Epigenetic regulation in neural crest development : role of DNA methyltransferases 3A and 3B

The neural crest is a group of migratory, multipotent stem cells that play a crucial role in many aspects of embryonic development. This uniquely vertebrate cell population forms within the dorsal neural tube but then emigrates out and migrates long distances to different regions of the body. These cells contribute to formation of many structures such as the peripheral nervous system, craniofacial skeleton, and pigmentation of the skin. Why some neural tube cells undergo a change from neural to neural crest cell fate is unknown as is the timing of both onset and cessation of their emigration from the neural tube. In recent years, growing evidence supports an important role for epigenetic regulation as a new mechanism for controlling aspects of neural crest development. In this thesis, I dissect the roles of the de novo DNA methyltransferases (DNMTs) 3A and 3B in neural crest specification, migration and differentiation. First, I show that DNMT3A limits the spatial boundary between neural crest versus neural tube progenitors within the neuroepithelium. DNMT3A promotes neural crest specification by directly mediating repression of neural genes, like Sox2 and Sox3. Its knockdown causes ectopic Sox2 and Sox3 expression at the expense of neural crest territory. Thus, DNMT3A functions as a molecular switch, repressing neural to favor neural crest cell fate. Second, I find that DNMT3B restricts the temporal window during which the neural crest cells emigrate from the dorsal neural tube. Knockdown of DNMT3B causes an excess of neural crest emigration, by extending the time that the neural tube is competent to generate emigrating neural crest cells. In older embryos, this resulted in premature neuronal differentiation. Thus, DNMT3B regulates the duration of neural crest production by the neural tube and the timing of their differentiation. My results in avian embryos suggest that de novo DNA methylation, exerted by both DNMT3A and DNMT3B, plays a dual role in neural crest development, with each individual paralogue apparently functioning during a distinct temporal window. The results suggest that de novo DNA methylation is a critical epigenetic mark used for cell fate restriction of progenitor cells during neural crest cell fate specification. Our discovery provides important insights into the mechanisms that determine whether a cell becomes part of the central nervous system or peripheral cell lineages.

Photo Degradation of Cotnaminants of Emerging concern (CECs) under Simulated Solar Radiation: Implications for their Environmental Fate

Contaminants of emerging concern (CECs) are continuously being released into the environment mainly because of their incomplete removal in the sewage treatment plants (STPs). The CECs selected for the study include antibiotics (macrolides, sulfonamides and ciprofloxacin), sucralose (an artificial sweetener) and dioctyl sulfosuccinate (DOSS, chemical dispersant used in the Deepwater Horizon oil spill). After being discharged into waterways from STPs, photo degradation is a key factor in dictating the environmental fate of antibiotics and sucralose. Photodegradation efficiency depends on many factors such as pH of the matrix, matrix composition, light source and structure of the molecule. These factors exert either synergistic or antagonistic effects in the environment and thus experiments with isolated factors may not yield the same results as the natural environmental processes. Hence in the current study photodegradation of 13 CECs (antibiotics, sucralose and dicotyl sulfosuccinate) were evaluated using natural water matrices with varying composition (deionized water, fresh water and salt water) as well as radiation of different wavelengths (254 nm, 350 nm and simulated solar radiation) in order to mimic natural processes. As expected the contribution of each factor on the overall rate of photodegradation is contaminant specific, for example under similar conditions, the rate in natural waters compared to pure water was enhanced for antibiotics (2-11 fold), significantly reduced for sucralose (no degradation seen in natural waters) and similar in both media for DOSS. In general, it was observed that the studied compounds degraded faster at 254 nm, while when using a simulated sunlight radiation the rate of photolysis of DOSS increased and the rates for antibiotics decreased in comparison to the 350 nm radiation. The photo stability of the studied CECs followed the order sucralose > DOSS > macrolides > sulfonamides > ciprofloxacin and a positive relationship was observed between photo stability and their ubiquitous presence in natural aquatic matrices. An online LC-MS/MS method was developed and validated for sucralose and further applied to reclaimed waters (n =56) and drinking waters (n = 43) from South Florida. Sucralose was detected in reclaimed waters with concentrations reaching up to 18 µg/L. High frequency of detection (> 80%) in drinking waters indicate contamination of ground waters in South Florida by anthropogenic activity.

Conjugated polymer nanoparticles for biological labeling and delivery

Cancer remains one of the world’s most devastating diseases, with more than 10 million new cases every year. However, traditional treatments have proven insufficient for successful medical management of cancer due to the chemotherapeutics’ difficulty in achieving therapeutic concentrations at the target site, non-specific cytotoxicity to normal tissues, and limited systemic circulation lifetime. Although, a concerted effort has been placed in developing and successfully employing nanoparticle(NP)-based drug delivery vehicles successfully mitigate the physiochemical and pharmacological limitations of chemotherapeutics, work towards controlling the subcellular fate of the carrier, and ultimately its payload, has been limited. Because efficient therapeutic action requires drug delivery to specific organelles, the subcellular barrier remains critical obstacle to maximize the full potential of NP-based delivery vehicles. The aim of my dissertation work is to better understand how NP-delivery vehicles’ structural, chemical, and physical properties affect the internalization method and subcellular localization of the nanocarrier. ^ In this work we explored how side-chain and backbone modifications affect the conjugated polymer nanoparticle (CPN) toxicity and subcellular localization. We discovered how subtle chemical modifications had profound consequences on the polymer’s accumulation inside the cell and cellular retention. We also examined how complexation of CPN with polysaccharides affects uptake efficiency and subcellular localization. ^ This work also presents how changes to CPN backbone biodegradability can significantly affect the subcellular localization of the material. A series of triphenyl phosphonium-containing CPNs were synthesized and the effect of backbone modifications have on the cellular toxicity and intracellular fate of the material. A mitochondrial-specific polymer exhibiting time-dependent release is reported. Finally, we present a novel polymerization technique which allows for the controlled incorporation of electron-accepting benzothiadiazole units onto the polymer chain. This facilitates tuning CPN emission towards red emission. ^ The work presented here, specifically, the effect that side-chain and structure, polysaccharide formulation and CPN degradability have on material’s uptake behavior, can help maximize the full potential of NP-based delivery vehicles for improved chemotherapeutic drug delivery.^

Women Creators: Artistry and Sacrifice in the Novels of Virginia Woolf

This thesis examines different facets of feminine artistry in Virginia Woolf's novels with the purpose of defining her conception of women artists and the role sacrifice plays in it. The project follows characters in "Mrs. Dalloway," "To the Lighthouse," and "Between the Acts" as they attempt to create art despite society's restrictions; it studies the suffering these women experience under regimented institutions and arbitrary gender roles. From Woolf’s earlier texts to her last, she embraces the uncertainty of identity, even as she portrays the artist’s sacrifice in the early-to-mid twentieth century, specifically as the creative female identity fights to adapt to male-dominated spaces. Through a close-reading approach coupled with biographical and historical research, this thesis concludes that although the narratives of Woolf's novels demand the woman artist sacrifice for the sake of pursuing creation, Woolf praises the attempt and considers it a crueler fate to live with unfulfilled potential.

Real-time Biosensor for the Assessment of Nanotoxicity and Cancer Electrotherapy

Knowledge of cell electronics has led to their integration to medicine either by physically interfacing electronic devices with biological systems or by using electronics for both detection and characterization of biological materials. In this dissertation, an electrical impedance sensor (EIS) was used to measure the electrode surface impedance changes from cell samples of human and environmental toxicity of nanoscale materials in 2D and 3D cell culture models. The impedimetric response of human lung fibroblasts and rainbow trout gill epithelial cells when exposed to various nanomaterials was tested to determine their kinetic effects towards the cells and to demonstrate the biosensor’s ability to monitor nanotoxicity in real-time. Further, the EIS allowed rapid, real-time and multi-sample analysis creating a versatile, noninvasive tool that is able to provide quantitative information with respect to alteration in cellular function. We then extended the application of the unique capabilities of the EIS to do real-time analysis of cancer cell response to externally applied alternating electric fields at different intermediate frequencies and low-intensity. Decreases in the growth profiles of the ovarian and breast cancer cells were observed with the application of 200 and 100 kHz, respectively, indicating specific inhibitory effects on dividing cells in culture in contrast to the non-cancerous HUVECs and mammary epithelial cells. We then sought to enhance the effects of the electric field by altering the cancer cell’s electronegative membrane properties with HER2 antibody functionalized nanoparticles. An Annexin V/EthD-III assay and zeta potential were performed to determine the cell death mechanism indicating apoptosis and a decrease in zeta potential with the incorporation of the nanoparticles. With more negatively charged HER2-AuNPs attached to the cancer cell membrane, the decrease in membrane potential would thus leave the cells more vulnerable to the detrimental effects of the applied electric field due to the decrease in surface charge. Therefore, by altering the cell membrane potential, one could possibly control the fate of the cell. This whole cell-based biosensor will enhance our understanding of the responsiveness of cancer cells to electric field therapy and demonstrate potential therapeutic opportunities for electric field therapy in the treatment of cancer.

By-catch and discarding practices in five Algarve (southern Portugal) metiers

The catches and discards of trawlers, seiners and trammel netters were studied in the Algarve (southern Portugal) using observers onboard commercial fishing vessels. Species diversity was high, with 236 species recorded as occasionally, frequently or regularly discarded. Mean discard rates per trip were 0.13, 0.20, 0.27, 0.62 and 0.70, respectively, for trammel nets, demersal purse seines, pelagic purse seines, fish trawls and crustacean trawls, with high variability in terms of discard volume and discard rate, This was especially so for se ncrs where from 0 to 100% of the total catch might be discarded. No significant relationships were found between discard quantity or discard rate and characteristics of the fishing vessels sampled within each metier (total length, TAB, hp, kW). Based on the above discard rates and the official landings, it was estimated that in 1996, Algarve trawlers discarded 9000-13000 tonnes (t) while seiners discarded 5500-8200 t. Discarding practices in these fisheries are reviewed and the reasons for discarding are presented by species and metier basis. The results support the need for more studies on the factors contributing to discarding, variability in discard quantities and rates, the fate of discards and their importance to the marine ecosystem, and on ways of reducing by-catch and discarding in these fisheries.

Fate of discards from deep water crustacean trawl fishery off the south coast of Portugal

Trawling for crustaceans takes place on the south coast of Portugal at depths between 200 and 800 m. Large amounts of discards are released back to sea, reaching the bottom in the general area where trawling occurs. The objective of this work was to study the time taken for decomposition of the discards, to identify the most important scavenging species involved, and to evaluate the impact on the species targeted by the fishery. We conducted a series of 22 trials, using traps baited with samples of the discards. The time of immersion varied between 1 and 40 h. The amount of tissue removed from the discards was evaluated on a qualitative scale of 1–3, and quantitatively. The species caught in the traps were identified. Considerable consumption of the bait had occurred after 5 h of immersion. After 24 h of immersion only fish bones were left; past 40 h, consumption was complete. The conger eel (Conger conger) was the most common fish species present in the traps. Two species, the amphipod Scopelocheirus hopei and the isopod Natatolana (Cirolana) borealis, were identified as most important for the recycling of organic matter in the system. The stomach contents of a sample of species caught in trawls were analysed for the presence of small scavengers. Preliminary results show that S. hopei and N. (C.) borealis are part of the diet of some of the target species of this fishery, such as the Norway lobster, Nephrops norvegicus and the rose shrimp Parapenaeus longirostris.

The representation of the northeastern migrant: interstices and culture in the figure of Francisco da Silva in "Liberdade", by Ruth Laus

It serves as the object of analysis of this article the representation of the northeastern migrant in the figure of the male character Francisco da Silva, protagonist of the short story "Liberdade", by Ruth Laus, as a fictional character who lives "in an interrogatory, interstitial space between the act of representation [...] and the presence of community itself" (Bhabha, 1998, p. 22) where he comes to enter and to settle. Chico, as he is known, embarks on a crowded truck, and his fate, like that of many other migrants, is the Southeast region of Brazil, where, between São Paulo and Rio de Janeiro, he chooses the sea. To the same extent, beyond the concept(s) of culture(s), and the need to (re)think the concept of [cultural] human community - here adding the cultural term to the proposal of Bhabha (1998), as a way of thinking about the role of the individual - are taken and analyzed, also, the cultural identity concepts based on the difference itself. It is thought in what way, then, and through what imposing character, the wealth and the values ​​of the Northeast, although managed to be spread on national home soil, yet not are conspicuous by their guarantee of permanence of its inhabitants in their homeland.