Investigating the role of histone deacetylase HDAC4 in long-term memory formation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Manawatu, New Zealand

Listed in 2017 Dean's List of Exceptional ThesesEpigenetic mechanisms are emerging as master regulators of cognitive abilities such as learning and memory. It has been previously shown that the histone deacetylase HDAC4 plays a critical role in memory formation in both mammals and insects although the specific mechanisms through which it acts have not yet been elucidated. HDAC4 undergoes nucleocytoplasmic shuttling and, in neurons, it is largely cytoplasmic implying it may play both nuclear and non-nuclear functions. To identify upstream regulators and downstream targets of HDAC4, a genetic interaction screen was performed in the fruit fly Drosophila melanogaster, a powerful model system to study the genetic mechanisms of neurological disease. Twenty-nine genes were found to interact with HDAC4 suggesting they are part of the same molecular pathway. Functional network analysis revealed that many of the genes could be grouped into three biological categories comprising transcriptional factors, SUMOylation machinery enzymes and cytoskeletal regulators/interactors. Within the latter, Ankyrin2 was selected for further analysis as it is implicated in synaptic stability and in human intellectual disability. In addition HDAC4 harbours a conserved ankyrin binding domain. Immunohistochemical analyses showed widespread distribution of Ankyrin2 throughout the adult brain and coincident distribution with HDAC4 was observed in the axons of the mushroom body, a key structure for memory formation in flies. Both HDAC4 and Ankyrin2 were also found to regulate mushroom body development. RNAi-mediated depletion of Ankyrin2 in the adult brain impaired long-term memory in the courtship suppression assay, a model of associative memory and preliminary evidence of a physical association between HDAC4 and Ankyrin2 was also demonstrated. The genes identified in the screen provide new avenues for investigation of the mechanisms through which HDAC4 regulates memory formation and preliminary analyses suggest that interaction with the cytoskeletal adaptor Ankyrin2 may involve remodelling of the actin/spectrin cytoskeleton, phenomenon that underlies memory related processes like synaptic plasticity and neuronal excitability

Investigation of the binding between olfactory receptors and odorant molecules in C. elegans organism

Abstract The molecular mechanisms regulating the complex sensory system that underlies olfaction are still not completely understood. The compounds formed from the interaction of Olfactory Receptors (ORs) with volatile molecules play a crucial role in producing the sense of olfaction. Therefore, it is necessary to investigate the binding mechanisms between these receptors and small ligands. In this work, we focus our attention on C.elegans, this is a particularly suitable model organism because it is characterized by a nervous system composed of only 302 neurons. To study olfaction in C.elegans, we select 21 ORs from its olfactory neurons, and present a pipeline, consisting of several computational methods, with the aim of proposing a set of possible candidates for binding the selected C.elegans ORs. This pipeline introduces an approach based on the selection of templates, and threading, that takes advantage of the structural redundancy among membrane receptors. This procedure is widely replicable because it is based on algorithms that are publicly available and are freely hosted on institutional servers

The long QT syndrome

Major progress has taken place, and at a very rapid pace, in the understanding of the congenital long QT syndrome (LQTS). This has been the direct consequence of the identification of several of the genes responsible for LQTS and of the studies that have followed, at both basic and clinical levels. A key issue is represented by the fact that all LQTS genes identified so far encode for ionic channels involved in the control of repolarization. The expression studies of the mutated genes have allowed identification of the specific electrophysiologic consequences of the specific mutations and have demonstrated alterations in the NA+ and in K+ currents sufficient to explain the prolongation of action potential duration and, hence, of the QT interval. Ongoing studies in the selected LQTS patients, for whom the specific mutations are known, are allowing a unique understanding of the complex genotype-phenotype correlation. These studies indicate the existence of what appear to be gene-specific patterns in many clinically important features such as the response to therapeutic interventions, the response to increases in heart rate, and in the factors that precipitate the life-threatening arrhythmias typical of this intriguing disease

Sympathetic activation, ventricular repolarization and Ikrblockade: Implications for the antifibrillatory efficacy of potassium channel blocking agents

AbstractObjectives. The aim of the present study was to test, in vivo and in vitro, the influence of adrenergic activation on action potential prolongation induced by the potassium channel blocking agent d-sotalol.Background. d-Sotalol is not effective against myocardial ischemia-dependent ventricular fibrillation in the presence of elevated sympathetic activity. Most potassium channel blockers, such as d-sotalol, affect only one of the two components of Ik(Ikr) but not the other (Iks). Iksis activated by isoproterenol. An unopposed activation of Iksmight account for the loss of anti-fibrillatory effect by d-sotalol in conditions of high sympathetic activity.Methods. In nine anesthetized dogs we tested at constant heart rate (160 to 220 beats/min) the influences of left stellate ganglion stimulation on the monophasic action potential prolongation induced by d-sotalol. In two groups of isolated guinea pig ventricular myocytes we tested the effect of isoproterenol (10−9mol/liter) on the action potential duration at five pacing rates (from 0.5 to 2., Hz) in the absence (n = 6) and in the presence (n = 8) of d-sotalol.Results. In control conditions, both in vivo and in vitro, adrenergic stimulation did not significantly change action potential duration. d-Sotalol prolonged both monophasic action potential duration in dogs and action potential duration of guinea pig ventricular myocytes by 19% to 24%. Adrenergic activation, either left stellate ganglion stimulation in vivo or isoproterenol in vitro, reduced by 40% to 60% the prolongation of action potential duration produced by d-sotalol.Conclusions. Sympathetic activation counteracts the effects of potassium channel blockers on the duration of repolarization and may impair their primary antifibrillatory mechanism. An intriguing clinical implication is that potassium channel blockers may not offer effective protection from malignant ischemic arrhythmias that occur in a setting of elevated sympathetic activity

O conceito de corpo

The purpose of this study was to analise and understand imaginary identification of their own body’s myths,enfasising the discussions about body aesthetics. The imstrument used for data collection was a questionnaire with a seven points semantic differential scale. The subjects were 28 undergraduate students enrolled at Biology, Ecology, and Physical Education programs at UNESP - Rio Claro. The data were descriptively analised and lead to the conclusion that body aesthetics notion do not differ among the areas studied. Among the results, only the subjects from the Ecology program showed response index slightly superior from the ones from the other programs. Nevertheless, this difference was not significant. The results pattern exibited by the subjects of the three program enfatizes the mythical sense of the perfect body.Este estudo teve por objetivo analisar e refletir a percepção e conscientização que permeiam as justificativas dos sujeitos, quanto ao nível de identificação imaginária dos próprios conceitos corporais, evidenciando as discussões a respeito da temática da estética corporal. O instrumento utilizado para a coleta de dados foi um questionário contendo uma escala de diferencial semântico de sete intervalos, aplicado a 28 sujeitos, estudantes de nível superior dos cursos de Biologia, Ecologia e Educação Física, da UNESP - Rio Claro. Os dados foram analisados descritivamente e levam à conclusão de que a noção de corpo estético não difere entre as áreas relacionadas. Entre os resultados obtidos, apenas o curso de ecologia apresentou um índice ligeiramente superior ao dos outros cursos, porém esta diferença não foi significativa. O padrão apresentado pelos três cursos evidencia um conceito de sentido mítico de corpo perfeito

A Shearless microfluidic device detects a role in mechanosensitivity for awcon neuron in Caenorhabditis elegans

AWC olfactory neurons are fundamental for chemotaxis toward volatile attractants in Caenorhabditis elegans. Here, it is shown that AWC(ON) responds not only to chemicals but also to mechanical stimuli caused by fluid flow changes in a microfluidic device. The dynamics of calcium events are correlated with the stimulus amplitude. It is further shown that the mechanosensitivity of AWC(ON) neurons has an intrinsic nature rather than a synaptic origin, and the calcium transient response is mediated by TAX-4 cGMP-gated cation channel, suggesting the involvement of one or more "odorant" receptors in AWC(ON) mechano-transduction. In many cases, the responses show plateau properties resembling bistable calcium dynamics where neurons can switch from one stable state to the other. To investigate the unprecedentedly observed mechanosensitivity of AWC(ON) neurons, a novel microfluidic device is designed to minimize the fluid shear flow in the arena hosting the nematodes. Animals in this device show reduced neuronal activation of AWC(ON) neurons. The results observed indicate that the tangential component of the mechanical stress is the main contributor to the mechanosensitivity of AWC(ON). Furthermore, the microfluidic platform, integrating shearless perfusion and calcium imaging, provides a novel and more controlled solution for in vivo analysis both in micro-organisms and cultured cells

Por que dançar? um estudo comparativo

Since the very beginning man uses movement and dance. With them he comunicates. expresses himself, satisfies himself, fascinates himself, educates himself Among several dance style. ballroom dance is an activity which complexity can be addapted to individual skills. it is approachable to either gender and to all ages and it can provide situations of optimal experience. Therefore, this study had the purpose to investigate the reasons why younger people and adults practice ballroom dance. Two groups were investigated: the first group with 60 subjects (25 males and 35 females) with average age equal to 52.18 years and the second group with 38 subjects (15 males and 23 females) with average age equal to 14.76 years. The results showed that firstly satisfaction was the strongest motive that guided adults and youthful to practice ballroom dance. Secondly, the results showed ballroom dance as a good option to fulfil free time. although adults enfasyzed this aspect a lot more than adolescents. Sociability as a motive to practice ballroom dance appeared in both groups with equivalent percentages. In accordance with the investigated subjects. ballroom dance provides satisfaction and it can be considered a leisure activity that facilitates socialization. These aspects are basic to the devepment of the individual. In this way. ballroom dance is suggested not only as an informal activity but also as an activity in the school curriculum.Desde os primórdios o homem se utiliza do movimento e da dança. Com isso, ele se comunica, se expressa, se satisfaz, se encanta, se educa. Dentre os vários tipos de dança, a dança de salão é uma atividade, cuja complexidade, pode se adaptar às habilidades individuais, é acessível a qualquer sexo e faixa etária podendo proporcionar situações de experiência máxima. Portanto, este estudo teve por objetivo investigar os motivos que levam os jovens escolares e adultos a praticarem a dança de salão. Dois grupos foram entrevistados: o 1º grupo com 60 sujeitos (25 homens e 35 mulheres) de idade média igual a 52,18 anos e o 2º grupo com 38 sujeitos (15 homens e 23 mulheres) de idade média igual a 14,76 anos. Os resultados mostram que primeiramente o prazer é o motivo mais forte que leva adultos e adolescentes à prática da dança de salão. Em segundo plano, mostra a dança de salão como uma boa opção para ocupação dó tempo livre. embora os adultos enfatizem bem mais este aspecto que os jovens. A sociabilidade, como motivo para a prática da dança, aparece em ambos os grupos etários com percentagens equivalentes. De acordo com a opinião dos sujeitos pesquisados, a dança de salão promove satisfação, pode ser considerada uma atividade de lazer e favorece a sociabilização, fatores fundamentais na formação do indivíduo. Sendo assim. sugere-se a dança de salão. não apenas como atividade informal, mas também como atividade curricular

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi003-A from a patient affected by an autosomal recessive form of Long QT Syndrome type 1

Abstract We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 51 years old female patient homozygous for the mutation c.535 G>A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1), not associated with deafness. The hiPSCs, generated using four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC, are pluripotent and can differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs)

The Mice Drawer System (MDS) Experiment and the Space Endurance Record-Breaking Mice

The Italian Space Agency, in line with its scientific strategies and the National Utilization Plan for the International Space Station (ISS), contracted Thales Alenia Space Italia to design and build a spaceflight payload for rodent research on ISS: the Mice Drawer System (MDS). The payload, to be integrated inside the Space Shuttle middeck during transportation and inside the Express Rack in the ISS during experiment execution, was designed to function autonomously for more than 3 months and to involve crew only for maintenance activities. In its first mission, three wild type (Wt) and three transgenic male mice over-expressing pleiotrophin under the control of a bone-specific promoter (PTN-Tg) were housed in the MDS. At the time of launch, animals were 2-months old. MDS reached the ISS on board of Shuttle Discovery Flight 17A/STS-128 on August 28th, 2009. MDS returned to Earth on November 27th, 2009 with Shuttle Atlantis Flight ULF3/STS-129 after 91 days, performing the longest permanence of mice in space. Unfortunately, during the MDS mission, one PTN-Tg and two Wt mice died due to health status or payload-related reasons. The remaining mice showed a normal behavior throughout the experiment and appeared in excellent health conditions at landing. During the experiment, the mice health conditions and their water and food consumption were daily checked. Upon landing mice were sacrificed, blood parameters measured and tissues dissected for subsequent analysis. To obtain as much information as possible on microgravity-induced tissue modifications, we organized a Tissue Sharing Program: 20 research groups from 6 countries participated. In order to distinguish between possible effects of the MDS housing conditions and effects due to the near-zero gravity environment, a ground replica of the flight experiment was performed at the University of Genova. Control tissues were collected also from mice maintained on Earth in standard vivarium cages