Friend or Foe: Exploring the Biological Function of the Mycobacterium Tuberculosis RecA Intein

Mycobacterium tuberculosis (Mtb) is the leading source of death due to a single infectious agent, and increasingly drug resistant strains threaten to quash progress in battling tuberculosis (TB) disease. Three of Mtb’s genes, the iron-sulfur cluster assembly protein sufB, the DNA gyrase dnaB and the recombinase recA, have been invaded by an intervening protein, or intein. Inteins are translated in frame with their host gene. The resulting precursor consists of the intein flanked by each half of the invaded protein, termed the N- and C-exteins (NE and CE). This hybrid protein is non-functional until the intein splices itself out, with concomitant ligation of the NE and CE to produce mature host protein, also called ligated exteins (LE). Splicing is coordinated by two catalytic residues: the first residue of the intein, which can be a cysteine or serine, and the first residue of the CE, which can be a cysteine, serine or threonine. In addition to productive splicing, off-pathway cleavage products can occur when the NE or CE are liberated (also called N-terminal or C-terminal cleavage: NTC and CTC respectively). Inteins were thought to be mobile genetic parasites that provided no benefit to the host, but recent evidence suggest that this paradigm is untrue for some inteins. Rather, they act as important post-translational regulators of their host proteins, generally by splicing in response to specific environments such as pH, temperature, or DNA damage. The RecA protein is an important hub for two DNA repair pathways: 1) as a regulator for the SOS DNA repair response, which has been implicated in generation of drug resistance, and 2) as the driver of homologous recombination, the high-fidelity double-strand break repair pathway. Mtb RecA is provided with a unique opportunity for post-translational regulation through the intein, which is specific to Mtb complex mycobacteria. Despite being the first bacterial intein discovered and well-studied in vitro using non-native expression systems, the role the Mtb RecA intein plays in Mtb biology is unknown. I leveraged the non-essentiality of Mtb’s recA to study the impact of the recA intein on regulation and production of RecA protein in Mtb. I followed RecA species production in various conditions (LE, intein and NE) via western blot analysis, and recA transcription via a green fluorescent protein-based recA transcriptional reporter. DNA damaging therapeutics mitomycin C and ofloxacin increased the amount of recA transcription, RecA splicing and NE production. Non-DNA damaging therapeutics isoniazid and rifampicin did not increase transcription, splicing or NTC. Treatment of cells grown in hypoxia impacted RecA splicing and NTC. Copper, a heavy metal used as an antimicrobial by macrophages, had no effect on RecA in ambient air conditions, but increased splicing and decreased NTC in hypoxia. Cisplatin, a chemotherapeutic that has been suggested as a potential splicing inhibitor and anti-Mtb therapeutic, had no effect on RecA in ambient air, but increased splicing in hypoxic conditions. This is the first report of NTC of a WT intein in its native environment and notably, NTC was affected by various conditions. I explored if NTC was biologically important. The NE retains all features needed to activate the SOS response: the ssDNA binding motifs, ATP binding motifs, interfaces for multimerization with other RecA monomers, and the key residues for activating cleavage of the SOS transcriptional repressor, LexA. By using western blot analysis, I found that the NE retained LexA-activating capability when heterologously expressed in E. coli. Interestingly, unspliced RecA precursor was not detected in Mtb under any environment tested, including those, such as copper and cisplatin, that inhibit RecA splicing in vitro. This runs counter to the initial paradigm that post-translational regulation by an intein would involve producing a pool of precursor protein, which could then have splicing triggered by specific environmental signals. RecA precursor was only detectable in Mtb when one or both catalytic cysteine residues were mutated, suggesting rapid splicing of the WT RecA intein. Rapid splicing was specific to the Mtb RecA intein, as splicing of a second Mycobacterial RecA intein, intein B, did not occur when expressed in Mtb. Rapid splicing of intein B could be induced by moving intein B to the position of the Mtb RecA intein. I also provide preliminary data on recA regulation in hypoxic air conditions and non-replicating persister populations, and how RecA may interact with the NucS mismatch repair system. These results provide the first evidence of how the Mtb RecA intein impacts regulation of RecA, and that regulation of the Mtb RecA intein in Mtb proper differs from previous in vitro results

Benjamin’s estranged Heim

This paper examines Walter Benjamin\u27s engagement with the motif of Heim (home) as it interweaves with his intellectual and personal experiences during the early 1920s. Through an analysis of Benjamin’s writings and correspondence, including his plans for the journal Angelus Novus and reflections on his friendship with Fritz Heinle, the study explores Heim as both a real and rhetorical space. Initially conceived as an idealized site of intellectual and spiritual communion, Heim is transformed by personal tragedy and historical crisis into an unattainable, speculative ideal—a rhetorical atopos. This evolution parallels Benjamin’s philosophical pursuit of “pure language” and his reimagining of translation as a generative act in the face of catastrophe. By situating Heim within Benjamin’s broader philosophical and political tensions, this paper argues that his work reflects a yearning for new forms of community while grappling with the irreducible fragmentation of language, memory, and belonging

RESOURCES TO STUDY THE ISOFORMS OF THE p53 FAMILY OF TRANSCRIPTION FACTORS

The p53 family of transcription factors regulates diverse gene regulatory networks such as those involved in DNA damage repair, epidermal commitment, and neuronal development. As a result, dysfunction in these pathways lead to numerous human health conditions, including multiple types of cancer and craniofacial developmental disorders. Each family member regulates a distinct gene network leading to varied cellular and organismal phenotypes, but all are capable of regulating programmed cell death, likely due to considerable similarity between protein structure and binding sites in DNA. Analysis of these shared and distinct functions is complicated by each p53 family member gene (TP53, TP63, and TP73) generating multiple N- and C-terminal isoforms with potential differences in activity. Here, we develop a novel p53 family expression library for comprehensively and uniquely interrogating their shared and distinct biological activities. The first collection allows for in vitro expression and transient expression of all 35 uniquely codon-shifted isoforms in a range of mammalian models. We used this library to demonstrate its utility in distinguishing isoforms based on their unique sequences enabling their unequivocal identification in complex biological settings. We also showcase the advantage of utilizing this collection for knockdown – rescue style experiments that are challenging due to the sequence homology present within this family. The second collection allows for stable integration and inducible expression of all 35 isoforms coupled with unique molecular identifiers (UMIs) suitable for use in pooled screens. We used this library to perform pooled drop-out screens to compare anti-proliferative activity between isoforms of this transcription factor family. We identified both known and novel activities that would have been difficult with conventional loss-of-function (LOF) approaches. This newly constructed tool provides the means to answer previously intractable questions about the p53 family and has the potential to significantly impact evolutionary biology, developmental biology, and cancer biology

USING CONVOLUTIONAL NEURAL NETWORKS TO IDENTIFY RARE WEATHER EVENTS: APPLICATION TO KONA LOW CLASSIFICATIONS WITH LARGE-SCALE WIND PATTERN

Extreme events pose challenges in prediction on account of their low frequency and erratic nature. Among these events, Kona lows are rare subtropical cyclones that significantly impact the weather pattern of Hawaii, sometimes causing heavy rainfall, strong winds, and coastal flooding. In the past, studies have used traditional statistical methods such as synoptic climatology, regression analysis, and manual classification that rely on expert judgment to identify Kona lows; however, they often struggle to capture the complex spatial patterns of the Kona storms. Convolutional Neural Networks (CNNs) exhibit a property called translation invariance, which allows them to learn and recognize Kona lows regardless of any changes in the spatial patterns. The goal of this study was to assess the CNN’s ability in correctly identifying extreme events. It uses the zonal wind data of Hawaii from the ERA5 reanalysis between the years 1990 and 2010 as an input. Several CNNs are trained using techniques like undersampling, oversampling, and manual weight distribution to overcome data imbalance, which occurs due to the high frequency of non-extreme events compared to the rare Kona lows. The best model achieved an Extreme Dependency Score (EDS) of 0.84, where 1 represents the best possible score reflecting perfect prediction with no false positives and a frequency bias of 0.90, which suggested slight underprediction. Combining EDS with the bias provides a more comprehensive picture where the scores indicate that the model is well calibrated and non-biased. The CNN model outperforms the reference random prediction and a simple artificial neural network model that uses the numerical PC1 ( First Principal Component ) and PC2 (Second Principal Component ) values derived from the Kona lows as an input. This is followed by a physical interpretation of the forecast using one of the Explainable AI (XAI) approaches. Randomized Input Sampling for Explanation (RISE) explains how the CNN model makes decisions by randomly masking a part of the input image to see how it affects the model output. The composite of the resulting heat map of the true positive events indicates the features of the zonal wind that are important for prediction .The heat map is consistent with the observed trend that Kona storms typically form to the southwest of Hawaii, as this area shows the highest importance, followed by the region surrounding the Hawaiian islands. These results demonstrate the potential of using deep learning techniques in forecasting rare events, promising avenues of future research in climate risk assessment

For The Love of Subtitling: Analysing The Challenges in Audio-Visual Translations of Rocky aur Rani kii Prem Kahani

Any form of audio-visual media utilises specific stylistic elements in order to represent the reality within which the media form is situated. These representations occur through the language and iconography of the media form, which works on stereotypical features that the audience can easily identify. Forming a pivotal part of the media, these representations must be rendered in the translations for a linguistically and culturally diverse audience. Audio-Visual Translation (AVT) has been used since the 1930s to make media accessible. Of the various modalities, subtitling has emerged as the most widespread mode of AVT due to time and cost benefits. However, the subtitling process is restricted by various challenges that can restrict the translation process, especially in terms of transferring culturally specific terms from the source. The verbal or linguistic aspect is merely one dimension of the process, as this form of translation deals not only with relaying information but also with the semiotics and pragmatics of meaning-making. The socio-cultural context of the linguistic element within media forms requires the translators to opt for a set of translation strategies that will best express this context to the audience. Through the examination of the subtitles of the Bollywood film Rocky Aur Rani kii Prem Kahani (2023), the paper will examine the process of AVT and the subtitling decisions made to reveal the cultural nuances embedded within the film that must be translated through subtitling. This forms an analysis framework, which the paper explores in terms of the narrative constructed on the cultural and socio-linguistic identity markers pertinent to audio-visual media

Symbols of Sovereignty and How They Speak to Us: Reading Contemporary Wampum Artwork in New York State Museums

Contemporary Haudenosaunee artworks in exhibit spaces across New York State are bringing cultural information and counter-historical narratives to the public that they might not encounter otherwise. Artwork that includes wampum depictions or materials has heuristic qualities that can engage viewers with Haudenosaunee ways of knowing and relating. Wampum heuristics and Haudenosaunee scholarship are employed to interrogate how wampum materials were collected, categorized, and researched by the New York State Museum during the salvage anthropology era. The narratives embedded in wampum artworks are traced from this salvage era history, throughout the century between the salvage era that began at the end of the 19th century and the passage of the Native American Graves Protection and Repatriation Act in 1990, up to the early 21st century, in which artworks incorporating images or themes of repatriated wampum are increasingly displayed in museum spaces. Museum professionals and Haudenosaunee artists collaborate on efforts to decolonize museum spaces by revisiting and reframing how Native American people, history, and material culture have been represented in Western institutions of learning. Within these efforts, there are many success stories but also a variety of tensions and problematic tendencies towards centering whiteness in cross-cultural environments, resulting in the Native American partners bearing an inequitable amount of the emotional and at times antagonizing strain of bringing about meaningful change. Examples from the lived experience of museum professionals, artists, and researchers demonstrate how epistemologically structuring relations around treaty frameworks can redress the traumas and inequities of our shared past, present, and future

Antibody-induced Multicellular Behaviors of Salmonella Typhimurium

Salmonella enterica is a globally distributed bacterial pathogen that is commonly acquired through consumption of contaminated food and water. The enterica subspecies includes over 1500 unique subvariants, or serovars, that are classified by antigen presentation. Due to its global environmental prevalence, broad host range, and increasing pathogenicity, serovar Typhimurium is of particular concern for human and animal health, as well as food safety. Once in the gastrointestinal tract, Salmonella Typhimurium (STm) exploits host immune mechanisms, implements an arsenal of virulence factors, and outcompetes the microbiota to invade the intestinal epithelium. While STm infections are generally self-limiting and relatively mild, emerging multidrug-resistant and hyper-invasive isolates of STm cause more severe and potentially systemic infections, as well as limit the efficacy of current treatment options. Secretory IgA (SIgA) antibodies represent the first line of defense at mucosal surfaces to protect against STm and thus represent a potential alternative to antibiotics. By virtue of its dimeric structure and high avidity, SIgA mediates a key effector function known as immune exclusion, a process in which invading bacteria are aggregated via antibody cross-linking, encased in mucus, and then cleared from the intestinal tract via peristalsis. While immune exclusion is essential for protection in vivo, the underlying mechanism of IgA-mediated agglutination, and the extent of active bacterial contribution in this process, remains unknown. The anti-lipopolysaccharide (LPS) monoclonal IgA Sal4 was generated from a mouse hybridoma in the early 1990s and demonstrates the capability to protect mice against STm infection through multiple routes of administration. In addition, previous studies have shown that Sal4 triggers motility arrest, inhibition of type 3 secretion, exopolysaccharide production, and agglutination of STm in vitro. Sal4 targets the O5 antigen component of LPS expressed on the surface of STm, and thus the impacts of Sal4 are dependent on expression of this epitope, which is directly and exclusively generated by the transacetylase encoded by the gene oafA. The results from these in vitro characterization studies of Sal4 collectively indicate that STm senses and adapts its behavior in response to Sal4 binding. From this, I hypothesized that Sal4 IgA acts as an environmental stress signal that drives STm to transition from a planktonic to sessile state, which leads to motility arrest, agglutination, and ultimately, biofilm formation. In Chapter 3, I developed a method known as the snow globe assay to visualize and quantify Sal4-mediated agglutination of STm. Bacterial aggregation was shown to occur in a time-, dose-, and O5-antigen expression-dependent manner in the snow globe assay, thus validating results from previous studies. Non-motile strains of STm did not agglutinate in homogenous culture with Sal4 treatment, however, aggregation was induced with a nucleating factor, such as agitation or the presence of motile cells, that could generate collisions and consequently, the formation of aggregates. Alternatively, selected cyclic-di-GMP metabolizing enzymes previously implicated in motility arrest and EPS production did not appear to play a significant role in the process of Sal4-mediated agglutination in the snow globe assay. This indicates that the cellular collisions caused by motility drive agglutination of STm in the presence of Sal4 IgA and establishes the snow globe assay as an approachable and effective technique for measuring antibody-induced aggregation of STm in vitro. In Chapter 4, I confirmed that a mutant strain of STm lacking oafA expression can outcompete WT over the course of four treatments with Sal4 IgA, even when outnumbered at a ratio of 1:6000. With this modified version of the snow globe assay, I performed a genome-wide CRISPR interference (CRISPRi) screen to identify genes that actively promote bacterial agglutination in response to repeated exposure to Sal4 IgA. A previously generated strain of STm 14028s containing a library of 36,651 plasmid-based spacers was treated twice with Sal4 IgA and next-generation sequencing was performed to determine the frequency of each spacer within the subpopulation present at the ALI. From this, I developed a custom analysis process to (1) exclude spacers below the cutoff for minimum read frequency, (2) calculate fold enrichment of spacers detected at the ALI between treatment groups, (3) align spacers to their target gene, and (4) identify genes that were targeted by more than one unique spacer. Ten percent of the de-enriched spacer list contained spacers that targeted genes linked to flagellar biosynthesis and complex assembly. Alternatively, oafA and fimW each had three unique spacers appear on the enriched list, indicating that silenced expression of these genes leads to enrichment of STm at the ALI. The gene product of fimW is a negative regulator of Type I Fimbriae (T1F) expression, and this function was confirmed with a quantitative mannose-sensitive yeast agglutination assay. The non-agglutinating phenotype of a fimW mutant was successfully complemented in trans, and further characterization revealed that this strain does not escape Sal4-mediated motility arrest or inhibition of epithelial cell invasion. However, the fimW mutant demonstrated enhanced biofilm formation relative to WT STm upon Sal4 treatment, indicating that overexpression of T1F can prime STm for the planktonic-to-sessile transition induced by Sal4. This study presents the snow globe assay as a standardized, approachable, and adaptable technique to quantify antibody-mediated bacterial agglutination. Furthermore, the results from this dissertation provide compelling evidence that the anti-LPS monoclonal IgA Sal4 acts as an environmental stressor of STm. I propose a model where Sal4 binding induces a stress response in STm that involves motility arrest, agglutination, and biofilm formation occurring sequentially. Future studies are necessary to elucidate the molecular steps that facilitate Sal4-induced biofilm formation of STm and the underlying mechanisms of Sal4-mediated protection against STm

The Effects of Mindfulness on Chronic Pain in Children and Adolescents: A Systematic Review

Mindfulness and mindfulness-based interventions have become increasingly popular in recent years. Typically, mindfulness-based interventions and their effects on mental health and/or adult populations have been explored in the literature. Limited research has been completed looking at mindfulness’ impacts on other facets, including chronic pain symptoms. Additionally, some research has been conducted to analyze mindfulness and its effects on school aged children and their day-to-day functioning with promising results. In this systematic literature review, mindfulness interventions and their effects on children and adolescents who experience chronic pain were explored. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) framework was used and resulted in the identification of 14 empirical studies, each of which were reviewed and coded to explore mindfulness’ effects on youth with chronic pain. The findings and implications of this review, the limitations of the research base, and future research directions are also discussed

Raw Data for Tailoring High Entropy Borides for Hydrogenation: The Influence of Crystal Morphology and the Exploration of Catalytic Pathways

The high entropy boride (HEB) Al0.2Nb0.2Pt0.2Ta0.2Ti0.2B2, with its unique crystal structure and high coordination (platinum coordinated to 12 borons), has been shown in our previous work to exhibit exceptional catalytic properties, especially in sulfur-rich environments, where traditional platinum catalysts would succumb to sulfur-poisoning. In this work, we investigate the mechanism of the HEB catalyst, first by comparing the synthesis by flux growth, as previously reported, to an arc melted preparation. It is evident that the aluminum flux growth synthesis encourages the growth of single crystals, with clear and defined crystal facets, whereas the arc melted sample results in poorly defined facets with non-uniform morphology. Here, we explore two potential mechanisms: hydrogen spillover effect (HSPE) and hydrogen atom transfer (HAT) by which the catalytic pathway is performed. Hydrogenation reactions were performed using WO3 and 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO), which highlight the ability of the heterogeneous HEB catalyst to perform the hydrogenation through a suspended solid solution in addition to a dissolved solution. We propose that the HEB Al0.2Nb0.2Pt0.2Ta0.2Ti0.2B2 goes through a hybrid HAT/HSPE mechanism, where H2 bonds to the platinum atoms on the edges of the HEB, dissociate, then the radical hydrogen departs to the substrate

Fabrication of Functional Microparticles for Biological Analysis

To better advance our knowledge of cellular behavior, development of an organism, and disease mechanisms understanding cellular heterogeneity in the functional proteome is essential. Bulk measurements across a cell population often fail to capture the variations which exist between individual cells, however single cell proteomic technology has increasingly demonstrated the characterization of cellular heterogeneity. A single cell functional proteomics assay needs to be both high-throughput and multiplexed to effectively identify biomarkers and provide a quantitative correlation to better understand the interrelationships within complex protein networks. Janus particles have the ability to be engineered to have good compatibility and stability in a complex biological environment. Functionalization or surface modification of a microparticle allows for a precise targeted detection of cytokines. The dual nature of a Janus particle allows for both surface interaction and detection in a cellular environment. In this first project a Janus particle is fabricated to demonstrate the asymmetric patterning and evaluate its capabilities in detecting cytokines in HL-60 cells. Fluorescent microscopy imaging confirms the Janus particle design and the ability to attach to a cell membrane, however the experimental yield is low for an adequate quantitative analysis with cytokines. In the second project a Multiplex In Situ Tagging (MIST) array is designed and the fluorescence ELISA sandwich technique is used to quantify the release of cytokines (P53, Total COX-2, TNF RI, Phospho-GSK-3, Cleaved Caspace-3, and Phospho-CREB) in THP1 cells. On an assembled microchip a multiplex DNA encoded microbead array with microwells has a high sensitivity to measure cytokine expression. The MIST array demonstrated shows the ability for a multiplex cytokine detection, however sensitivity demonstrated in this experiment would need to be optimized for a better understanding of the cellular heterogeneity of the cytokines expressed in the single cells