Nicotine exposure caused significant transgenerational heritable behavioral changes in Caenorhabditis elegans

Passive and active exposure to tobacco smoking among youth is directly associated with immediate as well as long-term health deterioration. Despite all public health policies and efforts, the percentage of teenage smokers is still relatively high, especially in developing countries. Very few, if any, studies have been done on the transgenerational effect of nicotine exposed during the more sensitive, early developmental stages. We employed C. elegans as a biological model to study the multigenerational impact of chronic nicotine exposure. Nicotine treatment was limited to N2 hermaphrodites of the F0 generation. Exposure was limited to the larval period L1-L4 (~31 hours) after which worms were transferred to a fresh NGM plate. N2 hermaphrodites at L4 developmental stage were used for behavioral analysis across three generations: F0, F1, and F2. Our results show that nicotine was associated with changes in sinusoidal locomotion, speed, and body bends in L4 larvae in all three tested generations. These behavioral alterations were not restricted to F0, but were observed in F1 and F2 generations which were never exposed to nicotine. Our study is the first to reveal that nicotine addiction is heritable using C. elegans as a model organism. These results underscored the sensitivity of early development stages, with hope to spread more awareness to encourage the avoidance of nicotine exposure, especially at a young age

Nicotine exposure caused significant transgenerational heritable behavioral changes in Caenorhabditis elegans

Passive and active exposure to tobacco smoking among youth is directly associated with immediate as well as long-term health deterioration. Despite all public health policies and efforts, the percentage of teenage smokers is still relatively high, especially in developing countries. Very few, if any, studies have been done on the transgenerational effect of nicotine exposed during the more sensitive, early developmental stages. We employed C. elegans as a biological model to study the multigenerational impact of chronic nicotine exposure. Nicotine treatment was limited to N2 hermaphrodites of the F0 generation. Exposure was limited to the larval period L1-L4 (~31 hours) after which worms were transferred to a fresh NGM plate. N2 hermaphrodites at L4 developmental stage were used for behavioral analysis across three generations: F0, F1, and F2. Our results show that nicotine was associated with changes in sinusoidal locomotion, speed, and body bends in L4 larvae in all three tested generations. These behavioral alterations were not restricted to F0, but were observed in F1 and F2 generations which were never exposed to nicotine. Our study is the first to reveal that nicotine addiction is heritable using C. elegans as a model organism. These results underscored the sensitivity of early development stages, with hope to spread more awareness to encourage the avoidance of nicotine exposure, especially at a young age

Three Generations, One Future: A Systematic Analysis on Nicotine's Effect across generations in C. elegans.

Tobacco smoking is a worldwide epidemic that is responsible for diseases and death rates that surpass those attributed to a combination of other causes (e.g. cancer, HIV, accidents). A major mediator of tobacco-smoke related negative consequences is nicotine. Nicotine is an addictive poison that entraps users in a vicious cycle of constant drug seeking and reinforcement. Despite the public health policies and laws enforced to decrease the habitual smoking, it is still prevalent, especially among adolescents. According to WHO, 40% of children and up to 60% of teenagers are passively and actively exposed to tobacco smoke. Early life stages are more vulnerable and sensitive to environmental and life experienced stresses. At that stage, stresses can have enduring effects that not only persist until adulthood, but are also inherited to the subsequent generations. With respect to nicotine, a wealth of studies have investigated the dose and time-dependent effects of this chemical on multiple systems including cell lines and model organisms. However, the transgenerational effect of nicotine exposed during post-embryonic stages has not been reported. On the molecular level, an increasing number of popular findings that show the involvements of certain microRNAs in physiological processes have expanded to include response to nicotine. Nevertheless, a systematic profiling of microRNA expression levels is yet to be determined. In our study, we employed C. elegans as our model to investigate the transgenerational effect of nicotine exposure limited to the post-embryonic larval stages of the parent F0 generation. Two concentrations (20[mu]M and 20mM) were chosen based on previous studies. We investigated the effect of nicotine on the behavior of L4 C. elegans (N2) across three generations (F0, F1, and F2). Here we report that nicotine altered the sinusoidal locomotion, body bends, and forward and backward speeds across three generations. Such represented an enduring and heritable addiction initiated by parental post-embryonic nicotine exposure. In addition our qRT-PCR results showed that direct nicotine exposure throughout the larval stages (30 hours), altered the systematic miRNA expression profiles in L4 C. elegans in a dose-dependent manner. Through target prediction analyses coupled with background research, fos-1 was predicted to be a key mediator of the addiction-like behavior in C. elegans larvae. Conclusively, our results offer novel insights on the sensitivity of early developmental stages to nicotine exposure. The behavioral transgenerational effect as well as the parental altered miRNA profiles will set the basis for future miRNA transgenerational analyses coupled with target and pathway validation. With this in mind, the need for suitable reference genes for normalization and reliable interpretations is necessary. We dedicated our last objective to identify reference gene candidates to serve this purpose. Based on results from five statistical approaches (geNorm, NormFinder, BestKeeper, dCt method, and RefFinder), we report that the expression levels of tba-1 and cdc-42 were the most stable among all of sixteen compiled genes. Taken together, our work is preliminary for a new research direction concerned with nicotine that would help support public health policies and awareness campaigns to further stress on the risks and dangers of tobacco addiction.M.S

Anàlisi de patrons funcional i estructurals en la regulació del calci en les cèl·lules cardíaques

Tesi sotmesa a embargament des de la data de defensa fins al dia 16 de setembre de 2022In 2019 the data published by the World Health Organization showed that the cardiac ischemia is the first cause of death worldwide. These diseases are caused by irregularities of the contractile mechanism’s function, starting from the cellular level till the organ dysfunction. To further understand the origin of these dysfunctions the thesis will focus on the molecular and cellular level. The small changes of the physiology at that level can lead to an anomalous cardiac electrophysiology. The previous studies suggest that there is a relation between the cardiac arrhythmia and the increase of global spontaneous calcium activity in cells. Ryanodine receptors have the major role in the calcium regulation. They release the calcium stored in the sarcoplasmic reticulum into the cytosol, then the calcium attaches to the actin and myosin to produce the mechanical contraction of the cell. The irregularities have been also related with an hyperphosphorylation of the ryanodine receptor channels, which increases the open probability of those. A large amount of data is generated in the biology and physiology laboratories, these have to be characterized, clustered and analysed to extract the meaning of the observed divergences. Thus, an interdisciplinary thesis has been developed in order to obtain experimental data with a confocal microscope in the laboratory and then creating new image processing tools in order to have an interpretable analysis of the experimental data. The main objective of the project is to characterize the spontaneous calcium activity through image processing tools and to observe its variations under different physiological conditions, such as the hiperphosphorylation of the ryanodine receptor channels or under cardiac arrhythmia conditions. The aim is to see how the modifications in the calcium activity affect to the propagation along the cell and report how the variations may lead to electric and contractile dysfunctions, and so, cause arrhythmias. To reach this goals the thesis is divided into three main sections or hypothesis: 1. Experimental data acquisition, define the best image acquisition method in order to develop the studies of calcium images and proposal of methods to detect subcellular structures and spontaneous calcium events. 2. Study of the calcium events substructure and the involved ryanodine receptor clusters. Comparison of the calcium activity properties of the cells at the basal level and under conditions of hyperphosphorylation or arrhythmia. 3. Study of the physiological modifications in the calcium activity through different phosphorylation pathways.The results explain the ryanodine receptor clusters distribution through the cells, allow to have the localization of the channels and the calcium activity simultaneously and show relevant details such as, the increase in the duration of the events or the increase in the volume of calcium released when more ryanodine receptor clusters are co-activated. These modifications lead to have bigger events and, thus, more potentially dangerous because can depolarize the neighbouring cells. The parameters reflect the relationship between the phosphorylation of the ryanodine receptors and the spontaneous calcium release in the arrhythmogenesis process.Segons les dades publicades per l'Organització Mundial de la Salut el 2019, la isquèmia cardíaca és la causa de mortalitat que ocupa el primer lloc a nivell mundial. Aquesta patologia és deguda a irregularitats en el funcionament del mecanisme contràctil, començant a nivell cel·lular, fins arribar a nivell d'òrgan. Per arribar a l'arrel d'aquestes disfuncions, en aquesta tesi es realitzarà un estudi a nivell cel·lular i molecular. Els petits canvis en aquest nivell poden arribar a escalar cap a una electrofisiologia cardíaca anòmala. Durant els darrers anys, els estudis de fisiologia cardíaca han relacionat les arítmies amb l'augment de l'activitat espontània de calci global a nivell cel·lular. Els principals encarregats de regular el calci en les cèl·lules dels miòcits cardíacs són els receptors de rianodina, que alliberen el calci emmagatzemat al reticle sarcoplasmàtic al citosol. Aquest s'uneix a l'actina i la miosina de la cèl·lula produint la contracció mecànica. Les irregularitats en l'activitat de calci han estat relacionades amb la fosforilació d'aquests canals, ja que un cop fosforil·lats la probabilitat d'obertura dels receptors augmenta. Als laboratoris de biologia i fisiologia es treballa amb volums relativament grans dades experimentals, que posteriorment s'han de caracteritzar, agrupar i donar un sentit als canvis observats. En vista d'aquest fet, s'ha volgut realitzar una tesi interdisciplinària, adquirint dades amb el microscopi confocal al laboratori i creant noves eines de processament d'imatges per facilitar una anàlisi interpretable de les dades experimentals. L'objectiu principal del projecte és caracteritzar l'activitat espontània del calci mitjançant tècniques de processament d’imatge i observar les variacions d'aquesta amb diferents condicions fisiològiques, com ara la fosforilació dels receptors de rianodina o amb cèl·lules de pacients amb arítmia. Veure com les modificacions en l'activitat de calci afecten a la propagació d'aquest al llarg de la cèl·lula i caracteritzar la perillositat de les variacions a l’hora d'induir un mal funcionament elèctric i contràctil i, per tant, donar a lloc arítmies. En motiu d'assolir aquest objectius la tesis es divideix en tres grans apartats o hipòtesis: 1. Obtenció de dades experimentals, definició del millor mètode per realitzar els estudis de les imatges de calci i proposta d’un mètode de detecció i quantificació d'estructures cel·lulars i esdeveniments de calci. 2. Estudi de la subestructura dels esdeveniments de calci i els clústers de receptors de rianodina involucrats. Comparació de les propietats de l'activitat de calci de les cèl·lules a nivell basal i sota condicions d'arítmia o fosforilació. 3. Estudi dels canvis electrofisiològics en l’activitat del calci emprant diferents vies de fosforilació dels canals de receptors de rianodina. Els resultats obtinguts expliquen la distribució dels receptors de rianodina en les cèl·lules, permeten tenir una visió de les activacions d'aquests canals durant l'activitat espontània de calci i mostren detalls rellevants, com ara l'augment de la durada, l'augment en el volum de calci alliberat i l'augment del nombre de clústers receptors de rianodina activats. Aquestes modificacions provoquen esdeveniments més grans i, per tant, més potencialment perillosos, ja que poden arribar a despolaritzar cèl·lules veïnes. Aquests paràmetres reflecteixen la relació entre la fosforilació dels receptors de rianodina i l'alliberament espontani de calci durant el procés d'aritmogènesi.Postprint (published version