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CHARACTERIZING HOST-PATHOGEN INTERACTIONS IN THE HEART IN A MOUSE MODEL OF AFRICAN TRYPANOSOMIASIS
No full textTrypanosoma brucei is a parasite which represents a threat to human and animal health throughout sub-Saharan Africa. Although T. brucei colonizes extravascular spaces, where it causes organ damage and inflammation, host-pathogen interactions in these spaces and their consequences remain poorly understood. There is evidence that T. brucei colonizes the heart, causing inflammation and cardiac dysfunction. However, despite evidence that the heart is an important focus of pathology in Human African Trypanosomiasis (HAT), the understanding of host-pathogen interactions in the heart during T. brucei infection remains to be thoroughly investigated. To investigate the host-pathogen interactions in the heart during T. brucei infection, we developed a mouse model of HAT. We demonstrate that in this model, T. brucei invades and colonizes the heart within a week of infection, resulting in myocarditis and cardiac dysfunction which is similar to that exhibited in natural infection. Using this model, we further investigated host-pathogen interactions in the heart.
On the host side, we find that T. brucei infection causes an intracardiac immune response characterized by increased numbers of most cell types—most notably including CD8+ T cells, Natural Killer cells, and pro-inflammatory macrophages, indicating an immunopathological component of the cardiac damage seen in T. brucei infection.
On the parasite side, we find that intracardiac parasites exhibit a unique transcriptome at both early and late infection timepoints, suggesting that they represent a unique population compared to intravascular parasites. In particular, intracardiac parasites are more proliferative and upregulate fewer stress-response genes than intravascular parasites, suggesting that the heart is a relatively protected parasite reservoir. This is in agreement with our findings on the host side, which indicate a relatively ineffective intracardiac immune response.
In our transcriptomic data, we identified two trypanosomal genes which we hypothesized were involved in cardiac colonization by T. brucei. On further investigation of these genes, we find that they are more generally involved in T. brucei pathogenesis. Knockout of these genes results in a decrease in T. brucei virulence, with mice exhibiting longer survival times and a less severe clinical phenotype. However, their function and specific contributions to pathogenesis remain to be fully characterized
On the Margins of 'Alle': Paralleling Feminist Theologies of Space in the Medieval Revelations of Julian of Norwich and the Antebellum Incidents of Harriet Jacobs
No full textOn the Margins of ‘Alle’ comparatively reads Julian of Norwich as the first woman to write in English alongside Harriet Jacobs, the first self-emancipated African-American woman to write her own autobiography without requiring an amanuensis. Ostensibly the two figures have little in common, yet both women are Christians and authors who enter the canon from enclosed spaces on the literal margins of patriarchy. The medieval Julian of Norwich was an English anchorite whose life spanned Norwich’s late fourteenth and early fifteenth centuries, while the antebellum African-American Harriet Jacobs was a self-emancipated Christian who escaped the chattel-slavery of 1840s North Carolina not by fleeing the slave-holding south, but by hiding in a disused garret in a dilapidated shed where she remained hidden for seven years. Both women sought the literal margins as an alternative to what this dissertation argues are the similar—though definitely not synonymous—objectifications of first the medieval English and then the United States’ enslaved woman as reproductive objects, intended to amplify the agency of masculine subjects. By transforming their respective margins into a divine platform from which both claim to speak with God’s authority, both the medieval Julian and the antebellum Harriet Jacobs claim the rights and roles of subjects: theologians whose ‘mystical’ vernacular theology grants them the authority to discourse on the divine, and the ability to redeem the reproductive body as a divinely ordained subject (rather than mere object). In arguing that the medieval Julian should be read as Jacobs’s literary and theological predecessor, this dissertation does not claim that Jacobs necessarily read Julian; nor does it argue that medieval unfreedom and antebellum enslavement posed identical oppressions. Rather, this dissertation uses a vocabulary of subjects, objects, and agency furnished by Materiality Studies to show how both figures enter the English canon similarly battling their respective objectifications, each redefining Christianity through the reproductive body with a vernacular theology that proves this faith practice as transtemporal as it is subversive. This allows new understandings of Julian as subversive and Jacobs as theological to come to the forefront in ways that reading their seminal texts separately has obscured
THERMODYNAMIC AND STRUCTURAL ANALYSES OF PERIPLASMIC CHAPERONE FKPA BINDING UNFOLDED OUTER MEMBRANE PROTEINS
No full textThe survival and virulence of gram-negative bacteria require proper biogenesis and maintenance of the outer membrane, which is densely packed with -barrel outer membrane proteins (OMPs). Before reaching the outer membrane, precursor unfolded OMPs (uOMPs) must cross the whole cell envelope. A network of periplasmic chaperones and proteases maintains unfolded but folding-competent conformations of these membrane proteins in the aqueous periplasm while simultaneously preventing off-pathway aggregation. Of the periplasmic chaperones involved in OMP biogenesis, the stress-responsive chaperone FkpA is perhaps the most understudied. This thesis investigates the thermodynamic and structural features of FkpA-uOMP interactions to understand the chaperone function of FkpA in the periplasm by focusing on the three species in the binding reaction: the uOMP, the chaperone FkpA, and the FkpA-uOMP complex. First, uOMP ensembles were created using coarse-grained molecular dynamics simulations in accordance with experimental properties. The resulting ensembles reflect the dynamic and diverse distribution of conformations accessible to uOMPs. The chaperone FkpA is also conformationally heterogenous and exhibits coupled folding and oligomerization at its dimer interface. However, FkpA must exist as a dimer in the periplasm barring an external source of perturbation. The FkpA-uOMP complex is tightly bound across an extensive binding interface, but FkpA still releases uOMPs to enhance their folding into membranes. All data is consistent with many uOMPs binding between the “arms” of the V-shaped chaperone via a shared interface. How concepts of binding promiscuity, multivalency, avidity, oligomerization, and conformational heterogeneity contribute to the FkpA-uOMP interaction are explored. The results within help define the role of FkpA in OMP biogenesis as a potent holdase that enhances overall OMP folding
Examining Social Perspective Taking Ability and Motivation in Monolinguals and Bilinguals
No full textThis dissertation examines the relationship between bilingualism and social perspective taking (SPT)—the social-cognitive capacity to discern what people think and feel, which is associated with significant academic and social benefits. Despite extensive research on SPT outcomes, less is known about the factors that enhance SPT, such as bilingualism. This dissertation considers both linguistic and cultural factors of bilingualism, and proposes that bilingualism as a complex construct may influence SPT ability and motivation,
Study 1 deconstructs SPT into its components, employing multiple measures to assess SPT in adults, finding that general SPT ability and motivation are positively correlated with context-specific abilities. However, self-reported SPT motivation did not align with performance-based measures, highlighting the complexity of measuring SPT.
Upon establishing robust measures to capture the multifaceted nature of SPT, Study 2 investigates the bilingual advantage in SPT, operationalizing bilingualism along multiple dimensions of linguistic experience and cultural identity. The study reveals that bilingual participants exhibit higher SPT abilities than monolinguals, while biculturalism appears to enhance SPT motivation. These findings suggest that language and culture significantly influence social cognition.
With evidence of bilingualism's advantageous role uncovered, Study 3 pivots to evaluate an intervention aimed at enhancing SPT through increased exposure to a second culture. Although the intervention did not yield significant effects on the main hypotheses, exploratory analyses revealed that it increased participants’ curiosity and cultural awareness. When compared to a non-intervention group, participants who received cultural exposure showed increased context-specific SPT ability.
Collectively, this research advances our understanding of SPT by offering a nuanced analysis of its facets, introducing a refined concept of bilingualism, and examining the real-world social-cognitive benefits of bilingual and bicultural experiences. The implications are profound, proposing that bilingualism, often undervalued in educational settings, could be a key asset in promoting understanding in a globalized society. The findings support the potential of bilingual education and cultural exposure interventions in fostering social and emotional competencies, essential for intercultural communication and global citizenship
TRANSCRIPTIONAL SIGNATURES OF TUMOR-SPECIFIC CD4+ CONVENTIONAL T CELLS IN NON-SMALL CELL LUNG CANCER
No full textImmune checkpoint blockade (ICB) therapy has primarily focused on unleashing the cytotoxic CD8+ T cell response due to their ability to directly recognize and kill tumor cells. Meanwhile, CD4+ T cells have largely been categorized as helpers for their CD8+ counterparts. Given the ability of anti-PD-1 treatment to also affect CD4+ T cell function, it is critical to also understand their role in the tumor microenvironment during ICB treatment, as this may be key in understanding why some patients respond to treatment while others do not. Our lab has acquired single-cell RNA sequencing and T cell receptor sequencing data from over 500,000 infiltrating T cells from tumor, adjacent normal lung tissue, tumor draining lymph nodes, and a distant metastasis acquired from a non-small cell lung cancer (NSCLC) clinical trial (NCT02259621). 15 patient samples were sequenced, in which 6 patients had a major pathologic response (MPR) to ICB, defined as less than or equal to 10% of viable tumor remaining at the time of resection, while 9 were classified as non-responders. From this study, our lab previously identified tumor-specific CD8+ T cells and discovered that the presence of these cells did not predict response to ICB therapy, indicating that other cells in the microenvironment, such as CD4+ T cells, are contributing to patient response.
Identifying which CD4+ TILs are tumor-specific remains a challenge. Therefore, this cell population has yet to be identified and analyzed in NSCLC at a single-cell level. We turned to an immunocompetent murine model for characterizing the transcriptomics of tumor-specific CD4+ T conventional (Tconv) cells. In murine tumors, tumor-specific CD4+ Tconv cells upregulate a Th1 and checkpoint-high program in the context of tumor antigen. Utilizing this model, we created a comprehensive gene score to extrapolate this signature onto our NSCLC clinical trial data for tumor-specific CD4+ T cell identification. The majority of tumor-specific cells in the human data are defined by their high CXCL13 expression. Looking a step further, characterization of gene expression differences between tumor-specific CD4+ T cells from the NSCLC clinical trial samples revealed that responders retain this high level of CXCL13 expression, as well as an overall programming consistent with an earlier stage of T cell exhaustion than non-responders. Taken together, this data provides insights into how tumor-specific CD4+ T cells contribute to the anti-tumor immune response and highlights the importance of their consideration in developing new ICB therapies and biomarkers for response prediction
Multiparametric prostate ultrasound imaging
Get PDFProstate cancer is the second most prevalent malignancy among men worldwide. The improvement of the rate of cure hinges upon accurate and early detection and treatment of aggressive prostate cancer. Current approaches suffer from low sensitivity (transrectal ultrasound), low specificity (prostate-specific antigen (PSA) test), lack of utility in treatment (PSA test and optical imaging), or cost-effectiveness and accessibility (radionuclide imaging and magnetic resonance imaging (MRI)). There exists a pressing need for an imaging modality that is fast, accurate, cost-effective, and capable of serving screening, active surveillance, and treatment purposes for prostate cancer.
In this dissertation, our focus is on exploring the potential of multiparametric ultrasound as a solution. While several ultrasound parameters such as Doppler imaging, contrast-enhanced ultrasound, and elastography have been extensively investigated for prostate cancer detection, the effectiveness of incorporating these into existing modalities remains controversial. Therefore, we propose to explore two novel pathways: photoacoustic (PA) imaging and ultrasound tomography (UST), to address the gaps in acoustic parameters crucial for functional, molecular, and quantitative imaging of prostate cancer.
Specifically, we leverage functional and molecular PA imaging to enable the measurement of prostate-specific membrane antigen (PSMA) expression that is associated with the aggressiveness of prostate cancer. We also employ quantitative UST to evaluate prostate’s speed of sound, acoustic attenuation, and reflection. We focus on addressing challenges of using PA and UST for prostate imaging regarding image reconstruction, contrast enhancement, instrumentation, and validation. We specifically investigated PA optical inverse problem, spectroscopic PA image enhancement, economic PA imaging solutions, realistic digital prostate phantoms for PA and UST, co-robotics UST system, and limited-angle UST reconstruction. Overall, our findings show promises of PA and UST imaging in enhancing prostate cancer diagnosis, with the potential of revolutionizing the field with their utility and widespread adoption
GENERATION OF A GENETICALLY ENGINEERED CELL LINE TO STUDY PROTEASOME DYNAMICS DURING STRESS
Get PDFProteasome function is vital to cell survival and impaired or inhibited proteasome function underlies many human diseases including cancer and neurodegenerative disease. Due to their critical importance, cells have evolved mechanisms to control and modulate proteasome function in response to environmental stressors. Utilizing CRISPR Cas-9 gene editing, we generated a stable Human Embryonic Kidney (HEK293T) cell line which contain fluorescently labeled proteasomes that can be used to visualize and study proteasome-mediated stress responses. Using live cell microscopy, we observe that proteasomes integrate into foci in response to osmotic stress and nutrient starvation in HEK293T cells. These foci are dynamic and reversible and can be resolved over time or with the removal of stressor, suggesting that foci formation is a pro-survival mechanism. A high thruput imaging and quantification pipeline was established to further study the molecular mechanisms underlying stress induced proteasome foci formation. Utilizing our cell line and image analysis platform to look at thousands of cells, we make the following observations 1) a small percentage of cells form detectable proteasome foci in untreated cells, independent of an exogenous stressor and 2) proteasome foci formed in HEK293 cells are not inhibited by pretreatment with the ubiquitin inhibitor MLN. These results may suggest that proteasome foci formation could be a general stress response with broad cellular function beyond what has previously been published, and that proteasome foci formation mechanisms in HEK293T cells may be molecularly distinct from other mammalian cell lines where this phenomenon has previously been observed. Future work will be aimed at determining if the proteasome foci formed in HEK293T cells are liquid phase-separated, comparing the proteins and RNA involved in osmotic stress foci vs. starvation stress foci, and identifying the minimal components required to form proteasome foci in vitro. The role of proteasome liquid-liquid phase separation during cellular stress responses could have important implications for neurodegenerative disease etiology
DEVELOPMENT AND UTILIZATION OF ARTIFICIAL ANTIGEN PRESENTING CELLS FOR MEMORY CD8+ T CELL EXPANSION
No full textAdoptive T cell therapy (ACT) relies on the ex vivo expansion of patient-derived T cells. While ACT has proven to be efficacious, factors such as cost, specialization requirements, and potency reduce its feasibility. T cell phenotype is an indicator of potency; central memory CD8+ T cells are more efficacious than effector CD8+ T cells as they persist longer in vivo. However, culturing conditions lead to an exhausted effector state. To improve ACT, artificial antigen presenting cells (aAPCs) were designed to mimic antigen presentation canonically done by dendritic cells. This allows aAPCs to expand T cells ex vivo and reduce the complexity of ACT. This research seeks to utlize aAPCs to develop synergistic and novel methods for preferential expansion of memory CD8+ T cells to address the issue of treatment potency within ACT.
To do this we sought to outline effects of Akt inhibition, a method known to increase the memory T cell population, on the T cell receptor (TCR) repertoire selection. To identify changes within the TCR repertoire paramagnetic aAPCs were used to preferentially expand and treat antigen specific CD8+ T cells with an Akt inhibitor. Akt inhibition was suspected to increase TCR diversity, subsequently increasing the number of CD8+ T cell clonotypes. These findings suggest that inhibition along the PI3-Akt-mTOR pathway may give rise to additional changes that lead to in vivo improvements.
In the absence of inhibition, Akt excludes memory forming transcription factors. This can be circumvented when oxidative stress accumulates within the cell and acts as a signal for antioxidant production, stress reduction, and promotion of survival and memory. Alginate, an antioxidant rich polysaccharide, has been shown to reduce stress and improve memory within T cells. We therefore sought to develop alginate-based aAPCs to expand memory CD8+ T cells. Our findings show that the cells expanded with alginate-based aAPCs showed superior in vivo survival, persistence, and exhibited transcriptional and phenotypical profiles likened to memory CD8+ T cells. This research outlines the novel use of material selection as a tertiary signal during T cell activation and expansion
Burned by the Sun: The Life, Teachings, and Controversies of Neḥemiah Ḥiya Ḥayon
Get PDFThe dissertation explores the career of Sabbatean kabbalist Nehemia Ḥiya Ḥayon, whose Strength to God and Inner Sanctum (Berlin, 1713), ignited an unprecedented controversy involving Jewish communities across Europe and the Ottoman Empire from 1713-1715. Ḥayon presented a trinitarian concept of the Deity and formulated a radical revision of the transmission of esoteric knowledge, advocating independent probing.
First, in contrast to focusing predominantly on the internal struggle against heterodoxy, I consider socio-economic factors. This study reveals that Ḥayon’s early confrontation with the Jerusalem rabbinate resulted from his attempt to establish a kabbalah school during an economic crisis in Palestine, which intensified the competition over depleted funds. Only after this plan failed did he turn his efforts to printing his books, taking advantage of philanthropic networks.
Second, whereas previous investigations have framed the controversy either as a clash between rabbinic authority and the communal leadership, or between proto-orthodox ‘zealots’ and Sabbatean ‘enthusiasts,’ I contend that the struggle was between two increasingly conflicting ideals: dissimulation and transparency. The study examines how Ḥayon and his opponents manipulated information and how information affected intra- and inner communal politics. The ability of Ḥayon’s adversaries to mobilize a substantial coalition depended on disseminating his book and giving it broad publicity through polemical publications. This strategy differed from the previous approach aiming to suppress potentially harmful information.
Third, I depart from well-established paths of kabbalah and Sabbateanism research dominated by hermetic kabbalistic discussion, seeking to establish a more nuanced understanding of Jewish religious discourse and connect it to broader cultural trends. Inter alia, this study examines anatomical depictions which Ḥayon employed to construct his theological arguments in internal discourse with other kabbalistic and Sabbatean systems, as well as his polemics against the Christian
Lastly, I explore the role of magic in Ḥayon’s kabbalistic doctrine, the reputation he built for himself, and eventually the outbreak of the controversy. Notably, this study claims that its initial stage was a clash over magic versus modern medicine, not theology.
In summary, this research adds a new chapter to the history of the Sabbatian movement and enhances our understanding of Jewish religiosity
UNFOLDING THE ROLE OF IRE1α-XBP1 SIGNALING IN CANCER
No full textMany common human ailments, including infectious, cardiovascular, metabolic, neurodegenerative diseases, and cancer, are characterized by an abnormal accumulation of misfolded proteins. An overabundance of non-functional proteins places significant physiological demands on the endoplasmic reticulum (ER), an organelle that balances protein translation and degradation. Following a pathological buildup of misfolded proteins, luminal stress sensors activate the Unfolded Protein Response (UPR)- an ancient eukaryotic mechanism to restore ER homeostasis. Prolonged or unresolved ER stress can eventually lead to cell death, making elements of the UPR attractive targets for therapeutic intervention.
The most conserved signaling component of the UPR is the endoribonuclease IRE1α and its transcriptional effector, XBP1s. IRE1α resolves ER stress in two unique ways: cleavage of a poorly defined subset of mRNAs to reduce global protein translation and splicing of XBP1 mRNA. In unstressed cells, an unstable protein of unknown function, XBP1u, is translated from the unspliced XBP1 transcript. UPR activation triggers excision of a 26-bp intron from the XBP1 transcript by the endoribonuclease moiety of IRE1α, creating a frame-shifted template for the translation of XBP1s, a transcription factor with a large c-terminal domain that is not present in XBP1u. The transcriptome of XBP1s is not clearly defined, and interpretation of past work is complicated by the use over-expression systems that retain endogenous XBP1u. In a variety of cancers, sustained IRE1α-XBP1s signaling is correlated with decreased overall survival and lower progression-free survival, though the mechanism for this is not well-understood.
In the following studies, evidence is presented for a novel role of XBP1s and XBP1u in regulating expression of an important drug target in cholesterol metabolism and cancer, PCSK9. The XBP1 and ERN1 (which encodes IRE1α) loci were disrupted in the human lung adenocarcinoma cell line A549 by CRISPR-Cas9 editing, resulting in a novel isogenic cell panel of XBP1 and IRE1 knockouts, and knock-in splice site mutants that caused constitutive expression of either XBP1s or XBP1u. Cells constitutively expressing endogenous XBP1s (hereafter referred to as XBP1∆26) had altered morphology and perturbed dynamics in vitro, and a loss of tumorigenicity when grafted into nude mice. RNAseq analysis of wild type and XBP1∆26 cells revealed strong upregulation of numerous genes, including many that had not previously been attributed to IRE1a-XBP1 signaling. Unexpectedly, a large magnitude of changes in down-regulated genes, including PCSK9. Using this unique cell pane was observed. Additionally, it was demonstrated that the level of PCSK9, a multifunctional protein involved in several disease processes, is jointly modulated by both XBP1 proteins