Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Gerald R. Reeck / Armet is a bifunctional protein that is apparently universally distributed among multicellular animal species, vertebrate and invertebrate alike. A member of the Unfolded Protein Response, (UPR) Armet promotes survival in cells that are under endoplasmic-reticulum (ER) stress. I have carried out biophysical studies on human Armet looking for compounds that bind to Armet and hence could reduce its anti-apoptotic function, thus potentially joining the growing class of pro-apoptotic drugs. Performed primarily with 1H-15N HSQC NMR, ligand studies showed that approximately 60 of the 158 residues are potentially involved with binding. The 60 residues are distributed throughout both domains and the linker suggesting multi-domain interaction with the ligand. Circular dichroism studies showed heat denaturation in a two-step unfolding process with independent unfolding of both domains of Armet with Tm values near 68°C and 83 C with the C-terminal domain unfolding first, as verified by 1H-15N HSQC NMR measurements. I also provide the first identification of UPR transcripts in pea aphids, Acyrthosiphon pisum, the genetic model among aphids. I measured transcript abundance with hope of finding future transcriptional targets for pest mitigation. I identified 74 putative pea aphid UPR components, and all but three of the components have higher transcript levels in aphids feeding on plants than those that fed on diets. This activated UPR state is attributed to the need for saliva proteins for plant feeding. Because aphids are agriculturally significant pests, and saliva is pivotal to their feeding on host plants, genes that encode saliva proteins may be targets for pest mitigation. Here I have sought the aphid’s saliva proteome by combining results obtained in several laboratories by proteomic and transcriptomic approaches on several aphid species. With these data I constructed a tentative saliva proteome for the pea aphid by compiling, collating, and annotating the data from several laboratories. I used RNA-seq to verify the transcripts in pea aphid salivary glands, thus expanding the proposed saliva proteome from approximately 50 components to around 130 components, I found that transcripts of saliva proteins are upregulated during plant feeding compared to diet feeding.

Philosophiae Doctor - PhD / Apple (Malus x domestica) production in the Western Cape, South Africa, is one of the major contributors to the gross domestic product (GDP) of the region. The production of apples is affected by a number of diseases. One of the economically important diseases is apple scab that is caused by the pathogenic fungus, Venturia inaequalis. Research to introduce disease resistance ranges from traditional plant breeding through to genetic manipulation. Parallel disease management regimes are also implemented to combat the disease, however, such strategies are increasingly becoming more ineffective since some fungal strains have become resistant to fungicides. The recently sequenced apple genome has opened the door to study the plant pathogen interaction at a molecular level. This study reports on proteomic and transcriptomic analyses of apple seedlings infected with Venturia inaequalis. In the proteomic analysis, two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify apple leaf proteins extracted from infected and uninfected apple seedlings. Using MelanieTM 2-DE Gel Analysis Software version 7.0 (Genebio, Geneva, Switzerland), a comparative analysis of leaf proteome expression patterns between the uninfected and infected apple leaves were conducted. The results indicated proteins with similar expression profiles as well as qualitative and quantitative differences between the two leaf proteomes. Thirty proteins from the apple leaf proteome were identified as differentially expressed. These were selected for analysis using a combination of MALDI-TOF and MALDI-TOF-TOF MS, followed by database searching. Of these spots, 28 were positively identified with known functions in photosynthesis and carbon metabolism (61%), protein destination and storage (11%), as well as those involved in redox/response to stress, followed by proteins involved in protein synthesis and disease/defence (7%), nucleotide and transport (3%). RNA-Seq was used to identify differentially expressed genes in response to the fungal infection over five time points namely Day 0, 2, 4, 8 and 12. cDNA libraries were constructed, sequenced using Illumina HiScan SQTM and MiSeqTM instruments. Nucleotide reads were analysed by aligning it to the apple genome using TopHat spliceaware aligner software, followed by analysis with limma/voom and edgeR, R statistical packages for finding differentially expressed genes. These results showed that 398 genes were differentially expressed in response to fungal infection over the five time points. These mapped to 1164 transcripts in the apple transcripts database, which were submitted to BLAST2GO. Eighty-six percent of the genes obtained a BLAST hit to which 77% of the BLAST hits were assigned GO terms. These were classed into three ontology categories i.e. biological processes, molecular function and cellular components. By focussing on the host responsive genes, modulation of genes involved in signal perception, transcription, stress/detoxification, defence related proteins, transport and secondary metabolites have been observed. A comparative analysis was performed between the Day 4 proteomic and Day 4 transcriptomic data. In the infected and uninfected apple leaf proteome of Day 4, we found 9 proteins responsive to fungal infection were up-regulated. From the transcriptome data of Day 4, 162 genes were extracted, which mapped to 395 transcripts in the apple transcripts. These were submitted to BLAST2GO for functional annotation. Proteins encoded by the up-regulated transcripts were functionally categorised. Pathways affected by the up-regulated genes are carbon metabolism, protein synthesis, defence, redox/response to stress. Up-regulated genes were involved in signal perception, transcription factors, stress/detoxification, defence related proteins, disease resistance proteins, transport and secondary metabolites. We found that the same pathways including energy, disease/defence and redox/response to stress were affected for the comparative analysis. The results of this study can be used as a starting point for targeting host responsive genes in genetic manipulation of apple cultivars.

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.

Orientador: Daniel Guariz Pinheiro / Resumo: O conjunto de microrganismos que vivem junto das plantas é chamado de microbioma ou fitomicrobioma, e exerce diversos papéis conjuntamente com seu hospedeiro inclusive para a manutenção da saúde conjunta, considerando que co-evoluíram juntos, como um holobionte e, portanto, podem ter desenvolvido mecanismos de adaptação mútua. No presente trabalho, exploramos a diversidade do microbioma de cana-de-açúcar pela mineração de dados de RNA-Seq: realizamos atribuição taxonômica às sequências daqueles organismos que foram sequenciados conjuntamente com as plantas, mas que não foram analisados em nenhum ponto. Dados de 15 acessos do SRA contendo 246 bibliotecas foram obtidos, totalizando 841 Gigabases. As leituras dessas bibliotecas foram processadas, montadas em sequências a fim de reconstruir as moléculas de RNA e passaram por processo de atribuição taxonômica com o Kraken2 usando um banco de referência expandido personalizado, gerando uma contagem de organismos por biblioteca, sendo que os \textit{taxa} microbianos foram quantificados a partir da identificação de 09 milhões de leituras correspondentes. Como prova de conceito do método, validamos diversas observações que já existiam sobre a diversidade do microbioma da cana-de-açúcar. Revelamos que em todas as plantas, há uma presença quase ubíqua de bactérias Gammaproteobacteria, especialmente Pseudomonas e Xanthomonas e de fungos Ascomycota. Além disso, que a diversidade é alterada pela natureza do estresse na planta, hídrico ou ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The set of microorganisms that live under the influence of its host is called microbiome or in case of plants, phytomicrobiome, and they play various roles alongside their host including in maintaining the health of the pair, considering that they co-evolved together as a holobiont and thus may developed mechanisms of mutual adaptation. In the present work, we explored the diversity of the sugarcane microbiome by mining RNA-Seq data: we performed taxonomic assignment to RNA sequences of those organisms that were sequenced together with the plants but were not analyzed at any point. As proof-of-concept, we were able to validate several known observations of the sugarcane microbiome. We revealed that in all plants there is an almost ubiquitous presence of Gammaproteobacteria, especially Pseudomonas and Xanthomonas and fungi Ascomycota. In addition, that diversity is altered the stress implied on the sugarcane plant, be it abiotic (drought) or biological (infection), and that the major modulators in the community are mostly the plant compartment and the type of stress involved. We also observed a set of original organisms negatively and positively correlated with smut disease, caused by Sporisorium scitamineum, potentially antagonistic to the disease agent. Additionally, we detected differentially abundant phytomicrobiome activity between treatments, including those in which plant-induced infection was performed. Finally, virus sequences were assembled, and we were able to retri... (Complete abstract click electronic access below) / Mestre

Neuroblastoma (NBL), the most common non-Central Nervous System (CNS) solid tumor of childhood, characteristically displays heterogeneous clinical presentation and biological behavior. Previous work has studied the genetic basis of the disease and revealed a low somatic mutation burden. In order to identify novel therapeutic targets and better understand the biology of high-risk NBLs, I investigated whole transcriptome profiles of two cohorts of metastatic NBLs using RNA sequencing. First, I studied changes in splicing pattern in a cohort of 29 patients. V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplified NBLs showed a distinct splicing pattern affecting multiple cancer hallmarks. Six splicing factors have altered expression patterns in MYCN-amplified tumors and cell lines, and binding motifs for these factors were significantly enriched in differentially-spliced genes. ChIP-seq analysis showed direct binding of MYCN to promoter regions of splicing factors PTBP1 and HNRNPA1, demonstrating that MYCN regulates splicing by directly regulating expression of key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage 4 NBL patients (p≤0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, a pro-tumor-growth isoform, resulted in repression of NBL cell growth. Second, I used whole transcriptome sequencing in a cohort of 150 patients to assess expressed mutations, fusion genes, and gene expression including long non-coding genes to provide clinically-relevant classification and to offer insights into NBL tumor biology. Twenty-four genes including ALK, ATRX and MYCN were recurrently mutated in NBL transcriptomes. In-frame FOXR1 fusions were detected in 4 samples, including 3 cases or 14% of stage 4S NBLs. Unsupervised gene expression analysis revealed four molecular subgroups. MYCN and tumor microenvironment were the primary discriminating signatures in these molecular subgroups. Fifty-eight percent of MYCN-not-amplified samples showed high MYCN signatures, which were potentially contributed by various genomic events such as MYCN activating mutations and FOXR1 fusions. High MYCN signature was significantly associated with poor overall survival in MYCN-not-amplified tumors (p=0.0017). In addition, the tumor microenvironment including stromal and immune cell infiltration significantly contributed to the NBL transcriptional landscape and tumor progression.

Obesity is characterized by the accumulation of excess adipose tissue and has become a global health burden as the rates of obesity in both adults and children continue an upward trend year after year. Obesity is an important public health concern as it has been linked to increased prevalence of comorbidities such as type 2 diabetes, fatty liver, cardiovascular complications and cancer. Adipocytes are exquisitely sensitive to energy demands, quickly responding by releasing fatty acids, storing excess calories as triglycerides and/or secreting adipokines. The ability of the adipocyte to carry out its important functions requires perpetual remodeling of the extracellular matrix (ECM) surrounding the adipocyte. Metabolic dysfunction and fibrosis arise when the healthy balance of remodeling becomes dysregulated. The long-term goal of this project was to gain a deeper understanding of the events occurring within an adipocyte in the context of weight gain. I hypothesized that the adipocyte would play an active role in remodeling of the ECM. To test this hypothesis, I fed C57/Bl6 mice either high fat diet (HFD) or chow diet for 8, 20 and 34 weeks, at which time, the perigonadal adipose tissue was digested to isolate the adipocyte fraction and RNA-seq analysis was performed. My data demonstrate that adipocytes responded to their obesogenic, pro-inflammatory environment through upregulation of ECM-related genes after only 8 weeks of HFD coupled with declining expression of mitochondrial genes and increasing genes associated with endoplasmic reticulum (ER) stress after 20 and 34 weeks of HFD. Overall, these data give a novel view into the dysfunctional state of isolated adipocytes over a time course of HFD and response to the changing microenvironment. MICAL2 is an atypical actin-modulating protein that has been shown to be involved in the regulation of MRTFA/SRF signaling and in cancer progression. I demonstrated for the first time that MICAL2 expression is enriched in the stromal vascular fraction of adipose tissue and is upregulated with HFD and pro-fibrotic stimulus, TGF-β1. I also demonstrated that MICAL2 is an anti-adipogenic and pro-fibrogenic protein. Altogether, the novel biology uncovered suggests a role for MICAL2 in adipose tissue remodeling which warrants further investigation. / 2021-06-14T00:00:00Z

Epilepsy, a disease characterized by recurring spontaneous seizures, affects over 65 million people, 2% of the world’s population. Over 30% of patients are refractory to all current medical therapy, and for those that can be treated, many suffer from severe drug side-effects. Understanding the molecular basis of epilepsy is vital to the advancement of better therapeutic options and an eventual cure. Upregulation of brain-derived neurotrophic factor (BDNF) is highly associated with epileptogenesis in human patients, as well as animal models. Our laboratory discovered that BDNF induces the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway in neurons and that inhibition attenuates spontaneous seizures in a temporal lobe epilepsy model. The mechanism behind JAK/STAT signaling in neurons and its relationship to epilepsy still remains to be elucidated and is the subject of my thesis. Surprisingly, even though BDNF is such a major signaling molecule, its full genomic impact has never been assessed. We conducted a high-density RNA-sequencing analysis of the BDNF transcriptome in cortical neurons and probed such regulation with selective JAK inhibitors. Results suggest that 68% of BDNF-induced changes in gene expression implicated in epilepsy are regulated by JAK/STAT signaling. Eighty percent of BDNF-induced changes coding for proteins involved in synaptic neurotransmission (receptor subunits and ion channels) involve JAK/STATs. Additionally, these datasets include genes that have never been associated with BDNF regulation (such as Dopamine Receptor D5 and Galanin Receptor 1). Most interestingly, the datasets reveal that BDNF-induced JAK/STAT signaling in neurons is non-canonical, as STAT3 phosphorylation at tyrosine 705 is not required for action. To directly examine STAT3’s role in epileptogenesis, we studied the transcriptome of transgenic mice that express lower levels of STAT3 specifically in neurons. Using the intrahippocampal kainic-acid (KA) model of epilepsy, our datasets suggest that STAT3 knockdown in vivo, and selectively in neurons, protects mice from KA-induced dysregulation of the sphingolipid metabolism pathway that is associated with the trafficking, sorting, and stability of membrane-bound proteins, including neurotransmitter receptors and ion channels. Finally, we discuss a model for JAK/STAT signaling in neurons that includes structural aspects of an intracellular BDNF receptor (p75NTR) associated with JAK2. / 2021-06-14T00:00:00Z

The symbiotic relationship between cnidarians and their photosynthetic dinoflagellate symbionts underpins the success of coral reef communities in oligotrophic, tropical seas. Despite several decades of study, the cellular and molecular mechanisms that regulate the symbiotic relationship between the dinoflagellate algae and the coral hosts are still not clear. One of the hypotheses on the metabolic interactions between the host and the symbiont suggests that ammonium assimilation by the host can be the underlying mechanism of this endosymbiosis regulation. An essential intermediate of the ammonium assimilation pathway is glutamate, which is also known for its glutamatergic signaling function. Interestingly, recent transcriptomic level and DNA methylation studies on sea anemone Aiptasia showed differences in metabotropic glutamate signaling components when comparing symbiotic and non-symbiotic animals. The changes in this process on transcriptional and epigenetic levels indicate the importance of glutamate signaling in regard to cnidarian symbiosis. In this study, I tested glutamatergic signaling effect on symbiosis in sea anemone Aiptasia using a broad-spectrum glutamate receptor inhibitor 7- CKA and glutamate. Significantly decreased cell density was observed in animals with inhibitor treatment suggesting a possible correlation between glutamate signaling and the establishment or maintenance of symbiosis. Using RNA-Seq, I was able to obtain transcriptional profiles of the animals under inhibitor and glutamate treatment. Differential gene expression and gene ontology analyses indicated changes in amino acid metabolism, lipid metabolism and such signaling pathways as MAPK, NF-kappa B and phospholipase C. Although amino acid and lipid metabolism could be a result of the reduced symbiotic state of inhibitor treated Aiptasia, the signaling pathways which are related to apoptosis and immune response provide an exciting venue for direct regulatory interaction between symbiosis and glutamatergic signaling. However, as these signaling pathways mainly act via signal transduction through protein phosphorylation, further studies looking at changes on a post-translational level might provide further insight into the mechanisms underlying the observed phenotype.

Indiana University-Purdue University Indianapolis (IUPUI) / We have previously demonstrated that disabling the transcription factor Nuclear Matrix Protein 4 (NMP4) improved parathyroid hormone (PTH)-induced trabecular bone gain in ovariectomized (OVX) and healthy mice. Here we evaluated whether loss of Nmp4 enhanced bone restoration in OVX mice under concurrent PTH combination therapies and anti-catabolic mono-therapies. Wild type (WT) and Nmp4-/- mice were OVX at 12wks of age followed by therapy regimens, administered from 16wks-24wks, and included individually or combined PTH, alendronate (ALN), zoledronate (ZOL), and raloxifene (RAL). Generally the PTH+RAL and PTH+ZOL therapies were more effective in restoring bone than the PTH mono-therapy. Loss of Nmp4 further improved the restoration of femoral trabecular bone under these treatments. RAL and ZOL mono-therapies moderately increased bone volume but unexpectedly the Nmp4-/- mice showed an enhanced RAL-induced increase in femoral trabecular bone. Immunohistochemical and flow cytometry analyses of the bone marrow and serum profiling for markers of bone formation and resorption indicated that the heightened osteoanabolism of the Nmp4-/- mice under these diverse osteoporosis treatments was partially attributed to an expansion of the osteoprogenitor pool. To address whether the enhanced bone formation observed in Nmp4-/- mice produced structurally sound tissue, mechanical testing was conducted on the femurs of healthy mice treated with intermittent PTH, RAL mono-therapy, or PTH+RAL. Nmp4-/- femurs showed modestly improved mechanical and material properties. At the cellular level, loss of Nmp4 accelerated mineralization in differentiating mesenchymal stem/progenitor cells (MSPCs). Transcriptomic and biochemical analyses indicated that loss of Nmp4 elevated ribosome biogenesis and expanded the capacity of the endoplasmic reticulum for processing protein. Preliminary data showed that disabling Nmp4 increased both aerobic glycolysis and oxidative phosphorylation in osteoprogenitors, which is an emerging hallmark of anabolic osteogenic cells. Transcriptomic analysis also suggested NMP4 targeted pathways driving bone formation. These included but not limited to BMP, IGF1, TGFβ and Wnt signaling pathways. Finally, transcriptomic profiling revealed that Nmp4-/- MSPCs showed a significant perturbation in numerous immunomodulatory pathways, particularly in the interleukin system. The heightened osteoanabolism of the Nmp4-/- skeleton enhances the effectiveness of diverse osteoporosis treatments, providing a promising target pathway for identifying barriers to pharmacologically-induced bone formation.

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