Marine ecosystems are already threatened by the effects of climate change through increases in ocean temperatures and pCO2 levels due to increasing atmospheric CO2. Marine fish living close to their thermal maximum have been shown to be especially vulnerable to temperatures exceeding that threshold, and even relatively small increases in elevated pCO2 levels have led to behavioral impairments with amplified predation risks. These ongoing threats highlight the need for further understanding of how these changes will impact fish and if any potential for adaptation or acclimation exists. The coral reef fish, Acanthochromis polyacanthus, has been well studied in response to singular environmental changes both through its phenotype and molecular expression profiles within and across generations. However, key questions regarding transgenerational heritability and molecular responses to multiple environmental changes have not been addressed. To further understand A. polyacanthus I examined the mechanisms behind heritability of behavioral tolerance to elevated pCO2 in an attempt to determine the maternal and paternal contributions to this phenotype. There was a strong impact of parental phenotype on the expression profiles of their offspring regardless of environmental exposure. Offspring from both parental pairs expressed mechanisms involved in tolerance to ocean acidification suggesting this phenotype is reliant on input from both parents. Creation of a new proteomic resource, a SWATH spectral library, delivered a closer examination of the link between phenotypic and expression changes. Analysis on different constructed libraries led to the use of an organism whole library combined with study specific data to analyze proteomic changes in A. polyacanthus under the combined environmental changes of ocean acidification and warming. With direct comparisons to transcriptomic changes in the same individuals I identified an additive effect of elevated pCO2 and temperature associated with decreases in growth and development. However, a strong role of parental identity on the expression profiles of offspring reinforced the high genetic variability of this species. This thesis provides novel insights into the heritability of phenotypic traits and the molecular responses to combined stressors in A. polyacanthus, as well as presenting a new resource for proteomic studies in this fish and other non-model species.

Modern advances in sequencing technologies have enabled exploration of molecular biology at unprecedented scale and resolution. Transcriptome sequencing (RNA-seq), in particular, has been widely adopted as a routine cost-effective method for assaying both genetic and functional characteristics of biological systems with resolution down to individual cells. Clinical research and applications leveraging these technologies have largely targeted tumor biology, where transcriptome sequencing can capture tumor genetic and epigenetic characteristics and aid with understanding the etiology or guide treatments. Specialized computational methods and bioinformatic software tools are essential for processing and analyzing RNA-seq to explore various aspects of tumor biology including driver mutations, genome rearrangements, and aneuploidy. With single cell resolution, such methods can yield insights into tumor cellular composition and heterogeneity. Here, we developed methods and tools to support cancer transcriptome studies for bulk and single cell tumor transcriptomes, focusing primarily on fusion transcript detection and predicting large-scale copy number alternations from RNA-seq. These efforts culminated in the development of STAR-Fusion for fast and accurate detection of fusion transcripts, FusionInspector for further characterizing predicted fusion transcripts and discriminating likely artifacts, and TrinityFusion for de novo reconstruction of fusion transcripts and tumor viruses. We also developed advanced methods for predicting copy number alterations and subclonal architecture from tumor and normal single cell RNA-seq data, as incorporated into our InferCNV software. In addition to these bioinformatic method and software developments, we applied our fusion detection methods to thousands of tumor and normal samples and gain novel insights that should further help guide researchers with clinical applications of fusion transcript discovery.

Large-scale transcriptomic studies are among of the most comprehensive accounts we have of the biological processes underlying human brain development and ageing. However, many analyses and descriptive models applied to gene expression data implicitly assume that developmental change is continuous and uninterrupted. Perhaps this bias is often overlooked because the emphasis is on what is changing during development rather than how development itself is changing. Indeed, despite the richness of transcriptomic data and its capacity to recapitulate higher-order functions, few have used it to understand the dynamics of brain development. Gene expression is determined by complex, high-dimensional interactions of the gene regulatory network. Dynamic systems theory states that the interactions of components in any complex systems will converge on certain stable patterns, also known as attractor states. To approximate these stable states, the current study leveraged robust and sparse k-means clustering to identify tissue samples with similar patterns of gene expression across the transcriptome. Sample ages were then used to visualize when in developmental time these stable patterns are present. The resulting model describes the developmental dynamics of the brain transcriptome as a series of non-linear, overlapping states that progress across the lifespan. / Thesis / Master of Science (MSc)

Indiana University-Purdue University Indianapolis (IUPUI) / Inner ear development requires the complex interaction of numerous cell types arising from multiple embryologic origins. Current knowledge of inner ear organogenesis is limited primarily to animal models. Although most mechanisms of cellular development show conservation between vertebrate species, there are uniquely human aspects of inner ear development which remain unknown. Our group recently described a model of in vitro human inner ear organogenesis using pluripotent stem cells in a 3D organoid culture system. This method promotes the formation of an entire sensorineural circuit, including hair cells, inner ear neurons, and Schwann cells. Our past work has characterized certain aspects of this culture system, however we have yet to fully define all the cell types which contribute to inner ear organoid assembly. Here, our goal was to reconstruct a time-based map of in vitro development during inner ear organoid induction to understand the developmental elements captured in this system. We analyzed inner ear organoid development using single-cell RNA sequencing at ten time points during the first 36 days of induction. We reconstructed the on-target progression of undifferentiated pluripotent stem cells to surface ectoderm, pre-placodal, and otic epithelial cells, including supporting cells, hair cells, and neurons, following treatment with FGF, BMP, and WNT signaling modulators. Our data revealed endogenous signaling pathwayrelated gene expression that may influence the course of on-target differentiation. In addition, we classified a diverse array of off-target ectodermal cell types encompassing the neuroectoderm, neural crest, and mesenchymal lineages. Our work establishes the Inner ear Organoid Developmental Atlas (IODA), which can provide insights needed for understanding human biology and refining the guided differentiation of in vitro inner ear tissue. / 2024-08-02

High-resolution computed tomography (HRCT) of the chest is commonly used in the diagnosis of a variety of lung diseases. Structural changes associated with clinical characteristics of disease may also define specific disease-associated physiologic states that may provide insights into disease pathophysiology. Gene expression profiling is potentially a useful adjunct to HRCT to identify molecular correlates of the observed structural changes. However, it is difficult to directly access diseased distal airway or lung parenchyma routinely for profiling studies. Previously, we have profiled bronchial airway in normal-appearing epithelial cells at the mainstem bronchus, detecting distinct gene expression alterations related to the clinical diagnosis of chronic obstructive pulmonary disease (COPD) and lung cancer. These gene expression alterations offer insights into the molecular events related to diseased tissue at more distal airways and in the parenchyma, which we hypothesize are due to a field-of-injury effect. Here, we expand this prior work by correlating airway gene expression to COPD and bronchiectasis phenotypes defined by HRCT to better understand the pathophysiology of these diseases. Additionally, we classified pulmonary nodules as malignant or benign by combining HRCT nodule imaging characteristics with gene expression profiling of the nasal airway. First, we collected brushing samples from the main-stem bronchus and assessed gene expression alterations associated with COPD phenotypes defined by K-means clustering of HRCT-based imaging features. We found three imaging clusters, which correlated with incremental severity of COPD: preserved, interstitial predominant, and emphysema predominant. 357 genes were differentially expressed between the normal and the emphysema predominant clusters. Functional analysis of the differentially expressed genes suggests a possible induction of inflammatory processes and repression of T-cell related biologic pathways, in the emphysema predominant cluster. We then discovered gene expression alterations associated with radiographic evidence of bronchiectasis (BE), an underdiagnosed obstructive pulmonary disease with unclear pathophysiology. We found 655 genes were differentially expressed in bronchial epithelium from individuals with radiographic evidence of BE despite none of the study participants having a clinical BE diagnosis. In addition to biological pathways that had been previously associated with BE, novel pathways that may play important roles in BE initiation were also discovered. Furthermore, we leveraged an independent single-cell RNA-sequencing dataset of the bronchial epithelium to explore whether the observed gene expression alterations might be cell-type dependent. We computationally detected an increased presence of ciliated and deuterosomal cells, as well as a decreased presence of basal cells in subjects with widespread radiographic BE, which may reflect a shift in the cellular landscape of the airway during BE initiation. Finally, we identified gene expression alterations within the nasal epithelium associated with the presence of malignant pulmonary nodules. A computational model was constructed for determining whether a nodule is malignant or benign that combines gene expression and imaging features extracted from HRCT. Leveraging data from single-cell RNA sequencing, we found genes increased in patients with lung cancer are expressed at higher levels within a novel cluster of nasal epithelial cells, termed keratinizing epithelial cells. In summary, we leveraged gene expression profiling of the proximal airway and discovered novel biological pathways that potentially drive the structural changes representative of physiologic states defined by chest HRCT in COPD and BE. This approach may also be combined with chest HRCT to detect weak signals related to malignant pulmonary nodules. / 2024-02-03T00:00:00Z

Methylmercury (MeHg) is a global pollutant that can elicit a range of adverse health effects in both humans and wildlife populations. Humans are often exposed to MeHg through the consumption of contaminated seafood. Developing fetuses are especially susceptible to the effects of MeHg as it can cross the blood-brain barrier and the placenta. At high doses in utero MeHg causes developmental disorders and congenital disabilities, but long-term low-dose effects are still not fully known. Using a culture model of cerebral cortex development, our lab has shown that low-dose MeHg promotes premature neuronal differentiation. Autism spectrum disorder (ASD) has been associated with prenatal MeHg exposure and is correlated with neuronal overproduction, but a cause-effect relationship has not been shown. In this thesis, I aim to test the hypothesis that prenatal exposure to low-dose MeHg can cause ASD-like symptoms in the offspring following premature neuronal differentiation. My results showed that adult mice prenatally exposed to MeHg exhibited key ASD characteristics including impaired communication, reduced sociability, and increased restrictive repetitive behaviours. Furthermore, I explored the underlying cellular and molecular mechanism that promotes premature neuronal differentiation caused by prenatal MeHg exposure. To reverse the MeHg-induced premature neuronal differentiation, I utilized metformin, an FDA-approved diabetes drug. Overall, these findings provide insights into the toxicology of MeHg and its relationship with ASD etiology, including the underlying mechanism, and a potential therapeutic strategy.

No description available.

Dihydrochalcones (DHCs) are specialised metabolites with a limited natural distribution, found in significant amounts in Malus x domestica Borkh. (cultivated apple) and wild Malus species. Among them, M. x domestica accumulates significant amounts of phloridzin, whilst trilobatin and sieboldin are abundant in some wild relatives. DHCs have demonstrated a wide range of bioactive properties in biomedical models. Some DHCs have also been reported to act as flavour enhancers. Phloridzin may act as an anti-diabetic compound by blocking sodium-linked glucose transport and renal reabsorption of glucose in kidneys. Despite the protective effects reported in mammal models, little is known about how these metabolites are biosynthesised and what is their function in planta, where it has been hypothesised a role for phloridzin in plant growth. The biosynthetic pathway leading to DHC formation has been proposed in apple, and some steps have been characterised recently. DHC pathway diverts from the main phenylpropanoid pathway most probably from 4-coumaroyl-CoA by the action of a yet unknown reductase that would produce 4-dihydrocoumaroyl-CoA. Then, chalcone synthase (CHS) catalyses its condensation to form phloretin. Phloretin can be directly glycosylated at position 2′- or 4′ by the previously characterised 2′- and 4′-O-UDP-glycosyltransferases PGT1 and PGT2, to produce phloridzin or trilobatin, respectively. However, sieboldin has been postulated to derive from hydroxylation in position 3 of phloretin before been glycosylated, and the key responsible enzyme producing 3-hydroxyphloretin has not been yet discovered. The main aim of this PhD proposal was to provide a better understanding of the physiological functions of DHCs in apple, as well as to contribute to the elucidation of the biosynthetic pathway as the molecular basis for future genetic engineering in apple. Towards this aim, functional characterisation was conducted in MdPGT1 knockdown apple lines by RNAi silencing and CRISPR/Cas9 genome editing to assess the physiological effect of targeting a key biosynthetic gene involved in phloridzin biosynthesis. In addition, molecular, transcriptomic and metabolomic analyses were integrated to evaluate candidate genes accounting for 3-hydroxylase activity involved in DHC biosynthesis in wild Malus species accumulating sieboldin. Moreover, a de novo transcriptome assembly was carried out in an intergeneric hybrid between M. x domestica and Pyrus communis L. known to accumulate intermediate levels of DHCs compared to apple, in order to identify additional genes potentially involved in DHC pathway. We compared the physiological effect of reducing phloridzin through PGT1 knockdown by RNAi silencing and CRISPR/Cas9 genome editing. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology as reported in literature, whereas genome edited lines exhibited normal growth despite reduced foliar phloridzin. Bioactive brassinosteroids and gibberellins were found to be key players involved in the contrasting effects on growth observed following phloridzin reduction. Moreover, a cytochrome P450 from wild M. toringo (K. Koch) Carriere syn. sieboldii Rehder, and M. micromalus Makino was identified as dihydrochalcone 3-hydroxylase (DHCH), proving to produce 3-hydroxyphloretin and sieboldin in yeast. Different DHCH allele isoforms found in domesticated apple and M. toringo and M. micromalus correlated with sieboldin accumulation in a Malus germplasm collection. Finally, the assembled de novo transcriptome of the intergeneric apple/pear hybrid integrated to functional annotation and metabolomic analysis resulted in the identification of genes potentially involved in DHC biosynthesis, providing the basis for future biochemical characterisation. Altogether these results contribute to get insight into the roles of DHCs in apple and to illustrate how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple. Furthermore, the present PhD thesis contributes to the state-of-the-art by elucidating key missing steps in the biosynthesis of DHCs, which could be relevant for future establishment of genetic engineered lines that contribute to assess physiological effects of altering DHCs content, as well as to establish heterologous expression systems to produce de novo DHCs.

<p dir="ltr">Nitrogen (N) is a macronutrient required for plant growth and is a major constituent of nucleic acids and proteins that are essential for several life processes. Plant response to N has been well understood at a molecular level but little is known about the chromatin or epigenetic level regulation of N response. Uncovering the epigenetic level regulation essential for plant N signaling and response is essential to improving our molecular understanding of N use efficiency (NUE). To fill this knowledge gap, we first performed a meta-analysis intersecting the published transcriptomic study of N-responsive genes in <i>Arabidopsis thaliana </i>with EpiNet, an extensive epigenetic regulatory network previously constructed in our lab through machine learning approaches, to identify a list of 18 potential epigenetic regulators that are predicted to control N response in plants. Next, by adopting a reverse genetics approach, we aimed to validate the <i>in-silico</i> prediction of these essential epigenetic regulators. To do this, we grew T-DNA insertional mutants for the genes encoding these epigenetic regulators, along with wild-type controls, under high and low N conditions, and compared them in various physiological traits. Our results indicate that 8 out of 10 confirmed knock-down mutants do show altered N-responsive phenotypes in comparison to the wild type. One of the mutants, <i>ashr2-1, </i>which is mutated in a gene encoding a putative SET-domain containing group protein (SDG) of putative histone methyltransferase, displayed reduced growth of primary root compared to WT in response to N. We performed RNA-sequencing to identify the differentially expressed genes that are induced or repressed by ASHR2 in N treatments to gain further insight into the molecular underpinnings of the ASHR2-mdediated N response in roots<i>.</i> In summary, our study has revealed knowledge on important epigenetic regulators in plant N responses, which has the potential to be extended to crop species as novel targets for enhancing NUE.</p>

Yes / Living longer without simultaneously extending years spent in good health ("health span") is an increasing societal burden, demanding new therapeutic strategies. Hydrogen sulfide (H2S) can correct disease-related mitochondrial metabolic deficiencies, and supraphysiological H2S concentrations can pro health span. However, the efficacy and mechanisms of mitochondrion-targeted sulfide delivery molecules (mtH2S) administered across the adult life course are unknown. Using a Caenorhabditis elegans aging model, we compared untargeted H2S (NaGYY4137, 100 µM and 100 nM) and mtH2S (AP39, 100 nM) donor effects on life span, neuromuscular health span, and mitochondrial integrity. H2S donors were administered from birth or in young/middle-aged animals (day 0, 2, or 4 postadulthood). RNAi pharmacogenetic interventions and transcriptomics/network analysis explored molecular events governing mtH2S donor-mediated health span. Developmentally administered mtH2S (100 nM) improved life/health span vs. equivalent untargeted H2S doses. mtH2S preserved aging mitochondrial structure, content (citrate synthase activity) and neuromuscular strength. Knockdown of H2S metabolism enzymes and FoxO/daf-16 prevented the positive health span effects of mtH2S, whereas DCAF11/wdr-23 - Nrf2/skn-1 oxidative stress protection pathways were dispensable. Health span, but not life span, increased with all adult-onset mtH2S treatments. Adult mtH2S treatment also rejuvenated aging transcriptomes by minimizing expression declines of mitochondria and cytoskeletal components, and peroxisome metabolism hub components, under mechanistic control by the elt-6/elt-3 transcription factor circuit. H2S health span extension likely acts at the mitochondrial level, the mechanisms of which dissociate from life span across adult vs. developmental treatment timings. The small mtH2S doses required for health span extension, combined with efficacy in adult animals, suggest mtH2S is a potential healthy aging therapeutic. / A.R.V., M.W., and T.E. were supported by the US Army Research Office (W911NF-19-1-0235). L.S., M.W., and T.E. were supported by the United Mitochondrial Disease Foundation (PI-19-0985). L.S. was also supported by the University of Exeter Jubilee Scholarship. M.C., N.J.S., and T.E. were supported by the UK Space Agency (ST/R005737/1). N.J.S. and T.E. were supported by BBSRC (BB/N015894/1). S.A.V. was supported by NASA (NNX15AL16G). N.J.S. was supported by grants from NASA [NSSC22K0250; NSSC22K0278] and acknowledges the support of the Osteopathic Heritage Foundation through funding for the Osteopathic Heritage Foundation Ralph S. Licklider, D.O., Research Endowment in the Heritage College of Osteopathic Medicine.