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Analysis of Young and Mature Thrombocytes in Zebrafish

Description: Eukaryotic platelets are small cell fragments that are released into the bloodstream from megakaryocytes, and their production is initiated in the bone marrow. They are mainly involved in blood hemostasis and thrombus formation. The newly synthesized platelets are called reticulated platelets or young platelets. Zebrafish thrombocytes are equivalent to mammalian platelets and have similar characteristics and functions. Likewise, zebrafish has both young and mature thrombocytes. Only young thrombocytes as reticulated platelets are labeled with thiazole orange. Similarly, labeling zebrafish thrombocytes with a specific concentration of DiI-C18 showed two populations of thrombocytes (DiI+ and DiI-). Again, only young thrombocytes showed DiI+ labeling. The mechanism of selective labeling of young thrombocytes by is unknown. Furthermore, there is no zebrafish line where young and mature thrombocytes are differentially labeled with fluorescence proteins. Therefore, in this study, we identified and confirmed that the RFP labeled cells of Glofish were young thrombocytes. In addition, we found that myosin light chain 2 (MLC2) promoter is expressed in young thrombocytes. We also generated a transgenic zebrafish line, GloFli fish, where the young and mature thrombocytes are labeled with red and green fluorescence proteins respectively. Furthermore, this study showed a two-fold increase in glycerol-phospholipids (GP) in mature thrombocytes compared to young thrombocytes suggesting the lipid composition may be important for differential labeling. Therefore, we tested the liposomes prepared with different ratios of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and observed that the lower amounts of PE favor the DiI-C18 labeling whereas higher concentrations of PC are less efficient. Also, in both PE and PC, increased concentrations of both resulted in decreased binding. These results are consistent with our observation that mature thrombocytes have higher concentrations GP and thus DiI-C18 may not bind to them efficiently compared to young thrombocytes.
Date: August 2018
Creator: Fallatah, Weam

Expression of G-protein Coupled Receptors in Young and Mature Thrombocytes and Knockdown of Gpr18 in Zebrafish

Description: In this study, a novel method based on biotinylated antibodies and streptavidin coated magnetic beads was used to separate the thrombocyte subpopulations from zebrafish whole blood. DiI-C18, a lipophilic dye, labels only young thrombocytes when used at low concentrations. Commercially available biotinylated anti-Cy3 antibody was used to label the chromophore of DiI-C18 on the young thrombocytes and streptavidin coated magnetic beads were added subsequently, to separate young thrombocytes. The remaining blood cells were probed with custom-made biotinylated anti-GPIIb antibody and streptavidin magnetic beads to separate them from other cells. Further, thrombocytes are equivalents of mammalian platelets. Platelets play a crucial role in thrombus formation. The G-protein coupled receptors (GPCRs) present on the platelet surface are involved during platelet activation and aggregation processes. So, thrombocytes were studied for the presence of GPCRs. The GPCR mRNA transcripts expressed in the young and mature thrombocytes were subjected to densitometry analysis and pixel intensities of the bands were compared using one way ANOVA. This analysis did not show significant differences between the young and mature GPCR mRNA transcripts but identified a novel GPCR, GPR18 that was not reported in platelets earlier. To study the function of this GPCR, it was knocked down using GPR18 specific antisense morpholino and vivo morpholino. The immunofluorescence experiment indicated the presence of GPR18 on thrombocytes. The results of the assays, such as, time to occlusion (TTO) and time to aggregation (TTA) in response to N-arachidonyl glycine (NAG) as an agonist, showed prolongation of time in GPR18 larval and adult morphants respectively, suggesting that GPR18 plays a role in thrombus formation in zebrafish. In conclusion, our results indicate that GPR18 may be present in zebrafish thrombocytes, it may be involved in thrombus formation and that NAG may be an agonist at GPR18 on thrombocytes.
Date: May 2013
Creator: Potbhare, Vrinda Nikhil

Forward Genetic Characterization of Medicago truncatula Tnt1 Insertion Mutants Defective in Nodule Development and Symbiotic Nitrogen Fixation

Description: Legumes are unique plants because they form special structures “nodules”, via symbiotic relationships with rhizobial bacteria present in the soil. Once rhizobia mature inside nodules, they fix atmospheric nitrogen providing a source of bioavailable nitrogen to the plant. To discover novel genetic components involved in the legume-rhizobia symbiosis by using forward genetic screening, we have isolated Medicago truncatula Tnt1 insertion mutants in the R108 ecotype, which are defective in nodule development and symbiotic nitrogen fixation in response to Sinorhizobium meliloti. Out of three mutants NF11044, NF11217 and NF8324, one of the mutants showed brown nodules and Fix- phenotype that is defective in symbiotic nitrogen fixation. The other two mutants showed white nodules and Fix- phenotype, also indicator of defects in symbiotic nitrogen fixation. To identify the underlying mutation causing the phenotype, we have developed molecular genetic markers by obtaining genomic sequences flanking the Tnt1 insertions by TAIL-PCR and Illumina sequencing. To carry out co-segregation analysis, back-crossed BC1F2 segregating populations were obtained. These are being phenotyped, genotyped and analyzed for co-segregation of the phenotype with the Tnt1 genetic markers. Back-crossing also has the effect of reducing the Tnt1 insertions, which are not linked to the nodulation defective phenotypes. Out of the three mutants, NF8324 harbors exactly the same insertion as in the rsd-1 Tnt1 mutant NF11265. The defect in NF11217 is caused by a Tnt1 insertion in the previously described PLC gene; the site of this insertion is close to that found in a different mutant, NF0217. For mutant NF11044, we developed linkage markers that place the defective locus on chromosome 7. To further characterize co-segregation in NF11044, a mapping population has been created by crossing the mutant with other ecotypes: A17 and A20. We tested mutants and wild type plants with linkage marker A20 X NF11044 BC1F2 that segregates 3:1(wild ...
Date: May 2015
Creator: Kadel, Khem L.

Homologs of Mammalian Lysosomal Lipase in Arabidopsis and Their Roles in Lipid Droplet Dynamics

Description: Lipid droplets (LDs) are organelles with many functions in cells and numerous protein interactors facilitate their biogenesis, maintenance, and turnover. The mammalian lipase responsible for LD turnover during lipophagy, LipA, has two candidate homologs in Arabidopsis: MPL1 and LIP1. One or both of these plant homologs may function in a similar manner to mammalian LipA, providing an LD breakdown pathway. To test this hypothesis, wild type (WT) Arabidopsis plants, MPL1 over-expressing (OE) mutants, and T-DNA insertion mutants of MPL1 (mpl1) and LIP1 (lip1) were examined for LD phenotypes in normal conditions and in environments where LD numbers are known to fluctuate. Plants to be imaged by confocal microscopy were exposed to heat stress and wounding to increase LD accumulation, senescence was induced in leaves to deplete lipids, and LDs were imaged throughout the day/night period to observe their diurnal regulation. The mutation of both MPL1 and LIP1 lead to an increase in LDs within the leaf mesophyll cells, although the spatial distribution of the LDs differed between the two mutants. mpl1 mutants had disrupted diurnal regulation of their LDs, but lip1 mutants did not. Alternately, lip1 mutants retained LDs during dark-induced senescence, and mpl1 mutants did not. Together these results suggest that MPL1 and LIP1 are likely both important for LD dynamics; however they appear have roles in different aspects of LD accumulation and turnover.
Date: December 2017
Creator: McClinchie, Elizabeth A

Identification of Hox Genes Controlling Thrombopoiesis in Zebrafish

Description: Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were both cumbersome or required breeding and production of fish where thrombocytes are GFP labeled. Therefore, I established a flow cytometry based method of counting the number of thrombocytes. I used mepacrine to fluorescently label the blood cells and used the white cell fraction. Standard antisense oligonucleotide designed to the central portion of each of the target hox mRNAs, was piggybacked by a control morpholino and intravenously injected into the adult zebrafish. The thrombocyte count was measured 48 hours post injection. In this study, I found that the knockdown of hoxc11b resulted in increased number of thrombocytes and knockdown of hoxa10b, ...
Date: December 2015
Creator: Sundaramoorthi, Hemalatha

Isolation and Characterization of Phages Infecting Streptomyces azureus

Description: Isolating novel phages using Streptomyces azureus, which produces antibiotic thiostrepton, as a host, and characterizing the genomes may help us to find new tools that could be used to develop antibiotics in addition to contribute to the databases of phages and specifically, Streptomyces phages. Streptomyces phages Alsaber, Omar, Attoomi, Rowa, and ZamZam were isolated using during this study. They were isolated from enriched soil and sequenced by Illumina sequencing method. They were isolated from three different geographical regions. They are siphoviridae phages that create small clear plaques with a diameter of approximately 0.5-1 mm, except for Rowa which has cloudy plaques, and they have varied sizes of their heads and tails. ZamZam was not characterized at this time. The sequencing shows that they are circular genome with 3' sticky overhang and various genomes' sizes with high percentage of GC content with the average of 66%. Alsaber was classified under sub-cluster BD3, while Omar was categorized under sub-cluster BD2. They share the same cluster of Cluster BD. Rowa was placed in Cluster BL and Attoomi is currently a singleton that does not fit into an established cluster. Alsaber yields 76 putative genes with no tRNA, Omar 81 putative genes with 1 tRNA. Attoomi 53 putative genes with no tRNA, and Rowa with 61 orfs and 7 tRNA. Rowa also was a putative temperate phage due to its lysogenic activity, and Row was not able to reinfect the lysogenic strain, S. azureus (Rowa). All of the isolated phages infected S. indigocolor, while only Attoomi and Rowa were able to infect S. tricolor. Upon completion of this project, we acquired more data and understanding of S. azureus phages and Actinobacteriophage in general, which will expand the scale of future research of Streptomyces bacteriophages.
Date: May 2018
Creator: Sulaiman, Ahmad M

Rapid Metabolic Response of Plants Exposed to Light Stress

Description: Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. So, in our study we have taken an metabolomics approach to identify the rapid response of plants to light stress. In the first part we have focused on the ultrafast (0-90 sec) metabolic response of local tissues to light stress and in the second part we analyzed the metabolic response associated with rapid systemic signaling (0-12 min). Analysis of the rapid response of Arabidopsis to light stress has revealed 111 metabolites that significantly alter in their level during the first 90 sec of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is dependent on an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for rapid metabolic responses. Taken together, our analysis could be used as an initial road map for the identification of different pathways that could be used to augment the rapid response of plants to abiotic stress. In addition, it highlights the important role of glutathione in initial stage of light stress response. Light-induced rapid systemic signaling and systemic acquired acclimation (SAA) are thought to play an important role in the response of plants to different abiotic stresses. Although molecular and metabolic responses to light stress have been extensively studied in local leaves, and to a lesser degree in systemic leaves, very ...
Date: May 2018
Creator: Choudhury, Feroza Kaneez

Revisiting the Neuroprotective Role of 17 Beta Estradiol: A Multi-Omics Based Analysis of the Rat Brain and Serum

Description: The ovarian hormone 17β-estradiol (E2) is one of the central regulators of the female reproductive system. E2 is also a pleiotropic regulator since it can exert its non-reproductive role on other organ systems. E2 is neuroprotective, it maintains body's energy homeostasis, participates in various repair mechanism and is required for neural development. However, there is a substantial evidence suggesting that there might be a molecular reprogramming of E2's action when it is supplied exogenously after E2 deprivation. Though the length of E2 deprivation and age has been linked to this phenomenon, the molecular components and how they activate this reprogramming is still elusive. Our main goal was to perform global proteomics and metabolomics study to identify the molecular components and their interaction networks that are being altered in the brain and serum after a short-term E2 treatment following ovariectomy (OVX) in Sprague Dawley rats. One of the strength of our global study is that it gave us extensive information on the brain proteome itself by identification of a wide number of proteins in different brain sections. By analyzing the differentially expressed proteins, our proteomics study revealed 49 different networks to be altered in 7 sections of the brain. Most of the perturbed networks were involved in cell metabolism, neural development, protein synthesis, cellular trafficking and degradation, and several stress response signaling pathways. We assessed the neuroenergetic status of the brain based on E2's response to various energy generating pathways, including glycolysis, TCA cycle, and oxidative phosphorylation, and several signaling pathways. All energetics pathways were shown to be downregulated in E2 treatment, which suggests that E2 exerts its neuroprotective role by restoring energy homeostasis in OVX rat model by regulating complex signaling and metabolic networks. Our second focus was to determine the metabolite response (amino acids and lipids) after E2 treatment ...
Date: August 2018
Creator: Zaman, Khadiza

Role of GPR17 in Thrombocyte Aggregation in Adult Zebrafish

Description: GPR17, a uracil nucleotide cysteinyl leukotriene receptor, belongs to the GPCR (G protein coupled receptor) family. It has been shown recently that inhibiting this protein in the nervous system in mice can lead to blockage of oligodendrocyte maturation, which supports myelin repair. Interestingly, our laboratory found GPR17 in thrombocytes. However, we do not know whether it has any function in thrombocyte aggregation or the nature of the ligand. In this paper, we studied the role of GPR17 in hemostasis, which is a fundamental defense mechanism in the event of injury. Using zebrafish as a model system, our laboratory has studied specifically thrombocytes, which play a significant role in hemostasis. The major reasons to use zebrafish as a model system are that their thrombocytes are functionally equivalent to human platelets, the adult fish are amenable to knockdown experiments, and they are readily available in the market. This study was performed by using a piggy back knockdown method where we used a chemical hybrid of control morpholino and an antisense oligonucleotide sequence leads to the degradation the mRNA for GPR17. After knockdown GPR17 in thrombocytes, the percent difference of the thrombocytes aggregation between the control and knockdown blood samples was measured by flow cytometry. We used various thrombocyte agonists to study differences in aggregation between the control and knockdown blood samples. The study showed that knockdown of GPR17 resulted in no significant differences in percent thrombocyte aggregation between control and agonist treated samples except for a slight increase in collagen-treated samples. Thus, it appears that GPR17 has no significant role in hemostasis.
Date: December 2015
Creator: Bohassan, Maruah Hejey

Studies in Trypsin as an Alarm Substance in Zebrafish

Description: Previous studies have shown that fish release alarming substances into the water to alert their kin to escape from danger. In our laboratory, we found that zebrafish produce trypsin and release it from their gills into the environment when they are under stress. By placing the zebrafish larvae in the middle of a small tank and then placing trypsin at one end of the tank, we observed that the larvae moved away from the trypsin zone and almost to the opposite end of the tank. This escape response was significant and did not occur in response to the control substances, bovine serum albumin (BSA), Russell's viper venom (RVV), and collagen. Also, previously, we had shown that the trypsin could act via a protease-activated receptor-2 (PAR2) on the surface of the cells. Therefore, we hypothesized that trypsin would induce a change in neuronal activity in the brain via PAR2-mediated signaling in cells on the surface of the fish body. To investigate whether the trypsin-responsive cells were surface cells, we generated a primary cell culture of zebrafish keratinocytes, confirmed these cells' identity by specific marker expression, and then incubated these cells with the calcium indicator Fluo-4 and exposed them to trypsin. By using calcium flux assay in a flow-cytometer, we found that trypsin-treated keratinocytes showed an increase in intracellular calcium release. To test whether PAR2 mediates the escape response to trypsin, we treated larvae with a PAR2 antagonist and showed that the trypsin-initiated escape response was abrogated. Furthermore, par2a mutants with knockdown of par2a by the piggyback knockdown method failed to respond to trypsin. Trypsin treatment of adult fish led to an approximately 2-fold increase in brain c-fos mRNA levels 45 mins after trypsin treatment, suggesting that trypsin signals may have reached the brain, probably via a spinothalamic pathway. Taken together, our ...
Date: August 2018
Creator: Alsrhani, Abdullah Falleh