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IDENTIFICATION OF FLUCONAZOLE RESISTANCE GENES IN CANDIDA GLABRATA USING TRANSPOSON INSERTION PROFILING: REDEFINING PDR1 AS A SENSOR OF CELLULAR STRESSES
No full textFungal infections are a growing health crisis worldwide. The increasing rate of drug resistant fungal strains and species is causing millions of infections, thousands of deaths, and billions of dollars’ worth of damage yearly. Fungal infections are difficult to treat and have high mortality and morbidity rates. In particular, the fungal pathogen C. glabrata has been rising in its incidence rate, partially due to its resistance to the frontline antifungal fluconazole. Due to its haploid nature and poor genetic tools, C. glabrata is relatively poorly studied despite its medical relevance. Currently only three different antifungal classes are available. Resistance is rising across the fungal kingdom to all three classes making it crucial that new therapies that enhance existing antifungal drugs or new antifungal drugs are developed. For new antifungals, the best possible targets are essential genes. Essential genes are necessary for cell survival or proliferation, and thus their inhibition will most effectively treat fungal infections.
In the following chapters I develop a transposon mutagenesis technique for usage in C. glabrata with the goal of saturating the genome with transposon disruptions. These pools of mutants will be used to identify essential genes for the targeting of new antifungals as well as further profiling and investigating Azole resistance, to understand how C. glabrata is able to evade the most common antifungal. I found previously well-characterized genes such as PDR1, CDR1, and UPC2A, but also identified new inputs into Pdr1 and Upc2A.
In chapter one I provide a review of the current understanding of Candidemia and C. glabrata virulence. I further detail the main classes of antifungals and the common resistance mechanisms used by the various Candida species. In particular I focus on Pdr1 and Azoles as those are most relevant in terms of treatment and C. glabrata pathology. Lastly, in chapter one I discuss how essential genes are defined and their relevance to treatment.
To aid in the identification of essential genes I help refine in Chapter 2 a machine learning algorithm that is able to identify essential genes in a diverse range of organisms using data from different transposons. To do this we employed the Hermes transposon to randomly insert into both haploid and diploid strains of S. cerevisiae to help further identify the important features for the training of the algorithm. In doing so I created the first genome wide survey for haploinsufficient genes and make several crucial observations on the strengths and weaknesses in the first-generation algorithm.
In Chapter 3 I implement the same technology in C. glabrata and perform the first whole genome survey to identify essential genes. Using this data and the data previously generated in Chapter 2 I perform the first high detailed comparison between the essentialomes of two related yeast species. In doing so I identify their core essentialome, the best target for new broad-spectrum antifungals, and identify several key changes in the evolvable essentialome.
In Chapter 4 I take the pools of insertion mutants generated in Chapter 3 and use them to screen for all of the genes involved in fluconazole resistance, as C. glabrata resistance to fluconazole and other Azole drugs are a key factor in its pathology. I found the two major known pathways, Upc2A/Ergosterol biosynthesis and Pdr1/Cdr1 efflux, as well as a number of minor pathways. Surprisingly, I found that ribosomes are a negative regulator of Pdr1, this pathway had not been previously implicated and was not thought to be surveyable due to its essential nature by standard genetic techniques or transposon mutagensis . Furthermore, in this chapter, I redefine Pdr1 as a sensor of cellular stresses and not xenobiotics. The final chapter in this thesis offers concluding remarks as to the overall impact of this thesis and how it will reshape the C. glabrata field
Debating and Constructing Legal Marriage and the State in New York and the United States, 1776-1860
No full textThis dissertation examines the contingent process by which marriage and state came to be intertwined in the United States, 1776-1860. Scholars across disciplines have often asked questions about the size and scope of function of federal and state governments in early America, as part of broader inquiries about the intended nature of early American governance. However, scholars have almost uniformly not done so through the lens of intimate relationships. This project centers questions surrounding intimacy to investigate how the United States formed. Through making decisions about how to regulate personal affairs, federal and state jurists actively constructed a particular mode of American governance that holds marriage at the center.
My dissertation, a legal and social history, reaches this conclusion through studying early American debates about whether and how jurists could make laws about marriage. I focus on New York, but frequently refer to similar processes in other states and at the federal level. State legislatures heatedly deliberated whether and when they had the right to intervene in couples’ intimate affairs.
This dissertation argues that by the 1830s, after a period of vigorous contestation, American state functions and matrimony came to reciprocally constitute each other. I ultimately theorize that jurists created a “marital state,” in which the New York and federal governments contingently came to privilege legally matrimony above all other relationships (influenced by the historian Margot Canaday’s term “straight state”). Further, beyond showing how the state has favored nuptials, I demonstrate how American governance itself, in its nascent years, formed to rely on marriage for its crucial functioning. New York state and federal governments created and maintained taxation, immigration, and welfare policies through understandings of the marital family. Simultaneously, state and federal governments came to deploy nuptials as a privatized solution to public problems surrounding poverty, as opposed to constructing a broader vision of state social assistance. In sum, this project denaturalizes assumptions surrounding intimate regulation in the past, while shedding new insights on the formation of American governance
The Effect of Poly(ADP-ribose) in Promoting Liquid-Liquid Phase Separation of C9ORF72 Arginine-Containing Dipeptide Repeats
No full textArginine-rich dipeptide repeats (R-DPRs) translated from the C9ORF72 GGGGCC hexanucleotide repeat expansions (HRE) is one of the most important causes of disease progression of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). These R-DPRs exhibit toxicity to neuronal cells, leading to compromised mitochondria function, neuronal DNA damage, and stress granule formation. An RNA-like polymer poly(ADP-ribose) (PAR) can promote phase separation of R-DPRs and the formation of stress granules. However, how PAR facilitates the phase separation of R-DPRs remains unknown. To address this, we develop a liquid-liquid phase separation assay (LLPS) to assess the potency and chain-length dependency of PAR-induced droplet formation of R-DPR, specifically poly(GR). Additionally, we employ Electrophoretic Mobility Shift Assays (EMSAs) to investigate the length-dependent binding affinity of poly(GR) to PAR. Our findings reveal that the phase separation process of PAR with poly(GR) is both dependent on the stoichiometry ratio and poly(GR) length, consistent with the EMSA analyses
TOWARDS MECHANISTIC UNDERSTANDING OF MITOCHONDRIAL BETA-BARREL BIOGENESIS: STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE SORTING AND ASSEMBLY MACHINERY COMPLEX
No full textMitochondria are essential eukaryotic organelles and play a vital role in many cellular processes, including ATP production, lipid synthesis, and apoptosis. The majority of the mitochondrial proteome is translated in the cytosol and imported into the mitochondria as unfolded precursors. Outer membrane β-barrel proteins are imported by the translocase of the outer membrane (TOM) complex then folded and inserted into the membrane by the sorting and assembly machinery (SAM) complex. The SAM complex is composed of three subunits: a β-barrel core (Sam50) that spans the mitochondrial outer membrane, and two accessory subunits (Sam35 and Sam37) that associate on the cytosolic side of the membrane. The SAM complex recognizes β-barrel substrate precursor proteins by a conserved C-terminal sequence motif, called the β-signal. The molecular mechanisms of SAM complex β-signal recognition, precursor folding, and membrane insertion are currently unknown.
Here I describe our work towards understanding mitochondrial β-barrel biogenesis through structural and functional studies of the Thermothelomyces thermophilus SAM complex. We solved the first high-resolution structures of the SAM complex using cryo-electron microscopy. Our structures confirmed that Sam50 is a sixteen-stranded β-barrel that associates with Sam35 and Sam37 from the cytosolic side of the membrane. We next investigated SAM complex interactions with β-signal peptides using a variety of biochemical and biophysical techniques. Our results show T. thermophilus SAM complex interacts transiently with the β-signal from a variety of substrate β-barrel proteins. Lastly, we characterized the interaction of the SAM complex with a small molecule inhibitor, darobactin A, that mimics a β-signal. We found that darobactin A inhibits SAM complex function by binding with nanomolar affinity to a region of Sam50 that prohibits the association of substrate β-signals. Together this work has advanced our understanding of the SAM complex functional mechanism and mitochondrial β-barrel biogenesis
LEARNING KERNELS WITH NONLOCAL DEPENDENCE IN MEAN FIELD EQUATIONS AND THE EXTENSION PROBLEM ON DIRICHLET SPACE
No full textThis thesis concludes the research conducted during the author’s Ph.D. in both applied and pure math topics.
The first part of the thesis introduces a nonparametric algorithm to learn interaction kernels of mean-field equations for 1st-order systems of interacting particles. The algorithm employs a probabilistic error functional derived from the likelihood of the mean-field equation’s diffusion process and achieves convergence in a weighted L2 space by using least squares with regularization on data-adaptive hypothesis spaces. The identifiability of the algorithm is also characterized. Under data-adaptive L2 spaces, the Fre ́chet derivative of the loss functional leads to a semi-positive integral operator. Thus identifiability holds on the eigenspaces with nonzero eigenvalues of the integral operator, and on the L2 spaces if and only if the integral operators are strictly positive. Therefore, the inverse problem is ill-posed, and subsequently, we propose RKHS-based regularization and numerically demonstrate its accuracy.
The second part investigates the problem of learning kernels in operators from data. The nonlocal dependence of kernels and singular inversion operators make the inverse problem ill-posed. We present Data Adaptive RKHS Tikhonov Regularization (DARTR). The data-adaptive RKHS (reproducing kernel Hilbert space) is the function space of identifiability, whose norm is viable for Tikhonov regularization. DARTR selects the optimal regularization parameter and is demonstrated to be accurate, robust, and convergent. Moreover, by the natural connection between Tikhonov regularization and
Bayesian priors, DARTR can also be considered as a data-adaptive prior, which proved to achieve a stable posterior whose mean has a small noise limit, both theoretically and computationally. Numerical tests show that a fixed prior can lead to a divergent posterior mean in the presence of errors, whereas the data-adaptive prior is convergent.
Finally, we prove the Ho ̈lder regularity and the Harnack principle for the weak solutions of the equation p ́Lqsu “ 0 defined on fractals in a quite general framework of Dirichlet spaces generated by L. The heat kernel of L is assumed to have sub-Gaussian estimates, which is common in the case of fractals. Our goal is to give a general setup for the investigation of the powers of generators of Dirichlet form.
Primary Readers and Advisors:
Yannick Sire, Fei L
INFORMAL ADVANCE CARE PLANNING WITH FAMILY IN BLACK ADULTS WITH END STAGE KIDNEY DISEASE
No full textEnd stage kidney disease (ESKD) prevalence has increased by 107% since the year 2000, and Black adults are 3.5 times more likely to be diagnosed with ESKD. The risk of morbidity and mortality are significant among Black adults across the kidney failure continuum making the process of advance care planning (ACP) important. Research examining advance care planning (ACP) in adults with kidney failure is primarily assessed by written advance directives. Black adults with kidney failure have limited representation in ACP research, and many prefer informal ACP conversations. We conducted a concurrent mixed-methods study that examined and explored the relationships between personal, interpersonal, and structural level factors and their association with informal ACP conversations with family among Black adults with kidney failure.
The study sample (N=301) had a mean age of 55.7 years, 52% were female, 24.6% received less than high school education, 48.8% had an income of $50,000 or less and 76% are currently receiving dialysis. Sixty-six percent of participants engaged in an informal ACP conversation and 36.5% completed a written advance directive. The personal factor of illness acceptance (OR 1.08, p=0.017) and the interpersonal factor of emotional support (OR 1.42, p=0.028) facilitated informal ACP conversations. The health experience factor of past experience as a surrogate decision maker (OR=1.86, p=0.035) facilitated informal ACP conversations with family. Qualitative data (n= 20 participants from the quantitative sample) confirmed and expanded on what was found quantitatively. Six subthemes emerged that facilitated Informal ACP conversations with family that include burden avoidance, faith, health event, experience with death, trusted family relationships, and incenter dialysis peer support. Barriers to Informal ACP conversations with family include the subthemes of avoidance or fear of discussing death, feeling stable, and the selected surrogate decision maker is not ready or is untrustworthy to receive ACP wishes.
This study demonstrates that Informal ACP conversations with family occur more frequently than written directives among Black adults with kidney failure. Future research should assess the patient’s social support network and intervention development should focus on those who are unbefriended or who have untrusted social support networks
Beyond The Standard Model Explorations
No full textPhysicists have made great progress in fitting the observed world: huge mess of data largely agree with the relatively simple Standard Model of particle physics. Extrapolating away from this mostly-understood regime, we find that the terrain of possibilities is vast and rugged with various open issues. These issues seem to point to a long list of new mechanisms that are likely to be part of nature. The past decades have seen a proliferation of theories designed to achieve such outcomes, along with a broad experimental program to search for their signatures. My job as a theorist is to maximize what we learn from each experiment and suggest the next compelling targets to aim for experimentally. Some of my attempts that led to published works are presented in this dissertation
Multiscale deformation and fragmentation in turbulent flows
No full textFrom breaking waves in the ocean to chemical reactors, the fragmentation of bubbles in turbulence can be found in various natural and industrial applications. To understand the complex interaction between bubbles and turbulence with multiple length scales, in this thesis, we experimentally investigate bubble breakup by leveraging 3D simultaneous measurements of both bubbles and their surrounding flows.
A phenomenological breakup model is first proposed to describe the time evolution of the bubble size spectrum as bubbles are broken into smaller sizes over time. Several criteria are extracted from the existing experimental data to constrain the key parameters of the model. In addition, to understand the breakup mechanisms, experiments were conducted to physically disentangle eddies of multiple length scales in turbulence using the flow generated by the head-on collision of two vortex rings. Surprisingly, we find that bubbles are preferentially broken by eddies of smaller size, challenging the classic Kolmogorov-Hinze framework which assumes that the bubble only interacts with eddies of similar size. A novel bubble breakup model which incorporates such sub-bubble-scale eddy contribution is proposed and shows excellent agreement with the experimental data. To further quantify the interactions with these sub-bubble-scale eddies, we examine the local deformation of the bubble interface, i.e., the interfacial velocity and curvature. Results suggest that bubble breakups typically comprise two distinct processes: an inertial deformation process and a final capillary-driven process. By modeling the time scale, it is found that the inertial deformation process is due to the collision with sub-bubble-scale, intermittent eddies, consistent with the conclusion from our previous study.
In addition, we also explore how such small-scale eddies in turbulence could interact with general deformable particles. In the second part of the thesis, we investigate the folding dynamics of fluid elements in turbulent flows. Through an analytical approach, we find that folding in turbulence is made up of two stages: an initial linear-growth stage driven by the velocity Hessian due to small-scale eddies, followed by an exponential-growth stage due to the stretching of already bent fluid elements. Such stretching motion is also found as the leading mechanism causing the intermittency of the folding
EXPLORATION OF CELL-BASED METHODS TO DISSECT LNCRNA-PROTEIN INTERACTIONS
Get PDFLong noncoding RNAs (lncRNAs) have been increasingly important in understanding regulations of gene expressions, thanks to the discovery of non-protein-coding genes and transcripts with the advent of high-throughput sequencing. It is known that most of the lncRNA functioning mechanisms involve interactions with RNA-binding proteins. Various in vitro experiments, such as in vitro transcribed RNA pulldown and electrophoretic mobility shift assay (EMSA), have been applied to study lncRNA-protein binding. However, the mechanisms of their interactions in the cell environment remain mysterious for most lncRNAs. Our goal is to explore cell-based methods to explore lncRNA-protein interactions. These include MPRNA-IP (Massively Parallel RNA Assay-Immunoprecipitation), a high throughput experiment developed by our lab, and CLIP-qPCR (Crosslinking Immunoprecipitation and qPCR) with RNA deletion mutants or RNase treatment. We focused on two lncRNA-protein interactions, cytosolic interaction NORAD-PUM2 and intranuclear interaction HOTAIR-EZH2. We detected a well-known PUM2-binding RNA sequence motif in NORAD, validating our methods for RNA-protein interactions. Regarding EZH2-HOTAIR interaction, our experiments showed that the EZH2 protein interacts with the 5’ end of lncRNA HOTAIR, as previous in vitro assays showed. But we unexpectedly identified that EZH2 also has a binding affinity with the middle region of HOTAIR while inside the cell nucleus. Taken together, our results present representative examples of applying multiple methods to study lncRNA-protein interactions inside the cells and provide new insights into the mechanism of lncRNA-protein interactions
Oral History of Loretta M. Kopelman
No full textThis interview with Loretta M. Kopelman, PhD, was conducted by Suzanne Snider on February 8, February 9, and March 4, 2023 in McLean, Virginia as part of Moral Histories: Voices and Stories from the Founding Figures of Bioethics, an oral history project of the Johns Hopkins Berman Institute of Bioethics. Prof. Kopelman is the Adjunct Professor of Family Medicine at Georgetown University Medical Center and Professor Emeritus at the Brody School of Medicine at East Carolina University. She was founding president of the American Society for Bioethics and Humanities (ASBH) and president of the Society for Health and Human Values (SHHV). Prof. Kopelman’s areas of expertise include research policy, ethics of human subjects research, research on children, and resource allocation.On Day 1 of the interview, Prof. Kopelman describes being raised in Queens, New York on the grounds of Creedmoor State Hospital, where her father was a psychiatrist. She reflects on her relationships and interactions on the hospital grounds as an early influence on her attention to patient care. Prof. Kopelman describes her undergraduate education at Syracuse University including her pivots from social work to psychology, and ultimately, to philosophy, which she first encountered through Descartes’ Meditations on First Philosophy. She went on to earn a PhD in philosophy at the University of Rochester. One of the few women in her graduate program, Prof. Kopelman describes her experiences of sexism while in graduate school.On Day 2, Prof. Kopelman spends substantial time describing the establishment of the American Society for Bioethics and Humanities (ASBH) in 1998, of which she served as founding president. Prof. Kopelman highlights notable tensions during the discussions that led to the ASBH’s founding. She tells a story of ASBH negotiating whether the organization should take stands on bioethics issues. Prof. Kopelman speaks about the flaws of the so-called Baby Doe regulations and contrasts them with the best interest standard. She describes her lifelong advocacy for ethically responsible research involving children.Prof. Kopelman discusses ethical questions related to different cultural practices, telling a story about forced marriage and comparative laws around child abuse protection in the UK. Prof. Kopelman describes Bioethics Summer Camp (started by Art Caplan in the 1990s) and developing close relationships with Baruch Brody and Marian Gray Secundy while there. She describes serving on a FDA panel evaluating the use of fentanyl as anesthesia for children and challenging the doctors from top institutions, citing this as part of her ethos: asking probing questions to get to the core of an issue. Prof. Kopelman reflects on the growth of bioethics and its increasing engagement with other disciplines, positing that this broadening is making bioethics a better field. Please note: Due to a significant loss of audio recorded on Day 1 and the project team's efforts to recover that lost material on Day 3, listeners may note thematic overlap between Day 3 and the previous two sessions, including discussions of the Baby Doe regulations, savior siblings, Jay Katz, and a growth hormone study.On interview Day 3, Prof. Kopelman describes: her interest in philosopher David Hume and his work on moral decisions; psychiatrist Stanley Walzer’s XXY syndrome study; bioethics as a second order discipline; and the case of Elizabeth Bouvia and end-of-life issues. Prof. Kopelman describes being recruited to the newly-formed East Carolina University School of Medicine and designing the medical humanities department there, where she became its first chair. She reflects on teaching ethics in medical school settings and the benefit of using literature as a teaching tool in both philosophy and medical humanities. Prof. Kopelman speaks in considerable depth about the 1990s NIH study which researched the effects of human growth hormone in children. Prof. Kopelman also speaks to her friendship with Jay Katz and the influence of his work around informed consent.This interview may be of interest to those seeking to learn more about: the field of bioethics and the history of bioethics in the US; gender justice; best interest standard and the Baby Doe regulations; philosophy; ethics across cultural practice; bioethics and interdisciplinarity; children as research subjects; bioethics and the humanities; experiences of founding bioethics departments in medical schools; and regional histories of bioethics (mid-Atlantic and New York State).Note about Day 1, File 3 audio: Due to technical difficulties faced by the production company and recordist, several hours of recorded audio were lost. The audio on this file terminates at time code 00:15:25. All efforts were made to review material recorded on Day 1 during an additional session scheduled a month later