Browsing by Subject "RNA-seq"
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Item Open Access Bioinformatics and Molecular Approaches for the Construction of Biological Artificial Cartilage(2018) Huynh, Nguyen Phuong ThaoOsteoarthritis (OA) is one of the leading causes of disability in the United States, afflicting over 27 million Americans and imposing an economic burden of more than $128 billion each year (1, 2). OA is characterized by progressive degeneration of articular cartilage together with sub-chondral bone remodeling and synovial joint inflammation. Currently, OA treatments are limited, and inadequate to restore the joint to its full functionality.
Over the years, progresses have been made to create biologic cartilage substitutes. However, the repair of degenerated cartilage remains challenging due to its complex architecture and limited capability to integrate with surrounding tissues. Hence, there exists a need to create not only functional chondral constructs, but functional osteochondral constructs, which could potentially enhance affixing properties of cartilage implants utilizing the underlying bone. Furthermore, the molecular mechanisms driving chondrogenesis are still not fully understood. Therefore, detailed transcriptomic profiling would bring forth the progression of not only genes, but gene entities and networks that orchestrate this process.
Bone-marrow derived mesenchymal stem cells (MSCs) are routinely utilized to create cartilage constructs in vitro for the study of chondrogenesis. In this work, we set out to examine the underlying mechanisms of these cells, as well as the intricate gene correlation networks over the time course of lineage development. We first asked the question of how transforming growth factors are determining MSC differentiation, and subsequently utilized genetic engineering to manipulate this pathway to create an osteochondral construct. Next, we performed high-throughput next-generation sequencing to profile the dynamics of MSC transcriptomes over the time course of chondrogenesis. Bioinformatics analyses of these big data have yielded a multitude of information: the chondrogenic functional module, the associated gene ontologies, and finally the elucidation of GRASLND and its crucial function in chondrogenesis. We extended our results with a detailed molecular characterization of GRASLND and its underlying mechanisms. We showed that GRASLND could enhance chondrogenesis, and thus proposed its therapeutic use in cartilage tissue engineering as well as in the treatment of OA.
Item Open Access Developing Quantitative Models in Analyzing High-throughput Sequencing Data(2021) Kim, Young-SookDiverse functional genomics assays have been developed and helped to investigate complex gene regulations in various biological conditions. For example, RNA-seq has been used to capture gene expressions in diverse human tissues, helping to study tissue-common and tissue-specific gene regulation. ChIP-seq has been used to identify the genomic regions bound by numerous transcription factors, thus helping to identify collaborative and competitive binding mechanisms of the transcription factors. Despite this huge increase in the amount and the accessibility of genomic data, we have several challenges to analyze those data with proper statistical methods. Some assays such as STARR-seq do not have a proper statistical model that detects both activated and repressed regulatory elements, making researchers depend on the statistical models developed for other assays. Some assays such as ChIP-seq and RNA-seq have limited joint analysis models that are flexible and computationally scalable, resulting in the limited statistical power in identifying the genomic regions or genes shared by multiple biological conditions. To solve those challenges in analyzing high-throughput assays, we first developed a statistical model called correcting reads and analysis of differential active elements or CRADLE to analyze STARR-seq data. CRADLE removes technical biases that can confound quantification of regulatory activity and then detects both activated and repressed regulatory elements. We observed the corrected read counts improved the visualization of regulatory activity, allowing for more accurate detection of regulatory elements. Indeed, through simulation study, we showed CRADLE significantly improved precision and recall in detecting regulatory elements compared to the previous statistical approaches and that improvement was especially prominent in identifying repressed regulatory elements. Based on our work on developing CRADLE, we adapted the statistical framework of CRADLE and developed a joint analysis model of multiple data for biology or JAMMY that can be applied to diverse high-throughput sequencing data. JAMMY is a flexible statistical model that jointly analyzes multiple conditions, identifies condition-shared and condition-specific genomic regions, and then quantifies the preferential activity of a subset of biological conditions for each genomic region. We applied JAMMY to STARR-seq, ChIP-seq, and RNA-seq data, and observed JAMMY overall improved the precision and recall in identifying condition-shared activity compared to the traditional condition-by-condition analysis. This gain of statistical power from the joint analysis led us to find a novel co-binding of two transcription factors in our study. Those results show the substantial advantages of using joint analysis model in integrating genomic data from multiple biological conditions.
Item Open Access Differential Expression of Coding and Long Noncoding RNAs in Keratoconus-Affected Corneas.(Investigative ophthalmology & visual science, 2018-06) Khaled, Mariam Lofty; Bykhovskaya, Yelena; Yablonski, Sarah ER; Li, Hanzhou; Drewry, Michelle D; Aboobakar, Inas F; Estes, Amy; Gao, X Raymond; Stamer, W Daniel; Xu, Hongyan; Allingham, R Rand; Hauser, Michael A; Rabinowitz, Yaron S; Liu, YutaoKeratoconus (KC) is the most common corneal ectasia. We aimed to determine the differential expression of coding and long noncoding RNAs (lncRNAs) in human corneas affected with KC.From the corneas of 10 KC patients and 8 non-KC healthy controls, 200 ng total RNA was used to prepare sequencing libraries with the SMARTer Stranded RNA-Seq kit after ribosomal RNA depletion, followed by paired-end 50-bp sequencing with Illumina Sequencer. Differential analysis was done using TopHat/Cufflinks with a gene file from Ensembl and a lncRNA file from NONCODE. Pathway analysis was performed using WebGestalt. Using the expression level of differentially expressed coding and noncoding RNAs in each sample, we correlated their expression levels in KC and controls separately and identified significantly different correlations in KC against controls followed by visualization using Cytoscape.Using |fold change| ≥ 2 and a false discovery rate ≤ 0.05, we identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas. Pathway analysis indicated the enrichment of genes involved in extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. Our correlation analysis identified 296 pairs of significant KC-specific correlations containing 117 coding genes enriched in functions related to cell migration/motility, extracellular space, cytokine response, and cell adhesion. Our study highlighted the potential roles of several genes (CTGF, SFRP1, AQP5, lnc-WNT4-2:1, and lnc-ALDH3A2-2:1) and pathways (TGF-β, WNT signaling, and PI3K/AKT pathways) in KC pathogenesis.Our RNA-Seq-based differential expression and correlation analyses have identified many potential KC contributing coding and noncoding RNAs.Item Open Access Elucidating Plasmodium Liver Stage Biology Through Transcriptomic Approaches(2018) Posfai, DoraMalaria is one of the leading causes of mortality attributed to infectious diseases worldwide. Every year, hundreds of thousands of children succumb to the disease and hundreds of millions more suffer the characteristic symptoms of malaria. It is caused by eukaryotic parasites of the genus Plasmodium and is transmitted to the human host via the bite of an Anopheles mosquito. Upon infection, the parasite must travel to the liver where it develops and replicates into merozoites, the parasite form that is able to infect red blood cells. It is only after release back into the blood stream as a merozoite that the parasite invades red blood cells, leading to the manifestation of disease.
The liver stage is clinically silent, yet an obligatory stage of the Plasmodium life cycle. Our knowledge of this portion of the life cycle is lagging compared to that of the blood stage because of inherent difficulties in experimental design. In particular, very little is known about the host and parasite gene expression during the early hours of infection. This work seeks to gain a greater understanding of the biological processes of host and parasite throughout the liver stage of infection through dual-RNA sequencing. We first utilize next-generation sequencing to map the global transcriptional state of the P. berghei-infected hepatocytes during the entire course of the liver stage infection. We find the most significant changes in gene expression occur early during infection and are primarily related to the host mounting an immune response. During mid to late time points of P. berghei infection of hepatocytes, genes related to host metabolism are enhanced among the differentially expressed genes, indicating a shift in active cellular processes later in infection.
From the host transcriptomic dataset, we identify aquaporin-3 (AQP3), a water and glycerol transporting membrane protein, as significantly induced upon P. berghei infection. Microscopic experiments reveal that the host AQP3 protein is trafficked to the parasitophorous vacuole membrane (PVM), the interface between the parasite and host cytosol. Through molecular genetic and chemical approaches, we show host AQP3 is essential for the proper development of the parasite during the liver and blood stages of the life cycle. Phenotypic studies suggest AQP3 is utilized by the parasite to obtain nutrients for growth.
Lastly, we also utilize target-based screens to identify novel antiplasmodial small molecules that have potential for treating liver stage malaria. We interrogate the species specificity of a panel of Hsp90 small molecules inhibitors and seek to understand the chemical moieties that determine species selectivity. We also utilize cell-based assays to screen for and identify compounds that act synergistically. The work presented herein sheds light on novel host-parasite interactions during the liver stage of Plasmodium infection and explores novel small molecules for malaria treatment.
Item Open Access Estrogen’s Impact on the Specialized Transcriptome, Brain, and Vocal Learning Behavior of a Sexually Dimorphic Songbird(2020) Choe, Ha NaThe song system of the zebra finch (Taeniopygia guttata) is highly sexually dimorphic, where only males develop the neural structures necessary to learn and produce learned vocalizations in adulthood. During early development, both males and females begin to develop their song system in a monomorphic manner, which diverges shortly after the onset of a critical sensory learning phase and results in reduced cell survival and proliferation in females, and accelerated cell proliferation in males. Estrogen has long been known to be involved in coordinating sexual development of the perinatal brain and nestling female zebra finches treated with estrogen do not exhibit this female-specific atrophy of the song system. How estrogen influences the development of the song system, and what it is doing at the molecular level has not been examined utilizing current generation sequencing technology.
In this dissertation, I tested whether estrogen manipulation impacts the transcriptomic profiles of telencephalic song learning nuclei in males and females. I treated animals with either vehicle, exemestane (an estrogen synthesis inhibitor), or 17-β-estradiol from the moment of hatching until time of sacrifice. I collected the song learning nuclei and their surrounding brain regions during the onset of sensory motor learning for transcriptomic analysis or during adulthood after collecting behavior. I found that of the 4 telencephalic song nuclei examined during the onset of the sensorimotor learning period at post hatch day 30, Area X was the most sexually dimorphic and the most impacted by estrogen administration. HVC was less sexually dimorphic and less impacted by estrogen manipulation. RA and LMAN had limited sexually dimorphic features, with little impact on their transcriptomes with estrogen manipulation. Additionally, I found that chronic estrogen depletion in males delayed male specific plumage development and resulted in impaired song learning. This supports the notion that while estrogen is sufficient in preventing atrophy of the song system in female zebra finches, it is not necessary for the gross development in males and may instead refine normal song development.
Item Open Access Molecular Interactions between Apicomplexan Parasites and their Host Cells(2021) Toro Moreno, MariaParasitic diseases caused by pathogens of the Apicomplexan phylum result in hundreds of thousands of deaths per year, in addition to being an immense socioeconomic burden on vulnerable populations. A notorious case is that of Plasmodium parasites, the causative agents of malaria and one of the most ancient and devastating diseases known to humankind. Prior to infecting red blood cells resulting in the symptomatic stage of infection, Plasmodium parasites infect liver cells and undergo one of the most rapid replication events known in eukaryotes. Given the asymptomatic nature and technical challenges associated with studying the liver stage, this portion of the Plasmodium life cycle remains poorly understood, hindering our ability to target this stage of the life cycle for disease prevention. In particular, the host and parasite pathways that are critical to parasite infection during this hepatic phase remain unknown. The lack of effective vaccines coupled with the widespread emergence of drug-resistant parasites necessitates our understanding of host-parasite infection biology to develop improved therapeutics. To elucidate host-parasite interactions in the Plasmodium liver stage, we implemented a forward genetic screen to identify host factors within the human druggable genome that are critical to P. berghei infection in hepatoma cells, as well as RNA-seq approaches to delineate host and Plasmodium gene expression regulation during infection. Through our genetic screen, we identified the knockdown of genes involved in host trafficking pathways to be detrimental to Plasmodium infection. We additionally pursued mechanistic studies using small molecules and imaging approaches and found that both P. berghei and the related apicomplexan parasite Toxoplasma gondii hijack host trafficking by rerouting host vesicles to their parasitophorous vacuole, although with differing specificities. Our extensive RNA-Seq analysis throughout the P. berghei liver stage revealed that hundreds of parasite genes, including some coding for putative exported proteins, are differentially expressed as early as 2 hpi and that multiple genes shown to be important for later infection are upregulated as early as 12 hpi. Using co-expression analyses, we examined potential regulation of gene clusters by ApiAP2 transcription factors and found enrichment of mostly uncharacterized DNA binding motifs. This finding indicates potential liver-stage targets for these transcription factors, while also hinting at alternative uncharacterized DNA binding motifs and transcription factors during this stage. We further explored regulatory mechanisms in the liver stage by identifying differentially expressed host lncRNAs in P. berghei-infected cells, and novel putative lncRNAs in P. vivax hypnozoites. Overall, our work uncovered critical host and parasite pathways in the Plasmodium liver stage and highlights the use of high-throughput genetic and transcriptomic approaches in combination with chemical biology and classic cell biology studies to uncover host-parasite interactions in challenging infection systems.
Item Open Access Using Single-Cell Analyses to Uncover Transcriptional Heterogeneity in Plasmodium falciparum(2018) Walzer, Katelyn AnnMalaria persists as a global health problem, with 212 million cases and 429,000 deaths worldwide in 2015 alone. It is caused by the apicomplexan parasite Plasmodium, which follows a complex life cycle that consists of multiple stages spanning from the human host to the mosquito vector. Among the Plasmodium parasites causing human malaria, the deadliest species is Plasmodium falciparum. Most P. falciparum parasites follow an asexual cycle in human erythrocytes that is characterized by a tightly synchronized continuous cascade of gene expression, although a small proportion commits to a sexual fate. Parasites committed to the sexual stage develop into male and female gametocytes over 9-12 days, with mature gametocytes being the only form of the parasite transmissible to the mosquito vector.
This commitment to a sexual fate is rare, and little is known about the transcriptional programs related to sexual commitment and mating-type determination. Furthermore, discrete changes that occur in these cells are largely undetectable in traditional bulk-cell analyses. Bulk-cell analyses were used to establish models for synchronous stage-specific transcriptional programming during the asexual intraerythrocytic developmental cycle (IDC) but left little resolved in terms of cellular heterogeneity and cell-fate decisions. Due to these limitations, we developed unbiased single-cell approaches on a microfluidic platform to analyze single parasites during late asexual and sexual stages. This work was divided into two main parts. The first focused on single-cell gene expression in male and female mid-to-late stage gametocytes. We captured 90 single parasites and compared the expression of 91 genes, including 87 gametocyte-specific genes. Our analysis identified distinct gene clusters whose expression associated with male, female, or all gametocytes. In addition, a small number of male gametocytes clustered separately from female gametocytes based on sex-specific expression independent of stage. RNA fluorescent in situ hybridization (RNA-FISH) validated the mutually exclusive expression pattern of male and female transcripts in gametocytes. These analyses uncovered novel male and female markers that are expressed as early as stage III gametocytogenesis, providing further insight into Plasmodium sex-specific differentiation previously masked in population analyses.
The second part of this work centered on single-cell RNA sequencing (scRNA-seq) of P. falciparum late asexual and sexual stages. First, we uncovered a large number of previously undefined gametocyte-specific genes. 46 asexual cells were then segregated into three separate clusters based on the differential expression of SERAs, rhoptries, and EXP2 plus transporters. RNA-FISH of cluster-specific genes validated this distinct stage-specific expression during the IDC and defined the highly variable transcriptional pattern of EXP2. Additionally, these analyses indicated huge variations in the stage-specific transcript levels among parasites. Overall, scRNA-seq and RNA-FISH of P. falciparum revealed distinct stage transitions and unexpected degrees of heterogeneity with potential impact on transcriptional regulation during the IDC and adaptive responses to the host.