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Several viruses from different virus families are known to cause ocular infections, e.g. members of the Adenoviridae, Picornaviridae and the Herpesviridae families. These infections are spread by contact and in the case of adenoviruses (Ads) and picornaviruses they are also highly contagious. The ocular infections caused by Ads and picornaviruses are called epidemic keratoconjunctivitis (EKC) and acute hemorrhagic conjunctivitis (AHC), respectively. Historically, EKC is caused mainly by three types of Ads from species D: Ad8, Ad19 and Ad37. The infection is characterized by keratitis and conjunctivitis but also involves pain, edema, lacrimation and blurred vision. AHC is caused mainly by two types of picornaviruses: coxsackievirus A24v (CVA24v) and enterovirus 70 (EV70), and is characterized by pain, redness, excessive tearing, swelling and subconjunctival hemorrhages. In addition, blurred vision, keratitis, malaise, myalgia, fever, headache and upper respiratory tract symptoms can also be experienced. Both infections are problematic in many parts of the world, affecting millions of people every year. Despite the great need, the only treatment available today is supportive treatment; no antiviral drugs are available to combat these common viral infections. Ad37 has previously been reported to use sialic acid (SA) as its cellular receptor. Since there is no antiviral treatment available against EKC we wanted to evaluate the inhibitory effect of SA-based antiviral compounds on Ad37 binding to and infection of ocular cells. We found that multivalent compounds consisting of SA linked to a globular carrier molecule, in this case human serum albumin, efficiently blocked Ad37 binding and infection at low concentrations. Further attempts were then made to improve the effect by chemically modifying SA monosaccharides. However, no enhanced inhibitory effect was accomplished and the conclusion was that the best inhibitors are based on unmodified SA. We next hypothesized that development of efficient SA-based binding inhibitors may require detailed knowledge about the structure of the SA-containing receptor. Using a battery of biological and biochemical experiments, including glycan array, binding and infection assays, X-ray crystallography and surface plasmon resonance (SPR); we identified a specific glycan involved in the binding and infection of Ad37. This glycan turned out to be a branched, di-SA-containing motif corresponding to the glycan motif of the ganglioside GD1a. However, the ganglioside itself did not function as a cellular receptor, as shown by a number of binding and infection assays. Instead, the receptor consisted of one or more glycoproteins that contain the GD1a glycan motif. This glycan docked with both its SAs into the trimeric Ad37 knob resulting in a very strong interaction as compared to most other protein-glycan interactions. Hopefully, this finding will aid development of more potent inhibitors of Ad37 binding and infection. The receptor for CVA24v, one of the main causative agents of AHC, has been unknown until now. We showed that this ocular virus, like Ad37, is also able to use SA as a receptor on corneal cells but not on conjunctival cells. This suggested that CVA24v may use two different receptors. As for Ad37, the receptor used by CVA24v on corneal cells also appears to be one or more sialic acid-containing glycoproteins. We believe that these findings may be a starting point for design and development of candidate drugs for topical treatment of AHC.

VEGF is a potent angiogenic and endothelial cell growth factor that is key for the development of the glomerulus, the main filtration unit of the kidney. It is continued to be expressed in the mature glomerulus, with podocytes being the major site of production. VEGF binds to two receptors, VEGFR-1 and VEGFR-2, which are expressed by the adjacent endothelial cells (ECs). VEGFR-2 is the primary mediator of VEGF signaling while VEGFR-1is thought to function as a ‘decoy’ receptor, sequestering VEGF away from VEGFR-2. Gene targeting studies in mice show that VEGF loss from the podocyte results in profound defects of the ECs, consistent with a paracrine signaling loop. However, the identification of VEGF receptors on podocytes in vitro suggests an additional autocrine signaling pathway for VEGF may exist. To further study the role of VEGF in the glomerulus and to address whether a VEGF autocrine loop is functional in vivo, we generated a transgenic mouse model with inducible VEGF upregulation in the podocyte and genetically deleted VEGFR-2 and VEGFR-1 from the podocyte using the Cre-loxP system. Increased VEGF production from the podocyte leads to increased glomerular permeability and ultrastructural changes in the glomerular filtration barrier depending on the time and length of induction. Podocyte-selective deletion of VEGFR-2 did not cause glomerular disease. In contrast, VEGFR-1 loss from the podocyte led to proteinuria and glomerular defects at 6 weeks of age with extensive podocyte foot process effacement. In keeping with the model that VEGFR-1 functions as a VEGF trap, similarities were observed between the glomerular lesions of VEGFR-1 mutant mice and transgenic mice that overexpress VEGF within podocytes. Strikingly, in vitro studies also revealed an increase in podocyte cell adhesion to sVEGFR-1, suggesting additional roles for sVEGFR-1. Together, these data suggest that a tight regulation of VEGF must be maintained in the adult glomerulus. Furthermore, these findings provide the first genetic evidence that VEGF autocrine signaling loop through VEGFR-2 is dispensable in normal glomeruli. In addition, podocytes express sVEGFR-1 and is required in podocytes in vivo to maintain glomerular integrity by regulating VEGF availability and podocyte cell adhesive properties.

VEGF is a potent angiogenic and endothelial cell growth factor that is key for the development of the glomerulus, the main filtration unit of the kidney. It is continued to be expressed in the mature glomerulus, with podocytes being the major site of production. VEGF binds to two receptors, VEGFR-1 and VEGFR-2, which are expressed by the adjacent endothelial cells (ECs). VEGFR-2 is the primary mediator of VEGF signaling while VEGFR-1is thought to function as a ‘decoy’ receptor, sequestering VEGF away from VEGFR-2. Gene targeting studies in mice show that VEGF loss from the podocyte results in profound defects of the ECs, consistent with a paracrine signaling loop. However, the identification of VEGF receptors on podocytes in vitro suggests an additional autocrine signaling pathway for VEGF may exist. To further study the role of VEGF in the glomerulus and to address whether a VEGF autocrine loop is functional in vivo, we generated a transgenic mouse model with inducible VEGF upregulation in the podocyte and genetically deleted VEGFR-2 and VEGFR-1 from the podocyte using the Cre-loxP system. Increased VEGF production from the podocyte leads to increased glomerular permeability and ultrastructural changes in the glomerular filtration barrier depending on the time and length of induction. Podocyte-selective deletion of VEGFR-2 did not cause glomerular disease. In contrast, VEGFR-1 loss from the podocyte led to proteinuria and glomerular defects at 6 weeks of age with extensive podocyte foot process effacement. In keeping with the model that VEGFR-1 functions as a VEGF trap, similarities were observed between the glomerular lesions of VEGFR-1 mutant mice and transgenic mice that overexpress VEGF within podocytes. Strikingly, in vitro studies also revealed an increase in podocyte cell adhesion to sVEGFR-1, suggesting additional roles for sVEGFR-1. Together, these data suggest that a tight regulation of VEGF must be maintained in the adult glomerulus. Furthermore, these findings provide the first genetic evidence that VEGF autocrine signaling loop through VEGFR-2 is dispensable in normal glomeruli. In addition, podocytes express sVEGFR-1 and is required in podocytes in vivo to maintain glomerular integrity by regulating VEGF availability and podocyte cell adhesive properties.

The establishment of neuronal polarity constitutes a central phase in neuronal development and synaptogenesis. In my thesis, I study factors that regulate the development of neuronal polarity and its relationship with neurotransmitter receptor expression. For my experiments, I have investigated the development of sensory neurons from neonatal rat nodose ganglia in culture. Sensory neurons have a pseudo-unipolar morphology, do not extend dendrites, and are devoid of synaptic connections on their somata. However, nodose neurons form synapses de novo in cultures, and I show that the neurons have retained the ability to extend dendrites. Extrinsic factors control dendrite extension by these neurons: the ganglionic satellite cells inhibit the growth of dendrites and induce the neurons to develop a unipolar morphology. In the absence of satellite cells, nodose neurons establish a new multipolar morphology and, in response to nerve growth factor (NGF), extend several dendrites. However, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) do not induce the neurons to extend dendrites, but promote the expression of properties typical of nodose neurons in vivo. / As nodose neurons acquire a new dendrite-axonal polarity in the presence of NGF, they increase the density of functional neuronal nicotinic acetylcholine receptors (nAChRs) on their somato-dendritic domains. To learn more about the relationship between dendrites extension and nAChR gene expression, I have examined the changes in transcript levels of nAChR subunits in neonatal rat sympathetic neurons developing in culture. I show that the developmental pattern of nAChR subunit expression in the cultured neurons follows closely that of sympathetic neurons developing in vivo, with the exception of one specific subunit $ alpha sb7$. I show that the increase in $ alpha sb3$ mRNA levels correlates well with an increase in the density of functional nAChRs on the neurons. In addition, my results suggest that these increases are regulated by mechanisms intrinsic to neonatal sympathetic neurons. On the other hand, the changes in $ alpha sb7$ gene expression, which correlate with changes in $ alpha$-bungarotoxin binding, are activity-dependent and regulated by a calcium/calmodulin-dependent protein kinase pathway. The results presented in this thesis provide insights on how neurons are influenced in their extension of dendrites and how this extension affects neurotransmitter receptor expression.

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity.

AntecedentsDurant els primers dies del desenvolupament postnatal les plaques motores es troben multiinnervades fins amb 5 axons. Aquesta multiinnervació decreix progressivament fins a l'adultesa on les plaques motores es troben innervades per un sol axó. En aquesta pèrdua axonal o eliminació sinàptica es troben implicats una sèrie de mecanismes presinàptics i postsinàptics relacionats amb el nivell d'activitat neuromuscular. En aquest context, poden estar implicats els autoreceptors muscarínics (mAChRs). En les sinapsis neuromusculars colinèrgiques existeixen autoreceptors muscarínics axonals sensibles al neurotransmissor que ell mateixos alliberen. Objectius S'estudia la participació dels diferents tipus de mAChRs en el procés d'eliminació sinàptica en la sinapsi neuromuscular de rata nounat mitjançant tècniques electrofisiològiques de registre intracel·lular convencional. També es vol determinar amb precisió els tipus de mAChRs presents en la sinapsi neuromuscular de la rata adulta, la seva relació amb els canals de calci dependents de voltatge (VDCC) i els mecanismes intraaxonals implicats.MetodologiaS'utilitza com a model les sinapsis neuromusculars del múscul Levator Auris Longus de rates adultes i nounats. Mitjançant el registre intracel·lular estudiem com diferents antagonistes muscarínics afecten els potencials de placa evocats (EPP) de la sinapsi neuromuscular adulta i de la sinapsi monoinnervada i doblement innervada de l'animal nounat. Per estudiar la possible relació dels muscarínics amb els VDCC incubem, prèviament, el múscul mitjançant blocadors dels canals de calci (Nitrendipina, -Conotoxina-GVIA i -Agatoxina-IVA) abans de determinar la resposta muscarínica. Per identificar una sinapsi doblement innervada en els animals nounats observem si a mesura que augmentem la intensitat de l'estímul elèctric apareix un segon EPP després del primer. Seguidament, les amplades dels potencials de placa més baixes i més altes es designen com a "EPP petit" i "EPP gran", respectivament.Principals resultatsEn l'adult, l'antagonista selectiu pel receptor muscarínic M1, Pirenzepina redueix la neurotransmissió evocada. Per altra banda, l'antagonista selectiu pel receptor muscarínic M2, Metoctramina incrementa l'alliberació evocada del neurotransmissor. Aquests dos mecanismes mediats pels muscarínics M1 i M2 depenen de l'entrada de calci per mitjà del canal P/Q. No trobem cap dada que ens indiqui la presència dels receptors muscarínics M3 (sensibles a 4-DAMP) i els receptors muscarínics M4 (sensibles a Tropicamida) en els músculs d'animals adults. En el nadó, la resposta dels agents muscarínics en el terminal nerviós de les sinapsis monoinnervades és similar a la resposta de l'EPP gran en les sinapsis doblement innervades. Aquestes respostes impliquen una inhibició de l'alliberació de l'ACh per mitjà de l'antagonista del receptor muscarínic M1, Pirenzepina i per mitjà de l'antagonista del receptor muscarínic M2, Metoctramina. L'EPP petit és també inhibit per l'antagonista del receptor muscarínic M2, Metoctramina però, és augmentat per l'antagonista del receptor M1, Pirenzepina i l'antagonista del receptor M4, Tropicamida.En relació als canals de calci dependents de voltatge, observem un canvi gradual des de la implicació dels VDCC tipus P/Q, N i L en totes les respostes mediades pels muscarínics M1, M2 i M4 en l'EPP petit, fins a la implicació d'un únic canal, el P/Q, en les respostes dels receptors M1 i M2 en les plaques monoinnervades. Aquest fet indica l'existència d'un progressiu tancament dels VDCC paral·lel a l'especialització del canal tipus P/Q de l'adult. Conclusió principalEls autoreceptors muscarínics poden, globalment, potenciar el terminal que genera l'EPP gran i inhibir el terminal que genera l'EPP petit col·laborant així en l'eliminació sinàptica del desenvolupament. / AntecedentsDuring first postnatal developing days the motor endplates are multiinnervated, this multiinnervation decrease at the next weeks until the adult age, when the motor endplates are monoinnervated. Is in this axonal losing or synaptic elimination where there are implicated presynaptic and postsynaptic mechanisms related with neuromuscular activity levels. In this context, muscarinic autoreceptors (mAChRs) can be involved. In the cholinergic neuromuscular junctions there are presynaptic muscarinic autoreceptors(mAChRs) with sensibility to neurotransmitter released by themselves. ObjectivesIn this work we investigate the participation of different subtypes of mAChRs in the synaptic elimination period in the newborn rat neuromuscular junction with electrophysiological recording techniques. We want previously determining, the presence of different subtypes, the functional behaviour and the voltage dependent calcium channels (VDCC)-dependence of the mAChRs in the adult motor nerve terminals.MethodsWe investigate presynaptic muscarinic autoreceptors involved in the modulation of neurotransmitter release and their VDCC-dependence, in the Levator Auris Longus muscle neuromuscular junction of newborn and adult rats. We use intracellular recording techniques to study who different muscarinic antagonists affect the endplate potentials (EPP) in the adult and newborn neuromuscular junctions.We previously incubated the muscle with VDCC blockers (Nitrendipine, -Conotoxin- GVIA, -Agatoxin-IVA) before determining the muscarinic response, to study the possible relation to mAChRs with VDCC.ResultsIn the adult, the M1 antagonist Pirenzepine (10 µM) decreases the evoked release. On the other hand, the M2 antagonist Methoctramine (1 µM) increases the evoked neurotransmitter release. Both mechanisms, depends on the calcium influx from the external media via P/Q calcium channels. We found no indices of M3 (4-DAMP-sensitive) or M4 (Tropicamide-sensitive) muscarinic receptors in the adult animals muscles.In the newborn, in dually innervated fibers, a second EPP can appear after the first when the intensity of the electrical stimulus is raised. The lowest and highest EPP amplitudes are designed as "the small- EPP" and "the large- EPP", respectively.The muscarinic agents response in the nerve terminal of monoinnervated synapses is similar to the large EPP response in dually innervated junctions. This response involved an ACh release inhibition by M1 muscarinic antagonist Pirenzepine and by M2 muscarinic antagonist Methoctramine. However, M1 antagonist Pirenzepine and M4 antagonist Tropicamide increase the small EPP.In relation to VDCC-dependence we observe a gradual change in the small EPP, from the P/Q, N, and L VDCC implication in all responses where the M1, M2 and M4 muscarinics are involved, to the implication of a lonely channel, the P/Q, in the M1 and M2 responses in the monoinnervated endplates.This indicates the existence of a progressive shutoff in parallel with maturation and specialization of the adult type P/Q channel.Main conclusionIn summary, muscarinic autoreceptors can directly modulate both, large-EPP generating ending potentiation, and small-EPP generating ending depression by their association with VDCC in developing neuromuscular junction.

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No description available.