May 10

Yusuke Koga
Advisor: Joshua Campbell
Title: Comparison of the tumor and lymph node immune microenvironment in early non-small cell lung cancer through multimodal single cell sequencing

Abstract:
Background/Purpose: Previous studies have analyzed the tumor and local immune microenvironments in lung cancers and suggest immune modulation is associated with worse clinical outcome. However, the tumor-immune microenvironment in early stage lung tumors and lymph nodes (LNs) have not been fully described. We aim to compare cell states in the immune microenvironments between lung tumors and LNs through multi-modal profiling of the transcriptome and surface proteins.

Methods: Needle biopsy samples were taken from 10 treatment-naive early stage lung cancer patients undergoing lung cancer resections. Tissues were obtained from normal lung, lung tumor, and multiple mediastinal LNs, and processed for  scRNA-seq  including labeling with Total-Seq C CITE-seq panel to quantify the levels of 130 cell surface proteins. In total, 76,721 cells (4,462 normal lung; 39,019 tumor; 33,240 LN) were identified with a median of 1,673 genes and 92 protein features detected per cell. Protein expression was decontaminated through the decontX algorithm. Weighted-Nearest Neighbor analysis from the Seurat R package was applied to integrate the CITE-seq and RNA-seq level data for clustering cells into subpopulations.

Results: Six broad cell populations were identified including T/NK, myeloid (CD14+), B (CD19+), mast (TPSAB1+), pDC (IRF8+), and epithelial (EPCAM+) cells. Among 8 CD4+ T lymphocyte subpopulations and 11 CD8+ T lymphocyte subpopulations observed through clustering, a naïve CD4+ and a CD8+ T subpopulation (LEF1+, TCF7+) was observed respectively. These naïve T lymphocyte populations displayed increased proportions in LNs in comparison to tumors. In addition, 5 of the 8 observed CD4+ T lymphocyte populations were enriched in LNs. Immune populations enriched in LNs were largely shared and uniform across different patients. In contrast, a single CD4+ and CD8+ T lymphocyte subpopulation displayed expression of T lymphocyte exhaustion markers (TIGIT+, LAG3+, PD-1+) and were enriched in tumors. 6 of the 11 observed CD8+ T lymphocyte populations were enriched in tumor samples in comparison to LNs. Several alveolar macrophage populations (MARCO+) were enriched in normal lung tissue, in which one showed a heightened stress response.

Conclusion: Single-cell profiling reveals diversity in immune cell populations between LNs, tumor, and adjacent normal tissue in early-stage LUAD. The results suggest the composition of immune cell type is fairly consistent across LNs but more heterogeneous in the tumor and adjacent normal tissue in early-stage patients. In the future, we aim to determine if these immune subpopulations are associated with survival, recurrence, aggressiveness, and predict responses for neoadjuvant treatments, which could improve prognosis and patient quality of life.

 
April 26

Tosin Olayinka
Advisors: Lindsay Farrer & Xiaoling Zhang
Title: Single-nucleus RNA Sequencing of Alzheimer’s Disease Post-Mortem Human Hippocampi Reveals Interactions Between Microglia and Endothelial Cells

Abstract:
Single-nucleus RNA sequencing (snRNA-seq) allows for the dissection of the cell type-specific transcriptional profile of brain tissue. This is of interest in the context of Alzheimer’s disease (AD) as multiple cell types play a role in its pathogenesis. AD can also co-occur with other orthogonal forms of dementia, further complicating the pathology of the disease. In this study we utilize snRNA-seq data from the hippocampi of a total of 12 patients, 11 of which have neuropathologically defined AD with or without an orthogonal form of dementia. We find that in those patients with both AD and vascular dementia there is a decrease in a specific set of genes related to immune activation in microglia and a decrease in genes which negatively regulate angiogenesis in endothelial cells. An association between these two sets of genes in the aforementioned respective cell types was found globally within the data and was thoroughly replicated in an independent snRNA-seq dataset of 465 samples. This provides preliminary evidence for microglial-endothelial interactions in the brain which may play a role in AD and vascular dementia pathology.

 
March 29

Nathaniel Borders
Advisor: Mo Khalil
Title: Expanding Access to Phage-Assisted Continuous Evolution (PACE) with the Development of a Miniature Bioreactor: The min-eVOLVER

Abstract:
Phage-assisted continuous evolution (PACE) is a powerful tool for directed evolution of macromolecules. However, this technology was previously inaccessible to many laboratories due to the bulky and expensive hardware required for performing just a few evolutions. The eVOLVER platform for continuous cell culture, with its 16 bioreactors, enabled directed evolution of microbes and greatly increased the throughput of PACE evolutions. However, many PACE evolutions do not require many concurrent evolutions, and the complexity and expense of the main eVOLVER platform still make it inaccessible for many researchers. To address this issue, the current work focuses on the development of a miniature bioreactor called the min-eVOLVER. The min-eVOLVER has only 2 bioreactors and a small benchtop footprint, making it simpler and more affordable for researchers to perform PACE evolutions. The development of the min-eVOLVER promises to expand the accessibility of PACE technology and increase the ability of researchers to explore the vast potential of directed evolution.

 
Zainab Khurshid
Advisors: Lindsay Farrer & Xiaoling Zhang
Title: Identification of Rare Coding Variants Associated with Alzheimer’s Disease

Abstract:
Alzheimer disease (AD) is a neurodegenerative disorder which manifests at age 65 or older. It’s a progressive disease characterized by memory loss and dementia. It is predicted that by 2050, the number of people in the US with AD will grow to 12 million. Despite its high heritability (h2 = 0.58–0.79), the genetic basis of AD is not fully understood. In addition to APOEwhich accounts for a substantial portion of the genetic variance of AD, more than 75 AD susceptibility loci have been identified through genome-wide association studies (GWAS). In addition, rare coding variants in several genes discovered by whole exome sequencing (WES) are known to have a high impact on AD risk [refs for AKAP9, TREM2, CASP7, etc.]. Rare biologically relevant variants are of special interest because they offer unique insight into potential disease mechanisms. Because previous WES and whole genome sequencing (WGS) studies for AD had limited power for detection of rare variant associations and focused primarily on persons of European ancestry, we sought novel AD-associated rare variant loci by performing an exome-wide study using large multi-ancestry WGS and WES datasets assembled by the Alzheimer Disease Sequencing Project.

 
March 15

Kelley Anderson
Advisors: Marc Lenburg & Jennifer Beane-Ebel
Title: Molecular Characterization of Precursors of Lung Adenocarcinoma

Abstract:
Lung cancer is the leading cause of cancer-related death. The five-year survival rate for lung cancer in the USA remained stagnant for decades, in large part due to disease detection at advanced stages. Although the last decade has seen significant progress in cancer treatment, the five-year survival rate for Stage I lung cancer is 60%. Adenocarcinoma (LUAD) is the most common form of lung cancer. Following surgical resection of pre-invasive adenocarcinoma in situ, the five-year survival rate approaches 100%, emphasizing the need to intervene before malignant potential is reached. In contrast to the well-characterized histopathologic sequence of lung squamous cell carcinoma, atypical adenomatous hyperplasia and adenocarcinoma in situ are the only known precursors in the sequence of LUAD pathogenesis. The features of precursors that lead to aggressive LUAD subtypes are poorly characterized. We sought to link transcriptomic and genomic changes in subsets of premalignant lesions to distinct clinicopathologic features of malignant disease. With this analysis, we discovered a subtype of premalignant lesion with features similar to aggressive LUAD histologic subtypes. This premalignant subtype was associated with increased accumulation of cancer driver mutations, indicating it might be more advanced and closer to malignant transformation. A unique pattern of gene expression with increased expression of immune-related pathways was also observed in this subtype. Molecular signatures measured in premalignant lesions may thus enhance histopathological grading and implicate immunotherapeutic strategies to halt lesion progression to cancer.

 
March 1

Lina Kroehling
Advisor:  Stefano Monti
Title:  Investigating the HNSCC TME through an integrated scRNAseq human atlas and GSEA graph tool.

Abstract:
Head and Neck Squamous Cell Carcinomas (HNSCC) are the sixth most prevalent form of cancer, with incidence rates continuing to rise and an annual death rate approaching half a million. Despite advances in characterizing genomic alterations in head and neck cancers, a better mechanistic understanding of molecular signaling and their contribution to intra-tumor phenotypes is needed.  Growing evidence has indicated that cell plasticity, including the loss of the epithelial state and the acquisition of a partial EMT (p-EMT) phenotype, as well as acquisition of stem-like features, contribute to cancer initiation and the progression to aggressive disease. The degree of immune infiltration in HNSCC has also been linked to EMT induction and tumor growth and dynamics, as interaction between cell types within the tumor microenvironment (TME) can drive EMT and confer stem cell-like phenotype to tumor cells by signaling through secreted factors. However, as tumors are highly heterogeneous, a complete understanding of the populations present in tumors of different phenotypes, and the interplay between them remains unclear. Currently, there are only a few human single cell RNA-seq datasets publicly available.  Through the integration of these datasets we obtain a dataset with unprecedented resolution with which we identify cell populations and inter-cellular communications that cause tumor progression, metastasis and treatment resistance. Additionally, through the development of a new tool that constructs a graphical representation of cell type interactions resulting downstream pathways, we are able to provide a snapshot of TME activity in HNSCC.

 
Marlene Tejeda
Advisors:  Lindsay Farrer & Mark Kon

Abstract:
Multiple infectious agents, including viruses, bacteria, fungi, and protozoa, have been linked to Alzheimer’s disease (AD) risk by independent lines of evidence. We explored this association by comparing the frequencies of viral species identified in a large sample of AD cases and controls. DNA sequence reads that did not align to the human genome in sequences were mapped to viral reference sequences, quantified, and then were tested for association with AD in in whole exome sequences (WES) and whole genome sequences (WGS) datasets.

 
February 15

Shruthi Bandyadka
Advisors:  Kim McCall & Josh Campbell
Title:  The role of V-ATPases in nurse cell phagoptosis in Drosophila melanogaster oogenesis

Abstract:
V-ATPases are trans-membrane protein complexes which form an essential component of the lysosomal machinery. They are multi-subunit mechanoenzymes that hydrolyze ATP to shuttle protons, helping maintain pH, and facilitating transport of ions across cellular and organellar compartments. During later stages of Drosophila melanogaster oogenesis, V-ATPase function is appropriated by somatic cells of the developing egg chamber, called Stretch Follicle Cells to acidify and kill germline-derived nurse cells, in a process known as phagoptosis. To understand how various subunits of the V-ATPase complex are regulated to promote nurse-cell phagoptosis, I investigated gene expression at both transcriptional and translational level, by integrating two previously published scRNA-seq atlases of the fly ovary and comparing the relative mRNA abundances from the composite dataset to the “translatome” of corresponding tissues. I identified a co-transcriptional mechanism by which Stretch Follicle Cells uniquely modulate the ‘a’ subunit of the complex, which may enable the complex to localize on the plasma membrane. Results from the computational analysis described herein, and in-situ hybridization and immunofluorescence experiments to validate the findings will be presented.

 
February 1

Zhaorong Li
Advisor: Juan Fuxman-Bass
Title: Studying how viral proteins affect host gene expression profiles in cancer using matrix decomposition method

Abstract:
Viral infections and their roles in the development and poor prognosis of cancers have been extensively studied since viruses such as Human Papillomavirus (HPV) and Hepatitis B/C virus (HBV/HCV) have been shown to cause cancers and lead to poor prognosis. The genomes of viruses encode multiple different viral products, such as capsid proteins, polymerase, and transcription factors, and it is shown that viral proteins have different expression patterns under different stages in the viral life cycle. Currently there are two main experimental approaches to study the effects of individual viral proteins on host gene expression. The first one is in-vitro experimental, which involves measuring gene expression in cells transduced with the viral protein of interest. The second one is in-silico computational, which involves studying infected cells or samples and comparing the host gene expression between cells/samples with low and high viral protein expression. By using these two approaches, the mechanisms of how several viral proteins affect host gene expressions have been revealed [5, 6]. These genes regulate important cancer prognosis related pathways such as epithelial-mesenchymal transition pathway and interferon signaling pathway. The first approach is based on data obtained from viral protein transduced cell lines, which means that the results cannot be generalized to the real-world scenario. The second approach uses hard cut-off to group samples by the expression of viral protein of interest, which will lose information during the process because the continuous expression is being binarized. The loss of information will lead to noisy and sporadic analysis results of the data. It would be very helpful if there is a method that can study the effects of viral proteins of interest using in-vivo data and at the same time does not lose information from categorizing continuous variables. In this project we propose an in-silico approach to study three questions: how viral proteins in cancer affect host gene expression profiles, whether viral proteins from different viruses affect host gene expression in different cancers differently and whether the effects of viral proteins over the host gene expressions are cell type specific. The result of this project will be a map of how different viral proteins affect host gene expression in different cancers, which will be useful to study how the viral proteins affect cancer prognosis and identify therapeutic targets.

 
Tong Tong
Advisor: W. Evan Johnson
Title: Blood-derived mitochondrial DNA copy number is associated with Alzheimer’s Disease through the regulation of plasma lipid and amino acid metabolism

Abstract:
Background: Blood-derived mitochondrial DNA copy number (mtDNA-CN) is the proxy measurement of mitochondrial function in the peripheral and central systems. Abnormal mtDNA-CN not only indicates impaired mtDNA replication and transcription machinery but also dysregulated biological processes such as energy and lipid metabolism. However, the relationship between mtDNA-CN and Alzheimer’s Disease (AD) and the underlying mechanisms of mitochondrial dysfunction in the pathogenesis of AD are still unclear.

Methods: To investigate the causal relationship between mtDNA-CN and AD, we performed two-sample Mendelian randomization (MR) using publicly available summary statistics from GWAS for mtDNA-CN and AD. Further, we estimated mtDNA-CN using whole-genome sequence data from blood samples of 1,521 ADNI participants and used a Cox proportional hazard model, adjusted for age, sex, and study phase, to assess the association between mtDNA-CN and AD risk. The association of AD biomarkers measured in brain and plasma metabolites with mtDNA-CN was evaluated using linear regression. We conducted causal mediation analysis to test the natural indirect effects of mtDNA-CN change on AD risk through the significantly associated biomarkers and metabolites.

Results: MR analysis using a profile likelihood approach suggested a causal relationship between decreased blood-derived mtDNA-CN and increased risk of AD (OR=0.71; P=0.023). Survival analysis showed that decreased mtDNA-CN was significantly associated with increased risk of conversion from mild cognitive impairment to AD (HR=0.80; P=0.0017). We also identified significant associations between mtDNA-CN and brain glucose metabolism (β=0.050; P=0.047) and amyloid-β (Aβ) (β=0.069; P=0.015) and CSF Aβ (β=-0.051; P=0.048). Plasma Aβ was, however, not associated. Several lipid species, amino acids, biogenic amines, and inflammatory proteins in plasma were also significantly associated with mtDNA-CN. Finally, causal mediation analyses showed that the effect of change in mtDNA-CN on AD risk is mediated by plasma neurofilament light (β=-0.055; P=0.010), glycine (β=0.016; P=0.044), and hexadecenoylcarnitine (β=-0.017; P=0.049).

Conclusions: Our study indicates that mtDNA-CN measured in blood is predictive of AD risk. Associations of mtDNA-CN with Aβ in CSF and brain, but not plasma, suggest cross-tissue regulation of mitochondria. Decreased mtDNA-CN might increase AD risk through lipid and amino acid metabolism. These results also suggest potential development of mtDNA-CN as a blood-based biomarker.

 
January 18

Aubrey Odom-Mabey
Advisor: W. Evan Johnson
Title:Characterization of longitudinal nasopharyngeal microbiome patterns in maternally HIV-exposed Zambian infants

Abstract:
Previous studies of infants born to HIV-positive mothers have linked HIV exposure to poor outcomes from gastrointestinal and respiratory illnesses, and to overall increased mortality rates. The mechanism behind this is unknown, but it is possible that differences in the nasopharyngeal (NP) microbiome between HIV-unexposed and HIV-exposed infants could play a role in perpetuating some outcomes. I conducted a longitudinal analysis of 170 NP swabs of healthy HIV-exposed, uninfected (HEU; n=10) infants and their HIV(+) mothers and HIV-unexposed, uninfected (HUU; n=10) infants and their HIV(-) mothers. These swabs were identified from a sample library collected in Lusaka, Zambia between 2015 and 2016. Using 16S rRNA gene sequencing, I characterized the maturation of the microbiome over the first 14 weeks of life to determine what quantifiable differences exist between HEU and HUU infants, and what patterns are reflected in the mothers’ NP microbiomes. These analyses indicate that the HEU infants in this study exhibit subtle differences in the NP microbial composition throughout the sampling interval. In both HEU and HUU infants, Staphylococcus and Corynebacterium began as primary colonizers of the NP microbiome but were in time replaced by Dolosigranulum, Streptococcus, Moraxella and Haemophilus. When studying differences between infants, the microbe Staphylococcus haemolyticus indicated a distinctive high association with HIV exposure at birth, even when accounting for the interaction between HIV exposure status and time of sampling. When comparing infants to their mothers with paired analyses, HEU infants’ NP microbiome composition was only slightly different from their HIV(+) mothers at birth or 14 weeks, including in their carriage of S. pneumoniae, H. influenzae, and S. haemolyticus. Given the results and study sampling limitations, further research must be conducted in order to confidently understand the relationship between HIV exposure and infants’ NP microbiomes.