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Setting-up an in vitro model to study the signaling mechanisms associated with intestine regeneration in the sea cucumber Holothuria glaberrima
(2019-12)
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....
Decreased hyperpolarization-activated cation current (Ih): a response mechanism to reduce cocaine-induced excitability in VTA DA neurons.
(2022-06-09)
The hyperpolarization-activated cation current (I<sub>h</sub>) is a determinant of intrinsic excitability in dopaminergic neurons (DA) of the ventral tegmental area (VTA). I<sub>h</sub> is a slowly activating cation inward current triggered by hyperpolarization. When elicited I<sub>h</sub> depolarizes the membrane to threshold for the generation of action potentials. Our laboratory has previously demonstrated that cocaine sensitization, a chronic cocaine administration model, significantly reduces I<sub>h</sub> amplitude in VTA DA neurons (Arencibia-Albite et al., 2012). Despite this current reduction, the spontaneous firing of VTA DA cells remained similar to control animals. The role of I<sub>h</sub> in controlling VTA DA excitability is poorly understood. Our hypothesis was that I<sub>h</sub> reduction could play a role as a homeostatic controller which compensates for cocaine-induced change in excitability. Our main goal was to understand how I<sub>h</sub> contributes to VTA DA neuronal excitability. In our first aim, we tried to elucidate if I<sub>h</sub> reduction can alter basal VTA DA neuronal excitability. We blocked I<sub>h</sub> and evaluated firing properties, such as rebound spiking, using whole-cell patch-clamp electrophysiology from naïve rats. Rebound spiking is defined as the production of one or more action potentials in response to the cessation of a prolonged hyperpolarizing current step. We also determined the effect of I<sub>h</sub> blockade on in vitro spontaneous excitability using cell-attached configuration from naïve rats. We hypothesized that I<sub>h</sub> blockade will reduce rebound and spontaneous firing of VTA DA cells. It was found that I<sub>h</sub> blockade can decrease evoked and spontaneous firing activity of VTA DA neurons. In addition, we evaluated the effect of I<sub>h</sub> blockade on spontaneous firing patterns using single-unit extracellular recordings from naïve rats. We found that I<sub>h</sub> blockade can decrease evoked and spontaneous firing activity, while increasing the interspike interval of VTA DA cells.<br /> <br /> To investigate how this current modulates cocaine-dependent excitability, we postulated that I<sub>h</sub> reduction serves as a homeostatic regulator (or controller) to oppose cocaine-induced excitability. We measured I<sub>h</sub> current using whole-cell patch-clamp electrophysiology, 2 and 24 hours after an acute cocaine injection. We found a progressive reduction of I<sub>h</sub> starting 24 hours after acute cocaine administration. Thus, this progressive decline observed from acute to chronic cocaine administration could serve as a homeostatic mechanism to reduce cocaine enhanced excitability. In addition, we quantified the increase in rebound action potentials, 2 and 24 hours after an acute cocaine injection. We found that after 2, 24 hours or seven days of cocaine administration, there is a significant increase in rebound action potentials of VTA DA neurons. Interestingly, there is a higher number of rebound action potentials after 2 hours of cocaine in comparison to 24 hours. When ZD, an I<sub>h</sub> blocker was perfused, there was a significant reduction in rebound action potentials. The progressive I<sub>h</sub> reduction may be a key factor in the decrease of rebound action potentials found from 2 to 24 hours after cocaine injections. The effect of I<sub>h</sub> blockade was also evaluated on in vitro spontaneous excitability using cell-attached configuration, 2 hours after an acute cocaine injection. When perfused with ZD, the spontaneous firing activity was also reduced, while significantly increasing the interspike interval providing further evidence in favor of an I<sub>h</sub> acting as a homeostatic regulator of VTA DA cell excitability.<br /> <br /> The overall cocaine effect can be influenced by the environmental context in which the drug is experienced (Caprioli et al., 2007). One question we addressed is how exposure to cocaine in a novel context interferes with chronic alterations on synaptic potentiation and neuronal excitability in the mesolimbic system (MLS). To evaluate this question an acute cocaine injection was administered to subjects in both a novel context or their home-cage. We found a significant increase in the AMPA-to-NMDA receptor-mediated EPSC ratio of VTA DA neurons in both the novelty and the homecage groups. Cocaine experienced in a novel environment produced a higher degree of synaptic potentiation. In addition, we found that I<sub>h</sub> blockade can also diminish the acute cocaine-induced increase in spontaneous firing activity in in vivo anesthetized rats. Again, the reduction in firing was correlated with an increase in the inter-spike interval.<br /> <br /> Lastly, we evaluated if I<sub>h</sub> can function as a homeostatic regulator of intrinsic excitability and can be modulated when an altered firing activity in VTA DA cells is present. We enhanced VTA DA excitability pharmacologically and recorded changes in evoked rebound action potentials and I<sub>h</sub>. VTA brain slices from naive rats were incubated with bicuculline methiodide (BIC) as a mechanism to enhance excitability and promote rebound excitation through GABAergic disinhibition. Our hypothesis was that I<sub>h</sub> will be decreased as a consequence of the pharmacological enhancement of firing activity. We found that BIC-induced excitability facilitates a reduction in I<sub>h</sub> conductance. Furthermore, I<sub>h</sub> blockade can significantly reduce this enhanced BIC-induced excitability.<br /> <br /> It has been reported that high frequency stimulation of the ventral subiculum (HFSvSUB) elicits in vivo persistent hyperactivity of VTA DA neurons (Glangetas et al. 2015). We elucidated the effect of I<sub>h</sub> blockade on HFSvSUB induced potentiation on in vivo spontaneous excitability. We hypothesized that I<sub>h</sub> blockade will significantly decrease HFSvSUB-induced firing patterns on VTA DA cells. We demonstrated that I<sub>h</sub> blockade significantly reduces firing activity after HFSvSUB. Additionally, I<sub>h</sub> blockade also significantly increased the interspike interval. These results suggest that I<sub>h</sub> is an intrinsic mechanism that can modulate the excitability of VTA DA neurons after an hyperexcitable state is present. <br /> <br /> The study of the regulatory role of this current can provide novel insights on how changes in intrinsic neuronal properties might establish cellular homeostasis in the VTA DA system and in conditions of altered excitability such as substance use disorder (SUD)....
A cross-talk between Transient Receptor Potential Vanilloid 1 and Cannabinoid Receptor 1 within the limbic system regulates depression and stress-induced anxiety-like behavior in rats.
(2022-05-08)
Clinical studies provide strong evidence that stress is an environmental risk factor that can trigger the onset of several neuropsychiatric disorders such as anxiety and depression in humans. Pre-clinical evidence suggests ...
Mechanisms of alcohol-induced sleep dysregulation in Drosophila melanogaster
(2022-06-06)
Alcohol consumption is known to disturb a variety of biological processes that affect normal physiological function. In the nervous system, alcohol is generally known to affect several molecular targets leading to an overall suppression of neuronal activity. As alcohol influences the nervous system, the organism produces a series of neuroadaptive changes that help restore neuronal homeostasis and that may lead to alcohol tolerance, dependence and ultimately addiction. Alcohol abuse can also disturb a number of biological processes, including a disruption of normal sleep patterns. As both alcohol addiction and sleep regulation are under homeostatic control, we hypothesize that these processes share a common mechanism. In this thesis, I explored the effect a set of circadian cells known as pdf neurons have in alcohol sensitivity and tolerance, using Drosophila melanogaster as a biological model. I found that these cells are important for the development of alcohol tolerance. Afterwards, I studied the effect alcohol has on sleep in general and found that alcohol affects not only sleep parameters but also the architecture of sleep and morning anticipation. Finally, we focused on understanding the role PDF neurons have in alcohol-induced sleep behaviors. As a result, we found that PDF neurons are important molecular mediators of alcohol-sleep interactions to occur....
Phytoestrogen coumestrol as an anti-cancer therapy against triple-negative inflammatory breast cancer
(2022-05-25)
Coumestrol (Cou) is a phytoestrogen present in soy and clover sprouts which is structurally similar to estrogen (E2) and has a cytotoxic effect in breast cancer cell lines. Nevertheless, the molecular mechanism by which ...
Fungi in paleomicrobiological samples reveal the flora and diets of ancient Caribbean cultures
(2022-09-01)
The gut microbiome plays essential functions in human health. Environmental disruptions such as changes in diet or lifestyle can exert a significant effect on a population’s microbiomes, resulting in several diseases. The study of the ancient microbiota preserved in archaeological samples (paleomicrobiology) is a window for characterizing these possible changes. Recent advances advocate for the consideration of the human microbiome while studying the evolution of humans. However, while more efforts have been made to incorporate microbiome in the evolution of humans, only bacterial communities have been evaluated, whilst fungal communities have been neglected. In addition to the fungal component, regional and temporal variations in dietary habits remain to be defined.<br />
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In this thesis, one of the missing pieces of the puzzle is considered: the mycobiome. Metagenomics approaches were applied for characterizing the fecal mycobiome in thousand-year-old coprolites from pre-Columbian Caribbean cultures, and to elucidate the diets and lifestyles of two pre-Columbian cultures, i.e., the Huecoid and Saladoid, prior to the arrival of Europeans. For this purpose, ancient DNA in coprolites retrieved from the pre-Columbian Huecoid and Saladoid deposits in Vieques, Puerto Rico were analyzed using shotgun metagenomic sequencing. In addition, ancient DNA sequences from the Huecoid and Saladoid coprolites were compared with those detected in coprolites from other ancient cultures, as well as extant feces from more modern cultures. To date, relatively little is known about the Huecoid and Saladoid ethnic groups and their cultural heritage.<br />
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The Saladoid gut mycobiome exhibited a higher alpha-diversity than that of the Huecoid. This result is further supported by the well-distributed relative abundance of fungal genera in the Saladoid coprolites compared to the Huecoid coprolites. The gut mycobiome of the Huecoid and Saladoid coprolites was similar at the phylum level, with Ascomycota representing the most abundant phyla, followed by Basidiomycota and Mucoromycota. However, the gut mycobiome composition at the genus level was highly different between the Huecoid and Saladoid coprolites, and the former resembled the ancestral gut mycobiome of Mexico. The gut mycobiome's α-diversity, as well as the composition and structure, distinguished the ancient and extant populations, with the pre-Columbian cultures harboring a lower total diversity and higher relative abundance of Aspergillus spp., whereas the extant populations were enriched with Mucor spp. and Malassezia spp. Despite differences in diet and lifestyles, certain fungal genera were present in most of the samples. Overall, these results suggest that the gut mycobiome reflects changes related to modern lifestyles. DNA from plants and phytopathogenic fungi from coprolites also showed that the Huecoid and Saladoid exhibited preferences in food items. The diet of the Huecoid culture included sweet potato, chili peppers, peanuts, and maize, and the edible maize smut, Ustilago spp., was likely consumed as well. In contrast, the Saladoid culture consumed chili peppers and papaya, and likely chewed tobacco (or ingested it in some way), for its narcotic and hallucinogenic effects. However, the Huecoid and the Saladoid diets were significantly more similar to each other than to the diets of present-day cultures. These results suggest that present-day diets diverge from ancient diets due to different available nutritional flora, social environments, and historical periods.<br />
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Our work revealed the gut mycobiome and dietary practices of pre-Columbian cultures, uncovered an unprecedented link between human lifestyles and ethnicity, and the diversity and composition of the gut mycobiome and diet. Results further support differences in diet and lifestyles among pre-Columbian Caribbean cultures (the Huecoid and Saladoid) with similar ecological conditions before the Spanish conquest, and these dietary differences were linked to shifts in the gut mycobiome. We demonstrate and emphasize that DNA sequence data from coprolites complement archaeological data and provide information otherwise impossible to obtain....
Using a native insect species and its neuromuscular system as models for assessing water toxicity in Puerto Rico: Chironomus sp. "Florida" (Diptera: Chironomidae)
(2022-09-16)
Different species of chironomids have been used as laboratory models to detect toxicity in aquatic environments. This is achieved by studying the response of different molecular and morphological markers in the larvae of exposed animals. The use of cell markers is rarely applied to assess toxicity within these animal models, but their implementation could help to assess toxicity in a more cost-effective way and to detect toxicity before morphological responses appear. With the finality to contribute to the field of toxicology and to emphasize the advantages of using cellular markers to assess toxicity, this thesis seeks to determine the efficiency of the Chironomus neuromuscular junction (NMJ) as a cellular marker. In order to evaluate the NMJ as a marker, the larvae of a chironomid species native to Puerto Rico, Chironomus sp."Florida";, was studied. First, the life cycle of this chironomid was described and a rearing protocol was presented to manage the larvae under laboratory conditions. Then, the neuromuscular system of the last larval stage of this chironomid was described in order to identify a model NMJ. Once the model NMJ was identified, its effectiveness as a marker of toxicity was evaluated by exposing larvae to high doses of two toxic aquatic pollutants: dibutyl phthalate and lead. The obtained results in this work indicates that the Chironomus NMJ is an excellent model for assessing toxicity since quantifiable changes at the number of boutons, muscle area, and density of boutons were observable when the larvae were exposed to contaminants. It is hoped that all the descriptions provided in this thesis will encourage the development of additional studies in the field of toxicology, but also in the field of neuroscience and comparative biology....
Protein-DNA interactomes of NKX2-5 and TBX5 mutations associated to Congenital Heart Defects
(2022-12-21)
Congenital Heart Diseases (CHD) are the most common disease found in neonates, with over 100,000 new reported cases each year. CHDs are characterized by malformations in the heart’s chambers, walls, and great vessels, ...
Evolution and DNA-binding specificity of the SIX class of transcription factors
(2022-12-21)
Transcription factors (TF) are critical for development and cellular processes and are found in all organisms. How their DNA-binding specificity changes through time has yet to be fully understood. TF DNA-binding specificity is determined by how their DNA-binding domain (DBD) interacts with DNA. TFs are identified by the sequence homology shared with described DBDs, which allows them to be classified into families. It is accepted that similar DBDs have the same DNA-binding specificity and bind to the same sequences. However, changes in a TF can lead to changes in its DNA recognition. TFs members of the sine oculis homeobox (SIX) homeodomain family are found from sponges to humans and are considered atypical members of the homeodomain (HD) family. They regulate numerous processes and phenotypic features, from eye development in flies and humans to red color patterning in Heliconius butterflies wings to human brain development. How evolutionary related TFs diversify has yet to be fully understood, especially diversification of their DNA-binding specificity. To understand the evolutionary history of this family, we performed phylogenetic inference that placed the first SIX within Porifera and the presence of the three canonical SIX (sine oculis, optix, and six4) in Cnidaria. In addition, we observe the presence of two major groups that show that optix and six4 are more evolutionary related. To determine changes in DNA-binding specificity, we performed in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX-seq) using full length SIX TF proteins from Drosophila melanogaster, Heliconius erato, and Homo sapiens. Our data shows the majority of SIX TFs bind to the canonical binding motif (5' -TGATAC-3' ), except for six4 members, which seem to prefer (5' -TGACAC-3' ).<br />
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Interestingly, the way they bind to these motifs differs. Both sine oculis and six4 homologs require a 5' -GA dinucleotide flanking the core motif on the 5' -end. In comparison, optix related members prefer a shorter flaking region and less dependence on 5' -GA. This is interesting since optix is more evolutionarily related to six4 than to sine oculis. We also found that Heliconius erato optix can bind DNA both as a monomer and as a homodimer with a preferred spacing of 2-bp between binding sites. Using the determined DNA-binding specificity of optix, we were able to predict optix binding to cis-regulatory elements (CRE) active during wing development. optix was capable to bind to all the predicted sites, including to its own promoter. Validation of optix binding to these CREs allows to expand the search of optix gene targets and contribute to our understanding of the mechanism of wing development and red color patterns in Heliconius butterflies....
Molecular mechanisms underlying temperature-dependent synaptic growth at the Drosophila melanogaster neuromuscular junction
(2023-08-17)
There is clear evidence that Earth's temperature is rising at an unprecedented rate. While consequences on ecosystems are being extensively studied, little is known about the consequences of temperature on the nervous system of ectothermic animals. Using the stereotyped synapse found at the Drosophila neuromuscular junction (NMJ), we asked how rearing temperature (15°C, 25°C, 29°C) can affect synaptic growth. We observed an increase in the number of synaptic boutons in animals reared at higher temperature. Indeed, animals reared at 29°C had a 100% increase in synaptic growth when compared to animals reared at 15°C. Interestingly, we found that from the two motor neurons that innervating the muscle, the number of boutons from the Is motor neuron increased with temperature while the boutons from the Ib motor neuron remained constant. This result indicates that motor neurons might be differentially sensitive to the changes in temperature.<br /> <br /> We looked for the molecular mechanisms that regulate temperature-dependent synaptic growth. We identified that autophagy was important for this regulation. Autophagy mutants showed a temperature-independent undergrowth, where there was a reduction in the Is boutons, while the Ib boutons remained constant through the rearing temperatures. In addition, with the use of Lysotracker and the p62 marker, we identified that the levels of autophagy changed at different rearing temperatures. Afterwards we found that the autophagy target, Highwire (Hiw), an E3 ubiquitin ligase, is a key regulator of temperature-dependent synaptic growth. Hiw is a negative regulator of synaptic growth, and its mutation induced a temperature-independent overgrowth phenotype. In addition, we found two important MAPKKK pathways that are important for regulation of synaptic growth at 15°C and 29°C. The first is the Wallenda-P38b pathway which is important for the addition of Is boutons of animals reared at 29°C. The second is the ASK1-JNK-c-Jun pathway, which is important for suppressing synaptic growth in animals reared at 15°C. Also, we found that constitutive activation of the insulin pathway in animals reared at 15°C increases the total number of synaptic boutons, but activation of the same pathway at 25°C does not affect synaptic growth. We hypothesize that temperature-dependent synaptic growth is regulated by global mechanisms like autophagy and the ubiquitin proteosome system that regulate the activation of stress response components, like the MAP kinase pathways. By changing the rearing temperature, we have identified novel roles for signaling pathways that were previously described to regulate synaptic growth in loss-of function or gain-of-function mutant backgrounds....