19 research outputs found

    Establishment of Larval Zebrafish as an Animal Model to Investigate <em>Trypanosoma cruzi</em> Motility <em>In Vivo</em>

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    Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique's relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal

    F-Spondin/spon1b Expression Patterns in Developing and Adult Zebrafish

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    F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b) promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF). F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate

    Veronica Akle

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    https://nsuworks.nova.edu/cnso_alumni/1000/thumbnail.jp

    Anatomical and functional significance of F-spondin and the role of circadian factors in adult neurogenesis in zebrafish

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    Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [email protected]. Thank you.Adult neurogenesis occurs in specific niches in the brain. Despite major interest in this process, many questions remain regarding the factors that regulate neurogenesis and maintain the stem cell niches. This dissertation investigates the contribution of two different factors: the extracellular matrix (ECM), with a focus on F-spondin, and the circadian system, with a focus on the hormone melatonin. Studies on the localization of F-spondin mRNA expression throughout zebrafish lifespan were conducted using a transgenic zebrafish line, in which GFP expression is under control of the f-spondin promoter. The results revealed that (1) F-spondin is expressed in critical areas involved in CNS patterning during development (e.g., notochord and floorplate) and in all sixteen adult neurogenic niches of zebrafish brain, suggesting a role in maintaining or modulating optimal microenvironment for developing neurons; (2) F-spondin is expressed by CNS neurons with long projections (e.g., habenula neurons), potentially releasing the protein at the axon terminals and stabilizing their distal synaptic connections through perineuronal nets; (3) F-spondin is expressed by diverse CSF-contacting neurons that sense and/or contribute to the contents of the CSF, thus potentially serving as a long-range signaling molecule, and (4) F-spondin is present in constantly renewing peripheral structures (e.g., pharyngeal teeth) and in actively regenerating tissue (e.g., caudal fin, following amputation). Assessment of the temporal 24-h pattern of F-spondin mRNA expression revealed a nighttime peak. Together, these data suggest an important role for F-spondin, and its periodic variation, in patterning and maintenance of neuronal and peripheral tissues during development and adulthood in the zebrafish. Studies addressing the circadian control of neurogenesis in adult zebrafish brain used both BrdU incorporation and quantitative RT-PCR approaches to study daily variation in cell division and expression of genes regulating the cell-cycle. The results demonstrated that (1) neurogenesis has a daily cycle, with an increased number of cells in S-phase during the night; (2) the exact timing of peak proliferation is location-dependent, and (3) melatonin promotes cell proliferation. The BrdU-based data was consistent with the expression pattern of several cell-cycle regulatory genes (eye/ins A2, B2, D, E), which undergo daily variation, with high mRNA levels present at night. This work, for the first time, demonstrates that F-spondin is important beyond developmental stages and further study is needed to elucidate its function in neurogenic niches of vertebrates. It also demonstrates that the cell-cycle regulatory genes and the rate of adult neurogenesis in zebrafish are controlled by circadian factors, including melatonin

    Investigation of Genetic Connectivity Among Marine and Freshwater Populations of the Atlantic Stingray (Dasyatis Sabina)

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    The Atlantic stingray is one of the few elasmobranchs to have conquered freshwater, establishing the only known permanent batoid population in the rivers and lakes of North America. The purpose of this project is to study the breeding and migration patterns of D. sabina over evolutionary time scales. It is hypothesized that the Florida freshwater populations were isolated in the St. Johns river basin when sea level fell during the Pleistocene. A major question we will address is whether marine and fresh water populations have been genetically isolated since that time. We are collecting mitochondrial DNA control region sequences from D. sabina in the following three locations: Florida west coast (Tampa Bay; marine animals), Florida east coast (Melbourne; marine animals), and the St. Johns River basin (fresh water animals). Comparing genetic variation wit hin and among these populations will reveal the population history and allow inferences on the extent of genetic connectivity among D.sabina from these environments. Using PCR protocols developed at NSU’s Oceanographic Center, we have successfully sequenced approximately 720 bases from three individuals from each geographic location. More animals are being sequenced to determine the utility of the chosen locus for population genetic studies. Our results will reveal the extent of genetic diversity present in D. sabina, and also have direct conservation and management applications because this stingray has been targeted as an environmental indicator species for anthropogenic factors in the St. Johns River basin system

    Patient-Derived Xenotransplant of CNS Neoplasms in Zebrafish: A Systematic Review

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    9 páginasGlioblastoma and neuroblastoma are the most common central nervous system malignant tumors in adult and pediatric populations. Both are associated with poor survival. These tumors are highly heterogeneous, having complex interactions among different cells within the tumor and with the tumor microenvironment. One of the main challenges in the neuro-oncology field is achieving optimal conditions to evaluate a tumor’s molecular genotype and phenotype. In this respect, the zebrafish biological model is becoming an excellent alternative for studying carcinogenic processes and discovering new treatments. This review aimed to describe the results of xenotransplantation of patient-derived CNS tumors in zebrafish models. The reviewed studies show that it is possible to maintain glioblastoma and neuroblastoma primary cell cultures and transplant the cells into zebrafish embryos. The zebrafish is a suitable biological model for understanding tumor progression and the effects of different treatments. This model offers new perspectives in providing personalized care and improving outcomes for patients living with central nervous system tumors

    Toxicity of Modified Magnetite-Based Nanocomposites Used for Wastewater Treatment and Evaluated on Zebrafish (<i>Danio rerio</i>) Model

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    Magnetite-based nanocomposites are used for biomedical, industrial, and environmental applications. In this study, we evaluated their effects on survival, malformation, reproduction, and behavior in a zebrafish animal model. Nanoparticles were synthesized by chemical coprecipitation and were surface-functionalized with (3-aminopropyl) triethoxysilane (APTES), L-cysteine (Cys), and 3-(triethoxysilyl) propylsuccinic anhydride (CAS). All these nanocomposites were designed for the treatment of wastewater. Zebrafish embryos at 8 h post-fertilization (hpf) and larvae at 4 days post-fertilization (dpf) were exposed to the magnetic nanocomposites Fe3O4 MNP (magnetite), MNP+APTES, MNP+Cys, MNP+APTES+Cys, and MNP+CAS, at concentrations of 1, 10, 100, and 1000 µg/mL. Zebrafish were observed until 13 dpf, registering daily hatching, survival, and malformations. Behavior was tested at 10 dpf for larvae, and reproduction was analyzed later in adulthood. The results showed that the toxicity of the nanocomposites used were relatively low. Exploratory behavior tests showed no significant changes. Reproduction in adults treated during development was not affected, even at concentrations above the OECD recommendation. Given the slight effects observed so far, these results suggest that nanocomposites at the concentrations evaluated here could be a viable alternative for water remediation because they do not affect the long-term survival and welfare of the animals

    <i>Spon1b:GFP</i> expression in the habenular nuclei and their projections.

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    <p><b>A-C.</b> Dorsal view of live zebrafish showing migration of ventral habenular nuclei from lateral to medial position: 3 dpf (B), 20 dpf (C), and 2 mpf (D). Asterisk indicates the position of the eye. <b>D.</b> Schematic of DCP in adult zebrafish showing areas of <i>spon1b:GFP</i> expression (E and F) and depicting the habenula (Hb), its afferent projections within the stria medullaris (sm), its efferent projection: fasciculus retroflexus (FR), and target nuclei: interpeduncular nucleus (NIn), and superior raphe (SR). <b>E-F.</b> Para-sagittal cut through fresh-frozen adult Tg(<i>spon1b</i>-GFP) brain. GFP fluorescence highlights all DCP structures, including vHb nuclei, FR and sm (E). Also, cells of the bed nucleus of the stria medullaris (BNSM, arrow in E), and projections to NIn and SR in F. Rostral to the left. <b>G.</b> Schematics of the relative shape and position of the <i>spon1b</i>-positive nuclei of the Hb, as shown in H-I. <b>H-L</b>. Coronal sections immunostained for GFP: rostral (H) and caudal (I) Hb, NIn (J), and rostral (K) and caudal (L) SR. Scale bars: A-B: 100 µm; D: 25 µm; H-L: 50 µm.</p
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