Dissertations / Theses on the topic 'Sensory receptors'

The main aim of this project was to define the neurotrophin survival requirements of sensory neurons during the early stages of their development both in vivo and in vitro. The in vitro survival of neural crest-derived but not placode-derived cranial sensory neurons is promoted by several different neurotrophins early in their development. Neural crest-derived neurons subsequently lose responsiveness to all neurotrophins except NGF. Loss of responsiveness of neural crest-derived sensory neurons to BDNF and NT3 is associated with a marked shift in the dose responses of these neurons to higher neurotrophin concentrations. Analysis of the timing of cell death in the trigeminal ganglia of mouse embryos that are homozygous for null mutations in the TrkA, TrkB and TrkC genes which encode high affinity receptors for NGF, BDNF and NT3 respectively, show that there is an early peak of apoptosis in TrkB and TrkC knockouts which is consistent with the early survival response of trigeminal neurons to BDNF and NT3 in vitro. The elevated peak of apoptosis in TrkA knockouts occurs at the same development stages as in wild type embryos which is consistent with the later response of trigeminal neurons to NGF in vitro. Furthermore, there is a high level of expression of TrkC mRNA in early trigeminal neurons which accords with the early survival response of these neurons to NTS. It is also shown that subsets of trigeminal neurons discriminate between neurotrophins at very high concentrations during the period of cell death, indicating that neurotrophin responses can be far more highly specific than previously thought. Taken together, these results show that neurotrophin switching is a physiologically relevant phenomenon in certain populations of developing sensory neurons.

O sistema olfatório de mamífero pode discriminar milhares de odores presentes no meio ambiente. Aproximadamente 1000 diferentes receptores olfatórios (ORs) são expressos no epitélio olfatório (OE) do nariz, Os ORs detectam os odores e transmitem os sinais resultantes para o bulbo olfatório (OB) no cérebro. Os ORs pertencem a super família dos receptores acoplados a proteína G (GPCR) e apresentam sete domínios transmembrânicos putativos. Por razões desconhecidas, os ORs são retidos no retículo endoplasmático quando expressos em linhagens de células de mamíferos heterólogas. Provavelmente, proteínas acessórias sejam requeridas para o endereçamento dos Ors para a superficie celular. No presente estudo, utilizamos o OR M93 para estudar os mecanismos de expressão de um ORo A dissertação teve como objetivos específicos: (l) construção de um vetor para expressão do OR M93 em fusão com GFP em levedura e análise de sua localização celular; (2) identificar proteínas expressas no epitélio olfatório de camundongo que interajam com os ORs. A análise por microscopia de fluorescência revelou que a expressão do OR M93 fusionado a GFP demonstrou um padrão de fluorescência que sugere a retenção do OR M93 no retículo endoplasmático. Nós utilizamos o sistema de duplo híbrido em levedura para varrer uma biblioteca de cDNA de epitélio olfatório de camundongo com uma isca correspondente à região N-terminal do OR M93. Quatro proteínas candidatas foram identificadas: HLA-B associado ao transcrito 3 (BAT-3/ Scythe), superfamília transmembrana 4 (membro CD82), superfamília transmembrana 4 (membro OAP-I) e sindecan (membro SDC2) (\"GenBank accession numbers\": BC026647, D14883, BC0430n e BC047144). A análise da hibridação in situ destas proteínas, revelou que a proteína OAP-1 é a melhor candidata a interação com OR M93. Dessa maneira, nós indicamos a proteína OAP-1 como possível proteína candidata a auxiliar o OR a ser expresso de maneira funcional em sistemas heterólogos.
The mammalian olfactory system can discrim inate thousands of odorants present in the environrnent. Approximately 1000 different olfactory receptors (ORs) are expressed in the olfactory epithelium (OE) of the nose. The ORs detect odorants and transmit the resulting signals to the olfactory bulb (OB) of the brain. ORs belong to the G-protein-coupled receptor (GPCR) super family and have seven putative transmembrane domains. For unknown reasons, the ORs are retained in the endoplasmatic reticulum when expressed in heterologous mammalian cell lines. Probably accessory proteins are required for the sorting of the ORs to the cell surface. In the present work, we used the OR M93 to study the mechanisms of OR expression. Our goals were to (1) construct an expression vector for OR M93 in fusion with GFP in yeast and (2) to identify proteins expressed in the mouse OE that interact with ORs. The analysis by fluorescence microscopy suggested that OR M93 in fusion with GFP was retained in the endoplasmic reticulum (ER) of yeast. We used the yeast two-hybrid system to screen a mouse OE cDNA library with a bait corresponding to the N-terminal region ofthe üR M93. Four potential candidates were identified: HLA-B associated transcript 3 (BAT-3/Scythe), transmembrane 4 superfamily (CD82 member), transmembrane 4 superfamily (TSPN-3 member) and syndecan (SDC2). In situ hybridization analysis suggests that OAP-l protein represents the best candidate for interaction with OR M93. We suggest the OAP-l protein could be an accessory protein required for the sorting of the ORs to the cell surface in heterologous cell lines.

The GABAergic system in adult sensory cortex is affected by sensory deprivation, but little is known about how this predominant inhibitory system is affected during ontogeny. The present study investigates developmental effects of whisker trimming on GABAa receptors in rat barrel cortex. Rats trimmed for 6 wk beginning at birth and adulthood showed similar decreases in [3H]muscimol binding in deprived relative to non-deprived barrels, suggesting absence of a critical period.

The role of nerve growth factor (NGF), a neurotrophic molecule, and its high-affinity receptor in intact and injured adult rat lumbar sensory neurons was examined at a cellular level using quantitative receptor radioautography to localize the NGF high-affinity receptor-positive subpopulation, in conjunction with histochemistry on adjacent sections. The 40-50% of sensory neurons displaying NGF receptors were characterized. Virtually all neurons containing substance P or CGRP were NGF receptor-positive, but not those with somatostatin or thiamine monophosphatase activity. The ability of a neuron to bind NGF with high-affinity correlates positively with growth-associated protein (GAP43) expression but not with neurofilament (NFM) expression. Following injury, sensory neurons atrophy, lose NGF receptors, decrease NFM expression, while GAP43 expression is elevated in all neurons irrespective of their ability to bind NGF. Infusion of NGF for 1 week, at the time of injury or 3 weeks following injury counteracts NGF receptor loss, cell atrophy, and decreased NFM expression, but only in those neurons bearing NGF receptors. GAP43 expression remained high in all neurons despite infusion. NGF's function in normal sensory neurons appears to be modulatory, permitting regulation of intrinsic properties. Injury disrupts this permissive state, which can be restored with exogenous NGF.

Sensory neurons are a heterogeneous group of cells that are specialized to detect stimuli acting on the skin such as touch, temperature, pain and itch. A major challenge in the sensory biology field is to isolate and characterize specific functional subsets of neurons as an exhaustive knowledge of many of them is still lacking. In my PhD project I established a protocol to analyse and sort by Fluorescently Activated Cell Sorting (FACS) peripheral sensory neurons, in order to isolate specific subsets and further perform gene expression profiling. In particular I applied these techniques to Avil-Cre::ReteGFP mice, where Ret-positive neurons express eGFP specifically in Dorsal Root Ganglia (DRG) neurons after Cre recombination. Ret is a receptor for the GDNF family expressed in at least 3 sensory neuron subsets: non-peptidergic nociceptors, Rapidly Adapting (RA) mechanoreceptors and C-fiber low-threshold mechanoreceptors. However, our immunohistochemical analysis suggested that there might be more Ret-positive subsets. I analysed Ret-positive neurons by FACS, combining the analysis of the pattern of endogenous eGFP expression with IB4-binding arrangement and I identified 5 diverse Ret-eGFP-positive subsets. I focused on two of them, Ret-eGFPLo:IB4Neg and Ret-eGFPHi:IB4Neg that did not bind to IB4 and expressed low and high levels of eGFP, respectively. Their expression profiles suggested that Ret-eGFPHi:IB4Neg neurons represent the previously described RA Mechanoreceptors, while the Ret-eGFPLo:IB4Neg subset constitutes a new Ret-positive class of sensory neurons involved in itch perception. To verify this assumption, we functionally characterized Ret-eGFPLo:IB4Neg neurons. We focused on three molecules whose receptors we found enriched in the subset: histamine, a well known pruritogen, IL-31, a cytokine that has been linked to the pathology of Atopic Dermatitis, and LY344864, a serotonin agonist whose receptors, 5-HT1f, was among the most expressed genes within the Ret-eGFPLo:IB4Neg subset. By calcium imaging we demonstrated that these substances are able to elicit a neuronal response mainly in Ret-eGFP/IB4 negative cells. Moreover, when injected in the nape of the neck of mice, they all cause scratching, substantiating a putative role of this population as itch receptors.. My data indicate that we have discovered a new Ret-positive subset of sensory neurons involved in itch perception. A more extensive characterization of these cells, for example with a Sst-ires-Cre line which we found to specifically mark Ret-eGFPLo:IB4Neg subset, will further clarify their role within the DRG in vivo.

Sensory biology of animals is studied throughout the world for the insight it provides to understanding ecosystems and improving how we manage species. In this research, I designed experiments to investigate the sensory biology and behaviour of two Australian species of freshwater crayfish from the genus Cherax, the yabby (Cherax destructor) and redclaw crayfish (Cherax quadricarinatus). Experimental apparatus were constructed and tailored to test specific questions on physiology, tactile (touch) sensitivity, observation techniques, aggressive behaviour and responses to electrical fields. The outcomes were:
• abdominal muscle mass was positively correlated to the size of the electrical fields produced by swimming crayfish,
• behaviour changed in response to contact with different structures and textures of wall surfaces,
• computer analysis of underwater behaviour was similar to that scored by a human observer,
• the level of aggression in groups of crayfish changed as group size increased, and
• two species of crayfish responded to electrical fields in the water by decreasing their locomotory movement.
These results reveal a way in which physiology relates to behaviour, how crayfish and other crustaceans may sense the invisible and behave in aquaculture ponds, as well as documenting methodology to further investigate these areas in the future.

The neurotransmitter L-Glutamate is the primary excitatory neurotransmitter of sensory neurons in the dorsal root ganglion (DRG). These neurons may also express ionotropic glutamate receptors, causing the potential for them to be directly excited by their own release of glutamate, from a neighboring neuron, or from other tissues. Glutamate is elevated in tissues after injury or inflammation, and iGluR signaling from the periphery has been shown to increase signaling in DRG neurons and contribute to the development of chronic pain. Targeting pharmacologic intervention of sensory neuron iGluRs present in peripheral terminals may constitute an attractive alternative or augmentation to chronic pain treatment regimens. A compartmented culture system was devised to enable the co-culture of sensory neurons and keratinocyte stem cells in discrete compartments to simulate a skin tissue in vitro, and allow focal agonist application to peripheral terminals. Activation of peripheral receptors with focal agonist application caused the propagation of signals towards somata of neurons in a fluidically separated compartment, causing excitatory post-synaptic currents (EPSC) that were observed and recorded via voltage-clamped whole-cell electrophysiology. EPSC responses observed exhibited statistically significant differences between the ? values of the EPSCs after respective agonist exposure. Immunofluorescent labeling and visualization of receptor expression showed that iGluR subunits are expressed in sensory neuron somata, sensory neuron peripheral processes, non-neuronal cells from the DRG, and keratinocyte stem cells. The implementation of this co-culture clamping facilitates the spatially discrete interaction of neuronal and non-neuronal cell types for the characterization of their interfaces, as well as for the discrete application of pharmacologic agents along axons to evaluate their spatially constrained influence on activity at a cellular, and intercellular level. The spatially restricted application of agonists represents a chemotransmissive instigation of electrochemical activity in neurons for studying EPSCs, instead of electrically stimulating a presynaptic cell, and so more faithfully represents what would occur in vivo. Using this system to test novel pharmaceuticals represents an intermediary step between the study of ligand interactions with receptors and systemic administration to experimental animals. The identification of the active receptors and their subunit-specific peripheral expression yield alternative therapeutic targets for chronic pain treatment.

Thesis (M.S.)--West Virginia University, 2007.
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Bone morphogenetic proteins (BMPs) are diffusible molecules involved in a variety of cellular interactions during development. In particular, Bmp4 expression accompanies the development of the ear sensory organs during patterning and specification of sensory cell fates, and it has been shown to play a role in inner ear development and morphogenesis. However, there is no understanding of the cellular effects of BMP4 in prosensory progenitors, and about its role in the process of sensory fate specification. The present thesis project was aimed at exploring the effects of BMP-signaling on the development of hair-cells, using the chick inner ear as a model.
The specific aims proposed were:
1- Analyze the cellular effects caused by addition of BMP4 in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.
2- Analyze the cellular effects caused by inhibition of BMP4 signaling in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.
3- Analyze the expression in the innear ear of downstream targets of BMP signalling, in particular, analyse the members of Id gene family.
4- Analyze the regulation of Id genes by BMP signalling in the inner ear.
5- Analyze the expression of genes involved in the process of terminal differentiation, in particular, Btg1 and Btg2 genes
6- Analyze the regulation of Btg1 and Btg2 gene by BMP signalling in the inner ear

The sense of taste is one of the most important factors in regulating ingestive decisions. This is central to a number of disease conditions including but not limited to obesity, diabetes, anorexia, hypertension, coronary artery diseases and malnutrition. The detection of the molecules eliciting taste qualities in food is mediated by the coordinated actions of distinct types of taste sensory cells (TSCs) housed in taste buds within specialized papillae throughout the oral cavity. Taste receptors in the taste sensory cells that detect food molecules are the key players in selecting dietary nutrients. One such example is L-amino acids, a critical part of one's diet. L-glutamate is the prototypical umami compound and is known to increase palatability of food. A unique characteristic of umami taste is the response potentiation of glutamate by 5' ribonucleotide monophosphates, such as inosine 5' monophosphate (IMP), which is also capable of eliciting an umami taste. Candidate receptors for umami taste include a heterodimer T1r1+T1r3, brain variants of mGluR1 and mGluR4, and the truncated variants of mGluR1 and mGluR4. Studies using heterogeneous expression of T1r1+T1r3 suggest it is an umami and a broadly tuned L-amino acid receptor. While much attention is devoted to understanding glutamate transduction, the detection mechanisms for other L-amino acids by TSCs are less well understood. Here calcium imaging of isolated TSCs and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice was performed to determine if other receptors are involved in the detection of L-amino acids and IMP. Ratiometric imaging with Fura-2 was used to study calcium responses to IMP and four L-amino acids (monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine) with and without IMP. The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across TSCs. Only a small subset of cells responded to all stimuli. Interestingly, L-amino acids other than glutamate elicited synergistic responses in a subset of TSCs. Additionally IMP alone elicited a response in a large number of TSCs. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex. Next the roles of mGluR1 and mGluR4 in the detection of the IMP and L-amino acids were investigated. Selective agonists for mGluR1, (RS)-3, 5-dihydroxyphenylglycine (DHPG; a group I mGluR agonist), and mGluR4, L-(+)-2-amino-4-phosphonobutyric acid (L-AP4; a group III mGluR4 agonist) elicited responses in TSCs. In addition, TSCs responsive to these agonists were also responsive to L-amino acids and IMP. More importantly, selective antagonists against different mGluRs such as (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; a group I mGluR antagonist), and (RS)-α-methylserine-O-phosphate (MSOP; a group III mGluR antagonist) significantly suppressed L-amino acid- and IMP-mediated responses in TSCs of T1r3 knockout mice. Collectively, these data provide evidence for the involvement of taste and the brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the hypothesis that multiple receptors contribute to the IMP and L-amino acid tastes.

Crickets provide a useful model system to study how animals analyze sound frequency. While much is known about how sound frequency is represented by central neurons and in behavior, little is yet known about auditory receptor neurons. I investigated physiological and anatomical properties of auditory receptor fibers (ARFs) and functional organization of their axon terminals, using single-unit recording and staining techniques. Behavioral experiments suggest that crickets are sensitive to two broad frequency ranges, centered at 4--5 kHz for acoustic communication and at 25--50 kHz for predator detection. However, cricket ARFs fall into three distinct populations, based on characteristic frequency (CF; low frequency, ∼3--5.5 kHz; mid frequency, 9--12 kHz; ultrasound, ≥18 kHz). One striking characteristic of single ARFs is the occurrence of multiple sensitivity peaks at different frequencies, which implies that the wide audible range of crickets is mediated by these multiple sensitivity peaks, even though CFs of ARFs are clustered at the three small ranges. To understand how populations of ARFs code sound intensity, level-response functions are examined. Physiological parameters derived from level-response functions are diverse, and are systematically related to threshold within each population. Low-frequency ARFs comprise two distinct anatomical types, based on the distributions of axon terminals, which also differ physiologically. Thus, based on CF and anatomy, cricket ARFs can be classified into four distinct populations. To understand how information flows from peripheral to central neurons, the positions of varicosities, i.e. output sites, of ARF axon terminals are mapped on a two-dimensional coordinate system. In crickets, the ARF axon terminals are functionally organized with respect to frequency and intensity. Anatomical organization with respect to threshold is related to physiological organization, which may reduce non-linear effects in postsynaptic

The purpose of this dissertation was to gain insight into spinal sensory regulation during locomotion. To this end, I developed a novel in vitro spinal cord-hindlimb preparation (SCHP) composed of the isolated in vitro neonatal rat spinal cord oriented dorsal-up with intact hindlimbs locomoting on a custom-built treadmill or instrumented force platforms. The SCHP combines the neural and pharmacological accessibility of classic in vitro spinal cord preparations with intact sensory feedback from physiological hindlimb movements. thereby expanding our ability to study spinal sensory function. I then validated the efficacy of the SCHP for studying behaviorally-relevant, sensory-modulated locomotion by showing the impact of sensory feedback on in vitro locomotion. When locomotion was activated by serotonin and N-methyl D-aspartate, the SCHP produced kinematics and muscle activation patterns similar to the intact rat. The mechanosensory environment could significantly alter SCHP kinematics and muscle activitation patterns, showing that sensory feedback regulates in vitro spinal function. I further demonstrated that sensory feedback could reinforce or initiate SCHP locomotion. Using the SCHP custom-designed force platform system, I then investigated how presynaptic inhibition dynamically regulates sensory feedback during locomotion and how hindlimb mechanics influence this regulation. I hypothesized that contralateral limb mechanics would modulate presynaptic inhibition on the ipsilateral limb. My results indicate that contralateral limb stance-phase loading regulates ipsilateral swing-phase sensory inflow. As contralateral stance-phase force increases, contralateral afferents act via a GABAergic pathway to increase ipsilateral presynaptic inhibition, thereby inhibiting sensory feedback entering the spinal cord. Such force-sensitive contralateral presynaptic inhibition may help preserve swing, coordinate the limbs during locomotion, and adjust the sensorimotor strategy for task-specific demands. This work has important implications for sensorimotor rehabilitation. After spinal cord injury, sensory feedback is one of the few remaining inputs available for accessing spinal locomotor circuitry. Therefore, understanding how sensory feedback regulates and reinforces spinally-generated locomotion is vital for designing effective rehabilitation strategies. Further, sensory regulation is degraded by many neural insults, including spinal cord injury, Parkinson's disease, and stroke, resulting in spasticity and impaired locomotor function. This work suggests that contralateral limb loading may be an important variable for restoring appropriate sensory regulation during locomotion.

Olfaction, the sense of smell, allows animals to perceive the multitude of volatile and nonvolatile molecules present in the environment. In many mammals, such as mice and rats, there are four unique chemosensory organs including the (1) main olfactory epithelium (MOE), (2) septal organ, (3) Grüneberg ganglion, and (4) vomeronasal organ (VNO). While the VNO detects some general volatile odorants, it is further specialized for the detection of behaviorally relevant nonvolatile odorants or pheromones. In rodents, the VNO is encased within a bony capsule and located at the base of the nasal cavity. Odorants are detected by vomeronasal sensory neuron (VSN)s, bipolar neurons with a single axon that projects to the accessory olfactory bulb of the brain and a single dendrite capped with microvilli that project into the lumen of the VNO. In the MOE, purinergic signaling through adenosine 5'-triphosphate (ATP) gated ionotropic P2X and G-protein coupled P2Y receptors contributes a neuroprotective and neuroregenerative pathway. As virtually nothing was known about purinergic signaling in the VNO, I set out to characterize the (1) presence of the purinergic receptors and (2) ATP release pathways. In isolated VSNs, ATP elicited an increase in intracellular calcium ([Ca2+]I) and an inward current with similar potency. Adenosine and the P2Y receptor agonists adenosine 5'-diphosphate (ADP), uridine 5'-triphosphate (UTP), and uridine 5'-diphosphate (UDP) were ineffective. The increase in [Ca2+]I was dependent upon extracellular calcium and the inward current elicited by ATP was partially blocked by the P2X receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS) and 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP). When coapplied with the natural stimulus dilute urine, ATP increased the inward current above that elicited by either dilute urine or ATP alone. Furthermore, ADP hyperpolarized the voltage dependence of steady state inactivation of voltage activated sodium current (INa) in a subset of VSNs. The hyperpolarization in the voltage dependence of steady state inactivation elicited by ADP was blocked in the presence of suramin, a purinergic receptor antagonist, but similar to that produced by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable protein kinase C (PKC) activator. Neither ATP nor ADP affected the voltage dependence of activation, fast inactivation, or time dependent recovery from inactivation. Interestingly, ADP reversibly increased spike frequency but did not change an action potential's amplitude, latency, halfwidth, or threshold voltage. Accordingly, we detected gene expression of the P2X1 and 3 as well as P2Y1, 2, and 6 receptors in the VNO and localized the P2Y1 and 2 receptors to isolated VSNs. Thus, excitability in VSNs can be enhanced by (1) ATP eliciting an inward current through P2X receptors and (2) ADP decreasing spike adaptation during persistent firing presumably through P2Y receptors. Moreover, one possible source of ATP may be from mechanical stimulation of the VNO that accompanies vasomotor pump activation.

Receptores purinérgicos são expressos em neurônios e glia e, por certo, participam dos processos de sinalização entre estes células. Nos gânglios da raiz dorsal o soma dos neurônios sensoriais é envolto por uma camada de células satélites (CSt), que são células de glia, cuja função é desconhecida. Até o momento a natureza dos receptores purinérgicos presentes nas células satélites foi pouco investigada. No presente trabalho demonstra-se a presença dos receptores P2Y1, P2Y2 e/ou P2Y4 e P2Y6 nas CSt de gânglios da raiz dorsal de ratos recém-nascidos. A prova da existência dos receptores foi a elevação na concentração intracelular do Ca2+, induzida pela aplicação de um agonista dos receptores. Nas culturas de mais de 24 horas, estimuladas por soro bovino fetal, proliferam de forma notável células de glia de aspecto predominantemente fusiforme. Determinou-se que as células fusiformes expressam receptores P2Y1, P2Y2 e/ou P2Y4 e P2Y6. As informações obtidas contribuem para o esclarecimento da sinalização entre neurônios e glia nos gânglios sensoriais da raiz dorsal.
Purinergic receptors are expressed and play role in the sinalization between neurons and glia. In dorsal root ganglia the soma of the sensory neurons is surrounded by a layer of satellite glial cells, whose function is unclear. There are evidences that ATP is released by neurons to act on receptors in satellite cells. So far, the nature of the purinergic receptors of satellite cells was not fully investigated. This study shows the presence of metabotropics purinergic receptors P2Y1, P2Y2 and/or P2Y4, e P2Y6 in satellite cells from dorsal root ganglia of newborn rats. The demonstration was carried on following the transient increases in intracellular calcium concentration induced by a purinergic agonist. As time goes by, in the presence of fetal bovine serum, there is a remarkable proliferation of glial cells, with predominant fusiform shapes. The fusiform cells also express P2Y1, P2Y2 and/or P2Y4, e P2Y6 receptors. These informations add on the understanding of the complex phenomena of neuron-glia interaction.

Transcranial direct current stimulation (tDCS) is a non-invasive form of brain stimulation which uses a very weak constant current to temporarily excite or inhibit activity in the brain area of interest via electrodes placed on the scalp, depending on the polarity and strength of the current. Presently, tDCS is being used as a tool to investigate frontal cognition in healthy controls and to improve symptoms in neurological and psychiatric patients. Relatively little research has been conducted with respect to tDCS and the auditory cortex (AC). The primary aim of this thesis was to elucidate the effects of tDCS on auditory sensory discrimination, assessed with the mismatch negativity (MMN) event-related potential (ERP). In the first pilot study, healthy participants were assessed in a randomized, double-blind, sham-controlled design, in which participants received anodal tDCS over the primary AC (2 mA for 20 minutes) in one session and ‘sham’ stimulation (i.e. no stimulation) in the other. Pitch MMN was found to be enhanced after receiving anodal tDCS, with the effects being evidenced in individuals with relatively low (vs. high) baseline amplitudes. No significant effects were seen with sham stimulation. A second study examined the separate and interacting effects of anodal and cathodal tDCS on MMN measures. MMN was assessed pre- and post-tDCS (2 mA, 20 minutes) in 2 separate sessions, one involving sham stimulation, followed by anodal stimulation, and one involving cathodal stimulation, followed by anodal stimulation. Only anodal tDCS over the AC increased pitch MMN in baseline-stratified groups, and while cathodal tDCS decreased MMN, subsequent anodal stimulation did not significantly alter MMNs. As evidence has shown that tDCS lasting effects may be dependent on N-methyl-D-aspartate (NMDA) receptor activity, a pharmacological study investigated the use of dextromethorphan (DMO), an NMDA antagonist, to assess possible modulation of tDCS’ effects on both MMN and working memory (WM) performance. The study involved four test sessions that compared pre- and post-anodal tDCS over the AC and sham stimulation with both DMO (50 mL) and placebo administration. MMN amplitude increases were only seen with anodal tDCS with placebo administration, not with sham stimulation, nor with DMO administration. In the sham condition, DMO decreased MMN amplitudes. Anodal tDCS improved WM performance in the active drug condition. Findings from this study contribute to the understanding of underlying neurobiological mechanisms mediating tDCS-sensory and memory improvements. As cognitive impairment has been proposed to be the core feature of schizophrenia disorder (Sz) and MMN is a putative biomarker of Sz, a pilot study was conducted to assess the effects of pre- and post-tDCS on MMN measures in 12 Sz patients, as well as WM performance. Temporal, frontal and sham tDCS were applied in separate sessions. Results demonstrated a trend for pitch MMNs to increase with anodal temporal tDCS, which was significant in a subgroup of Sz individuals with auditory hallucinations, who had low MMNs at baseline. Anodal frontal tDCS significantly increased WM performance, which was found to positively correlate with MMN-tDCS effects. The findings contribute to our understanding of tDCS effects for MMN-indexed sensory discrimination and WM performance in healthy participants and individuals with Sz disorder and may have implications for treatment of sensory processing deficits in neuropsychiatric illness.

Drosophila es un modelo versátil para entender las bases genéticas de la señalización celular. En particular, el ojo compuesto de Drosophila proporciona un tejido ideal para estudiar los mecanismos de integración de señales que dirigen la formación de la red de neuronas fotorreceptoras. La vía de Ras/MAPK está involucrada en la determinación de todos los fotorreceptores, pero es particularmente necesaria para la especificación del fotorreceptor R7, ya que su ausencia determina la transformación del R7 en una célula no neural. De manera que durante el desarrollo del ojo, Ras media la decisión entre la diferenciación neural y no neural. Sin embargo, se desconoce el perfil de genes activados por Ras. Con el objetivo de describir este perfil, hemos realizado microarrays comparando diferentes alelos del receptor tirosina quinasa Sevenless (Sev) que participa en la especificación del R7 mediante la activación de la vía de Ras / MAPK. Uno de los genes que responden a la activación de Sev es el gen amyloid protein precursor-like (Appl). El incremento de la expresión de Appl detectado en los microarrays, también se confirmó por hibridación in situ. En un estudio detallado de localización de la proteína, observamos que aunque está presente en todos los fotorreceptores, hay más proteína Appl en R7 y R8, que en los otros. Para evaluar si Ras es necesario para la activación de Appl, realizamos clones con el alelo dominante negativo del gen Epithermal Growth factor Receptor (DERDN). En estos clones no se detectó Appl, mientras que los clones del alelo constitutivamente activo de Ras; RasV12 dieron lugar a la sobreexpresión de Appl. En cualquier otro tejido, RasV12 no produjo expresión ectópica, indicando que Ras es necesario y suficiente para la expresión de Appl aunque esta regulación es dependiente del dominio del ojo. Para ver si el factor de transcripción de la vía; Pnt, es capaz de unirse a Appl para activar su expresión realizamos clones con el alelo de pérdida de función pntΔ88. Estos clones mostraron que la falta de pnt tiene como resultado la ausencia de Appl. En segundo lugar evaluamos la posible unión directa de Pnt a Appl mediante transgénicos de regiones supuestamente enhancers de Appl unido a un promotor mínimo y al gen reportero lacZ. Ninguna de las construcciones fue capaz de dirigir la expresión de lacZ en ojo, sin embargo dos de ellas fueron capaces de dirigirla en cerebro, sugiriendo que estas dos secuencias actúan como unidades reguladoras de la expresión de Appl. Ya que no produjeron expresión en ojo, decidimos sensibilizar la respuesta de las construcciones a Ras, incrementando la actividad de la vía mediante clones RasV12. En este nuevo contexto, los dos ETS que tenían expresión en cerebro, fueron capaces de dirigir la expresión de lacZ. Además la unión de pnt a Appl se confirmó mediante experimentos de InmunoPrecipitación de la Cromatina (CHIP). Ras en ojo es responsable de la determinación de casi todos los fotorreceptores, sin embargo observamos que mientras que la determinación no está afectada en mutantes Appld, sí lo está el funcionamiento del R7. Este fotorreceptor es el único capaz de ver la luz ultravioleta. Mediante experimentos de comportamiento observamos que las moscas Appld tienen disminuida la capacidad de discernir la luz UV. Esta reducción es debida en parte porque el 2% de los axones de R7 de moscas Appld no llegan a hacer la sinapsis. Para tratar de incrementar los efectos observados y poder describir con mayor claridad la función de Appl en ojo, testamos la habilidad de los mutantes Appld de discernir el UV, en combinación con mutantes heterocigotos de proteínas descritas en el proceso de guía de axones. La perdida de función de Appl combinada con el mutante heterocigoto del gen neurotactina (nrt), produjo un claro deterioro de la capacidad del R7 para discernir UV y de los axones pare llegar a hacer la sinapsis demostrando que Appl es necesario para la correcta función del R7.
In a genome wide expression profile search for genes that characterize the Drosophila R7 photoreceptor specification we found Appl, the ortholog of human APP and a key factor in the pathogenesis of Alzheimer’s disease. We analyzed Appl expression in the eye imaginal disc and found that is highly accumulated in R7 photoreceptor cells. The R7 photoreceptor is responsible for UV light detection. To explore the link between high expression of Appl and R7 function, we have analyzed Appl null mutants and found reduced preference for UV light, likely due to mistargeted R7 axons. Moreover, axon mistargeting and inappropriate light discrimination are enhanced in combination with neurotactin mutants. R7 differentiation is triggered by the inductive interaction between R8 and R7 precursors, which results in a burst of Ras1/MAPK activated by the tyrosine kinase receptor Sevenless. Thus, we have studied whether Ras1/MAPK is responsible for the high Appl expression. Inhibition of Ras1 signaling leads to reduced Appl expression, whereas constitutive activation drives ectopic Appl expression. We show that Appl is directly regulated by the Ras/MAPK pathway through a mechanism mediated by PntP2, an ETS transcription factor that specifically binds ETS sites in the Appl regulatory region. Also, the zebrafish appb expression increased after ectopic fgfr activation in the neural tube of zebrafish embryos, suggesting a conserved regulatory mechanism.

ABSTRACT: Carotid bodies (CB) are peripheral chemoreceptor organs sensing changes in arterial blood O2, CO2 and pH levels. Hypoxia and acidosis or hypercapnia activates CB chemoreceptor cells, which respond by releasing neurotransmitters in order to increase the action potential frequency in their sensory nerve, the carotid sinus nerve (CSN). CSN activity is integrated in the brainstem to induce a fan of cardiorespiratory reflex responses, aimed at normalising the altered blood gases. Exogenously applied adenosine (Ado) increases CSN chemosensory activity inducing hyperventilation through activation of A2 receptors. The importance of the effects of adenosine in chemoreception was reinforced by data obtained in humans, in which the intravenous infusion of Ado causes hyperventilation and dyspnoea, an effect that has been attributed to the activation of CB because Ado does not cross blood-brain barrier and because the ventilatory effects are higher the closer to the CB it is injected. The present work was performed in order to establish the functional significance of adenosine in chemoreception at the carotid body in control and chronically hypoxic rats. To achieve this objective we investigated: 1) The release of adenosine from a rat carotid body in vitro preparation in response to moderate hypoxia and the specificity of this release. We also investigated the metabolic pathways of adenosine production and release in the organ in normoxia and hypoxia; 2) The modulation of adenosine/ATP release from rat carotid body chemoreceptor cells by nicotinic ACh receptors; 3) The effects of caffeine on peripheral control of breathing and the identity of the adenosine receptors involved in adenosine and caffeine effects on carotid body chemoreceptors; 4) The interactions between dopamine D2 receptors and adenosine A2B receptors that modulate the release of catecholamines (CA) from the rat carotid body; 5) The effect of chronic caffeine intake i.e. the continuous blockage of adenosine receptors thereby simulating a caffeine dependence, on the carotid body function in control and chronically hypoxic rats. The methodologies used in this work included: molecular biology techniques (e.g. immunocytochemistry and western-blot), biochemical techniques (e.g. neurotransmitter quantification by HPLC, bioluminescence and radioisotopic methods), electrophysiological techniques (e.g. action potential recordings) and ventilatory recordings using whole-body plethysmography. It was observed that: 1) CB chemoreceptor sensitivity to hypoxia could be related to its low threshold for the release of adenosine because moderate acute hypoxia (10% O2) increased adenosine concentrations released from the CB by 44% but was not a strong enough stimulus to evoke adenosine release from superior cervical ganglia and arterial tissue; 2) Acetylcholine (ACh) modulates the release of adenosine/5’-adenosine triphosphate (ATP) from CB in moderate hypoxia through the activation of nicotinic receptors with α4 and ß2 receptor subunits, suggesting that the excitatory role of ACh in chemosensory activity includes indirect activation of purinergic receptors by adenosine and ATP, which strongly supports the hypothesis that ATP/adenosine are important mediators in chemotransduction; 3) adenosine increases the release of CA from rat CB chemoreceptor cells via A2B receptors; 4) the inhibitory effects of caffeine on CB chemoreceptors are mediated by antagonism of postsynaptic A2A and presynaptic A2B adenosine receptors indicating that chemosensory activity elicited by hypoxia is controlled by adenosine; 5) The release of CA from rat CB chemoreceptor cells is modulated by adenosine through an antagonistic interaction between A2B and D2 receptors, for the first time herein described; 6) chronic caffeine treatment did not significantly alter the basal function of CB in normoxic rats assessed as the dynamics of their neurotransmitters, dopamine, ATP and adenosine, and the CSN chemosensory activity. In contrast, the responses to hypoxia in these animals were facilitated by chronic caffeine intake because it increased the ventilatory response, slightly increased CSN chemosensory activity and increased dopamine (DA) and ATP release; 7) In comparison with normoxic rats, chronically hypoxic rats exhibited an increase in several parameters: ventilatory hypoxic response; basal and hypoxic CSN activity; tyrosine hydroxylase expression, CA content, synthesis and release; basal and hypoxic adenosine release; and in contrast a normal basal release and diminished hypoxia-induced ATP release; 8) Finally, in contrast to chronically hypoxic rats, chronic caffeine treatment did not alter the basal CSN chemosensory activity. Nevertheless, the responses to mild and intense hypoxia, and hypercapnia, were diminished. This inhibitory effect of chronic caffeine in CB output is compensated by central mechanisms, as the minute ventilation parameter in basal conditions and in response to acute hypoxic challenges remained unaltered in rats exposed to chronic hypoxia. We can conclude that adenosine both in acute and chronically hypoxic conditions have an excitatory role in the CB chemosensory activity, acting directly on adenosine A2A receptors present postsynaptically in CSN, and acting presynaptically via A2B receptors controlling the release of dopamine in chemoreceptor cells. We suggest that A2B -D2 adenosine / dopamine interactions at the CB could explain the increase in CA metabolism caused by chronic ingestion of caffeine during chronic hypoxia. It was also concluded that adenosine facilitates CB sensitisation to chronic hypoxia although this effect is further compensated at the central nervous system.-------- RESUMO: Os corpos carotídeos (CB) são pequenos orgãos emparelhados localizados na bifurcação da artéria carótida comum. Estes órgãos são sensíveis a variações na PaO2, PaCO2, pH e temperatura sendo responsáveis pela hiperventilação que ocorre em resposta à hipóxia, contribuindo também para a hiperventilação que acompanha a acidose metabólica e respiratória. As células quimiorreceptoras (tipo I ou glómicas) do corpo carotídeo respondem às variações de gases arteriais libertando neurotransmissores que activam as terminações sensitivas do nervo do seio carotídeo (CSN) conduzindo a informação ao centro respiratório central. Está ainda por esclarecer qual o neurotransmissor (ou os neurotransmissores) responsável pela sinalização hipóxica no corpo carotídeo. A adenosina é um neurotransmissor excitatório no CB que aumenta a actividade eléctrica do CSN induzindo a hiperventilação através da activação de receptores A2. A importância destes efeitos da adenosina na quimiorrecepção, descritos em ratos e gatos, foi reforçada por resultados obtidos em voluntários saudáveis onde a infusão intravenosa de adenosina em induz hiperventilação e dispneia, efeito atribuído a uma activação do CB uma vez que a adenosina não atravessa a barreira hemato-encefálica e o efeito é quanto maior quanto mais perto do CB for a administração de adenosina. O presente trabalho foi realizado com o objectivo de esclarecer qual o significado funcional da adenosina na quimiorrecepção no CB em animais controlo e em animais submetidos a hipoxia crónica mantida. Para alcançar este objectivo investigou-se: 1) o efeito da hipóxia moderada sobre a libertação de adenosina numa preparação in vitro de CB e a especificidade desta mesma libertação comparativamente com outros tecidos não quimiossensitivos, assim como as vias metabólicas de produção e libertação de adenosina no CB em normoxia e hipóxia; 2) a modulação da libertação de adenosina/ATP das células quimiorreceptoras do CB por receptores nicotínicos de ACh; 3) os efeitos da cafeína no controlo periférico da ventilação e a identidade dos receptores de adenosina envolvidos nos efeitos da adenosina e da cafeína nos quimiorreceptores do CB; 4) as interacções entre os receptores D2 de dopamina e os receptores A2B de adenosina que modulam a libertação de catecolaminas (CA) no CB de rato e; 5) o efeito da ingestão crónica de cafeína, isto é, o contínuo bloqueio e dos receptores de adenosina, simulando assim o consumo crónico da cafeína, tal como ocorre na população humana mundial e principalmente no ocidente, na função do corpo carotídeo em ratos controlo e em ratos submetidos a hipoxia crónica. Os métodos utilizados neste trabalho incluíram: técnicas de biologia molecular como imunocitoquímica e western-blot; técnicas bioquímicas, tais como a quantificação de neurotransmissores por HPLC, bioluminescência e métodos radioisotópicos; técnicas electrofisiológicas como o registro de potenciais eléctricos do nervo do seio carotídeo in vitro; e registros ventilatórios in vivo em animais não anestesiados e em livre movimento (pletismografia). Observou-se que: 1) a especificidade dos quimiorreceptores do CB como sensores de O2 está correlacionada com o baixo limiar de libertação de adenosina em resposta à hipóxia dado que a libertação de adenosina do CB aumenta 44% em resposta a uma hipóxia moderada (10% O2), que no entanto não é um estímulo suficientemente intenso para evocar a libertação de adenosina do gânglio cervical superior ou do tecido arterial. Observou-se também que aproximadamente 40% da adenosina libertada pelo CB provém do catabolismo extracelular do ATP quer em normóxia quer em hipóxia moderada, sendo que PO2 reduzidas induzem a libertação de adenosina via activação do sistema de transporte equilibrativo ENT1. 2) a ACh modula a libertação de adenosina /ATP do CB em resposta à hipoxia moderada sugerindo que o papel excitatório da ACh na actividade quimiossensora inclui a activação indirecta de receptores purinérgicos pela adenosina e ATP, indicando que a adenosina e o ATP poderiam actuar como mediadores importantes no processo de quimiotransducção uma vez que: a) a activação dos receptores nicotínicos de ACh no CB em normóxia estimula a libertação de adenosina (max 36%) provindo aparentemente da degradação extracelular do ATP. b) a caracterização farmacológica dos receptores nicotínicos de ACh envolvidos na estimulação da libertação de adenosina do CB revelou que os receptores nicotínicos de ACh envolvidos são constituídos por subunidades α4ß2. 3) a adenosina modula a libertação de catecolaminas das células quimiorreceptoras do CB através de receptores de adenosina A2B dado que: a)a cafeína, um antagonista não selectivo dos receptores de adenosina, inibiu a libertação de CA quer em normóxia quer em resposta a estímulos de baixa intensidade sendo ineficaz na libertação induzida por estímulos de intensidade superior; b) o DPCPX e do MRS1754 mimetizaram os efeitos da cafeína no CB sendo o SCH58621 incapaz de induzir a libertação de CA indicando que os efeitos da cafeína seriam mediados por receptores A2B de adenosina cuja presença nas células quimiorreceptoras do CB demonstramos por imunocitoquímica. 4) a aplicação aguda de cafeína inibiu em 52% a actividade quimiossensora do CSN induzida pela hipóxia sendo este efeito mediado respectivamente por receptores de adenosina A2A pós-sinápticos e A2B pré-sinápticos indicando que a actividade quimiossensora induzida pela hipóxia é controlada pela adenosina. 5) existe uma interacção entre os receptores A2B e D2 que controla a libertação de CA do corpo carotídeo de rato uma vez que: a) os antagonistas dos receptores D2, domperidona e haloperidol, aumentaram a libertação basal e evocada de CA das células quimiorreceptoras confirmando a presença de autorreceptores D2 no CB de rato que controlam a libertação de CA através de um mecanismo de feed-back negativo. b) o sulpiride, um antagonista dos receptores D2, aumentou a libertação de CA das células quimiorreceptoras revertendo o efeito inibitório da cafeína sobre esta mesma libertação; c) a propilnorapomorfina, um agonista D2 inibiu a libertação basal e evocada de CA sendo este efeito revertido pela NECA, um agonista dos receptores A2B. O facto de a NECA potenciar o efeito do haloperidol na libertação de CA sugere que a interacção entre os receptores D2 e A2B poderia também ocorrer ao nível de segundos mensageiros, como o cAMP. 6) a ingestão crónica de cafeína em ratos controlo (normóxicos) não alterou significativamente a função basal do CB medida como a dinâmica dos seus neurotransmissores, dopamina, ATP e adenosina e como actividade quimiossensora do CSN. Contrariamente aos efeitos basais, a ingestão crónica de cafeína facilitou a resposta à hipóxia, dado que aumentou o efeito no volume minuto respiratórioapresentando-se também uma clara tendência para aumentar a actividade quimiossensora do CSN e aumentar a libertação de ATP e dopamina.7) após um período de 15 dias de hipóxia crónica era evidente o fenómeno de aclimatização dado que as respostas ventilatórias à hipóxia se encontram aumentadas, assim como a actividade quimiossensora do CSN basal e induzida pela hipóxia. As alterações observadas no metabolismo da dopamina, assim como na libertação basal de dopamina e de adenosina poderiam contribuir para a aclimatização durante a hipoxia crónica. A libertação aumentada de adenosina em resposta à hipóxia aguda em ratos hipóxicos crónicos sugere um papel da adenosina na manutenção/aumento das respostas ventilatórias à hipóxia aguda durante a hipóxia crónica. Observou-se também que a libertação de ATP induzida pela hipóxia aguda se encontra diminuída em hipóxia crónica, contudo a ingestão crónica de cafeína reverteu este efeito para valores similares aos valores controlo, sugerindo que a adenosina possa modular a libertação de ATP em hipóxia crónica. 8) a ingestão crónica de cafeína em ratos hipóxicos crónicos induziu o aumento do metabolismo de CA no CB, medido como expressão de tirosina hidroxilase, conteúdo, síntese e libertação de CA. 9) a ingestão crónica de cafeína não provocou quaisquer alterações na actividade quimiossensora do CSN em ratos hipóxicos crónicos no entanto, as respostas do CSN à hipóxia aguda intensa e moderada e à hipercapnia encontram-se diminuídas. Este efeito inibitório que provém da ingestão crónica de cafeína parece ser compensado ao nível dos quimiorreceptores centrais dado que os parâmetros ventilatórios em condições basais e em resposta à hipoxia aguda não se encontram modificados em ratos expostos durante 15 dias a uma atmosfera hipóxica. Resumindo podemos assim concluir que a adenosina quer em situações de hipoxia aguda quer em condições de hipoxia crónica tem um papel excitatório na actividade quimiossensora do CB actuando directamente nos receptores A2A presentes pós-sinapticamente no CSN, assim como facilitando a libertação de dopamina pré-sinapticamente via receptores A2B presentes nas células quimiorreceptoras. A interacção negativa entre os receptores A2B e D2 observadas nas células quimiorreceptoras do CB poderia explicar o aumento do metabolismo de CA observado após a ingestão crónica de cafeína em animais hipóxicos. Conclui-se ainda que durante a aclimatização à hipóxia a acção inibitória da cafeína, em termos de resposta ventilatória, mediada pelos quimiorreceptores periféricos é compensada pelos efeitos excitatórios desta xantina ao nível do quimiorreceptores centrais.------- RESUMEN Los cuerpos carotídeos (CB) son órganos emparejados que están localizados en la bifurcación de la arteria carótida común. Estos órganos son sensibles a variaciones en la PaO2, en la PaCO2, pH y temperatura siendo responsables de la hiperventilación que ocurre en respuesta a la hipoxia, contribuyendo también a la hiperventilación que acompaña a la acidosis metabólica y respiratoria. Las células quimiorreceptoras (tipo I o glómicas) del cuerpo carotídeo responden a las variaciones de gases arteriales liberando neurotransmissores que activan las terminaciones sensitivas del nervio del seno carotídeo (CSN) llevando la información al centro respiratorio central. Todavía esta por clarificar cual el neurotransmisor (o neurotransmisores) responsable por la señalización hipóxica en el CB. La adenosina es un neurotransmisor excitatório en el CB ya que aumenta la actividad del CSN e induce la hiperventilación a través de la activación de receptores de adenosina del subtipo A2. La importancia de estos efectos de la adenosina en la quimiorrecepción, descritos en ratas y gatos, ha sido fuertemente reforzada por resultados obtenidos en voluntarios sanos en los que la infusión intravenosa de adenosina induce hiperventilación y dispnea, efectos estés que han sido atribuidos a una activación del CB ya que la adenosina no cruza la barrera hemato-encefalica y el efecto es tanto más grande cuanto más cercana del CB es la administración. Este trabajo ha sido realizado con el objetivo de investigar cual el significado funcional de la adenosina en la quimiorrecepción en el CB en animales controlo y en animales sometidos a hipoxia crónica sostenida. Para alcanzar este objetivo se ha estudiado: 1) el efecto de la hipoxia moderada en la liberación de adenosina en una preparación in vitro de CB y la especificidad de esta liberación en comparación con otros tejidos no-quimiosensitivos, así como las vías metabólicas de producción y liberación de adenosina del órgano en normoxia y hipoxia; 2) la modulación de la liberación de adenosina/ATP de las células quimiorreceptoras del CB por receptores nicotínicos de ACh; 3) los efectos de la cafeína en el controlo periférico de la ventilación y la identidad de los receptores de adenosina involucrados en los efectos de la adenosina y cafeína en los quimiorreceptores del CB; 4) las interacciones entre los receptores D2 de dopamina y los receptores A2B de adenosina que modulan la liberación de catecolaminas (CA) en el CB de rata y; 5) el efecto de la ingestión crónica de cafeína, es decir, el bloqueo sostenido de los receptores de adenosina, simulando la dependencia de cafeína observada en la populación mundial del occidente, en la función del CB en ratas controlo y sometidas a hipoxia crónica sostenida. Los métodos utilizados en este trabajo incluirán: técnicas de biología molecular como imunocitoquímica y western-blot; técnicas bioquímicas, tales como la cuantificación de neurotransmissores por HPLC, bioluminescencia y métodos radioisotópicos; técnicas electrofisiológicas como el registro de potenciales eléctricos del nervio do seno carotídeo in vitro; y registros ventilatórios in vivo en animales no anestesiados y en libre movimiento (pletismografia). Se observó que: 1) la sensibilidad de los quimiorreceptores de CB esta correlacionada con un bajo umbral de liberación de adenosina en respuesta a la hipoxia ya que en respuesta a una hipoxia moderada (10% O2) la liberación de adenosina en el CB aumenta un 44%, sin embargo esta PaO2 no es un estimulo suficientemente fuerte para inducir la liberación de adenosina del ganglio cervical superior o del tejido arterial; se observó también que aproximadamente 40% de la adenosina liberada del CB proviene del catabolismo extracelular del ATP en normoxia y en hipoxia moderada, y que bajas PO2 inducen la liberación de adenosina vía activación del sistema de transporte equilibrativo ENT1. 2) la ACh modula la liberación de adenosina /ATP del CB en respuesta a la hipóxia moderada lo que sugiere que el papel excitatório de la ACh en la actividad quimiosensora incluye la activación indirecta de receptores purinérgicos por la adenosina y el ATP, indicando que la adenosina y el ATP pueden actuar como mediadores importantes en el proceso de quimiotransducción ya que: a) la activación de los receptores nicotínicos de ACh en el CB en normoxia estimula la liberación de adenosina (max 36%) que aparentemente proviene de la degradación extracelular del ATP. Se observó también que este aumento de adenosina en el CB en hipoxia ha sido antagonizado parcialmente por antagonistas de estos mismos receptores; b) la caracterización farmacológica de los receptores nicotínicos de ACh involucrados en la estimulación de la liberación de adenosina del CB ha revelado que los receptores nicotínicos de ACh involucrados son constituidos por sub-unidades α4ß2. 3) la adenosina modula la liberación de CA de las células quimiorreceptoras del CB a través de receptores de adenosina A2B ya que: a) la cafeína, un antagonista no selectivo de los receptores de adenosina, ha inhibido la liberación de CA en normoxia y en respuesta a estímulos de baja intensidad siendo ineficaz en la liberación inducida por estímulos de intensidad superior; b) el DPCPX y el MRS1754 ha mimetizado los efectos de la cafeína en el CB y el SCH58621 ha sido incapaz de inducir la liberación de CA lo que sugiere que los efectos de la cafeína son mediados por receptores A2B de adenosina que están localizados pré-sinapticamente en las células quimiorreceptoras del CB. 4) la aplicación aguda de cafeína ha inhibido en 52% la actividad quimiosensora del CSN inducida por la hipoxia siendo este efecto mediado respectivamente por receptores de adenosina A2A pós-sinápticos y A2B pré-sinápticos lo que indica que la actividad quimiosensora inducida por la hipoxia es controlada por la adenosina. 5) existe una interacción entre los receptores A2B y D2 que controla la liberación de CA del CB de rata ya que: a) el sulpiride, un antagonista de los receptores D2, ha aumentado la liberación de CA de las células quimiorreceptoras revertiendo el efecto inhibitorio de la cafeína sobre esta misma liberación; b) los antagonistas de los receptores D2, domperidona y haloperidol, han aumentado la liberación basal e evocada de CA de las células quimiorreceptoras confirmando la presencia de autorreceptores D2 en el CB de rata que controlan la liberación de CA a través de un mecanismo de feed-back negativo; c) la propilnorapomorfina, un agonista D2, ha inhibido la liberación basal e evocada de CA sendo este efecto revertido por la NECA, un agonista de los receptores A2B. Ya que la NECA potencia el efecto del haloperidol en la liberación de CA la interacción entre los D2 y A2B puede también ocurrir al nivel de segundos mensajeros, como el cAMP. 6) la ingestión crónica de cafeína en ratas controlo (normóxicas) no ha cambiado significativamente la función basal del CB medida como la dinámica de sus neurotransmisores, dopamina, ATP y adenosina y como actividad quimiosensora del CSN. Al revés de lo que pasa con los efectos básales, la ingestión crónica de cafeína facilitó la respuesta a la hipóxia, ya que ha aumentado la respuesta ventilatória medida como volumen minuto presentando también una clara tendencia para aumentar la actividad quimiosensora del CSN y aumentar la liberación de ATP y dopamina. 7. Después de un período de 15 días de hipoxia crónica se puede observar el fenómeno de climatización ya que las respuestas ventilatórias a la hipoxia están aumentadas, así como la actividad quimiosensora del CSN basal e inducida por la hipoxia. Los cambios observados en el metabolismo de la dopamina, así como en la liberación basal de dopamina y de adenosina podrían contribuir para la climatización en hipoxia crónica. El aumento en la liberación de adenosina en respuesta a la hipoxia aguda en ratas sometidas a hipoxia crónica sugiere un papel para la adenosina en el mantenimiento/aumento de las respuestas ventilatórias a la hipoxia aguda en hipoxia crónica sostenida. Se ha observado también que la liberación de ATP inducida por la hipoxia aguda está disminuida en hipoxia crónica y que la ingestión crónica de cafeína reverte este efecto para valores similares a los valores controlo, sugiriendo que la adenosina podría modular la liberación de ATP en hipoxia crónica. 8. la ingestión crónica de cafeína ha inducido el aumento del metabolismo de CA en el CB en ratas hipóxicas crónicas, medido como expresión de la tirosina hidroxilase, contenido, síntesis y liberación de CA. 9. la ingestión crónica de cafeína no ha inducido cambios en la actividad quimiosensora del CSN en ratas hipóxicas crónicas sin embargo las respuestas do CSN a una hipoxia intensa y moderada y a la hipercapnia están disminuidas. Este efecto inhibitorio que es debido a la ingestión crónica de cafeína es compensado al nivel de los quimiorreceptores centrales ya que los parámetros ventilatórios en condiciones básales y en respuesta a la hipoxia aguda no están modificados en ratas expuestas durante 15 días a una atmósfera hipóxica. Resumiendo se puede concluir que la adenosina en situaciones de hipoxia aguda así como en hipoxia crónica tiene un papel excitatório en la actividad quimiosensora del CB actuando directamente en los receptores A2A localizados pós-sinapticamente en el CSN, así como controlando la liberación de dopamina pré-sinaptica vía receptores A2B localizados en las células quimiorreceptoras. Las interacciones entre los receptores A2B y D2 observadas en las células quimiorreceptoras del CB podrían explicar el aumento del metabolismo de CA observado después de la ingestión crónica de cafeína en animales hipóxicos. Por fin, pero no menos importante se puede concluir que durante la climatización a la hipoxia la acción inhibitoria de la cafeína, medida como respuesta ventilatória, mediada por los quimiorreceptores periféricos es compensada por los efectos excitatórios de esta xantina al nivel de los quimiorreceptores centrales.

A neuropatia diabética causa diminuição ou perda da sensibilidade protetora do pé, tornando-o mais vulnerável ao trauma mecânico e térmico. A profilaxia das complicações neuropáticas tem início pela identificação da perda de sensibilidade e, portanto, do comprometimento neurológico. O Pressure Specified Sensory Device(TM) (PSSD) é um equipamento desenvolvido para quantificar o limiar de pressão, aplicada sobre a pele, necessária para que o paciente perceba o estímulo provocado por: um ponto estático, um ponto em movimento, dois pontos estáticos e dois pontos em movimento. Denominamos grupo estudo, aos trinta e quatro pacientes diabéticos do tipo 2, sem história prévia de feridas e/ou amputações nos pés que foram submetidos à avaliação de sensibilidade cutânea utilizando-se o PSSD(TM). Foram realizados testes nos territórios cutâneos dos nervos fibular profundo, plantar medial e ramo calcâneo do nervo tibial posterior. Estímulos foram provocados segundo as modalidades: um ponto estático (1 PE), um ponto em movimento (1 PD), dois pontos estáticos (2 PE) e dois pontos em movimento (2 PD), para as duas últimas modalidades. Previamente às modalidades 2PE e 2PD determinou-se o limiar de discriminação entre dois pontos estáticos (D2PE) e em movimento (D2PD). Foram realizados apenas no grupo estudo, testes com o monofilamento de Semmes-Weisntein nº 5,07 (MSW) e com o diapasão de 128 Hz. Vinte e oito pacientes não-diabéticos, submetidos aos mesmos testes, formaram o grupo controle. Para os limiares de sensibilidade, encontramos valores superiores no grupo estudo (p < 0,05). Ao compararmos os limiares de sensibilidade alcançados pelos pacientes diabéticos sensíveis e não sensíveis ao estímulo promovido pelo MSW nº 5,07 verificamos que o p-valor variou entre 0,018 < p < 0,113 para 1 PE e 0,002 < p < 0,083 para 2 PE, conforme o território cutâneo estudado. Na comparação dos limiares de sensibilidade da modalidade 1 PD entre diabéticos sensíveis e insensíveis à vibração do diapasão de 128 Hz, as diferenças não foram estatisticamente significantes (p = 0,183). Os resultados obtidos nos permitiram sugerir que o dispositivo PSSD(TM) seja utilizado como forma de acompanhamento do comprometimento da fibra nervosa.
Neuropathy is a severe progressive loss of protective sensation on the feet, making the patient more vulnerable to mechanical trauma and consequently more suitable to the development of chronic wounds, major distortion of the foot bone architecture and eventually to limb amputation. Prophylaxis should be enforced to avoid foot ulceration and for that, evaluation of the degree of loss of sensation on the skin is essential. The PSSD (Pressure Specified Sensory Device(TM)) was developed in order to quantify the threshold of pressure applied to the skin that could be recognized as positive by the patient. Pressure of one or two points is tested both statically and with movement, thus assessing the function of fast and slow response nerve fibers. Threshold of two-point discrimination was also measured in mm. Thirty four (n = 34) diabetic patients, type II, with no previous history of wounds on the lower extremity were studied using the tests, one point static (1PE), one point moving (1PD) and two points static (2 PE), and moving (2 PD) on the cutaneous territory of the fibular nerve and posterior tibial nerve (two territories - medial plantar and calcaneous nerves). The control group (28 non diabetic patients) was assessed by the same exams and the results were compared. In the diabetic group the cutaneous territories were also evaluated using the conventional Semmes-Weinstein filament nº 5,07 e vibrometer of the 128 Hz. Altered values were observed for the static and dynamic tests over the three studied nerve territories. The differences were statically significant (p < 0,05). Comparing the threshold of sensibility between sensitive and non sensitive diabetic patients to MSW nº 5,07 test, we observed that p-value range was 0,018 0,113 when 1PE test was applied, and 0,002 0,083 when 2PE test was applied, according to the cutaneous territories evaluated. Numeric quantification of the threshold of pressure allows us to determine the status of the fiber/receptor structures as well as the functional deficit of nerve fibers. Our findings suggest that PSSD(TM) is an adjuvant tool to evaluate the degree of loss of sensation on the skin.

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.

Philosophiae Doctor - PhD
Tsetse flies are the biological vectors of human and animal trypanosomiasis and hence representant medical and veterinary importance. The sense of smell plays a significant role in tsetse and its ecological interaction, such as finding blood meal source, resting, and larvicidal sites and for mating. Tsetse olfactory behaviour can be exploited for their management; however, olfactory studies in tsetse flies are still fragmentary. Here in my PhD thesis, using scanning electron microscopy, electrophysiology, behaviour, bioinformatics and molecular biology techniques, I have investigated tsetse flies (Glossina fuscipes fuscipes) olfaction using behaviourally well studied odorants, tsetse repellent by comparing with attractant odour. Insect olfaction is mediated by olfactory sensory neurons (OSNs), located in olfactory sensilla, which are cuticular structures exposed to the environment through pore and create a platform for chemical communication.

This thesis is concerned with describing and experimentally investigating the nature of perceptual learning. Ecological psychology defines perceptual learning as a process of educating attention to structural properties of stimuli (i.e., invariants) that specify meaning (i.e., affordances) to the perceiver. Although such definition comprehensively describes the questions of what humans learn to perceive, it does not address the question of how learning occurs. It is proposed in this thesis that the principles of classical and operant conditioning can be used to strengthen and expand the ecological account of perceptual learning. The perceptual learning of affordances is described in terms of learning that a stimulus is associated with another stimulus (classical conditioning), and in terms of learning that interacting with a stimulus is associated with certain consequences (operant conditioning). Empirical work in this thesis investigated the effect of conditioning on pitch and speech perception. Experiments 1, 2, and 3 were designed to modify pitch perception in Shepard tones via tone-colour associative training. During training, Shepard tones were paired with coloured circles in a way that the colour of the circles could be predicted by either the F0 (pitch) or by an F0-irrelevant auditory invariant. Participants were required to identify the colour of the circles that was associated with the tones and they received corrective feedback. Hypotheses were based on the assumption that F0-relevant/F0- irrelevant conditioning would increase/decrease the accuracy of pitch perception in Shepard tones. Experiment 1 investigated the difference between F0-relevant and F0- irrelevant conditioning in a between-subjects design, and found that pitch perception in the two conditions did not differ. Experiments 2 and 3 investigated the effect of F0- relevant and F0-irrelevant conditioning (respectively) on pitch perception using a within subjects (pre-test vs. post-test) design. It was found that the accuracy of pitch perception increased after F0-relevant conditioning, and was unaffected by F0-irrelevant conditioning. The differential trends observed in Experiments 2 and 3 suggest that conditioning played some role in influencing pitch perception. However, the question whether the observed trends were due to the facilitatory effect of F0-relevant conditioning or the inhibitory effect of F0-irrelevant conditioning warrants future investigation. Experiments 4, 5, and 6 were designed to modify the perception of McGurk syllables (i.e., auditory /b/ paired with visual /g/) via consonant-pitch associative training. During training, participants were repeatedly presented with /b/, /d/, and /g/ consonants in falling, flat, and rising pitch contours, respectively. Pitch contour was paired with either the auditory signal (Experiments 4 and 5) or the visual signal (Experiment 6) of the consonant. Participants were required to identify the stop consonants and they received corrective feedback. The perception of McGurk stimuli was tested before and after training by asking participants to identify the stop consonant in each stimulus as /b/ or /d/ or /g/. It was hypothesized that conditioning would increase (1) /b/ responses more in the falling than in the flat/ rising contour conditions, (2) /d/ responses more in the flat than in the falling/ rising contour conditions, and (3) /g/ responses more in the rising than in the falling/flat contour conditions. Support for the hypotheses was obtained in Experiments 5 and 6, but only in one response category (i.e., /b/ and /g/ response categories, respectively). It is suggested that the subtlety of the observed conditioning effect could be enhanced by increasing the salience of pitch contour and by reducing the clarity of auditory/visual invariants that specify consonants.

Philosophiae Doctor - PhD
Tsetse flies are the biological vectors of human and animal trypanosomiasis and hence representant medical and veterinary importance. The sense of smell plays a significant role in tsetse and its ecological interaction, such as finding blood meal source, resting, and larvicidal sites and for mating. Tsetse olfactory behaviour can be exploited for their management; however, olfactory studies in tsetse flies are still fragmentary. Here in my PhD thesis, using scanning electron microscopy, electrophysiology, behaviour, bioinformatics and molecular biology techniques, I have investigated tsetse flies (Glossina fuscipes fuscipes) olfaction using behaviourally well studied odorants, tsetse repellent by comparing with attractant odour. Insect olfaction is mediated by olfactory sensory neurons (OSNs), located in olfactory sensilla, which are cuticular structures exposed to the environment through pore and create a platform for chemical communication. In the sensilla shaft the dendrite of OSNs are housed, which are protected by called the sensillum lymph produced by support cells and contains a variety of olfactory proteins, including the odorant binding protein (OBP) and chemosensory proteins (CSP). While on the dendrite of OSNs are expressed olfactory receptors. In my PhD, studies I tried to decipher the sense of smell in tsetse fly. In the second chapter, I demonstrated that G. f. fuscipes is equipped with diverse olfactory sensilla, that various from basiconic, trichoid and coeloconic. I also demonstrated, there is shape, length, number difference between sensilla types and sexual dimorphism. There is a major difference between male and female, while male has the unique basiconic sensilla, club shaped found in the pits, which is absent from female pits. In my third chapter, I investigated the odorant receptors which are expressed on the dendrite of the olfactory sensory neurons (OSNs). G. f. fuscipes has 42 ORs, which were not functionally characterised. I used behaviourally well studied odorants, tsetse repellents, composed of four components blend. I demonstrated that tsetse repellent is also a strong antifeedant for both G. pallidipes and G. f. fuscipes using feeding bioassays as compared to the attractant odour, adding the value of tsetse repellent. However, the attractant odour enhanced the feeding index. Using DREAM (deorphanization of receptors based on expression alterations of mRNA levels). I found that in G. f. fuscipes, following a short in vivo exposure to the individual tsetse repellent component as well as an attractant volatile chemical, OSNs that respond to these compounds altered their mRNA expression in two opposite direction, significant downregulation and upregulation in their number of transcripts corresponding to the OR that they expressed and interacted with odorant. Also, I found that the odorants with opposite valence already segregate distinctly at the cellular and molecular target at the periphery, which is the reception of odorants by OSNs, which is the basis of sophisticated olfactory behaviour. Deorphanization of ORs in none model insect is a challenge, here by combining DREAM with molecular dynamics, as docking score, physiology and homology modelling with Drosophila a well-studied model insects, I was able to predict putative receptors of the tsetse repellent components and an attractant odour. However, many ORs were neutral, showing they were not activated by the odorants, demonstrating the selectivity of the technique as well as the receptors. In my fourth chapter, I investigated the OBPs structures and their interaction with odorants molecules. I demonstrated that OBPs are expressed both in the antenna, as well as in other tissues, such as legs. I also demonstrated that there are variations in the expression of OBPs between tissues as well as sexes. I also demonstrated that odorants induced a fast alteration in OBP mRNA expression, some odorants induced a decrease in the transcription of genes corresponding to the activated OBP and others increased the expression by many fold in OBPs in live insect, others were neutral after 5 hours of exposure. Moreover, with subsequent behavioural data showed that the behavioural response of G. f. fuscipes toward 1-octen-3-ol decreased significantly when 1-octen-3-ol putative OBPs were silenced with feeding of double-stranded RNA (dsRNA). In summary, our finding whereby odorant exposure affects the OBPs mRNA, their physiochemical properties and the silencing of these OBPs affected the behavioural response demonstrate that the OBPs are involved in odour detection that affect the percept of the given odorant. The expression of OBPs in olfactory tissues, antenna and their interaction with odorant and their effect on behavioural response when silenced shows their direct involvement in odour detection and reception. Furthermore, their expression in other tissues such as legs indicates they might also have role in other physiological functions, such as taste.

Pest control strategies targeting insect olfaction represent a promising venue for control of tortricid insects (Lepidoptera: Tortricidae). Among tortricids, the grapevine moth Lobesia botrana (Denis and Schiffermüller) and the codling moth Cydia pomonella (L.) are serious pests for worldwide production of fruit crops. We employed several approaches to the olfactory system, from electrophysiological and behavioral studies in the grapevine moth, to bioinformatic and molecular studies of olfactory sensory proteins in the codling moth. At the receptor level, we studied both the Olfactory Receptors (ORs), the most common class of sensory proteins mediating detection of odors in insect antennae, and the Transient Receptor Potential (TRP) channels, a novel family of receptor, that recently were also found in the antennae of lepidopterous species. We demonstrated electrophysiological and behavioral responses of the grapevine moth to volatiles emitted by a non-host, Perilla frutescens, previously known to activate TRPs in the rat, Rattus norvegicus. In the codling moth, we characterized a novel TRP channel (TRPA pyrexia-like) and we confirmed activation of its human orthologue to the same non-host compounds active on the olfactory system of the grapevine moth. ORs were heterologously expressed in vivo and in vitro, for identification of their ligands among host and non-host plant volatiles and pheromones (deorphanization). Among several ORs of codling moth, we deorphanized a candidate pheromone receptor (PR) to plant synergists, an OR to non-host volatiles and another PR candidate to a pheromone antagonist of the insect. Our study thus opens for refinement of existing pest control, or novel applications. The behavioral response of the grapevine moth to volatiles from a nonhost plant, and the identification of a novel TRP channel in the codling moth may have perspectives for application in agriculture, targeting the somatosensory system of these tortricids. The evolutionary implications of the responses of the human orthologue of TRPA pyrexia-like to volatiles active on the grapevine moth olfactory system could imply a large degree of conservation of the receptor function. In the codling moth, identification of synergist and antagonist ligands for candidate PRs and deorphanization of an OR to non-host plant volatiles suggest a possible role of these receptors in reproductive and ecological isolation. This could lead to further refinement of existing semiochemicalbased control techniques, by enabling a better understanding of mate- and host-finding in this species.

Thesis (Ph.D.)--University of Western Sydney, 2007.
A thesis submitted to the University of Western Sydney, College of Arts, School of Psychology in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliography.

Le bisphénol A (BPA) est une molécule dérivée du plastique, considéré comme un perturbateur endocrinien (PE) pour sa capacité de liaison aux récepteurs nucléaires (RNs). Il est associé à des troubles neurodéveloppementaux, probablement en raison de la présence de certains RNs dans le cerveau des vertébrés. Les PEs peuvent également affecter les invertébrés marins, dont les ascidies, mais leur mode d'action reste inconnu. Mon projet de thèse a pour but d’étudier la toxicité du BPA sur le développement embryonnaire de l'ascidie Phallusia mammillata. Tout d’abord, j’ai évalué l’expression des RNs de P. mammillata et j’ai trouvé que 5 d’entre eux (COUP, ERR, PPAR, PXR/VDR) sont exprimés dans le cerveau de l’ascidie (= vésicule sensorielle) ou à proximité (TR). Ils correspondent majoritairement aux orthologues humains connus pour lier le BPA. Puis, j’ai évalué la toxicité du BPA au cours du développement et constaté qu’il est toxique de façon dose-dépendante. En effet, j’ai montré que de faibles doses de BPA induisent une toxicité neurodéveloppementale en affectant la différenciation de l'organe sensoriel pigmenté de l’ascidie. Ce phénotype est spécifique aux bisphénols. Enfin, l’étude d’agonistes et d’antagonistes de l’estrogen-related receptor (ERR) a montré des phénotypes correspondant à ceux obtenus avec le BPA. J’ai trouvé que Pm-ERR est exprimé dans la vésicule sensorielle de la larve, autour de l'organe sensoriel pigmenté. Il semble donc que Pm-ERR est impliqué dans ce phénotype. Toutefois, la présence des autres RNs que j’ai identifiés soulève la possibilité de leur implication dans le développement de l’ascidie et/ou de la toxicité de PEs. Ils ne doivent donc pas être négligés
Bisphenol A (BPA) is a plastic-derived molecule that is now considered an endocrine disruptor (ED). BPA impair hormonal systems via binding to nuclear receptors (NRs) and it has been linked to neurodevelopmental disorders, possibly due to the presence of some NRs in the vertebrate brain. BPA has been reported to affect marine invertebrates such as ascidians, but how is BPA acting is not known. My PhD project is aimed at deciphering the toxicity of BPA on embryonic development of the marine invertebrate chordate Phallusia mammillata (Tunicata). Firstly, I assessed the embryonic expression of P. mammillata NRs and found that 5 are expressed within the ascidian brain (also called sensory vesicle, SV) (COUP, ERR, PPAR, PXR/VDR) or nearby (TR). Interestingly, the human orthologues of most of these NRs are known to bind BPA. Secondly, I assessed BPA toxicity during P. mammillata embryonic development and found that BPA is toxic in a dose-dependent manner. I also show that at micromolar doses BPA induces neurodevelopmental toxicity by impairing differentiation of the ascidian pigmented sensory organ (PSO). I further show that this phenotype is specific to bisphenols. Finally, estrogen-related receptor (ERR) agonists and antagonists partially phenocopied BPA phenotype. Interestingly, I found that Pm-ERR is expressed in the larval SV close to the ascidian PSO, thus suggesting an involvement of Pm-ERR in the BPA phenotype. Furthermore, the complex pleiotropic action of BPA together with the presence of other NRs in the ascidian larval brain raises the possibility that these NRs are involved both in ascidian brain development and EDs toxicity, thus they should not be overlooked

Although deafness is a universal problem, effective treatments have remained elusive. In order to develop potential treatments, an overall understanding of the cellular process of auditory hair cell regeneration, which occurs in fish but not mammals, must be established. A previous microarray analysis and qRT-PCR validation of noise-exposed zebrafish showed that growth hormone (GH) was significantly upregulated during the process of auditory hair cell regeneration. Thus, GH may play an important role during hair cell regeneration. However, cellular effects of exogenous GH in the zebrafish auditory hair cell regeneration have not been examined after noise exposure. To understand the effect of GH in hair cell regeneration, adult zebrafish were exposed to a 150 Hz pure tone at a source level of 179 dB re 1 μPa RMS for 36 hours. Afterward the fish were immediately injected intraperitoneally with carp recombinant GH (20 μg/gram of body mass) or buffer (0.1 M, pH 7.4 phosphate buffer) and then placed in a recovery tank. The effect of GH on apoptosis in fish inner ear end organs were examined using TUNEL-labeling. Cell proliferation was measured by BrdU incorporation assay. Hair cell regeneration was determined by phalloidin-labeling to allow visualization of hair cell stereociliary bundles. After GH injection, the numbers of TUNEL-labeled cells showed a significant decrease in all three inner ear end organs (saccule, lagena, utricle), suggesting GH may suppress hair cell death induced by acoustic trauma. Higher levels of cell proliferation were also observed in the ears of GH-injected fish, indicating that GH is capable of activating cell mitosis in the zebrafish auditory system. Following sound exposure, the GH-injected group exhibited greater numbers of saccular hair cell bundles compared to the buffer-injected group. These results indicate that GH promotes hair cell regeneration following acoustic damage. Future studies are needed to examine the potential therapeutic benefits of GH in the mammalian ear.

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2007.
Title from title page screen (viewed on February 29, 2008). Research advisor: Robert S. Waters, Ph.D. Document formatted into pages (xix, 179 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 152-178).

Le bisphénol A (BPA) est une molécule dérivée du plastique, considéré comme un perturbateur endocrinien (PE) pour sa capacité de liaison aux récepteurs nucléaires (RNs). Il est associé à des troubles neurodéveloppementaux, probablement en raison de la présence de certains RNs dans le cerveau des vertébrés. Les PEs peuvent également affecter les invertébrés marins, dont les ascidies, mais leur mode d'action reste inconnu. Mon projet de thèse a pour but d’étudier la toxicité du BPA sur le développement embryonnaire de l'ascidie Phallusia mammillata. Tout d’abord, j’ai évalué l’expression des RNs de P. mammillata et j’ai trouvé que 5 d’entre eux (COUP, ERR, PPAR, PXR/VDR) sont exprimés dans le cerveau de l’ascidie (= vésicule sensorielle) ou à proximité (TR). Ils correspondent majoritairement aux orthologues humains connus pour lier le BPA. Puis, j’ai évalué la toxicité du BPA au cours du développement et constaté qu’il est toxique de façon dose-dépendante. En effet, j’ai montré que de faibles doses de BPA induisent une toxicité neurodéveloppementale en affectant la différenciation de l'organe sensoriel pigmenté de l’ascidie. Ce phénotype est spécifique aux bisphénols. Enfin, l’étude d’agonistes et d’antagonistes de l’estrogen-related receptor (ERR) a montré des phénotypes correspondant à ceux obtenus avec le BPA. J’ai trouvé que Pm-ERR est exprimé dans la vésicule sensorielle de la larve, autour de l'organe sensoriel pigmenté. Il semble donc que Pm-ERR est impliqué dans ce phénotype. Toutefois, la présence des autres RNs que j’ai identifiés soulève la possibilité de leur implication dans le développement de l’ascidie et/ou de la toxicité de PEs. Ils ne doivent donc pas être négligés
Bisphenol A (BPA) is a plastic-derived molecule that is now considered an endocrine disruptor (ED). BPA impair hormonal systems via binding to nuclear receptors (NRs) and it has been linked to neurodevelopmental disorders, possibly due to the presence of some NRs in the vertebrate brain. BPA has been reported to affect marine invertebrates such as ascidians, but how is BPA acting is not known. My PhD project is aimed at deciphering the toxicity of BPA on embryonic development of the marine invertebrate chordate Phallusia mammillata (Tunicata). Firstly, I assessed the embryonic expression of P. mammillata NRs and found that 5 are expressed within the ascidian brain (also called sensory vesicle, SV) (COUP, ERR, PPAR, PXR/VDR) or nearby (TR). Interestingly, the human orthologues of most of these NRs are known to bind BPA. Secondly, I assessed BPA toxicity during P. mammillata embryonic development and found that BPA is toxic in a dose-dependent manner. I also show that at micromolar doses BPA induces neurodevelopmental toxicity by impairing differentiation of the ascidian pigmented sensory organ (PSO). I further show that this phenotype is specific to bisphenols. Finally, estrogen-related receptor (ERR) agonists and antagonists partially phenocopied BPA phenotype. Interestingly, I found that Pm-ERR is expressed in the larval SV close to the ascidian PSO, thus suggesting an involvement of Pm-ERR in the BPA phenotype. Furthermore, the complex pleiotropic action of BPA together with the presence of other NRs in the ascidian larval brain raises the possibility that these NRs are involved both in ascidian brain development and EDs toxicity, thus they should not be overlooked

Nerve growth factor (NGF) is a known factor in the development of persistent pain, a condition which affects approx. 20% of the UK population. Current therapeutics available for pain exhibit limited effectiveness and therefore better targeted and more effective therapeutics are essential. Clinical trials using anti-NGF have been successful in consistently alleviating pain of patients suffering from chronic pain however the main mechanism of action of NGF is unknown. The main aim of this PhD was to investigate the role for TrkA and p75 receptors for NGF in the development of persistent pain, specifically targeted to primary sensory afferents. The first aim was to determine whether NGF acts directly or indirectly (via other cell types reported to express receptors for NGF) on sensory neurons in the development of pain. To address this we optimised and characterised a protocol for purifying sensory neuronal cultures from dorsal root ganglia (DRG) using magnetic assisted cell sorting (MACS), a method we found to reliably produce viable 95% pure neuronal cultures. The second major question surrounding the mechanism of NGF is whether NGF acts mainly through the TrkA or the p75 receptor, or do the two receptors work in synergy. In order to address this we used two different methods. First, we chose to use an approach using viral vectors, both lenti and adeno-associated virus, to introduce Cre recombinase into DRG neurons with floxed regions of the NTRK1 and NGFR genes to knockout expression of TrkA or p75 respectively. Secondly, we bred a new transgenic mouse lines for conditional knockout of TrkA or p75 under the control of tamoxifen by crossing the above mentioned floxed mouse lines with an Advillin CreERT2 transgenic mouse, where Cre activity is limited to sensory and sympathetic neurons.

Background Trigeminal ganglion (TG) is a key player in processing noxious stimuli. Among many ligand-gated ion channels, trigeminal sensory neurons express on their membranes purinergic P2X3 receptors and capsaicin-sensitive transient receptor potential vanilloid 1 channels (TRPV1). These receptors are thought to be involved in pain transduction and pathophysiology of different pain syndromes, including migraine disorders. P2X3 and TRPV1 channels are continuously regulated by a variety of endogenous modulators, which, upregulating these receptors, can cause sensitization and promote development of pathological pain conditions. Although positive P2X3 and TRPV1 regulators are well studied, not much is known about those which might restrain the activity of these receptors. One candidate for the role of endogenous negative regulator of sensory ganglion activity is the brain natriuretic peptide (BNP). In fact, BNP was recently reported to downregulate inflammatory pain and firing frequency of small neurons in dorsal root ganglia via its receptor NPR-A. Aims In order to investigate the role of BNP/NPR-A system in trigeminal ganglion in control conditions and in migraine pathology we used wild-type (WT) mice and transgenic R192Q KI mice of the familial hemiplegic migraine type 1 (FHM1) model. First we characterized BNP and NPR-A expression and functional properties of the BNP/NPR-A pathway in trigeminal ganglions of WT and KI mice. To understand if this pathway can affect the properties of sensory neurons in TG we studied the effects of endogenous and exogenous BNP on P2X3 and TRPV1 receptors responses in vitro. Investigating molecular mechanisms underneath P2X3 receptor modulation we carefully examined changes in P2X3 phosphorylation and membrane distribution and considered involvement of particular kinases and phosphatases in this process. Firing activity of the WT and KI trigeminal neurons were also evaluated to find out if the modulatory effects of BNP/NPR-A system on the P2X3 channels are reflected in neuronal excitability. Additionally, in search for new potent P2X3 antagonists a variety of diaminopurine derivatives as well as several adenosine nucleotide analogues were evaluated on recombinant P2X3 receptors in HEK cells and on native P2X3 receptors of TG sensory neurons. Results We found abundant expression of NPR-A in trigeminal ganglion along with low levels of BNP itself; the BNP/NPR-A pathway in both WT and KI neurons proved to be functional. Exogenously applied BNP inhibited TRPV1-mediated responses in WT and KI trigeminal neurons without any changes in the receptor’s expression level. On the other hand, P2X3 receptors were not sensitive to additional exogenous BNP, but appeared to be downregulated by the low amount of endogenous BNP already present in WT TG cultures. This negative modulation included P2X3 serine phosphorylation and receptor redistribution to the non-lipid raft membrane compartments. Both mechanisms were dependent on the activity of protein kinase G. Interestingly, in KI mice NPR-A-mediated P2X3 inhibition could not be seen and receptors remained upregulated, most probably due to the increased activity of P/Q calcium channels and high concentration of calcitonin gene related peptide (CGRP). Considering firing properties of trigeminal neurons, inactivation of BNP/NPR-A system with NPR-A antagonist anantin caused a hyperexcitability phenotype of WT cultures, which was very similar to what is typical for KI neurons. KI cultures remained unaltered, consistent with lack of BNP/NPR-A regulation over P2X3 activity. Experiments with new diaminopurine compounds and adenosine nucleotide derivatives resulted in molecules which showed antagonistic behavior towards P2X3 receptors with IC50 values in low micromolar and nanomolar range, respectively. Conclusion The main result of the present study is the identification of BNP/NPR-A pathway as an intrinsic negative modulatory system for P2X3 and TRPV1 receptors activity in sensory neurons of mouse trigeminal ganglion and related neuronal excitability. However, in a mouse FHM1 migraine model BNP/NPR-A lacked the inhibitory effect on P2X3 receptors due to the overall amount of activation these receptors undergo in KI neurons. Modifications of diaminopurine and adenosine scaffold could serve as a promising strategy in search for new potent antagonists of P2X3 receptors.

INTRODUÇÃO: O tratamento das neoplasias malignas anorretais exige ressecções que podem levar ao surgimento de defeitos perineais extensos. Esses defeitos necessitam de reconstrução que deve ser realizada, preferencialmente, com retalhos. Dentre eles, destacamos o retalho perfurante da artéria pudenda interna, localizado no sulco glúteo, vascularizado por vasos perfurantes cutâneos da artéria pudenda interna, e inervado por ramos do nervo pudendo e do nervo cutâneo femoral posterior. Esse retalho apresenta diversas vantagens em comparação com os outros utilizados para reconstrução perineal, e os dados relacionados à avaliação de sua sensibilidade cutânea são escassos, discrepantes e sujeitos a críticas metodológicas. OBJETIVO: Avaliar a sensibilidade cutânea do retalho perfurante da artéria pudenda interna após 12 meses da reconstrução perineal em cirurgias de amputação abdominoperineal de reto, e compará-la com a sensibilidade cutânea pré-operatória do sulco glúteo (área doadora do retalho). MÉTODOS: Estudo prospectivo com 25 pacientes submetidos à amputação abdominoperineal de reto por neoplasias malignas anorretais e reconstruídos com o retalho perfurante da artéria pudenda interna de avanço VY bilateral. As modalidades de sensibilidade tátil, dolorosa, térmica e vibratória foram analisadas em quatro áreas do sulco glúteo no pré-operatório e nas quatro áreas correspondentes do retalho 12 meses após a cirurgia. A avaliação da sensibilidade tátil foi realizada com o Pressure Specified Sensory Device(TM) (PSSD(TM)), aparelho que quantifica a pressão aplicada à pele, estática ou em movimento. As outras modalidades de sensibilidade foram analisadas com o método de escolha forçada, utilizando ponta de agulha para sensibilidade dolorosa, contato quente/frio para sensibilidade térmica e diapasão de 128 Hz para sensibilidade vibratória. RESULTADOS: Os limiares de sensibilidade tátil medidos com o PSSD(TM) no retalho perfurante da artéria pudenda interna após 12 meses da reconstrução perineal foram semelhantes aos limiares de sensibilidade tátil no sulco glúteo no pré-operatório, tanto no teste de pressão estática quanto em movimento. A comparação entre esses limiares não apresentou diferença estatisticamente significante, com valores de p superiores a 0,05 nas quatro áreas avaliadas, para ambos os testes. Todos os pacientes apresentaram sensibilidade dolorosa, térmica e vibratória nas quatro áreas testadas, tanto no sulco glúteo no pré-operatório quanto no retalho após 12 meses da cirurgia. CONCLUSÃO: Nas reconstruções perineais após amputação abdominoperineal de reto, espera-se que a sensibilidade cutânea do retalho perfurante da artéria pudenda interna seja mantida
INTRODUCTION: The treatment of anorectal malignancies requires resection that can lead to extensive perineal defects. These defects require reconstruction which should be performed, preferably, with flaps. Among them, we highlight the internal pudendal artery perforator flap, located on the gluteal fold, vascularized by cutaneous perforator vessels from the internal pudendal artery, and innervated by branches from the pudendal nerve and the posterior femoral cutaneous nerve. This flap has many advantages compared to others used for perineal reconstruction, and data related to the evaluation of its cutaneous sensibility are scarce, discrepant and subject to methodological criticisms. OBJECTIVE: To evaluate cutaneous sensibility of the internal pudendal artery perforator flap 12 months after perineal reconstruction in abdominoperineal resection of rectum, and compare it with the preoperative cutaneous sensibility of the gluteal fold (flap donor area). METHODS: A prospective study of 25 patients undergoing abdominoperineal resection of rectum for anorectal malignancies, and reconstruction with the internal pudendal artery perforator flap, in bilateral VY advancement. The modalities of tactile, pain, thermal and vibration sensibility were analyzed in four areas of the gluteal fold preoperatively and in the four corresponding areas of the flap 12 months after surgery. Tactile sensibility was assessed using the Pressure Specified Sensory Device(TM) (PSSD(TM)), a device that measures the pressure applied to the skin, static or moving. The other types of sensibility were analyzed with the forced-choice method, using a needle for pain sensibility, hot/cold contact for thermal sensibility and 128 Hz tuning-fork for vibration sensibility. RESULTS: The tactile sensibility thresholds measured with PSSD(TM) on the internal pudendal artery perforator flap 12 months after perineal reconstruction were similar to tactile sensibility thresholds of the gluteal fold preoperatively, both in static and moving pressure tests. The comparison between these thresholds showed no statistically significant difference, with p values greater than 0.05 in the four areas evaluated, for both tests. All patients presented pain, thermal and vibration sensibility in all four areas tested, on the both the gluteal fold preoperatively and the flap 12 months after surgery. CONCLUSION: In perineal reconstructions after abdominoperineal resection of rectum, it is expected that the cutaneous sensibility of the internal pudendal artery perforator flap is maintained

Esta tesis se ha hecho principalmente para estudiar e investigar polímeros impresos (MIPs) con la intención de usarlos como sensores de larga vida. La línea de investigación de esta tesis es la dirigida a conseguir la integración de estas formaciones impresas dentro de una lengua electrónica (ET), que es la rama de especialización en la que se ha desarrollado principalmente este proyecto. Después de hacer una revisión de la literatura, que inicialmente se centraba en la aplicación de MIPs a un equipo electroquímico, un sensor voltamétrico impreso y un procedimiento sensitivo complementario, el procedimiento se creó a través de una combinación de protocolos tomados de la literatura. Este sensor, descrito en el Artículo 1, presentaba una buena selectividad hacia el analito primario, teofilina, además de la especificidad requerida frente a sus análogos estructurales. Aunque el diseño del sensor permitía una mejor regeneración de la superficie respecto a otros sistemas parecidos encontrados en la literatura, el comportamiento de los polímeros usados en el MIP retardaba la tasa de transferencia de electrones en la superficie del sensor. Por culpa de este fenómeno, la sensibilidad del sensor se reducía. Justo después de estos experimentos iniciales, se hizo una colaboración con el grupo del Profesor Sergey Piletskey en la Universidad de Leicester (UoL) de Reino Unido. Durante este período, se realizó un estudio intensivo del proceso de diseño de impresión molecular asistido por un sistema computacional de modelización molecular ‘inhouse’. Se puso énfasis en el diseño de un receptor impreso para la molécula de baja solubilidad melanina, que se toma como ‘template modelo’. El MIP resultante se caracterizó y usó para la detección de melanina en muestras de leche, tal y como se describe y detalla en el Artículo 2. Más tarde, utilizando los conocimientos adquirido durante la estancia en Leicester, se desarrollaron nuevas técnicas de modelización computacional para la evaluación de los métodos utilizados en la modelización de MIPs, con el objetivo de obtener una técnica de evaluación virtual para el diseño de receptores impresos, optimizados para los requerimientos necesarios para su posterior aplicación en un sensor ET, tal como se detalla en el Artículo 3. Tal y como se detalla en el capítulo final de esta tesis, la experiencia y conocimientos adquiridos durante la investigación, se usaron para diseñar un grupo de sensores que funcionan asociados a ET. Este desarrollo podría ampliarse profundizando en la selección computacional de polielectrolitos, que luego serían inmovilizados en la superficie de un electrodo voltamétrico mediante una tinta de grafito conductora, de elevada robustez y estabilidad. En este sentido, también se proponen otras recomendaciones para lograr la mejora de la capacidad de regeneración de los MIPs utilizados, por ejemplo mediante la separación de MIP y electrodo. Finalmente, se presentan algunas sugerencias para colaboraciones institucionales, con el propósito de crear un sistema ET móvil, que permita recoger y analizar muestras en campo.
This thesis was predominantly undertaken to study and investigate molecular imprinted polymers (MIPs) with a view to their use as high longevity sensing elements in sensor arrays. The research line of the thesis was intended to lead to the integration of these imprinted arrays into an Electronic Tongue (ET) sensing system which is the area of expertise of the research group in which this project was primarily executed. Having initially executed a review of the literature, focusing initially on the application of the MIPs to an electrochemical device, an imprinted voltammetric sensor and a complimentary sensing procedure was developed using a combination of protocols extracted from the literature. This sensor, described in Article 1, had good selectivity toward the primary analyte, theophylline, and specificity against structural analogues. Though the design of the sensor allowed for significantly improved regeneratibility of the sensor relative to similar systems in the literature, the insulating nature of the polymers used in the MIP reduced the electron transfer rate at the sensor surface and thus resulted in a reduction in sensitivity. Following this initial experimental study, a secondment was undertaken in the University of Leicester under the supervision of Professor Sergey Piletsky. During this period, an intensive study of the design process of molecular imprinting, aided by an in-house computational molecular modelling platform, was conducted focusing on the design of an imprinted receptor for the low solubility 'model template', melamine. This MIP was successfully synthesised, characterised and used in the detection of melamine in milk samples, as detailed in Article 2. Further development of computational modelling techniques for the evaluation of MIP modelling techniques was also achieved with a view to create a virtual evaluation technique for the design of imprinted receptor sites optimised for the requirements of their application to an ET sensor array using the skills acquired during the Leicester secondment as detailed in Article 3. As detailed in the final chapter of this thesis, the insight into the imprinting process which was acquired during the research has been used to design a sensor array system which meets the specifications of ET experimental runs. This takes the form of the introduction of the research topic computationally selected polyelectrolytes, immobilised onto a voltammetric electrode surface via highly robust conducting graphite ink. Additional recommendations are also made to further enhance the on-going MIP projects within the laboratory, such as the separation of the MIP and the electrode to increase MIP regeneratibility. Some final suggestions for some other inter-institutional collaboration are also presented which aim to creating portable ET system for in-field sample collection and analysis.