ELUCIDATING GENE SIGNATURES THAT CONTROL THE CIRCADIAN RHYTHM IN CYANOBACTERIA USING BIOINFORMATICS METHODS

poster abstractBackground: The daily light-dark cycle govern rhythmic changes in the behavior and physiology of most species. This circadian rhythm, or bi-ological “clock,” allows the organism to anticipate and prepare for the changes in the physical environment that are associated with day and night, thereby ensuring that the organism carry our specific processes at the right time of the day. Studies have found that the internal clock con-sists of an array of genes and the protein products they encode, which regulate various physiological processes throughout the body. Cyanothece sp. ATCC 51142 is an organism that has both photosynthetic (producing oxygen) and nitrogen fixing ability. The N2-fixing enzyme, nitrogenase, is highly sensitive to oxygen for which it has developed a temporal regula-tion in which N2 fixation and photosynthesis occur at different times throughout a diurnal cycle with very high levels of CO2 fixation during the light and high levels of N2 fixation in the dark. The mechanisms underly-ing the circadian rhythm and the signature genes elucidating this mecha-nism are addressed in this research. Objective: The objective is to integrate gene expression data with da-ta and knowledge from prior studies using bibliomics techniques, in the de novo construction of quasi-complete transcriptional regulatory networks to identify gene signatures in functional motifs and elucidate their role in circadian rhythms in cyanothece sp. ATCC 51142. Methodology: The sequence data of Transcription profiling time se-ries of cyanothece sp. ATCC 51142 grown in 12-hour light/12 hour dark then 24 h light from Array Express was used to construct the initial global regulatory network. Different network topological features (degree, betweeness and eccentricity) are used to identify the signature pathways during the day and night. The genes of the global regulatory network were used to construct networks of homologous species. The functions of the already known genes in well-studied homologous species were mapped to the function of the unannotated genes of cynaothece sp. ATCC 51142. Results: We have identified significant (p<0.05) signature pathways like photosynthesis, pantothenate and CoA biosynthesis and Glyoxylate and dicarboxylate metabolism that operate during the day. And during the night, pathways such as ribosome, riboflavin metabolism, and fatty acid biosynthesis sulfur metabolism were found to be significant (p<0.05). We will further investigate the genes that were already known to be significant using cyanobase database in a particular biological path-way and the novel genes that are identified by bibliomics approach

Quantification of Circadian Rhythms in Single Cells

Bioluminescence techniques allow accurate monitoring of the circadian clock in single cells. We have analyzed bioluminescence data of Per gene expression in mouse SCN neurons and fibroblasts. From these data, we extracted parameters such as damping rate and noise intensity using two simple mathematical models, one describing a damped oscillator driven by noise, and one describing a self-sustained noisy oscillator. Both models describe the data well and enabled us to quantitatively characterize both wild-type cells and several mutants. It has been suggested that the circadian clock is self-sustained at the single cell level, but we conclude that present data are not sufficient to determine whether the circadian clock of single SCN neurons and fibroblasts is a damped or a self-sustained oscillator. We show how to settle this question, however, by testing the models' predictions of different phases and amplitudes in response to a periodic entrainment signal (zeitgeber)

THE SKELETAL MUSCLE MOLECULAR CLOCK REGULATES THE TIMING OF SUBSTRATE METABOLISM AND THE CIRCADIAN EXPRESSION OF TITIN-CAP

Skeletal muscle is a major contributor to whole-body metabolism as it serves as a depot for both glucose and amino acids, and is a highly metabolically active tissue. An intrinsic molecular clock mechanism exists within skeletal muscle that regulates the timing of physiological processes. A key function of the clock is to regulate the timing of metabolic processes to anticipate time of day changes in environmental conditions. The purpose of this study was to identify metabolic genes that are expressed in a circadian manner and determine if these genes are regulated downstream of the intrinsic molecular clock by assaying gene expression in an inducible skeletal muscle-specific Bmal1 knockout mouse model (iMS-Bmal1−/−). The skeletal muscle circadian transcriptome we analyzed was highly enriched for metabolic processes. Acrophase (time of peak expression) analysis of circadian metabolic genes revealed a temporal separation of genes involved in substrate utilization and storage over a 24-h period with many differentially expressed in the skeletal muscle of the iMS-Bmal1−/− mice compared to wildype. However, the iMS-Bmal1−/− mice displayed circadian behavioral rhythms indistinguishable from iMS-Bmal1+/+ mice. We also observed a gene signature indicative of a fast to slow fiber-type shift and a more oxidative skeletal muscle in the iMS-Bmal1−/− model. These data provide evidence that the intrinsic molecular clock in skeletal muscle temporally regulates genes involved in the utilization and storage of substrates independent of circadian activity. Disruption of this mechanism caused by phase shifts (that is, social jetlag) or night eating may ultimately diminish skeletal muscle’s ability to efficiently maintain metabolic homeostasis over a 24-h period. The molecular-clock targets genes for circadian expression in a tissue-specific manner, possibly through interactions with tissue-specific factors. In order to identify novel mechanisms responsible for driving circadian gene expression of muscle-specific genes we focused our study on the molecular regulation of the Titin-cap gene. We choose this gene as it was highly circadian in the skeletal muscle circadian transcriptome, and has previously been shown to be modulated by the clock factor BMAL1 in heart-tissue, and the myogenic regulatory factor MYOD1 in skeletal-muscle. Promoter-reporter experiments demonstrated that BMAL1:CLOCK and MYOD1 work in a synergistic fashion to transactivate the Titin-cap gene in skeletal-muscle. Circadian expression of Titin-cap relied on the normal function of MYOD1 as mutant vectors altered the rhythmic oscillation and expression. We provided evidence that BMAL1 and MYOD1 bind to a tandem E-Box element in the proximal promoter element, and that this element is required for the circadian expression of Titin-cap in skeletal-muscle. These data provide a novel mechanism in which the molecular-clock works with a tissue specific transcription factor to drive circadian gene expression

Stochasticity and Synchrony in the Mammalian Circadian Network

A vast majority of life on Earth exists in an environment where resource availability and environmental conditions are temporally periodic. There is therefore an evolutionary advantage for organisms to partition behavior into certain times of day. Circadian rhythms, endogenous near-24 hour oscillations in gene expression, perform this task. These rhythms exert control over a large fraction of biological processes, and as such are implicated in a wide range of diseases, especially metabolic and mental disorders. Circadian rhythms are generated at a single-cell level through a complex set of interlocked genetic feedback loops. Individual components of the circadian network are considered “sloppy” due to stochastic noise, and it is only through the interaction of cellular oscillators at a network level that precise rhythms are generated. Medically treating or reverse-engineering this complicated genetic architecture necessitates mathematical understanding at multiple physical scales, from cells to tissue. This thesis seeks to describe the complex dynamics and hierarchical organization of circadian rhythms in mammals through systems dynamics and mathematical approaches. The overarching theme of this work will be the interplay between stochasticity and synchronization in circadian rhythms. Stochastic noise and precise oscillation are not completely at odds, however. In this thesis, I first develop a model of the circadian oscillator which incorporates the core negative feedback loop and an important neuropeptide coupling pathway. I use this model to investigate claims about the roles of Cryptochrome isoforms within the core circadian clock, and show that despite seemingly-different roles, experimental data is consistent with a parallel role for Cryptochrome isoforms. Next, I present a method for inferring functional connections within the suprachiasmatic nucleus (SCN), the mammalian “master clock,” and describe the network structure within the SCN. Finally, I examine growth and development of the SCN in utero

\u3ci\u3eAnaplasma Phagocytophilum\u3c/i\u3e Modulates Mammalian and Arthropod Signaling for Its Survival and Transmission

Vector-borne diseases (VBDs) are illnesses transmitted to humans and other animals by arthropods such as ticks, mosquitoes, and fleas. These arthropod vectors transmit infectious pathogens such as viruses, bacteria, and protozoa, to humans during blood-feeding. We have very few control strategies to treat or control these diseases. Human anaplasmosis, caused by the bacterium Anaplasma phagocytophilum, is the second most common tick-borne disease in the United States. This work defines three studies elucidating Anaplasma phagocytophilum-mediated modulation of cell signaling in mammalian cells and arthropod vector Ixodes scapularis ticks. The first study focused on mammalian PI3 kinases signaling in regulating cell cycle gene expression during A. phagocytophilum infection. Using the human megakaryocytic cell line, MEG-01, we observed a differential expression of cell cycle genes in these cells upon A. phagocytophilum infection. Both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class I PI3 kinases and phosphorylated protein kinase B (Akt/PKB) and IκB were higher at early and late stages of A. phagocytophilum infection. Inhibition of PI3 kinases with LY294002 treatment resulted in a significant reduction in the bacterial load and the expression of cell cycle gene expression. These results suggest a role for PI3K-Akt-NF-κB signaling in the modulation of megakaryocyte cell cycle genes upon A. phagocytophilum infection. The second study showed that A. phagocytophilum uses tick transcriptional activator protein-1 (AP-1) as a molecular switch in the regulation of the arthropod antifreeze gene, iafgp. RNAi-mediated silencing of ap-1 significantly affected iafgp gene expression and bacterial burden in ticks during acquisition from the murine host. The electrophoretic mobility shift assays (EMSAs) revealed that both the bacterium and AP-1 protein influence iafgp promoter and expression. The luciferase assays demonstrated that a 700 bp upstream region of the antifreeze gene is sufficient for AP-1 binding to drive iafgp gene expression. Furthermore, survival assays revealed that ap-1 deficient ticks were more susceptible to cold than the mock control ticks. These data show that AP-1 acts as an upstream transcriptional activator to drive the iafgp expression that is critical for A. phagocytophilum survival in I. scapularis ticks. The third study identified and characterized the circadian components in I. scapularis. The identification of the core clock genes in ticks was made using bioinformatic analysis from the Ixodes scapularis genome. Core clock genes like clock1 and bmal1 were upregulated upon tick feeding on the murine host. RNAi-mediated knockdown of the arthropod clock1 gene resulted in an increased bacterial transmission from ticks to the murine host. These results indicate that arthropod clock-mediated signaling is essential for transmitting A. phagocytophilum from tick to the vertebrate host. Taken together, these studies highlight several undefined mechanisms that A. phagocytophilum modulates for its survival in mammalian cells and ticks

Diel patterns and tissue-specificity of environmental responses in fish

Humans are profoundly changing aquatic environments through climate change and the release of nutrients and chemicals. To understand the effects of these changes on natural populations, knowledge on individuals’ environmental responses is needed. At the molecular level, the environmental responses are partly mediated by chances in messenger RNA and protein levels. In this thesis I study messenger RNA and protein responses to an assortment of environmental stressors in fish. As daily (diel) rhythms are known to be ubiquitous in different tissues, I particularly focus on diel patterns in the responses. The studied species are the three-spined stickleback (Gasterosteus aculeatus L.) and the Arctic char (Salvelinus alpinus L.), both of which have circumpolar distribution in the Northern hemisphere. In the first two studies, three-spined sticklebacks were exposed to both the non-steroidal anti-inflammatory drug diclofenac and low-oxygen conditions (hypoxia), and their responses measured at separate time points in the liver and gills. The results show how the seemingly unrelated environmental stressors, hypoxia and anti-inflammatory drugs, can have harmful combined effects that differ from the effects of each stressor alone. Moreover, both stressors disturbed natural diel patterns in gene expression. In the third study, I studied the responses of three-spined sticklebacks to two test chemicals: one used in hormonal medicine (17α-ethinyl-oestradiol) and one used as a plasticizer and solvent chemical (di-n-butyl phthalate). The results suggest that the phthalate can affect genes related to spermatogenesis in fish testes, while estrogen-mimicking compounds can lead to numerous disturbances in the endocrine system. In the final study, the temperature-dependence of diel rhythms in messenger RNA levels were evaluated in the liver tissue of the Arctic char, a cold-adapted salmonid. The results show that cold acclimation repressed diel rhythms in gene expression compared to warm-acclimated fish, in which the expression of hundreds of genes was rhythmic, suggesting the circadian clock of the Arctic fish species can be sensitive to temperature. Overall, the results of the thesis indicate that fishes’ responses to abiotic factors interact with their diel rhythms, and more studies on the consequences of these interactions are needed to comprehensively understand human impacts on ecosystems.Ihmistoiminnan tuottamat kasvihuonekaasut sekä kemikaali- ja ravinnepäästöt muuttavat ympäristöä voimakkaasti. Muutosten vaikutusten ymmärtämiseksi tarvitsemme tietoa eri lajien ympäristövasteista. Molekyylitasolla vasteita säätelevät proteiinit, joiden määrä soluissa on osin riippuvainen tuotetun lähetti-RNA:n määrästä. Tässä väitöskirjassa käsittelen eri ympäristömuutosten vaikutuksia kaloihin lähetti-RNA:n määrän ja entsyymiaktiivisuuden tasolla eri kudoksissa. Vuorokausirytmien tiedetään säätelevän monien geenien ilmenemistä, joten keskityin etenkin ajalliseen vaihteluun kalojen ympäristövasteissa. Tutkitut kalalajit olivat lauhkeaan ilmastoon sopeutunut kolmipiikki (Gasterosteus aculeatus L.) ja arktisiin olosuhteisiin sopeutunut nieriä (Salvelinus alpinus L.). Kummatkin lajit elävät laajoilla alueilla pohjoisella pallonpuoliskolla. Kahdessa ensimmäisessä työssä testasin kolmipiikkien vasteita jätevesissä esiintyvälle tulehduskipulääke diklofenaakille ja vähähappisille olosuhteille (hypoksialle). Maksa- ja kiduskudoksista saadut tulokset osoittavat hypoksialla ja diklofenaakilla olevan mahdollisesti haitallisia yhteisvaikutuksia kaloihin, ja että kiduksissa ja maksassa tapahtuvat muutokset voivat olla erisuuntaisia. Sekä diklofenaakin että hypoksian havaittiin myös muuttavan entsyymien luontaista päivärytmiä. Kolmannessa osatyössä tutkin kolmipiikkien vasteita kahdelle hormonitoimintaa häiritseville yhdisteelle, keinotekoiselle estradiolille sekä muoviyhdisteissä käytetylle dibutyyliftalaatille. Havaitsin ftalaatin vaikuttavan siittiöiden toimintaa säätelevien geenien luentaan ja estradiolin estävän useiden steroidihormonien tuotantoa säätelevien geenien ilmenemistä kalojen sukuelimissä. Viimeisessä osatyössä tutkin lämpenemisen vaikutuksia geeniluennan päivärytmiin lohikaloihin kuuluvalla nieriällä. Tulokset osoittivat, että korkeassa lämpötilassa nieriän geenien luenta vaihteli voimakkaasti päivän aikana, mutta viileässä lämpötilassa vaihtelu oli huomaamatonta, mikä viittaa siihen, että arktisten kalojen biologinen kello voi olla herkkä lämpötilamuutoksille. Kokonaisuudessaan väitöskirjan tulokset osoittavat, että meidän on vaikea ennustaa ympäristömuutosten yhteisvaikutuksia luonnon populaatioihin, jos emme ymmärrä ympäristövasteiden ja biologisten rytmien vuorovaikutuksia.Siirretty Doriast

Comparative transcriptome analysis reveals key pathways and regulatory networks in early resistance of Glycine max to soybean mosaic virus

As a high-value oilseed crop, soybean [Glycine max (L.) Merr.] is limited by various biotic stresses during its growth and development. Soybean mosaic virus (SMV) is a devastating viral infection of soybean that primarily affects young leaves and causes significant production and economic losses; however, the synergistic molecular mechanisms underlying the soybean response to SMV are largely unknown. Therefore, we performed RNA sequencing on SMV-infected resistant and susceptible soybean lines to determine the molecular mechanism of resistance to SMV. When the clean reads were aligned to the G. max reference genome, a total of 36,260 genes were identified as expressed genes and used for further research. Most of the differentially expressed genes (DEGs) associated with resistance were found to be enriched in plant hormone signal transduction and circadian rhythm according to Kyoto Encyclopedia of Genes and Genomes analysis. In addition to salicylic acid and jasmonic acid, which are well known in plant disease resistance, abscisic acid, indole-3-acetic acid, and cytokinin are also involved in the immune response to SMV in soybean. Most of the Ca2+ signaling related DEGs enriched in plant-pathogen interaction negatively influence SMV resistance. Furthermore, the MAPK cascade was involved in either resistant or susceptible responses to SMV, depending on different downstream proteins. The phytochrome interacting factor-cryptochrome-R protein module and the MEKK3/MKK9/MPK7-WRKY33-CML/CDPK module were found to play essential roles in soybean response to SMV based on protein-protein interaction prediction. Our findings provide general insights into the molecular regulatory networks associated with soybean response to SMV and have the potential to improve legume resistance to viral infection