Le neuroblastome est cancer du jeune enfant se développant au sein du système nerveux périphérique sympathique. Cette tumeur est caractérisée par sa grande hétérogénéité clinique : allant de formes régressant spontanément aux tumeurs de haut-risque, réfractaires aux traitements les plus agressifs. La survie à long terme des patients présentant un neuroblastome de haut-risque reste par ailleurs inférieure à 50%, ce qui souligne la nécessité de trouver de nouveaux traitements afin d’améliorer leur prise en charge thérapeutique.Récemment, en définissant le paysage épigénétique des cellules de neuroblastome, nous avons observé la présence de super-enhancers (SE). La caractérisation du paysage des SE dans les lignées de neuroblastome nous a permis de révéler l’hétérogénéité cellulaire du neuroblastome, composée de deux identités distinctes : noradrénergique et mésenchymateuse. Chacune des identités cellulaires est caractérisée par un circuit de régulation transcriptionnelle (CRC) : les facteurs PHOX2B, HAND2 et GATA3 définissent l’identité noradrénergique alors que les facteurs de la famille AP-1 gouvernent l’identité mésenchymateuse. Nous avons par ailleurs montré la différence de sensibilité aux chimiothérapies classiquement utilisées en clinique entre ces deux types cellulaires, avec une résistance accrue des cellules mésenchymateuses.Mon travail de thèse porte sur la caractérisation du rôle des facteurs de transcription PHOX2B et GATA3 dans l’établissement et le maintien de l’identité noradrénergique des cellules de neuroblastome. J’ai réalisé leur knock-out par CRISPR-Cas9 dans la lignée noradrénergique SH-SY5Y. L’inactivation de PHOX2B ne modifie ni le programme transcriptionnel ni le phénotype des cellules, arborant une identité noradrénergique. En revanche, les cellules inactivées pour GATA3 possèdent un phénotype cellulaire mésenchymateux ainsi que des capacités de migration, d’invasion et de résistance aux chimiothérapies. Le knock-out de PHOX2B et GATA3 entraine une diminution de la prolifération cellulaire, traduisant le phénomène d’addiction transcriptionnelle des cellules cancéreuses. La caractérisation du paysage épigénétique des cellules inactivées pour GATA3 démontre leur reprogrammation de l’identité noradrénergique vers l’identité mésenchymateuse avec l’effondrement des SE noradrénergiques ainsi que l’acquisition de SE mésenchymateux. GATA3 est donc indispensable pour le maintien de l’identité noradrénergique in vitro.Les résultats générés lors de ma thèse montrent que les facteurs de transcription impliqués dans un même CRC possèdent des rôles distincts dans l’identité cellulaire. La caractérisation de la dynamique de reprogrammation ainsi que des facteurs impliqués dans ce processus nous permettrons de mieux comprendre les phénomènes de plasticité cellulaire à l’origine de la progression tumorale et de la rechute thérapeutique des patients. / Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system characterized by its diversity of clinical presentations from spontaneous regression to highly aggressive tumors. Currently, the overall survival of high-risk neuroblastoma patients remains under 50% which highlight the need to find new therapeutic approaches to improve patient outcome.Recently, we defined the epigenetic landscape of neuroblastoma cell lines and observed the presence of super-enhancers (SE). The characterization of the SE landscape let us to define the heterogeneity of neuroblastoma cell identity with the presence of noradrenergic and mesenchymal cells. Both cell identities are governed by a core regulatory circuitry (CRC), composed by PHOX2B-HAND2-GATA3 in the noradrenergic cells and by AP-1 transcription factors in the mesenchymal cells. We also demonstrate the different behaviors of the cells regarding chemotherapy treatments with a higher resistance of the mesenchymal cells.My thesis aimed at deciphering the role of PHOX2B and GATA3 transcription factors in the establishment and the maintenance of the noradrenergic identity of neuroblastoma cells. To do this, PHOX2B and GATA3 were knock-out by CRISPR-Cas9 in the noradrenergic SH-SY5Y cell line. PHOX2B knock-out has no major impact neither on the transcriptomic profile nor the phenotype of the cells. PHOX2B knock-out cells still maintain their noradrenergic identity. In contrast, GATA3 knock-out cells harbor a mesenchymal phenotype showing higher ability to migrate, invade and being pore resistant to chemotherapy than control SH-SY5Y cells. Both PHOX2B and GATA3 knock-out decrease the SH-SY5Y cell proliferation in vitro and in vivo, which highlight the transcriptional dependency of the noradrenergic cells for their identity-related transcription factors. The characterization of the epigenetic landscape of GATA3 knock-out cells revealed their reprograming from the noradrenergic to the mesenchymal identity with the loss of noradrenergic SE and the acquisition of mesenchymal SE. These results demonstrate that GATA3 is essential for the maintenance of the noradrenergic identity in vitro.Altogether, these results show that transcription factors involved in a CRC can have distinct role in the cell identity. The characterization of the reprogramming dynamics as well as the factors involved in this process will allow us to better understand the cellular plasticity involved in the tumor progression and patient relapse.

In the field of neuroscience, being in love and feeling romantically attached to a partner is described as a dynamic process. Romantic love may be viewed as a motivational system, changing throughout time and place, fluctuating on the interest and motivation of the individual. Early memories and attachment towards a caregiver, lay the foundation for later attachment behavior, also known as attachment styles. In this thesis, an exploratory approach is present. The thesis aims to introduce and describe the neural correlates of romantic love and romantic attachment. Brain regions concerned with reward, emotion and thought processing, such as the reward circuitry network of the brain and the limbic system, are being investigated. So are other brain areas involved in romantic love and romantic attachment. Research findings suggest that brain areas responsible for affection, emotional control, learning, memory and social judgment are all involved in the complex processes of being in love and feeling romantically attached. These findings are represented by the involvement of the frontal lobe, cerebral cortex, limbic system, orbitofrontal cortex, and hippocampus, anterior cingulate cortex (ACC), ventral tegmental area (VTA), caudate tail, including the reward pathways of the brain. Distribution and regulation of neurotransmitters such as; vasopressin, oxytocin, dopamine, corticosterone and serotonin are all present in the state of romantic  attachment and romantic love. Overlapping evidence confirms the involvement of the reward circuitry network, together with the limbic system as crucial in the formation and maintenance of a romantic relationship.

The ability of animals to sense, interpret, and respond appropriately to social stimuli in their environment is essential for identifying and distinguishing between members of their own species. In mammals, social interactions both within and across species play a key role in determining if an animal will live to pass on its genes to the next generation or else be removed from the gene pool. The result of this selection pressure can be observed in specialized neural circuits that respond to social stimuli and orchestrate appropriate behavioral responses. This highly conserved network of brain structures is often referred to as the Social Behavior Network (SBN). The medial amygdala (MeA) is a central node in the SBN and has been shown to be involved in transforming information from olfactory sensory systems into social and defensive behavioral responses. Previous research has shown that individual neurons in the MeA of anesthetized mice respond selectively to different chemosensory social cues, a characteristic not observed in its upstream relay, the accessory olfactory bulb (AOB). However, the cause of this stimulus selectivity in the MeA is not yet understood. Here, I hypothesize that a subpopulation of neurons in the MeA that express the enzyme aromatase are involved in the sensory representation of social stimuli in awake, behaving animals. To test this hypothesis, I designed and built a novel behavioral apparatus that allows for discrete presentations of social stimuli in a highly controllable and reproducible environment. I then injected the adeno-associated virus (AAV) AAV-Syn-Flex-GCAMP6s into the MeA of Aromatase:Cre transgenic mice and implanted a fiber optic cannula slightly above the injection site. The combination of this transgenic mouse line and conditional AAV caused GCaMP6s expression to be exclusive to aromatase-expressing neurons. By coupling my novel behavioral apparatus to a fiber photometry system, I successfully recorded the moment-to-moment activity of aromatase neurons in the MeA of awake, behaving animals as they investigated various social stimuli. Aromatase neurons in the MeA of adult male mice respond strongly to conspecific social stimuli, including live adult mice, mouse pups, and mouse urine samples. Sniffing and investigative behaviors correlated strongly with increased GCaMP6s signal in aromatase neurons, reflecting increases in their neural activity. Interestingly, after repeated investigations of the same stimuli the activity of aromatase neurons gradually diminished. Presenting a novel stimulus following repeated investigations of a familiar stimulus reinstated some, but not all of the initial GCaMP6s signal. This points to the potential role that aromatase neurons may play in the habituation to social stimuli that are consistently present in their environment. Investigations of predator stimuli did not evoke significant responses from aromatase neurons, nor did investigations of non-social stimuli. These results demonstrate that aromatase expressing neurons in the MeA of awake, behaving animals encode the sensory representation of conspecific social stimuli, and their responses are highly selective to the type of stimulus presented.

The addition of powered components for active assays into paper-based analytical devices opens new opportunities for medical and environmental analysis in resource-limited applications. Current battery designs within such devices have yet to adopt a ubiquitous circuitry material, necessitating investigation into printed circuitry for scalable platforms. In this study, a microfluidic battery was mated with silver-nanoparticle conductive ink to prototype an all-printed sensing platform. A multi-layer, two-cell device was fabricated, generating 200 μA of direct electrical current at 2.5 V sustained for 16 minutes with a power loss of less than 0.1% through the printed circuitry. Printed circuitry traces exhibited resistivity of 75 to 211 10-5 Ω m. Resistance of the printed traces increased upwards of 200% depending on fold angle and directionality. X-ray diffraction confirmed the presence of face-centered cubic silver after sintering printed traces for 30 minutes at 150°C in air. A conductivity threshold was mapped and an ink concentration of 0.636 μL mm-3 was identified as the lower limit for optimal electrical performance.

Visual attention improves sensory processing, as well as perceptual readout and behavior. Over the last decades, many proposals have been put forth to explain how attention affects visual neural processing. These include the modulation of neural firing rates and synchrony, neural tuning properties, and rhythmic, subthreshold activity. Despite the wealth of knowledge provided by previous studies, the way attention shapes interactions between cortical layers within and between visual sensory areas is only just emerging. To investigate this, we studied neural signals from macaque V1 and V4 visual areas, while monkeys performed a covert, feature-based spatial attention task. The data were simultaneously recorded from laminar electrodes disposed normal to cortical surface in both areas (16 contacts, 150 μm inter-contact spacing). Stimuli presentation was based on the overlap of the receptive fields (RFs) of V1 and V4. Channel depths alignment was referenced to laminar layer IV, based on spatial current source density and temporal latency analyses. Our analyses mainly focused on the study of Local Field Potential (LFP) signals, for which we applied local (bipolar) re-referencing offline. We investigated the effects of attention on LFP spectral power and laminar interactions between LFP signals at different depths, both at the local level within V1 and V4, and at the inter-areal level across V1 and V4. Inspired by current progress from literature, we were interested in the characterization of frequency-specific laminar interactions, which we investigated both in terms of rhythmic synchronization by computing spectral coherence, and in terms of directed causal influence, by computing Granger causalities (GCs). The spectral power of LFPs in different frequency bands showed relatively small differences along cortical depths both in V1 and in V4. However, we found attentional effects on LFP spectral power consistent with previous literature. For V1 LFPs, attention to stimuli in RF location mainly resulted in a shift of the low-gamma (∼30-50 Hz) spectral power peak towards (∼3-4 Hz) higher frequencies and increases in power for frequency bands above low-gamma peak frequencies, as well as decreases in power below these frequencies. For V4 LFPs, attention towards stimuli in RF locations caused a decrease in power for frequencies < 20 Hz and a broad band increase for frequencies > 20 Hz. Attention affected spectral coherence within V1 and within V4 layers in similar way as the spectral power modulation described above. Spectral coherence between V1 and V4 channel pairs was increased by attention mainly in the beta band (∼ 15-30 Hz) and the low-gamma range (∼ 30-50 Hz). Attention affected GC interactions in a layer and frequency dependent manner in complex ways, not always compliant with predictions made by the canonical models of laminar feed-forward and feed-back interactions. Within V1, attention increased feed-forward efficacy across almost all low-frequency bands (∼ 2-50 Hz). Within V4, attention mostly increased GCs in the low and high gamma frequency in a 'downwards' direction within the column, i.e. from supragranular to granular and to infragranular layers. Increases were also evident in an ‘upwards’ direction from granular to supragranular layers. For inter-areal GCs, the dominant changes were an increase in the gamma frequency range from V1 granular and infragranular layers to V4 supragranular and granular layers, as well as an increase from V4 supragranular layers to all V1 layers.

It is of interest to understand how new neurons incorporate themselves into the existing circuitry of certain neuronal populations. One such population of neurons is that which are born in the subventricular zone (SVZ) and migrate to the olfactory bulb where they differentiate into granule cells. Another area of interest is the role of brain-derived neurotrophic factor (BDNF) on the survival and overall health of these neurons. This study aimed to test whether or not BDNF is a survival factor for adult-born granule cells. Here were utilized a transgenic mouse model over-expressing BDNF under the α- calcium/calmodulin-dependent protein kinase II (CAMKIIα) promoter, and tested its effect on olfactory granule cells under sensory deprived conditions. Results from this experiment indicated that there was no significant difference in cell death or cell survival when comparing transgenic and wild type animals. We concluded that BDNF is not a survival factor for adult-born granule cells. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

How are neuromodulatory networks organised to adapt sensory discrimination for different contexts? I hypothesised that neurons within a sensory circuit express different neuromodulatory receptors for differential modulation. Here I aimed to use the simple and genetically amenable Drosophila larval Mushroom Body (MB) calyx, a higher order processing area involved in learned odour discrimination, as a model to map octopamine (OA) neuromodulatory circuitry. I first identified olfactory projection neurons (PNs), a GABAergic feedback neuron and cholinergic extrinsic neurons as putative postsynaptic partners to OA neurons in the MB calyx using GFP reconstitution across synaptic partners. Next, I used novel EGFP-tagged OA receptors generated from recombination-mediated cassette exchange with MiMIC insertions in receptor genes to visualise endogenous expression patterns of OA receptors. Most notably, this is the first report of α2-adrenergic-like OA receptor localisation in any insect. For the first time, I showed that the α1-adrenergic-like OAMB localised to PN presynaptic terminals in the calyx; while Octβ1R localised diffusely in the calyx, resembling the innervation pattern of MB neuron dendrites. I detected EGFP-tagged Octα2R and Octβ2R in some PN cell bodies but not in neuron terminals - suggesting that Octα2R and Octβ2R may be expressed in some PNs, provided the misfolded fusion proteins are retained in the cell bodies of the neurons they are normally expressed in. Furthermore, I found that Octα2R and GABAAR fusion proteins localised to OA cell bodies but not to neuronal terminals, suggesting that OA neurons are subjected to inhibition, again given that these are not artefacts of the fusion proteins. To obtain tools to study OA modulation in the larval calyx, I then confirmed the expression patterns of driver lines that more specifically labelled calyx-innervating OA and extrinsic neurons, and tested the efficacy of three OAMB receptor knockdown lines. This initial attempt of mapping OA receptors, while subjected to further verification and development, is consistent with my hypothesis that a single neuromodulatory source can regulate multiple neuronal types in the same circuit through the distribution of different types of neuromodulatory receptors. This provides a new perspective in how the anatomical organisation of neuromodulation within a sensory network may translate to flexible outputs.

Orientador: Eduardo Tavares Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-11T02:41:16Z (GMT). No. of bitstreams: 1 Zanella_FabioPieroni_M.pdf: 8365446 bytes, checksum: c1fa57572fdaf7e48b08a6a5fb58a524 (MD5) Previous issue date: 2006 / Resumo: O ultra-som na medicina tem passado por enorme evolução nas últimas décadas e ocupado posição de destaque cada vez maior como ferramenta para terapia e diagnóstico. Isso é devido principalmente ao fato de que os equipamentos de diagnóstico por ultra-som são de relativo baixo custo, o ultra-som é uma radiação não-ionizante e permite realização de exame por método não-invasivo e as imagens são geradas e visualizadas em tempo real. Na geração de imagens deste tipo, é comum a utilização de transdutores matriciais. Entretanto, o Brasil apresenta defasagem tecnológica com respeito à construção destes transdutores e à eletrônica envolvida em sua operação. O objetivo deste trabalho consistiu no desenvolvimento de circuitos eletrônicos com 12 canais de geração e de recepção de ondas ultra-sônicas para operação com transdutor matricial linear. O sistema é capaz de excitar transdutores piezoelétricos e receber ecos ultra-sônicos na faixa de 0,5 a 30 MHz e tem seus circuitos de recepção protegidos contra a alta tensão dos pulsos gerados para a excitação do transdutor. Os disparos dos elementos do transdutor e o tempo de corte dos sinais nos circuitos de recepção, para evitar receber sinais indesejáveis referentes ao período inicial de oscilação do transdutor, são controlados via circuito com microcontrolador PIC 16F877 que, juntamente com o programa de controle, foram desenvolvidos para conectar o sistema a um microcomputador. Os 12 canais foram caracterizados eletricamente e verificou-se seu funcionamento utilizando um transdutor piezoelétrico linear de 12 elementos com 1 MHz de freqüência central, especialmente desenvolvido para este trabalho. Os resultados mostraram que o sistema funciona adequadamente, gerando imagem de um phantom construído em nosso laboratório / Abstract: Ultrasound in medicine has gone through great evolution in the last few decades and has occupied important position as a tool for therapy and diagnosis. This is due to the ultrasound equipment be of relatively low-cost, ultrasound is a non-ionizing radiation, is a non-invasive imaging method, and the images are created and seen in real time. It is common the use of transducer arrays in order to generate this kind of image. There is a lack of know how in Brazil relative to the construction of these transducers and the involved electronics in their operation. The objective of this work was the development of a multi-purpose 12 channel pulser/receiver electronic circuitry to operate with linear transducer arrays. The system is able to fire ultrasound piezoelectric transducers and to receive ultrasound echo signals in the range 0.5-30 MHz. The system has reception circuits with protection against high voltage pulses. The firing of transducer elements and cutting time of the reception circuits, to avoid unwanted signals of natural initial transducer oscillations, can be controlled via PIC 16F877 hardware and software designed to connect the system to a microcomputer. The electrical characteristics of the 12 channel pulser/receiver and its use in firing a specially constructed 1 MHz 12 element PZT transducer array has been carried out and the images of a specially constructed phantom showed that it can be used in laboratory conditions / Mestrado / Engenharia Biomedica / Mestre em Engenharia Elétrica

No description available.

Anxiety is an affective state defined by heightened arousal and unease in the absence of a clear and present fear-inducing stimulus. Chronic and inappropriate anxiety leads to anxiety disorders, the most common class of human mental disorder. Recent work suggests projections to the ventral tegmental area (VTA), are critical for anxiety behavior expression. However, the relationship between efferent VTA projections and anxiety is unclear. This thesis resolves anxiety circuitry connecting the dopaminergic (DAergic) VTA to the interpeduncular nucleus (IPN), coined the mesointerpeduncular circuit. I hypothesize the mesointerpeduncular circuit affects anxiety through the release of anxiogenic corticotropin releasing factor (CRF) during nicotine withdrawal and anxiolytic dopamine (DA) during drug naïve behavior. Electrophysiological and pharmacological data suggest CRF release from the DAergic VTA during nicotine withdrawal activates CRF receptor 1 (CRFR1) potentiating the glutamatergic activation of “Type 2” neurons and anxiety-like behavior in mice. However, in nicotine naïve conditions CRF production is negligible. Instead, in vivo DA release is anticorrelated with anxiety-like behaviors. Optogenetic stimulation and inhibition drives decreased and increased anxiety-like behaviors, respectively. Electrophysiological experiments reveal a complex interpeduncular microcircuit where D1-like DA receptor expressing “Type C” neurons in the caudal IPN (cIPN) regulate glutamatergic release in the ventral IPN (vIPN) through presynaptic GABA receptors. The result is propagation of the signal to excite “Type A” and inhibit “Type B” vIPN neurons. Finally, pharmacological activation or inhibition of interpeduncular D1-like DA receptors is sufficient to decrease and increase anxiety-like behaviors respectively. Thus, this circuit is important for modulating anxiety-like behavior.