Calcium ions are one of the most versatile signalling molecules. They are essential tothe proper functioning of various cellular processes in many different types of cells.The calcium signals have been studied in the past using ratiometric dyes like Fura-2.We showed that the genetically encoded calcium indicator GCaMP6m displays highersignal to noise ratio than Fura Red and therefore used it to study the calcium cytosolicconcentration in MDCK II cells. We use fluorescence microscopy to record and Pythonto analyse calcium signals in individual cells. Cellpose was used for automating thesegmentation. The cells were treated with ouabain, a cardiotonic steroid shown toincrease the intercellular communication between cells through gap junctions. Thecells were also transfected with connexins 43. We showed that ouabain does nothave an impact on the number of calcium peaks. We observed higher correlationsin the calcium between adjaçent transfected cells, but the results are not statisticallysignificant. We also observed clearly defined oscillations in one low confluencerecording with a period of around two minutes. / Kalciumjonen är en av de mest mångsidiga signalmolekylerna. De är nödvändiga förfunktionen av diverse cellulära processer i mångaolika typer av celler. I projektet studerades den cytosoliska kalciumkoncentrationeni MDCK II-celler. Fluorescensmikroskopi användes för att registrera, och Python föratt analysera, kalciumsignaler i enskilda celler. Cellpose användes för att automatiserasegmenteringen. Cellerna behandlades med ouabain, en kardiotonisk steroid somvisat sig öka den intercellulära kommunikationen mellan celler genom ”gap junctions”.Cellerna transfekterades också med connexin-43. Det påvisades att ouabain intehar någon inverkan på antalet kalciumtoppar. Högre korrelationer observeradesi kalcium mellan intilliggande celler som transfekterats, men resultaten är intestatistiskt relevanta. Tydligt definierade oscillationer observerades i en inspelning medlåg konfluens i en period på cirka två minuter. Slutligen visades att GCaMP6m har etthögre signal-brusförhållande än Fura Red.

Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / Exposure to oxidative stress leads to accumulation of reactive oxygen species and this stimulates protective cellular functions as a compensatory response to prevent the spread of apoptotic signal and prevent cell death. The purpose of this dissertation is to understand the importance of PKCγ activation and regulation of the retinal gap junction protein Cx50, and what role PKCγ plays in this neuro-protective effect. Through electron microscopy we were able to show that PKCγ knockout mice retinas had incomplete cellular organization in the outer plexiform layer (OPL) of the retina, the layer of retina where Cx50 plays an important role in retinal cellular synapses. Electroretinograms confirmed that this structural disorganization also led to loss of functional response to light stimuli in PKCγ knockout mice retinas. In vivo exposure to 100% hyperbaric oxygen (HBO) caused significant degradation of the retina in knockout mice compared to control mice. Thicknesses of the inner and nuclear and ganglion cell layers were increased, with complete disruption of OPL in PKCγ KO mice retinas. Damage to the outer segments of the photoreceptor layer and ganglion cell layer was significantly more apparent in the central retinas of HBO-treated knockout mice. Cx50 immunolabeling showed significant reduction to HBO treatment of PKCγ control mice retinas, HBO treatment failed to produce reduction of Cx50 immunolabeling in KO mice retinas. In the R28 retinal cell line, PKCγ enzyme was shown to be activated by phorbol ester (TPA) and hydrogen peroxide. This resulted in translocation to the cellular membrane as confirmed by western blot and confocal microscopy. Suppression of PKCγ by siRNA rendered R28 cells more sensitive to oxidative stress-induced cell apoptosis, the process of apoptosis started earlier, and this resulted in cell death. R28 treatment with phorbol esters and hydrogen peroxide led to reduction in gap junction activity and Cx50 gap junction cell disassembly. This dissertation shows that PKCγ plays an important role in structural organization of retina and has a neuro-protective effect in response to oxidative stress, in part because of its control of Cx50.

Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / Connexin proteins are the principle structural components of gap junction channels that connect the cytoplasm of two cells and maintain direct intercellular communication through the exchange of ions, small molecules and cellular metabolites. Colocalization and tissue-specific expression of diverse connexin molecules are reported to occur in a variety of organs. Impairment of gap junctional intercellular communication, caused by mutations, gain of function or loss of function of connexins, is involved in a number of diseases including the development of cancer. Here the functions of a gap junction protein, connexin46 (Cx46), have been investigated in two hypoxic tissues, lens and breast tumor. We show that human breast cancer cells, MCF-7 and breast tumor tissues express connexin46 (Cx46) and it plays a critical role in protecting cells against hypoxia-induced death. Interestingly, I find that Cx46 is upregulated in MCF-7 breast cancer cells and human breast cancer tumors. Downregulation of Cx46 by siRNA promotes cell death of human lens epithelial cells (HLEC) and MCF-7 cells under hypoxic conditions. Furthermore, direct injection of anti-Cx46 siRNA into xenograft tumors prevents tumor growth in nude mice. Our result suggests that both normal hypoxic tissue (lens) and adaptive hypoxic tissue (breast tumor) utilize the same protein, Cx46, as a protective strategy against hypoxia. In the last part of the dissertation, we show that over expression of Cx46 induces the degradation of another connexin, connexin43, in rabbit lens epithelial NN1003A cells. Over expression of Cx46 increases ubiquitination of Cx43. Moreover, the Cx46-induced Cx43 degradation is counteracted by inhibitors of proteasome. Taken together, these data indicate that the degradation of Cx43, upon Cx46 over expression, is mediated by the ubiquitin-proteasome pathway. I also provide evidence that that C-terminal tail of Cx46 is essential to induce degradation of Cx43. Therefore, our study shows that Cx46 has a novel function in the regulation of Cx43 turnover in addition to its conventional role as a gap junction protein. This may contribute to protection from hypoxia in both the lens and tumors.

Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / The avascular lens tissue depends on the gap junction channels to facilitate intercellular communication for supplying cells deep within the lens with nutrients and removing waste products of cellular metabolism. In the absence of the protein synthesis machinery in the inner lens fiber cells, the proper regulation of gap junction channels becomes extremely important as disturbance of the lens homeostasis can lead to cataract development. Phosphorylation of gap junction subunit connexin proteins has been shown to play an important channel-modulating role in a variety of tissue. Protein kinase C-[Gamma] (PKC[Gamma]) has been implicated in the phosphorylation of connexins in the lens. Here the role of PKC[Gamma] in the regulation of gap junction coupling in the mouse lens has been investigated. We have compared the properties of coupling in lenses from wild type (WT) and PKC[Gamma] knockout (KO) mice. Western blotting, confocal immunofluorescence microscopy, immunoprecipitation, RT-PCR and quantitative real time PCR were used to study gap junction protein and message expression; gap junction coupling conductance and pH gating were measured in intact lenses using impedance studies. PKC[Gamma]was found to regulate the amount and distribution of Cx43 in the lens. Gap junction coupling conductance in the differentiating fibers (DF) of PKC[Gamma] KO lenses was 34% larger than that of WT. In the mature fiber (MF), the effect was much larger with the KO lenses having an 82% increase in coupling over WT. Absence of PKC[Gamma] in the KO mice also caused abnormal persistence of nuclei in the typical nucleus-free region in the DF. These results suggest a major role for PKC[Gamma] in the regulation of gap junction expression and coupling in the normal lens mediated by phosphorylation of the lens connexins. This becomes very vital in the diabetic lenses which contain a depleted amount of PKC[Gamma] and people suffering from spinocerebellar ataxia type-14 (SCA14) who have a mutated inactive form of PKC[Gamma]. Prolonged exposure of lenses to oxidative stress in these patients can lead to cataract formation. In cultured human lens epithelial cells (HLECs), 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated the depletion of Cx43 protein level via PKC-mediated phosphorylation of Cx43. At the same time Cx46 protein and message levels were upregulated in response to TPA treatment. So, the PKC activator regulates Cx43 and Cx46 in opposing ways. The possible mitochondria localization of Cx46 reported here could help in finding the non-junctional roles for Cx46.

As junções comunicantes medeiam a comunicação entre células e são fundamentais para o desenvolvimento e homeostase em organismos multicelulares. Nos invertebrados as junções são formadas por proteínas transmembrana denominadas inexinas. As junções permitem a passagem de pequenas moléculas através de um canal intercelular, entre uma célula e outra adjacente. O díptero Rhynchosciara americana tem contribuído para o estudo da biologia dos invertebrados, bem como para o estudo da interação entre genes, regulação gênica e desenvolvimento biológico. A partir de um banco de ESTs foram identificadas algumas mensagens com homologia as inexinas. Deste modo, o presente trabalho pretende estudar a inexina-2 de R. americana pela: caracterização molecular; análise do perfil expressão; e localização celular. Os nossos resultados de caracterização confirmam que a mensagem é de uma proteína de junção comunicante e as análises do perfil de expressão e localização celular mostram que a inexina-2 pode participar de diversos processos fisiológicos ao longo do desenvolvimento de R. americana. / The gap junctions mediate communication between cells and are fundamental to the development and homeostasis in multicellular organisms. In invertebrates the gap junctions are formed by transmembrane proteins called innexins. The gap junctions allow the passage of small molecules through an intercellular channel, between a cell and other adjacent. The dipteran Rhynchosciara americana has contributed to the study of the biology of invertebrates, as well as for the study of the interaction between genes, gene regulation and biological development. As from a ESTs bank identified some messages with homology connexins. Therefore, this paper aims to study the innexin R. americana by: molecular characterization; analysis of the expression profile; and cellular localization. Our molecular characterization results confirm that the message is from a gap junction protein and analysis of the expression and cellular localization profile show that innexin can participate in many physiological processes during the development of R. americana.

Dans les dernières décennies, l'impact dû à l'altération de la microstructure du tissu cardiaque dans la survenue de troubles arythmiques (syndrome de Brugada, fibrillation auriculaire, syndromes de repolarisation précoce…) est de plus en plus étudié. Les données expérimentales relatives au fonctionnement et aux régulations intervenant aux échelles cellulaires et subcellulaires (jonctions communiquantes, rôle de certains canaux ioniques) sont de plus en plus nombreuses, et fournissent un cadre adapté aux numériciens pour développer ou affiner des modèles et en valider les comportements. Dans cette thèse, nous proposons le développement et l'étude d'un modèle « microscopique » prenant en compte la géométrie individuelle des cellules et les jonctions communiquantes entre elles. Le modèle vise à comprendre la propagation du potentiel d'action au sein d'un réseau de cellules. Nous établissons ce modèle via une étude du comportement des ions dans les cellules. Ce comportement, décrit par diverses équations de la physique microscopique (électrostatique...), fournit un cadre à partir duquel, en effectuant quelques analyses dimensionnelles et une étude asymptotique, nous dérivons le modèle susmentionné. Puis, nous démontrons l'existence d'une solution à ce modèle à l'aide d'un processus de discrétisation en temps « semi-implicite » et de théorèmes de compacité. Nous proposons ensuite un ensemble de simulations dont l'objet est de comprendre la propagation des potentiels d'action entres cellules au sein d'un réseau, et en particulier le rôle des jonctions communiquantes. Nous étudions différents modèles de jonctions communiquantes, dont un non-linéaire et dépendant du temps. Cette thèse ouvre de nombreuses perspectives, à courte échéance des comparaisons à des observations expérimentales chez la souris, et à plus long terme de recherche sur les mécanismes de propagation à l'échelle cellulaire et leurs impact sur les troubles du rythme cardiaque. / During the last decades, studies regarding the prospective impact of the alterations at the microscopic scale of the heart tissue in the appearance of arrhythmias (Brugada's syndrome, atrial fibrillation, early repolarization syndrome...) have been more numerous. The amount of experimental data regarding the behaviors and regulations that occur at a cellular and a subcellular (gap junctions, role of specific ionic channels) is increasing and these data provide an adapted frame for the computational mathematicians to develop or improve models and confirm their behaviour. In this thesis, we developed and studied a ``microscopic'' model taking into account the individual geometry of the cells and the gap junctions between them. This model is designed to enhance our understanding of the action potential propagation in a network of cells. We extracted this model using a study of the ions movements in the cells. These movements, described by various microscopic physics equations (electrostatic...), and some dimensional analysis, including an asymptotic study, allow us to derive the model. We then show that the problem described by such a model has a solution, via a semi-implicit time discretization process and compacity arguments. Afterwards, we offer numerous simulations in order to enhance our understanding of the action potential propagation between the cells of various networks. We specifically customize the gap junction models we use (a geometric one, a linear one and a non-linear one) to enhance our comprehension. This thesis introduces many questions. On the short-term, on the comparison between experimental data observed on mice cells and our results. On the long-term regarding the mechanisms regulating the action potential propagation, and their impact on the alterations of the cardiac rhythm.

Large parts of the cortex and the thalamus project into the striatum,which serves as the input stage of the basal ganglia. Information isintegrated in the striatal neural network and then passed on, via themedium spiny (MS) projection neurons, to the output stages of thebasal ganglia. In addition to the MS neurons there are also severaltypes of interneurons in the striatum, such as the fast spiking (FS)interneurons. I focused my research on the FS neurons, which formstrong inhibitory synapses onto the MS neurons. These striatal FSneurons are sparsely connected by electrical synapses (gap junctions),which are commonly presumed to synchronise their activity.Computational modelling with the GENESIS simulator was used toinvestigate the effect of gap junctions on a network of synapticallydriven striatal FS neurons. The simulations predicted a reduction infiring frequency dependent on the correlation between synaptic inputsto the neighbouring neurons, but only a slight synchronisation. Thegap junction effects on modelled FS neurons showing sub-thresholdoscillations and stuttering behaviour confirm these results andfurther indicate that hyperpolarising inputs might regulate the onsetof stuttering.The interactions between MS and FS neurons were investigated byincluding a computer model of the MS neuron. The hypothesis was thatdistal GABAergic input would lower the amplitude of back propagatingaction potentials, thereby reducing the calcium influx in thedendrites. The model verified this and further predicted that proximalGABAergic input controls spike timing, but not the amplitude ofdendritic calcium influx after initiation.Connecting models of neurons written in different simulators intonetworks raised technical problems which were resolved by integratingthe simulators within the MUSIC framework. This thesis discusses theissues encountered by using this implementation and gives instructionsfor modifying MOOSE scripts to use MUSIC and provides guidelines forachieving compatibility between MUSIC and other simulators.This work sheds light on the interactions between striatal FS and MSneurons. The quantitative results presented could be used to developa large scale striatal network model in the future, which would beapplicable to both the healthy and pathological striatum. / QC 20100720

<p>The basal ganglia form an important structure centrally placed in the brain. They receive input from motor, associative and limbic areas, and produce output mainly to the thalamus and the brain stem. The basal ganglia have been implied in cognitive and motor functions. One way to understand the basal ganglia is to take a look at the diseases that affect them. Both Parkinson's disease and Huntington's disease with their motor problems are results of malfunctioning basal ganglia. There are also indications that these diseases affect cognitive functions. Drug addiction is another example that involves this structure, which is also important for motivation and selection of behaviour.</p><p>In this licentiate thesis I am laying the groundwork for a detailed model of the striatum, which is the input stage of the basal ganglia. The striatum receives glutamatergic input from the cortex and thalamus, as well as dopaminergic input from substantia nigra. The majority of the neurons in the striatum are medium spiny (MS) projection neurons that project mainly to globus pallidus but also to other neurons in the striatum and to both dopamine producing and GABAergic neurons in substantia nigra. In addition to the MS neurons there are fast spiking (FS) interneurons that are in a position to regulate the firing of the MS neurons. These FS neurons are few, but connected into large networks through electrical synapses that could synchronise their effect. By forming strong inhibitory synapses on the MS neurons the FS neurons have a powerful influence on the striatal output. The inhibitory output of the basal ganglia on the thalamus is believed to keep prepared motor commands on hold, but once one of them is disinhibited, then the selected motor command is executed. This disinhibition is initiated in the striatum by the MS neurons.</p><p>Both MS and FS neurons are active during so called up-states, which are periods of elevated cortical input to striatum. Here I have studied the FS neurons and their ability to detect such up-states. This is important because FS neurons can delay spikes in MS neurons and the time between up-state onset and the first spike in the MS neurons is correlated with the amount of calcium entering the MS neuron, which in turn might have implications for plasticity and learning of new behaviours. The effect of different combinations of electrical couplings between two FS neurons has been tested, where the location, number and strength of these gap junctions have been varied. I studied both the ability of the FS neurons to fire action potentials during the up-state, and the synchronisation between neighbouring FS neurons due to electrical coupling. I found that both proximal and distal gap junctions synchronised the firing, but the distal gap junctions did not have the same temporal precision. The ability of the FS neurons to detect an up-state was affected by whether the neighbouring FS neuron also received up-state input or not. This effect was more pronounced for distal gap junctions than proximal ones, due to a stronger shunting effect of distal gap junctions when the dendrites were synaptically activated.</p><p>We have also performed initial stochastic simulations of the Ca²⁺-calmodulin-dependent protein kinase II (CaMKII). The purpose here is to build the knowledge as well as the tools necessary for biochemical simulations of intracellular processes that are important for plasticity in the MS neurons. The simulated biochemical pathways will then be integrated into an existing model of a full MS neuron. Another venue to explore is to build striatal network models consisting of MS and FS neurons and using experimental data of the striatal microcircuitry. With these different approaches we will improve our understanding of striatal information processing.</p>

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Medical Sciences

Early experimental work suggested that the retina’s main role was to detect changes in brightness and contrast, namely working as a light detector, and that most of the complex computations in the visual system happened upstream in the brain. In reality, there is a growing wealth of literature indicating that the retina itself processes multiple channels of visual information (contrast, motion, orientation, etc.), making it much more complex than it originally appeared. For instance, there now appear to be over 20 types of retinal ganglion cells. To this end, the work in this thesis will focus on the identification and characterization of a single type of retinal ganglion cell in the mouse retina. In the first section of my results, I will show that this cell type, identified as the only GFP+ ganglion cell in the transgenic Hb9::eGFP retina, is a directionally selective ganglion cell (DSGC), that preferentially responds to objects moving upward through the visual field. This cell has a pronounced morphological asymmetry that helps it to synergistically (along with asymmetric inhibition) generate directionally selective responses. In the second results section, I will describe a novel phenomenon exhibited by Hb9+ DSGCs: Thanks to gap junction mediated signals, Hb9+ cells are able to anticipate moving stimuli and correct for lags that are inherent in visual signals generated by photoreceptors. In the third results section I will elucidate the mechanisms for the gap junction mediated anticipatory signals outlined in the second results section. Together, these results provide a significant advancement in our understanding of how the retina processes moving stimuli and provide a compelling example of how chemical and electrical synapses interact to allow for exquisite signal multiplexing.