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.

No description available.

No description available.

Corn (Zea mays) is frequently infected by a soil fungal pathogen Aspergillus flavus. The fungus produces aflatoxins, which cause liver cancer. Maize inbred lines that are resistant to infection by A. flavus have been developed, and these inbred lines provide excellent models for studying molecular mechanisms of maize resistance to the fungus. MicroRNA-like RNAs (milRNAs) recently identified in A. flavus had been found to be correlated with aflatoxin production conditions, suggesting that the milRNAs might play a role in the regulation of aflatoxin production. In this research, small RNAs were isolated from kernels of maize (resistant Mp719 and susceptible Va35) inoculated with A. flavus NRRL 3357 (aflatoxigenic) and NRRL 21882 (nonaflatoxigenic) and then subjected to RNA sequencing. Sequencing had identified 69 A. flavus milRNAs and 691 Z. mays miRNAs. The differential expression of some maize miRNAs revealed their potential role in response to inoculation, A. flavus growth, and aflatoxin production.

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No description available.

Improving the efficiency by which crops use nutrients is critical for maintaining high crop productivity while reducing fertility management costs and eutrophication related to fertilizer runoff. The native crucifer and halophyte, Yukon Eutrema salsugineum, was used in this study. Yukon E. salsugineum is closely related to important Brassica crops and thrives in its native habitat on soil that is low in available phosphate (Pi) and high in sulfur (S). To determine how Yukon E. salsugineum copes with low Pi, leaf transcriptomes were prepared from four week-old plants grown in controlled environment chambers using soil lacking or supplemented with Pi and/or S. This thesis focused on using bioinformatic approaches to assemble, analyze and compare the transcriptome profiles produced by the Yukon E. salsugineum plants undergoing four nutrient combinations of high and/or low Pi and S. The objective of the study was to identify traits associated with altered S and/or Pi with the prediction based on other species that low Pi, in particular, would pose the greatest stress and hence elicit the greatest transcriptional reprogramming. Transcriptome libraries were generated from four treatment groups with three biological replicates each. Reads in each library were mapped to 23,578 genes in the E. salsugineum transcriptome with an average unique read mapping ratio of 99.52%. Surprisingly, pairwise comparisons of the transcriptomes showed little evidence of Pi-responsive reprogramming whereas treatments differing in soil S content showed a clear S-responsive transcriptome profile. Principal Component Analysis revealed that the low variance quaternary Principal Component distinguished the transcriptomes of plants undergoing low versus high Pi treatments with differential gene expression analysis only finding 11 Pi-responsive genes. This outcome suggests that leaf transcriptomes of Yukon E. salsugineum plants under low Pi are largely undifferentiated from plants provided with Pi and is consistent with Yukon E. salsugineum maintaining Pi homeostasis through fine-tuning the expression of protein-coding and non-coding RNA rather than large-scale transcriptomic reprogramming. Previous research has shown Yukon E. salsugineum to be very efficient in its use of Pi and this work suggests that the altered expression of relatively few genes may be needed to develop Pi-efficient crops to sustain the crop demand of a growing population. / Thesis / Master of Science (MSc)

Glicocorticoides (GCs) modulam a reprodução, interferindo na produção de hormônios gonadais, estradiol (E2) e progesterona (P4). Concomitantemente, E2 e P4 também influenciam a liberação de cortisol. Além disso, em altas concentrações os GCs interferem na via de sinalização da insulina prejudicando o funcionamento dos órgãos, incluindo os do sistema reprodutivo bem como a produção de P4 pelo corpo lúteo (CL). O CL utiliza glicose para manter sua atividade, e o cortisol interfere na via de sinalização da glicose em diversos tecidos, porém ainda não existem estudos quanto à sua atividade no CL canino, tampouco quanto à presença de genes relacionados à via de sinalização dos GCs neste órgão. Os objetivos deste estudo foram caracterizar a expressão dos genes relacionados à via de sinalização dos GCs e da insulina em CL de cadelas cíclicas e identificar expressão diferencial destes genes entre os dias 20 e 60 do diestro, que correspondem à fase final de crescimento do CL e à fase de regressão luteínica, respectivamente. Ainda, avaliar as concentrações basais de cortisol salivar (CS) e metabólitos fecais de glicocorticoides (MGFs) em diferentes dias do diestro (10, 20, 30, 40, 50 e 60) de cadelas cíclicas, comparando com os níveis de E2 e P4 já publicados; correlacionar os valores obtidos nas duas técnicas; e estabelecer um intervalo de valores basais de CS e MFGs para o diestro. Para esta pesquisa foram selecionadas 28 cadelas cíclicas saudáveis; foi realizado o sequenciamento de nova geração (RNA-Seq) dos CLs coletados de 18 animais em dias específicos do diestro (dia 10, 20, 30, 40, 50 e 60 pós-ovulação); das outras 10 cadelas foram coletadas amostras de fezes e saliva, durante todo o diestro, e realizada mensuração da concentração de MFGs e CS. Nos resultados de RNA-Seq foi identificada a expressão dos genes NR3C1, HSD11B1, HSD11B2, SLC2A4, INSR e IRS1. Os genes NR3C1 (p>0,3; FDRCortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,6) e SLC2A4 (pCortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,02; FDRCortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,1) não apresentaram expressão diferencial no diestro. Os genes HSD11B1 (p=0,0001; FDR=0,0021), HSD11B2 (p=0,013; FDR=0,06), INSR (p=0,002; FDR=0,01) mostraram maior expressão no dia 20 do diestro, quando comparado ao dia 60. O gene IRS1 (p=0,0006; FDR= 0,006) estava mais expresso no dia 60 do diestro em relação do dia 20. As dosagens hormonais demonstraram que no dia 10 do diestro (CS=0,0656 ± 0,0237µg/dL e MGFs=110,41 Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 46,51pg/ng) as duas mensurações apresentaram concentrações menores que nos demais dias do diestro (CS=0,1027 Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,0496µg/dL e MFGs=220,22 Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 183,74pg/ng). A concentração média de CS=0,0972µg/dL (0,011-0,246µg/dL) e MFGs=189,875pg/ng (9pg/ng-1067,2pg/ng) foi definida para o diestro canino. Estes resultados sugerem que o CL canino também é influenciado pelo cortisol circulante da mesma forma que outros tecidos já estudados, podendo interferir na via de sinalização da insulina e, consequentemente, prejudicar seu funcionamento e o sucesso reprodutivo. Além disso, em relação aos resultados das dosagens hormonais, os níveis baixos de CS e MFGs no dia 10 do diestro corroboram com os achados em literatura e com a queda de E2 neste mesmo período, o que sugere uma associação entre a produção de ambos os hormônios. / Glucocorticoids (GCs) modulate reproduction by interfering in the gonadal hormones production, estradiol (E2) and progesterone (P4). Likewise, E2 and P4 influence release of cortisol. Furthermore, high concentrations of GCs may harm the functioning of organs, including reproductive organs and progesterone (P4) production by the corpus luteum (CL). CL utilizes glucose to maintain its own activity and cortisol interferes with the glucose signaling pathway in several tissues. Studies characterizing cortisol activity and expression of genes related to GC signaling pathways in canine CL are still scarce. The aims of this study were to characterize the expression of genes related to GCs and insulin signaling pathways in cyclic canine CL and to identify differential expression of these genes between day 20 and day 60 of diestrus which correspond, respectively to luteal final growth and regression phases. Moreover to evaluate the basal concentrations of salivary cortisol (SC) and fecal glucocorticoid metabolites (FGMs) on different days of diestrus (10, 20, 30, 40, 50 e 60) in cyclic bitches, moreover to compare E2 and P4 levels already published. As well as to correlate the values of SC and FGMs, and also to establish an interval of basal concentrations of SC and FGMs for diestrus. For this research 28 healthy cyclic bitches were selected; RNA-Seq of the corpora lutea from 18 animals on specific days of diestrus (day 10, 20, 30, 40, 50, 60) was made. Saliva and fecal samples were collected from the other 10 bitches during diestrus phase. Enzyme immunoassay was made to measure the concentrations of SC and FGMs. The RNA-Seq results identified the expression of the genes NR3C1, HSD11B1, HSD11B2, SLC2A4, INSR e IRS1. No difference on the expression of NR3C1 (p>0,3; FDR>0,6) and SLC2A4 (p>0,02; FDR>0,1) was observed during diestrus. Nevertheless HSD11B1 (p=0,0001; FDR=0,0021), HSD11B2 (p=0,013; FDR=0,06), INSR (p=0,002; FDR=0,01) showed a higher expression on day 20 of diestrus and the gene IRS1 (p=0,0006; FDR= 0,006) presented higher expression on day 60 of diestrus. Results related to hormonal evatuations showed lower SC and FGMs concentrations on day 10th (SC=0,0656 ± 0,0237µg/dL and FGMs=110,41 ± 46,51pg/ng) than on the other days of diestrus (SC=0,1027 ± 0,0496 µg/dL e FGMs=220,22 ± 183,74 pg/ng). Average concentrations of SC=0,0972µg/dL (0,011-0,246µg/dL) and FGMs=189,875pg/ng (9pg/ng-1067,2pg/ng) were defined for canine diestrus. These data suggest that canine CL is influenced by cortisol similarly to other tissues in which cortisol may interfere in the insulin signaling pathway and, consequently, its function. Besides that, lower levels of SC and FGMs on day 10th of the diestrus corroborate with the literature data and with the decrease in E2 production at the same period. This data suggest that the production of both hormones are associated.

Inúmeros aspectos da reprodução em Apis mellifera já foram extensamente divulgados, no entanto, os mecanismos reguladores da manutenção do estado estéril das operárias, bem como aqueles que permitem a ativação de seus ovários, ainda estão para serem descobertos. Por exemplo, a organização dos folículos ovarianos em crescimento e a arquitetura e papel das células foliculares neste processo. Além disso, para compreender o processo de ativação dos ovários em um contexto mais amplo, também é necessária uma investigação da síntese e maturação de diferentes classes de RNAs as quais modelam redes de interações gênicas extremamente complexas. Portanto, neste doutorado, tivemos como objetivo realizar 1- uma análise morfológica dos ovários ativos de operárias de A. mellifera obtidos em condições orfandade, com ênfase nas células foliculares e 2- um estudo aprofundado da regulação da expressão gênica (genes estruturais e reguladores) que é de fundamental importância para ligar os genótipos aos fenótipos. A análise morfológica dos ovários de operárias de A. mellifera foi realizada em microscópio de fluorescência ou confocal (priorizou a contagem das células foliculares) e microscópio eletrônico de transmissão, que permitiu a descrição e caracterização, pela primeira vez, da patência em ovários de operárias A. mellifera. Paralelamente, por meio da técnica de RNAseq, foi possível analisar o transcriptoma (miRNAs e mRNAs) de amostras específicas de ovários, em diferentes estados fisiológicos, em rainhas e operárias. Os mRNAs e miRNAs que se destacaram em nossas análises in silico foram validados experimentalmente por RT-PCR com alto grau de reprodutibilidade e em harmonia com o estado fisiológico dos ovários. Os transcritos altamente expressos nos ovários ativados foram: fpps5, cad, obp7, yellow-g e aqueles representados pelo GB42182 e GB44975. Acreditamos estes genes possam fazer parte da rede que regula o processo de ativação dos ovários em A. mellifera. Os miRNAs que se destacaram em nossas análises foram: A) miR-306 e miR-317 - altamente expressos nas amostras de ovários funcionais e B) miR-71 pelo fato de, nas análises in silico, ser o mais forte candidato a alvejar a vitelogenina, e na análise experimental, apresentarem, microRNAs e mRNAs, perfis de expressão antagônicos. A construção de bibliotecas de microRNAs e mRNAs a partir de ovários funcionais e não funcionais de abelhas operárias e rainhas, a análise de expressão, bem como a predição de uma rede de integração nos deu um retrato do sensível equilíbrio reprodutivo que mantém ambas as castas em aparente harmonia dentro da colônia aonde elas assumem, no momento certo, seus papéis nesta sofisticada sociedade empreendendo ou não a reprodução. / Countless aspects of reproduction in Apis mellifera have been widely published, however, the regulatory mechanisms for the maintenance of the sterile state of workers as well as those that allow the activation of their ovaries are still to be discovered, as much as the organization of growing ovarian follicles, the architecture and the role of follicular cells during this process. Furthermore, to understand the activation process of the ovaries in a broader context, it is also necessary to investigate the synthesis and maturation of different classes of RNAs which exemplify networks of gene interactions, extremely complex. Therefore, PhD project, we aimed to approach: 1 - A morphological analysis of active ovaries of A. mellifera workers obtained in queenless conditions, with emphasis on the follicular cells and 2 - A detailed study of the regulation of gene expression (structural and regulatory genes) that is crucial for linking genotypes to phenotypes. Morphologic analysis of workers ovaries of A. mellifera was performed under a fluorescence microscope or confocal (prioritized follicular cell count) and transmission electron microscope, which allowed, for the first time, a description and characterization of the patency of worker ovaries in A. mellifera. Similarly, by RNAseq technique, it was possible to analyze the transcriptome (miRNAs and mRNAs) of specific samples of ovaries at different physiological states, in queens and workers. mRNAs and miRNAs that stood out in our in silico analysis were experimentally validated by RT-PCR with high reproducibility and in harmony with ovaries physiologic state. Transcripts highly expressed in activated ovaries were fpps5, cad, obp7, yellow-g and those represented by GB42182 and GB44975. We believe these genes may be part of the network that regulates ovaries activation process in A. mellifera. miRNAs that stood out in our analysis were: - a) - miR-306 and miR-317 - highly expressed in samples of active ovaries and b) -miR-71 by the fact that the in silico analysis, was the strongest candidate to target vitellogenin, and in experimental analysis, presented antagonistic profile of expression when microRNAs and mRNAs were contrasted. The construction of microRNAs and mRNAs libraries from active and inactive ovaries of worker bees and queens, the analysis expression, as well as the prediction of a integrative network has given us a portrait of the sensitive reproductive balance that keeps both castes of bees in apparent harmony within the colony, where they take each one, at the right time, their roles in this sophisticated society, undertaking or not the reproduction.