal adhesions (FAs)— well-characterized protein complexes at the cell-ECM contact site seen in vitro—in a living embryo and whether cell-ECM adhesion is required for primordium migration. I found that the primordium cells in vivo do not form FAs, rather they form short-lived nascent adhesion-like structures.
Next, I depleted components of cell-ECM adhesions from primordium cells with a new protein degron approach and found them to be required for efficient migration. To further investigate the traction stress generation on a tissue scale, I decided to develop a novel approach to bring the traction force microscopy technique to the embryo to visualize and quantify the traction stresses that the primordium generates on the BM. Combining direct stiffness measurements of the BM by atomic force microscopy with a software pipeline to track the optical marks painted on the BM, I collaborated with Daniele Panozzo’s group to calculate the BM deformation and the traction stresses that the primordium exerts on its surrounding. Interestingly, I found that the rear of the primordium generates greater stresses and physically deforms the BM, likely through the higher activity of Myosin II in its rear.
In summary, I identified the substrate, quantified the traction stresses that the primordium generates, and uncovered how the primordium cells mechanistically migrate on the BM. Interestingly, observation of in vivo traction stress suggests that the primordium has a rear engine to propel itself forward.
Casey Vieni— Bhabha and Ekiert Labs
Date: September 14, 2021
Abstract: The MCE protein family is nearly ubiquitous in double-membraned bacteria, and MCE proteins are believed to act as transporters that facilitate lipid movement between the inner and outer membranes. My thesis work compiles structural and mechanistic studies of MCE proteins from Escherichia coli and Mycobacterium tuberculosis. In E. coli, the MCE protein LetB is a tunnel-forming protein thought to directly link the inner and outer membranes, facilitating transport of substrates between the two membranes. I have combined Cryo EM and functional studies in E. coli to dissect the role of different domains in the LetB tunnel. In Mycobacterium tuberculosis (Mtb) MCE proteins play a critical role in virulence, and throughout the course of Mtb infection the expression of MCE transporters is differentially regulated by MCE transcriptional regulators (MceRs). I used Cryo EM toward gaining a structural understanding of Mce3R, a TetR transcriptional regulator. Cryo EM of Mce3R bound to DNA suggests that this protein exhibits a unique pseudo dimer structure of two fused TetR domains and effectively “tetramerizes'' in order to recognize its cognate operator sequence. My thesis work begins to unravel how MCE transport is modulated at both the transcriptional and protein level.
Frank Yeung—Cadwell Lab
Title: The Role of Commensal Fungi in Immune Development and Disease Susceptibility
Date: May 25, 2021
Abstract: The gut microbiota has a fundamental role in the development and stability of the host immune system. Colonization by certain bacteria, and even individual species, can alter the course of many infectious and inflammatory diseases. Our understanding of the immune consequences of colonization by members of the gut microbiota is based primarily on laboratory mouse models. Although this conventional approach has enabled detailed mechanistic studies on the immune system, laboratory mice may not reflect the more complex diseases of humans and free-living mammals in a natural environment.
In this thesis, we present the use of a unique outdoor facility that allows us to “rewild” and adapt established mouse models to study disease risk in a more natural environment. Our data suggests that introduction of laboratory C57BL/6 mice to our facility restores deficient immune cell populations coincident with microbiome changes. The increase in circulating neutrophils and CD4+/CD8+ memory cells, reduction in naive T cells, and increased expression of costimulatory molecules on antigen presenting cells all occur in the absence of viral, bacterial, or parasitic pathogen exposure. Rather, colonization by fungi isolated from rewilded mice or Candida albicans was sufficient to increase circulating granulocytes and enhance granulopoiesis in the bone marrow. Colonization of laboratory mice by Candida albicans protected the host from infection against staphylococcus and pneumococcal infections. These findings establish a model to investigate how the natural environment impacts immune development and show that sustained fungal exposure impacts granulocyte numbers.
Douglas Brawley—Traaseth and Wang Labs
Title: Structure and Mechanism of the Staphylococcus aureus Multidrug Efflux Pump NorA
Date: January 19, 2021
Abstract: Microbial resistance to antibiotics is a worldwide medical problem. Among the most persistent human pathogens is methicillin-resistant Staphylococcus aureus (MRSA), which displays resistance to beta-lactams, fluoroquinolones, and other antibiotics. MRSA utilizes a diverse array of molecular mechanisms to circumvent the effects of antimicrobial agents, including expression of multidrug efflux pumps. These transporters are membrane-embedded proteins that extrude toxic antibiotics and biocides out of the cytoplasm.
NorA is the best characterized multidrug efflux pump in MRSA and confers multidrug resistance by expelling bactericidal substrates, including fluoroquinolone antibiotics. As a member of the major facilitator superfamily (MFS) of secondary active transporters, NorA uses energy from the proton gradient to drive substrate efflux across the membrane. However, a more detailed characterization of the transport mechanism of NorA, such as the molecular basis for substrate polyspecificity, conformational exchange, and proton-coupling, remains unclear.
To address these important questions, we elucidated the atomic structure of two NorA-Fab complexes (3.2 and 3.7 Å) using single particle cryo-electron microscopy (Cryo-EM) aided by synthetic antigen-binding fragments (Fabs). These structures reveal NorA to adopt a canonical MFS family fold and show the composition of residues lining the drug binding cavity. NorA residues suspected to serve important functional roles based on structural analysis and sequence homology were mutated and tested in MRSA. From our structure-function studies, we uncovered several irreplaceable residues in NorA involved in substrate specificity, conformational exchange, and proton-coupling. All together these results provide new and important insights into the multidrug efflux mechanism used by NorA and other homologous multidrug efflux pumps in pathogenic bacteria.
Furthermore, our NorA-Fab structures unexpectedly revealed that both Fabs used as fiducial markers for Cryo-EM could also serve as potential inhibitors of NorA function. Namely, both Fabs were discovered to bind NorA predominantly through a single loop that inserts into the substrate binding cavity. This mode of Fab binding likely blocks drug binding and halts conformational exchange, and therefore inhibits drug resistance conferred by NorA. Accordingly, this structural observation also suggests that peptidomimetics mimicking the Fab loop may serve as promising scaffolds for a novel class of efflux pump inhibitors.
Doctoral Thesis Defenses 2020
Below are abstracts defended during 2020.
Mary Rossillo—Ringstad Lab
Title: Gene Regulatory Mechanisms Required for Neuronal Chemosensitivity
Date: September 16, 2020
Abstract: During development, the nervous system generates neurons that perform a variety of functions and accordingly possess distinct molecular and physiological characteristics. How transcription factors regulate the expression of molecules required for the function of specific neuron-types remains a critical question in neurobiology. I have selected the chemosensory BAG neurons of C. elegans as a model to study this question.
BAGs are highly specialized sensory neurons that detect the respiratory gas carbon dioxide (CO2) to mediate attraction or avoidance behaviors in a context-dependent manner. For their function BAG neurons require ETS-5, a conserved ETS-domain containing transcription factor. My studies of ETS-5 have revealed mechanisms that pattern ETS-5 expression in the developing nervous system, and target-genes that ETS-5 regulates to support sensory neuron physiology.
The first study revealed distinct roles for different isoforms of the C. elegans homolog of PAX6, VAB-3, in regulating expression of a BAG-cell fate. A homeodomain-only short isoform of VAB-3 is expressed in BAG chemosensory neurons, where it promotes expression of factors required for their chemosensory function. A different, longer isoform of VAB-3, comprising a homeodomain and a paired homology domain, represses expression of ETS-5 in lineages not fated to generate BAG neurons. This repressor function requires the eyes absent homolog EYA-1 and the Six-class homeodomain protein CEH-32. Together, these data indicate that a promoter selection system together with cell-specific expression of accessory factors allows VAB-3/PAX6 to either promote or repress expression of a BAG-cell fate depending on lineage.
The second study sought to identify new factors required for the physiology and function of BAG neurons by identifying and characterizing ETS-5 target-genes. This approach revealed that nhr-6, which encodes the sole C. elegans NR4A-type nuclear receptor, is an ETS-5 target gene that is required for BAG-mediated avoidance of CO2. NHR-6 is a nuclear receptor, and our data indicate that NHR-6 regulates expression of a subset of BAG-specific genes. Unlike ets-5 mutants, which are defective for both attraction and avoidance to CO2, nhr-6 mutants are fully competent for attraction. These data indicate that the remarkable ability of BAGs to adaptively assign positive or negative valences to a chemosensory stimulus requires a gene-regulatory program supported by an evolutionarily conserved type of nuclear receptor.
I have also performed experiments to biochemically and genetically identify factors that cooperate with ETS-5 to promote expression of a BAG neuron fate, and I have tried to detect a CO2 response in induced serotonergic neurons. My studies of ETS-5 and its function in the nematode nervous system might inform understanding of how its mammalian homolog PET1 functions to promote development of brainstem circuits, some of which mediate sensation of respiratory gases to control breathing. My findings will hopefully illuminate conserved aspects of mechanisms that promote the development of specialized neuron-types and the molecular basis of mechanisms that monitor respiratory gases to control animal behavior and physiology.
Jennifer Schiavo—Froemke Lab
Title: Innate and Plastic Mechanisms in Rodent Auditory Cortex for Maternal Behavior
Date: August 18, 2020
Abstract: To what extent are our behaviors driven by “nature” (innate or hardwired processes) or “nurture” (learned from experience)? For my thesis work, I examined how innate and learned processes interact for the onset of maternal behavior in mice. Specifically, we took advantage of an auditory-driven maternal behavior, retrieval of lost pups in response to distress calls, to examine the extent to which auditory cortex is intrinsically tuned to vocal features prior to parental experience, and what neuroplastic mechanisms underlie auditory learning for the recognition of pup vocalizations.
Mouse mothers readily retrieve isolated pups back to the nest based on ultrasonic distress calls emitted from the lost pups. In contrast, pup-naive virgins do not recognize the meaning of these calls, but retrieve isolated pups to the nest following co-housing with a mother and litter. We found that the onset of maternal behavior in virgins resulted from experience-dependent plasticity that built on an intrinsic sensitivity to the most common pup call repetition rate. In maternal females, calls with inter-syllable intervals (ISIs) from 75 to 375 ms elicited pup retrieval, and cortical responses generalized across these ISIs. In contrast, pup-naive virgins were behaviorally sensitive only to the most common (“prototypical”) ISIs, and excitatory neurons were narrowly tuned to “prototypical” pup calls.
Inhibitory and excitatory neuronal and synaptic tuning were initially mismatched in naive cortex, with untuned inhibition and overly narrow excitation. During co-housing, excitatory neurons broadened their tuning to represent a wider range of ISIs, while inhibitory tuning sharpened to form a perceptual boundary. We presented synthetic calls during co-housing and observed that neurobehavioral responses adjusted to match these statistics, a process requiring auditory cortical activity and the hypothalamic oxytocin system. Neuroplastic mechanisms therefore build on an intrinsic sensitivity in mouse auditory cortex, enabling rapid plasticity for reliable parenting behavior.
Kameron Azarm—Smith Lab
Title: Persistent Telomere Cohesion Protects Aged Cells from Premature Senescence
Date: June 19, 2020
Abstract: Human telomeres are bound by the telomere repeat binding proteins TRF1 and TRF2. TRF2 protects chromosome ends from DNA damage response (DDR) through formation of a “t-loop” structure. TRF1 promotes resolution of sister telomere cohesion through its recruitment of the poly(ADP-ribose) polymerase, tankyrase 1. Due to the “end-replication problem” for linear chromosomes and to nucleolytic processing that generates the 3’ overhang, human telomeres shorten following each round of replication. The loss of telomeric DNA ultimately leads to insufficient chromosome-end protection and to activation of a DDR at telomeres that signals checkpoint-dependent replicative senescence.
While insufficient loading of TRF2 at shortened telomeres contributes to the DDR in senescence, the contribution of TRF1 to senescence induction has not been determined. I show that counter to TRF2 deficiency-mediated induction of DNA damage, TRF1 deficiency serves a protective role to limit induction of DNA damage induced by subtelomere recombination. Shortened telomeres recruit insufficient TRF1 and as a consequence, inadequate tankyrase 1 to resolve sister telomere cohesion, resulting in a delayed resolution in mitosis known as persistent telomere cohesion. Forcing resolution of persistent telomere cohesion by overexpression of wild type TRF1, but not a tankyrase-binding deficient mutant, leads to excessive RAD51-dependent subtelomere copying, which induces a DDR and checkpoint-dependent senescence arrest.
My findings suggest that the persistent cohesion protects short telomeres from inappropriate recombination. I demonstrate that it is the shortened telomeres per se in any cellular context (aged normal cells, ALT cancer cells, or telomerase-inhibited telomerase positive cancer cells) that induce persistent telomere cohesion, providing an inherent protective mechanism that accompanies telomere shortening. Ultimately, in the final division prior to senescence, aged cell telomeres are no longer able to maintain cohesion and as a result, subtelomere copying ensues. Thus, the gradual loss of TRF1 and concomitant persistent cohesion that occurs with telomere shortening ensures a measured approach to replicative senescence.
Hannah Fehlner-Peach—Littman Lab
Title: Genomic and Functional Variation of Intestinal Commensals and Their Interactions with the Host
Date: May 19, 2020
Abstract: The intestinal microbiome provides essential services for the host, influencing immune function, nutrient absorption, and susceptibility to pathogens. Dietary history, antibiotics treatment, and disease status have been associated with the bacterial community present within an individual’s gut. Additional microbial functions may be revealed through strain-level analysis of intestinal bacteria. The intestinal microbiome is therefore an intriguing target for therapeutic intervention. Through exploration of the genomic and functional diversity of the disease- and diet-associated intestinal bacterium Prevotella copri, and assessment of T-cell responses to the model commensal Bacteroides thetaiotaomicron, I address open questions of intestinal commensal strain-level diversity and immune responses.
High abundance of intestinal Prevotella copri has been associated with both new onset rheumatoid arthritis and a diet high in plant fiber. In a new cohort of patients and healthy controls, we demonstrate that a greater percentage of new onset rheumatoid arthritis (NORA) patients than healthy controls harbor the intestinal bacterium P. copri, confirming a trend reported in a previous study (Scher et al., 2013). We describe the genomic diversity of 83 P. copri isolates from 11 human donors, including 8 NORA patients and 3 healthy controls. We demonstrate that isolates with distinct genomes, which can be categorized into different P. copri complex clades, utilize defined sets of plant polysaccharides. Mice colonized with P. copri isolates from NORA patients belonging to a divergent clade had more severe arthritis scores than mice colonized with isolates from the main clade.
In a mouse model of chemically induced colitis, mice colonized with certain P. copri strains have worse outcomes than mice colonized with other P. copri strains. These results suggest that distinct strains of P. copri may play a role in exacerbating disease. The assembly of 83 novel P. copri genomes provides a basis for investigating genes in this specific intestinal microbe that are associated with disease or health and for interrogation of the intricacies of intestinal microbial diversity.
Naïve CD4+ T-cells develop into pro- or anti-inflammatory helper T-cells in response to signals from antigen-presenting cells and the microorganisms they encounter in the periphery. Specific properties of intestinal bacteria may dictate pro- or anti-inflammatory T-cell responses in the gut. We studied intestinal T-cell responses to the model intestinal commensal Bacteroides thetaiotaomicron that was engineered to express the SFB-3340 antigen (Bt-SFB), a peptide of which is presented to transgenic 7B8 T cells in vivo (Yang et al., 2014). Although transgenic T-cells proliferated in mice colonized with Bt-SFB, they did not differentiate into known CD4+ subsets. Instead, proliferating T-cells upregulated genes suggesting exposure to Type I IFN. In another model using engineered B. thetaiotaomicron strains, we found that regulatory T-cells expand in the colon of mice colonized with UDCA-producing bacteria, suggesting a possible mechanism for the anti-inflammatory properties of this secondary bile acid. Overall, the work presented here lays the groundwork for interrogation of T-cell responses to intestinal commensals.
Stephanie Lau—Knaut Lab
Title: Molecular Control of Chemokine-Guided Cell Migration
Date: May 18, 2020
Abstract: Chemokine-guided cell migration is a fundamental process in development and immunity. One prominent chemokine is CXCL12 (also known as SDF1), which binds and signals through the G-protein coupled receptor CXCR4 to guide the migration of many cell types, including neuronal, immune, germ, and vascular cells. CXCL12-guided cell migration is normally very precise and robust but irregularities in CXCL12 concentrations and CXCR4 signaling can affect cell migration and lead to pathology such as a rare disease in humans called WHIM syndrome whereby patients develop varying symptoms of warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis.
To understand how cells in vivo extract directional behavior from CXCL12, we took advantage of the optical accessibility and genetic amenability of the zebrafish posterior lateral line primordium (primordium), a tissue that uses CXCL12-guided migration, to measure endogenous CXCL12 signaling concentrations and CXCR4 receptor behavior. We found that cells sense CXCL12 concentrations around the dissociation constant, Kd, with its signaling receptor CXCR4. At concentrations around the Kd, the signal-to-noise is greatest and cells are most sensitive to the small differences in chemokine concentrations.
We found that the CXCL12 concentrations were maintained around the Kd by a bona fide negative feedback loop with the second CXCL12 receptor, ACKR3 (also known as CXCR7), which does not signal and instead scavenges and degrades CXCL12. When the CXCL12-CXCR4 Kd was mismatched or the CXCL12-ACKR3 negative feedback loop was broken, the signal-to-noise became less optimal and cells migrated less directionally and slower. In humans, increased CXCR4 signaling through a mutation in the cytoplasmic tail of CXCR4 is thought to be the molecular pathogenesis of WHIM syndrome. We investigated the function of CXCR4WHIM and found that the CXCR4WHIM receptor was more sensitive to CXCL12 because it stayed on the cell membrane whereas the wild-type receptor was internalized and degraded. Overall, these results demonstrate new molecular mechanisms regulating chemokine levels and receptor signaling to guide precise cell migration in a living animal.