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Uracil Landscape Of Mouse B Cell Genome Undergoing Class-Switch Recombination
No full textActivation-induced cytosine deaminase (AID) is required for both somatic hypermutation (SHM) and class switch recombination (CSR) within mammalian antibody genes. Uracils created by AID in the Ig loci are recognized and processed by base-excision and mismatch repair pathways leading to antibody diversification. This involves a hypermutation process restricted to the V(D)J segments of the immunoglobulin genes, SHM, and an error-prone end-joining process that causes isotype switching, CSR, but the molecular details of these processes have remained obscure for the past 20+ years. Most studies of SHM and CSR have used AID-promoted mutations as a proxy for the uracils, but their results are distorted by error-prone and error-free repair of uracils. To directly map uracils created by cytosine deaminase family enzymes, Bhagwat lab developed a technique where the uracils are excised by a uracil-DNA glycosylase, and a biotinylated alkoxyamine is used to covalently tag and sequence uracil-containing DNA fragments (UPD-Seq). Previously, this method has been used to map uracils created by human AID in the E. coli genome and created a uracilation map, i.e., uracilome. Here we report the first uracilome of a mammalian genome undergoing CSR. The murine CH12F3 cell line undergoes robust isotype switching from IgM to IgA upon stimulation with cytokines but does not undergo SHM. Using UPD-seq, we determined the uracilome of AID in the entire murine genome. An analysis of the uracilome showed uracilation peaks in the switch μ (Sμ) and switch α (Sα) regions of the IgH gene. No peaks were observed in any other switch regions or in the V(D)J exons. This shows that the creation of uracils by AID is a prerequisite for isotype switching and the lack of SHM in CH12F3 cells is caused by the lack of creation of uracils by AID in the V(D)J regions. Furthermore, the uracilation peaks in Sμ and Sα coincide with previously reported switch junctions suggesting that most, perhaps all, uracils created in the switch regions have the potential of being processed into strand breaks. There is a direct relationship between the occurrence of potential R-loop forming and G-quadruplex forming sequences and uracil accumulation in Sμ, but not in Sα. In contrast there is no direct relationship between the occurrence of WRCY sequences and uracil accumulation. Interestingly, the uracilation peak within Sμ and Sα regions have a correlation with the transcription stalling. This correlation is consistent with the cytosine to uracil conversion by AID occurring concurrently with transcription elongation and stalling. Intriguingly, the uracilation peak within Sμ has a sharp boundary on the 3\u27 side. These and other observations based on results of UPD-seq clarify the mechanism of CSR in mammalian cells
Development Of Microfluidic Thermal Gel Electrophoresis For Biological Analysis
Get PDFABSTRACTDEVELOPMENT OF MICROFLUIDIC THERMAL GEL ELECTROPHORESIS FOR BIOLOGICAL ANALYSIS by MARIO AMBROCIO CORNEJO May 2024 Advisor: Dr. Thomas H. Linz Major: Chemistry (Analytical Chemistry) Degree: Doctor of Philosophy The molecular biology that takes place within cells is vital to the over function of an organism. Healthy cellular function is crucial to a wide web of interconnected processes that need to be balanced and regulated. When these processes become abnormal, disease states are developed. The mapping of biological networks has led to the development of gene therapy and more accurate diagnostic tests. However, there are still vital biomolecules that due to their physical properties are difficult to analyze. In this dissertation, microfluidic thermal gel electrophoresis (TGE) has been developed for cell enrichment, analysis of cellular content (specifically for microRNAs), and improved selectivity by incorporating high-temperature analysis. These methods were all developed with the goal of improving upon cellular analysis by reducing sample dilution, improving upon limit of detection, and preventing probe promiscuity during multiplex analysis. Microfluidic TGE is also developed to perform label-free detection to reduce sample preparation times and cost. We have demonstrated a wide range of compatibility from TGE analysis and made fundamental characterizations needed for future method development
Benzene Exposure In Reproductive Biology: A Comprehensive Analysis Of Placental Response And Molecular Mechanisms In Trophoblast Function
Get PDFThe increase in environmental challenges and exposure to pollutants like benzene poses pressing concerns for reproductive health. This dissertation investigates the impact of benzene exposure during gestation on placental function, a key determinant for fetal development and pregnancy outcomes. Our work establishes that benzene exposure during pregnancy induces maternal immune activation, leading to increased rates of fetal resorptions, compromised fetal growth, and abnormal placental development. Intriguingly, we identify a sexual dimorphic response in male and female placentas to benzene exposure, attributable to inherent differences between male and female placental biology. On a molecular level, benzene is metabolized into hydroquinone in the body, which disrupts trophoblast invasion and migration by activating the aryl hydrocarbon receptor pathway. We elucidate that hydroquinone induced AhR activation suppresses Twist1, a critical transcription factor for trophoblast function, subsequently leading to the downregulation of Ifitm1, an essential regulator of extravillous trophoblast migration and invasion. Collectively, our findings suggest a critical AhR-Twist1-IFITM1 axis that is disrupted by benzene exposure. These results offer critical insights into the detrimental effects of environmental pollutants on reproductive health and underscore the urgency for comprehensive investigations on the influence of volatile organic compounds during gestation
Answering For Their Visibility: Gendered And Racialized Muslim Experiences At Work
No full textThe current U.S. political climate places Muslims as antithetical to U.S. ideologies and a threat to national security. Muslim women who wear a veil are easily identifiable and targeted. The project explores how racialized religious visibility (the hijab, beards) affects Muslim women and men in the workplace. This project uses qualitative interviews (total n=84) to compare three sets of employees in professional jobs: Muslim women who veil, Muslim women who do not veil, and Muslim men. This research has four goals: (1) to show how race, gender, and religion intersect to perpetuate subgroup differences among Muslim professionals, (2) to explore the strategies Muslims use to accommodate/challenge workplace discrimination and heightened visibility, (3) to determine how Muslims navigate career advancement given their religious identity, and (4) to examine the impact of workplace location on gender, racial, and religious disparities at work. By focusing on the social construction of Muslim subgroups, strategies, career advancement, and workplace location, this research investigates the gendered processes that undergird the racial demarking of Muslims, and how women who wear a veil may be further racialized by the hijab. This study engages with theories of orientalism and racial formation to analyze how Muslim experiences at work set them apart from non-Muslims and each other. This study also extends our theorizing of how partial tokenization affects Muslims. Experience of looking \u27ethnic\u27 highlights the hijab in ways that may (re)produce a particular kind of femininity that creates differences among Muslim women. In an era of political wrangling and social discontent, it is critical to understand the complexities of Muslim work experiences, a primary setting where social mobility and assimilation occur among immigrant groups. As there are no official statistics on Muslims in the workforce, this study provides insight into how religion, race, and gender distinctly affect their experiences at work. The research may help educators and the wider public understand the experiences of Muslims living and working in the U.S. It may also inform public policymakers and employers about workplace inequalities