The sea cucumber Holothuria glaberrima is a mighty model to study organ regeneration. Our group has described the cellular mechanisms underlying intestine re-growth in H. glaberrima and is exploring the signaling mechanisms involved in this process. One of the limitations to our studies has been the lack of suitable cell culture methodologies required to advance the regeneration studies. I have now established in vitro systems where individual cells or explants can be kept and studied. Cultured cells and explants were analyzed using various techniques that include light, fluorescence, and electron microscopy. Cells isolated by enzymatic dissociation from regenerating guts of H. glaberrima could be maintained in the right conditions for up to two weeks in vitro. Different phenotypes were identified using cellular markers established previously in our lab. However, no mature muscle cells or neurons were found, which is not suitable to perform studies about cell dedifferentiation/redifferentiation. Additionally, isolated cells showed deficient proliferative activity. For that reason, I characterized gut explants as an alternative. Remarkably, the histological characteristics, including the presence of mature muscle cells and the proliferative rate, in cultured explants resembled more accurately the in vivo conditions compared to the dissociated cultures. Thus, I decided to use gut explants to gain new insights about the signaling mechanisms associated with the intestine regeneration in H. glaberrima.<br /> <br /> Initial in vivo studies using small molecules that are putative disruptors of the Wnt pathway supported the involvement of the Wnt pathway on intestine regeneration. These studies showed that iCRT14, a Wnt pathway inhibitor, decreased the size of the regenerating intestine, while LiCl, a Wnt pathway activator, increased its size. The possible cellular mechanisms by which the signaling disruptors affected the gut rudiment size were further studied in vitro using additional pharmacological agents. Among them, those that inhibited the GSK-3 enzyme (a component of the Wnt pathway) were found to increase muscle cell dedifferentiation. However, these agents also induced a reduction in cell proliferation, suggesting that cell dedifferentiation can be decoupled from cell proliferation during intestinal regeneration.<br /> <br /> Interestingly, the drop of cell proliferation in explants was also caused by small molecules that block the Wnt pathway in other points different to GSK-3. For that reason, I propose that GSK-3 is the mediator of the cellular dedifferentiation response and that it takes place by a signaling pathway that is independent of Wnt. Differently, cellular proliferation appears to be controlled by the canonical Wnt pathway during intestine regeneration. My results open the door for future studies where the signaling pathways involved at each regeneration stage can be determined during intestine regeneration....

Adolescence is a transitional stage characterized by unique neurobiological changes in the brain. The brain's maturation during this stage, particularly in the frontal cortex and the limbic system, is responsible for ...

Nuclear protein localization-3 (Npl3) is a highly dynamic protein involved in mRNA biogenesis. Previous studies showed that the npl3-95 allele encodes a protein that can aggregate into high molecular weight complexes. Besides, two different bioinformatics analysis scored Npl3 as a potential prion protein in yeast. Npl3 protein was characterized as a novel prion-like protein in Saccharomyces cerevisiae using genetic and biochemical criteria. Results presented in this doctoral dissertation show that; (1) Npl3 harbors a putative prion domain in its RGG domain, (2) the paromomycin sensitivity phenotype associated with the npl3-95 mutant allele is dominant and shows non-mendelian inheritance (cytoplasmic inheritance), (3) GndHCl treatment and HSP104 deletion eliminate the paromomycin sensitivity phenotype, and (4) over-expression of the Npl3 wild type protein induces prion-like aggregates. Interestingly, the presence of the Npl3 prion-like form confers the acquisition of an adenine prototrophy phenotype and a decrease in flocculation in the yeast Saccharomyces cerevisiae. Altogether, the results presented in this dissertation provide new evidence of the biological significance of yeast prions and their regulatory role in gene expression....

Coffee is the second largest export product of developing countries and is one of the ten most important cultivated crops in Puerto Rico, especially in the mountainous region. The coffee berry borer (CBB) Hypothenemus hampei Ferrari (1967), which arrived in Puerto Rico in 2007, is considered the most destructive pest in all coffee producing areas worldwide. The control of CBB is an enormous challenge because this insect spends most of its life inside the coffee berry, even, the use of insecticides has proved ineffective. Therefore, the development and implementation of biological control alternatives, as part of integrated pest management (IPM), is very important for the sustainability of the coffee industry. In this dissertation, I combine the spatial distribution of suitable habitats for CBB, detection of entomopathogenic nematodes (EPNs), a potential biocontrol agent and determining soil factors that affect the natural occurrence of EPNs in any given site. Species distribution model models (Chapter II) was performed using a total of 97 (241 sites sampled) georeferenced CCB presence and nineteen bioclimatic variables and altitude. Distribution maps were generated illustrating the suitable area for coffee berry borer in Puerto Rico. CBB distribution is favored by precipitation of wettest quarter, highest altitude and precipitation seasonality. In order to validate the model result, field percent of infestation was also calculated by the total of borer berries divided on total of berries in selected 3 branches in each sample site and positive relationship between model suitable index and field infestation of CBB was found. Furthermore, we sampled a total of 32 farms throughout the coffee production area in Puerto Rico using the insect bait method to extract EPNs (Chapter III). A total of 143 EPN isolates were recovered. I also made the first ix report of the presence of the recently recognized as an EPN Oscheius myriophila which was identified, based on molecular and morphometrical analysis, in 90.85% of the sample site. Finally, each nematode sampled site was characterized, firstly, according to coffee agroecosystem (full sun or under shade) and second, by soil physicochemical characteristics (Chapter IV). Using a generalized linear model (GLM), I determine the interaction of soil parameters such as pH, texture and elevation coupled with shade coffee to predict O. myriophila occurrence. In conclusion, dissertation provides a basic framework to develop integrated pest management for CBB integrating better knowledge about its preferences in climatic condition in order to prevent outbreak and the use of native EPN O. myriophila....

The current position of the islands that make up the Caribbean Region, such as the Greater Antilles, the Lesser Antilles and the Bahamas, was a very dynamic one. Our interest in understanding the evolution of anuran communication becomes more important due to the fact these species within the Puerto Rican Bank have been isolated from each other by physical barriers for approximately 8,000- 10,000 years by the rising sea levels after the last glacial maximum, creating different niches augmenting the diversity in the Archipelago; which provides a privileged location to study evolution in a microscale for many taxa and amphibians. This study focuses in anuran communication, of two species: Eleutherodactylus coqui and Eleutherodactylus antillensis. Common species such as the ones under study gives the opportunity to explore variation across populations, these species can be found from the lowest elevations to the highest. The fact that these species cover many habitats expose them to different environmental temperatures. This variation in temperature if its beyond specific thermal thresholds may constrain physiological processes associated with sound production to such an extent that calling behavior is inhibited. To better understand these patterns, we studied two main components: acoustic communication and the anatomical features of the larynx. The results varied among species, each population's calls differ not only in acoustical parameters such as frequency and duration, but also the sound producing organ was variable within populations across the island and body size was not correlated with laryngeal morphology. Furthermore, acoustic frequencies varied with laryngeal size and shape....

Worldwide, ~3,400 species of snakes are recognized. These reptiles are an important component of tropical and temperate ecosystems because of their role as high level predators. Many snake species worldwide are threatened by various stressors. Particularly vulnerable are those that inhabit the Antilles due to the tropical climate of the Islands, topographical conditions, dense human populations, habitat loss, plus fear and misconceptions due to cultural traditions. The Puerto Rican racer (Borikenophis portoricensis) is a species that inhabits the Puerto Rican archipelago and shares many similarities with other Antillean colubrids. Thus, this species has the potential to be used as a model for evaluating stressors that affect colubrid populations in the Antilles. In the first chapter of this work I present the Spanish validation and subsequent use of an instrument designed to measure fear and knowledge relating snakes. I found a low but significant negative correlation between the knowledge and fear of these reptiles, suggesting that fear of snakes can be somewhat mitigated with knowledge. I also found that women have greater fear of snakes, but that males and females did not differ in their knowledge about these animals. In addition, people who had handled or manipulated a snake had less fear and more knowledge of these reptiles. In the second chapter, I present the results of a study in which cameras traps were used to assess the presence and potential threat of invasive species in three secondary karst forests in northern Puerto Rico that differed in anthropogenic activity, and where the Puerto Rican racer was common. Four invasive and one native predators were detected by the cameras, with rats being the most abundant. Significant differences were found between the forests in the detection of vertebrates, including invasive species. The third chapter presents the results of a study in which I used radiotelemetry to measure movement patterns, habitat and microhabitat use, and home range size of male and female racers in the dry/cold and rainy/hot seasons. I found significant seasonal differences between males and females in movement and home range size. Males exhibited larger home ranges in the dry/cold season than during the wet/hot season and their home ranges were larger than females in both seasons. Females were found hidden more frequently than males, were detected more often in leaf litter on the crests or tops of the mogotes. Males spent more time moving or basking and preferred fallen branches and tree roots than females. In conclusion, invasive predators are a threat to the Puerto Rican racer and, more harsh and prolonged dry seasons, as predicted by climate change models for the Caribbean, could negatively affect the survival of males and mating success. Finally, fear of snakes can be reduced by knowledge and handling of live snakes....

Historical legacies can be important determinants of the structure, composition and dynamics of secondary forests recovering from agricultural conversion. Tracing the effects of historical processes on secondary tropical ...

Although neurons are long-lived cells little is known about the mechanisms responsible for maintaining their properties and cellular stability. Here, we investigate the pair-rule transcription factor Gooseberry (Gsb), ...

Global climate is changing at unprecedented rates, causing detrimental effects on vulnerable organisms and forcing them to cope with unfavorable conditions. Ectotherms often adapt to changing abiotic variables by altering their distribution ranges. Amphibians are known to alter their distribution because of rising temperatures. In Puerto Rico we have observed a range-shift to higher elevations in one species of direct-developing frog. Here we considered two species that occur sympatrically in part of their range at El Yunque tropical montane reserve. One species, Eleutherodactylus portoricensis, is presently limited to high elevations above 700 m.a.s.l., although it was previously found down to 400 m.a.s.l. Contrastingly, its congeneric E. coqui, can be found at all elevations (0-1,300 m.a.s.l.). Our objective was to characterize both species&rsquo; thermo-physiological profiles and question if the observed range-shift to higher elevations of E. portoricensis was associated to higher physiological sensitivity. We evaluated critical thermal minima and maxima, and thermal preference for three populations: E. portoricensis (850m), and low (180m) and highland (850m) E. coqui. We also evaluated dehydration stress during jumping performance by reducing 5% of their body mass using dry-air chambers. Lastly, we modeled E. portoricensis current and future distribution range under various scenarios of climate change. Our results show that E. portoricensis has a narrower thermal tolerance range and prefers lower temperatures when given a gradient. Also, E. portoricensis showed lower performance levels than E. coqui when dehydrated. Our results suggest that higher physiological sensitivity may explain elevational range contraction on this species. In addition, distribution models show a decrease in occurrence probability later this century in comparison to current projections. Our models also predict a range contraction towards higher elevations. This range shift would most likely result in the extirpation of most E. portoricensis populations. This research highlights the vulnerability of an endemic tree frog in Puerto Rico as a consequence of anthropogenic climate change and the urge to develop effective conservation efforts....

Pathogenesis often involves the formation of biofilms and adaptation to low oxygen environments to survive in the human body. Aspergillus fungi are opportunistic pathogens that can form biofilms and adapt to microenvironments that vary in oxygen. However, interactions between biofilms and low oxygen environments are not well understood, The first chapter, we studied biofilm formation in vitro by A. fumigatus, A. niger, A. flavus, and A. terreus in microaerophilic conditions. All species formed biofilms in microaerophilic conditions. In the second chapter, we compared biofilm formation vs. planktonic cells of Aspergillus spp. in microaerophilic vs. aerobic conditions and compared resistance to Amphotericin B in different conditions. A. flavus was more resistant to AMB than A. fumigatus in microaerophilic conditions. In the third chapter, we studied the roles of melanin in biofilm formation and virulence. We evaluate biofilms formation in microaerophilic vs. aerobic conditions in A. fumigatus mutants deficient in pyomelanin (hppd) and DHN-melanin (alb1). Melanin was not crucial for biofilm formation. The alb1 mutants showed increased metabolic activity in a microaerophilic environment. Also, the alb1-mutant was more virulent in Drosophila melanogaster than several wild type strains of A. fumigatus. These data support why aspergillosis infections are challenging to eradicate....