Foram utilizados 160 leitões recém-desmamados, com peso médio inicial de 6,43 ± 0,71 kg, com o objetivo de avaliar os efeitos dos nucleotídeos sobre o desempenho, a frequência de diarreia, a morfometria de órgãos, a histologia do epitélio intestinal e a microbiota intestinal. Foram testados cinco tratamentos em um experimento em blocos casualizados, com oito repetições (blocos) por tratamento e quatro animais por unidade experimental (dois machos castrados e duas fêmeas). Os tratamentos foram: dieta basal à base de milho, farelo de soja, derivados lácteos e plasma com inclusão de 120 ppm de clorohidroxiquinolina (antimicrobiano) e dieta basal contendo 0, 100, 150 e 200 ppm de nucleotídeos. Para a determinação das variáveis de desempenho, os animais foram pesados ao 1º, 14º e 34º dia de experimentação e foram quantificadas as rações fornecidas e desperdiçadas. Para avaliar a frequência de diarreia, foram observadas presença e ausência de diarreia na baia, diariamente, no período da manhã. Ao final do experimento, um animal de cada baia (unidade experimental) foi abatido para avaliação da morfometria de órgãos (estômago vazio, intestino delgado vazio, pâncreas, fígado e baço) e da histologia do epitélio intestinal (altura e largura de vilosidade, profundidade de cripta, relação altura de vilosidade: profundidade de cripta), além da coleta de amostras do conteúdo do duodeno e do jejuno para avaliação da microbiota intestinal dos leitões (mesófilos, Gram positivos totais, Gram negativos totais, Lactobacillus spp., Escherichia coli, Salmonella spp., Staphylococcus aureus e Clostridium perfringens). No período de 1 a 14 dias de experimentação, as variáveis de desempenho não foram influenciadas (P>0,05) pelos tratamentos. Para o período total (1 a 34 dias), houve efeito linear benéfico da inclusão dos nucleotídeos no peso final (P=0,005; P34 = 0,0033X + 23,657, R2 = 0,87) e no ganho diário de peso (P=0,008; GDP = 0,0002X + 0,4955, R2 = 0,83) dos animais. Os leitões alimentados com o antibiótico tiveram menor (P=0,049) frequência de diarreia comparados aos animais alimentados com nucleotídeos no período de 1 a 14 dias de experimentação. Por outro lado, no período de 1 a 34 dias de experimentação, os tratamentos não afetaram (P>0,05) a frequência de diarreia, a morfometria de órgãos, a histologia do epitélio intestinal e a microbiota intestinal. Assim, de maneira geral, a inclusão de até 200 ppm de nucleotídeos em dietas complexas para leitões recém-desmamados melhora o desempenho dos animais, sem afetar a frequência de diarreia, a morfometria de órgãos, a histologia do epitélio intestinal e a microbiota intestinal de leitões na fase de creche. / The purpose of this study was to evaluate the effects of dietary nucleotide levels on performance, occurrence of diarrhea, organ morphometry, intestinal histology and intestinal microbiota of weanling pigs fed complex diets containing corn, soybean meal, milk products, and spray-dried plasma. One hundred and sixty 21d-weaned pigs, averaging 6.43 ± 0.71 kg BW, were used in a randomized complete block design experiment with 5 treatments, 8 replications (blocks) per treatment and 4 animals per experimental unit (pen). The treatments were: basal diet with 120 ppm of chloro-hydroxyquinoline (antimicrobial treatment), and basal diet with 0, 100, 150, and 200 ppm of nucleotides. The ADG, ADFI, G:F and occurrence of diarrhea were calculated during 1 to 14 and 1 to 34 d of experimental period. A day after the end of the experimental period, an animal from each pen was slaughtered to evaluate of organ morphometry (empty stomach, empty small intestine, pancreas, liver and spleen), intestinal histology (villus height, villus width, crypt depth and villus height-to-crypt depth ratio), and intestinal microbiota (mesophiles, Gram-positive bacteria, Gram-negative bacteria, Lactobacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp., and Clostridium perfringens). During 1-14 d of experimental period, performance was not affected (P>0.05) by treatments. For the total experimental period (1-34 d), beneficial linear effects of dietary levels of nucleotides on final BW (P=0.005; BW = 0.0033X + 23.657, R2 = 0.87) and ADG (P = 0.008; ADG = 0.0002X + 0.4955, R2 = 0.83) were observed, but not (P>0.05) on ADFI and G:F. Pigs fed antimicrobial treatment had lower (P=0.049) occurrence of diarrhea from d 1 to 14 than those fed nucleotide treatments. However, for the total experimental period (1-34 d), treatments did not affect (P>0.05) occurrence of diarrhea, organ morphometry, intestinal histology and intestinal microbiota. Therefore, adding up to 200 ppm of dietary nucleotides to complex diets for weanling pigs showed beneficial effect on growth performance, without affecting organ morphometry, intestinal histology, intestinal microbiota and occurrence of diarrhea of nursery pigs.

Pacientes sépticos podem evoluir para choque séptico, destes 40% sobrevivem. Caracterizamos o modelo experimental, avaliamos fatores envolvidos na inflamação e avaliamos a modulação causada por purinas (ATP/ADP) na quantificação de superóxido (O2-) e na reatividade vascular da aorta isolada. Os resultados sugerem que na aorta isolada de animais endotoxêmicos, ATP e ADP aumentam a síntese de óxido nítrico (NO), porém somente o ATP reduz a biodisponibilidade de O2-, provavelmente pelo reacoplamento da NO sintase endotelial / Septic patients can evolve for septic shock and 40% of these survive. We characterize the experimental model we evaluate involved factors in the inflammation and evaluate the modulation caused by purines (ATP/ADP) in the superoxide quantification (O2-) and in the vascular reactivity of isolated aorta. The results suggest that in isolated aorta of endotoxemics rats, ATP and ADP increase the endothelial nitric oxide synthase (NOS) however just ATP reduces the bio availability of O2-, probably for the re-couples of the endothelial NOS synthase

O objetivo deste trabalho foi avaliar os efeitos de níveis de nucleotídeos nas dietas de leitões recém-desmamados sobre o desempenho, a morfometria de órgãos e a histologia do epitélio intestinal. Foram utilizados 144 leitões desmamados aos 21 dias de idade e com peso médio inicial de 5,80 ± 0,16 kg em um experimento inteiramente ao acaso, com seis tratamentos, seis repetições por tratamento e quatro animais por baia (unidade experimental). Os tratamentos foram: Am (antimicrobiano) dieta basal com inclusão de 40 ppm de sulfato de colistina, assim como dieta basal com 0, 150, 300, 450 e 600 ppm de nucleotídeos. Ao final do experimento, foi abatido um animal de cada baia para coleta das amostras para avaliação da morfometria de órgãos e da histologia do epitélio intestinal. No período de 1 a 14 dias de experimentação, houve piora linear (P<0,01) das variáveis de desempenho, enquanto que no período total de 34 dias, houve redução linear (P=0,03) do peso final dos animais com o aumento dos níveis de nucleotídeos na dieta. Os leitões alimentados com a dieta com o antibiótico colistina apresentaram maior (P<0,01) comprimento do intestino delgado e menor (P<0,01) relação altura de vilosidade:profundidade de cripta (AV:PC) no duodeno do que aqueles que receberam nucleotídeos. Foi observado, também, aumento linear (P<0,01) no peso relativo do baço, assim como redução linear (P<0,01) da relação AV:PC e redução linear (P<0,01) da profundidade de cripta no duodeno dos animais em função da adição de nucleotídeos na dieta. Assim, embora os níveis de até 600 ppm de nucleotídeos em dietas complexas de leitões recém-desmamados não tenham proporcionado melhora no desempenho, eles acarretaram benefícios à morfometria de órgãos e à histologia do epitélio intestinal dos animais. / The purpose of this work was to evaluate the effects of dietary nucleotide levels on performance, organs morphometry and intestinal histology of weanling pigs. One hundred and forty-four pigs weaned at 21 days of age and 5.80 ± .16 kg initial live weight were used a completely randomized design experiment with six treatments, six replications per treatment and four animals per pen (experimental unit). The treatments were: Am antimicrobial: basal diet with 40 ppm of colistin sulfate and basal diet with 0, 150, 300, 450 and 600 ppm of nucleotides. At the end of experimental period, an animal of each pen was slaughtered to evaluate of organs morphometry and intestinal epithelium histology. For the 1 to 14 days of experimental period, dietary nucleotides depressed linearly (P<.01) performance traits, while for the total experimental period of 34 days linear reduction of final live weight (P=.03) was observed with added nucleotides. Pigs fed diet with colistin showed greater (P<.01) length of small intestine and lower (P<.01) villus height:crypt depth ratio (AV:PC) of duodenum than those fed nucleotides. Also, a linear increase (P<.01) of relative weight of spleen, as well as a linear reduction (P<.01) of AV:PC and linear reduction (P<.01) of crypt depth of duodenum were observed with added dietary nucleotides. Therefore, although added nucleotides up to 600 ppm in complex diet did not improve performance, they showed some beneficial effects on organs morphometry and intestinal epithelium histology of weanling pigs.

L'effet protecteur des HDL sur les pathologies cardio-vasculaires est principalement attribué à leur rôle central dans le Transport Retour du Cholestérol (TRC). Ce processus assure l'efflux du cholestérol excédentaire des cellules périphériques vers le foie, au niveau duquel il est éliminé dans les sécrétions biliaires. Dans ce contexte, notre équipe a identifié à la surface des cellules hépatiques la présence d’un complexe enzymatique, très proche de l’ATP synthase mitochondriale, comme étant un récepteur de haute affinité pour l’apoA-I (protéine majoritaire des HDL). Cette ATP synthase de surface, également appelée ecto-F1-ATPase, joue un rôle clé dans l’endocytose hépatique des HDL. En effet, la liaison de l’apoA-I stimule l’activité ATPasique de l’enzyme, entrainant la production d’ADP extracellulaire puis l’activation spécifique du récepteur nucléotidique P2Y13, aboutissant in fine à l’endocytose des HDL. Ainsi, l’équipe a montré le rôle clé de la voie ecto-F1-ATPase/P2Y13 dans l’endocytose hépatique des HDL et par conséquent dans les effets protecteurs de ces derniers dans l’athérosclérose.Les travaux de thèse présentés ici visent à déterminer les mécanismes de régulation de cette ecto-F1-ATPase. Compte tenu de l’importance de la régulation des taux d’ADP et d’ATP extracellulaires dans l’endocytose des HDL, nous nous sommes intéressés dans un premier temps aux acteurs moléculaires qui pourraient réguler le métabolisme nucléotidique à la surface cellulaire. Nous avons mis en évidence la présence, à la surface des cellules HepG2, de l’adénine nucléotide translocase (ANT), une autre protéine classiquement localisée à la mitochondrie. Nous avons montré que l’ecto-ANT est impliquée dans la régulation des taux des nucléotides adényliques ADP et ATP extracellulaires et que son fonctionnement est lui-même dépendant du taux de ces derniers dans le milieu extracellulaire. / The cardioprotective effect of high-density lipoprotein cholesterol (HDL-C) is mostly attributed to their metabolic functions in reverse cholesterol transport (RCT), a process whereby excess cell cholesterol is taken up from peripheral cells and processed in HDL particles, and later delivered to the liver for further metabolism and bile excretion. ATP synthase, classically known to be located in the mitochondrial inner membrane, has been unexpectedly found expressed at the plasma membrane of hepatocytes, as a receptor for apoA-I, playing a role in HDL-cholesterol uptake. On hepatocytes, apoA-I binding to ecto-F1-ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y13-mediated HDL endocytosis pathway. The strict dependence of HDL endocytosis on extracellular ADP level led us to study first, whether other plasma membrane proteins than ecto-F1-ATPase could regulate extracellular ADP level. We highlighted the presence on hepatocytes cell surface of Adenine Nucleotide Translocase (ANT), another transmembrane protein of the inner mitochondrial membrane. We showed that ecto-ANT activity could increase or reduce extracellular ADP level, depending on the extracellular ADP/ATP ratio. Furthermore, we demonstrated that pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F1-ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis in human hepatocytes. We then sought to explore the molecular mechanisms involved in targeting ecto-F1-ATPase to the plasma membrane. Indeed, F1-ATPase ectopic expression at the plasma membrane has been described on several cell types and has been related to several physiological and pathophysiological processes however, the pathway involved in its transport to the cell surface remains unknown.

Including inherited genetic diseases, like lipoprotein lipase deficiency, and acquired diseases, such as cancer and HIV, gene therapy has the potential to treat or cure afflicted people by driving an affected cell to produce a therapeutic protein. Using primarily viral vectors, gene therapies are involved in a number of ongoing clinical trials and have already been approved by multiple international regulatory drug administrations for several diseases. However, viral vectors suffer from serious disadvantages including poor transduction of many cell types, immunogenicity, direct tissue toxicity and lack of targetability. Non-viral polymeric gene delivery vectors (polyplexes) provide an alternative solution but are limited by poor transfection efficiency and cytotoxicity. Microfluidic (MF) nano-precipitation is an emerging field in which researchers seek to tune the physicochemical properties of nanoparticles by controlling the flow regime during synthesis. Using this approach, several groups have demonstrated the successful production of enhanced polymeric gene delivery vectors. It has been shown that polyplexes created in the diffusive flow environment have a higher transfection efficiency and lower cytotoxicity. Other groups have demonstrated that charge-stabilizing polyplexes by sequentially adding polymers of alternating charges improves transfection efficiency and serum stability, also addressing major challenges to the clinical implementation of non-viral gene delivery vectors. To advance non-viral gene delivery towards clinical relevance, we have developed a microfluidic platform (MS) that produces conventional polyplexes with increased transfection efficiency and decreased toxicity and then extended this platform for the production of ternary polyplexes. This work involves first designing microfluidic devices using computational fluid dynamics (CFD), fabricating the devices, and validating the devices using fluorescence flow characterization and absorbance measurements of the resulting products. With an integrated separation mechanism, excess polyethylenimine (PEI) is removed from the outer regions of the stream leaving purified polyplexes that can go on to be used directly in transfections or be charge stabilized by addition of polyanions such as polyglutamic acid (PGA) for the creation of ternary polyplexes. Following the design portion of the research, the device was used to produce binary particle characterization was carried out and particle sizes, polydispersity and zeta potential of both conventional and MS polyplexes was compared. MS-produced polyplexes exhibited up to a 75% reduction in particle size compared to BM-produced polyplexes, while exhibiting little difference in zeta potential and polydispersity. A variety of standard biological assays were carried out to test the effects of the vectors on a variety of cell lines – and in this case the MS polyplexes proved to be both less toxic and have higher transfection efficiency in most cell lines. HeLa cells demonstrated the highest increase in transgene expression with a 150-fold increase when comparing to conventional bulk mixed polyplexes at the optimum formulation. A similar set of experiments were carried out with ternary polyplexes produced by the separation device. In this case it was shown that there were statistically significant increases in transfection efficiency for the MS-produced ternary polyplexes compared to BM-produced poyplexes, with a 23-fold increase in transfection activity at the optimum PEI/DNA ratio in MDAMB-231 cells. These MS-produced ternary polyplexes exhibited higher cell viability in many instances, a result that may be explained but the reduction in both free polymer and ghost particles.

DNA damage can cause genome instability, which may lead to human cancer. The most common form of DNA damage is DNA base damage, which is efficiently repaired by DNA base excision repair (BER). Thus BER is the major DNA repair pathway that maintains the stability of the genome. On the other hand, BER mediates DNA demethylation that can occur on the promoter region of important tumor suppressor genes such as Breast Cancer 1 (BRCA1) gene that is also involved in prevention and development of cancer. In this study, employing cell-based and in vitro biochemical approaches along with bisulfite DNA sequencing, we initially discovered that an oxidized nucleotide, 5’,2-cyclo-2-deoxyadenosine in DNA duplex can either cause misinsertion by DNA polymerase β (pol β) during pol β-mediated BER or inhibit lesion bypass of pol β resulting in DNA strand breaks. We then explored how a T/G mismatch resulting from active DNA demethylation can affect genome integrity during BER and found that pol β can extend the mismatched T to cause mutation. We found that AP endonuclease 1 (APE1) can use its 3'-5' exonuclease to remove the mismatched T before pol β can extend the nucleotide preventing a C to T mutation. The results demonstrate that the 3'-5' exonuclease activity of APE1 can serve as a proofreader for pol β to prevent mutation. We further explored the effects of exposure of environmental toxicants, bromate and chromate on the DNA methylation pattern on the promoter region of BRCA1 gene with bisulfite DNA sequencing. We found that bromate and chromate induced demethylation of 5-methylcytosines (5mC) at the CpG sites as well as created additional methylation at several unmethylated CpG sites at BRCA1 gene in human embryonic kidney (HEK) 293 cells. We further demonstrated that the demethylation was mediated by pol β nucleotide misinsertion and an interaction between pol β and DNA methyltransferase 1 (DNMT1) suggesting a cross-talk between BER and DNA methyltransferases. We suggest that DNA base damage and BER govern the interactions among the environment, the genome and epigenome, modulating the stability of the genome and epigenome and disease development.

Chronic, noncommunicable diseases such as chronic fatigue syndrome (also known as myalgic encephalomyelitis) are rapidly becoming a worldwide epidemic that profoundly affects public health and productivity. Chronic fatigue syndrome (CFS) is characterised by severe and debilitating fatigue and although its etiology is still unknown, recent studies have found considerable evidence that mitochondrial dysfunction and oxidative stress might be responsible for the underlying energy deficit in these patients. Adenine and pyridine nucleotides could be used as potential biomarkers for energy related disorders such as chronic fatigue syndrome because of their various functions in the energy and redox pathways. The first part of this study focussed on developing a liquid chromatography electrosprayionisation tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of these nucleotides in blood samples. Due to the instability of nucleotides in biological matrices it was also necessary to find a suitable extraction method that would be able to stop enzymatic activity via protein precipitation. Out of the four extraction methods investigated during this study, deproteinisation of whole blood samples with perchloric acid produced the highest nucleotide abundances. Although nucleotide standards were found to be stable in perchloric acid, nucleotide levels in blood samples were not stabilised by addition of perchloric acid. The second part of this study consisted of measuring the nucleotide levels in blood samples of controls and possible CFS patients in order to test the proof of concept of the new LCESI- MS/MS method. Despite changes in the nucleotide levels due to perchloric acid and problems with nucleotide instability, it was still possible to distinguish between the two groups based on the results obtained with the new LC-ESI-MS/MS method. The newly developed LC-ESI-MS/MS method proved to be reliable and adequate for nucleotide quantification in whole blood samples, thus the aim of this study was achieved. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

The origin of the first RNA polymers is central to most current theories regarding the origin of life. However, difficulties associated with the prebiotic formation of RNA have lead many researchers to conclude that simpler polymers, or proto-RNAs, preceded RNA. These earlier polymers would have been replaced by RNA over the course of evolution. A remaining difficulty for this theory is that the de novo synthesis of a feasible proto-RNA has not yet been demonstrated by plausible prebiotic reactions. This thesis focuses on two problems associated with prebiotic proto-RNA synthesis: The formation of nucleosides and the necessity of reversible backbone linkages for error correction in nucleic acid polymers. "The Nucleoside Problem", or the lack of success in forming pyrimidine nucleosides by plausible prebiotic reactions, represents a significant stumbling block to the RNA world hypothesis. Nearly four decades ago Orgel and coworkers demonstrated that the purine nucleosides adenosine and inosine are synthesized by heating and drying their respective bases and ribose in the presence of magnesium, but these reaction conditions do not yield the pyrimidine nucleosides uridine or cytidine from their respective bases. In this thesis a potential solution to The Nucleoside Problem is hypothesized based upon a proposed chemical mechanism for nucleoside formation. This hypothesis is supported by the successful synthesis of 2-pyrimidinone nucleosides by a plausible prebiotic reaction in good yield, demonstrating that pyrimidine nucleosides could have been available in the prebiotic chemical inventory, but that uridine and cytidine were likely not abundant. Reversible backbone linkages are necessary to provide a mechanism for error correction in non-enzymatic template-directed syntheses of proto-RNAs. In this thesis, acetals are explored as low-energy, reversible linkage groups for nucleosides in polymers. The synthesis of glyoxylate-acetal nucleic acids (gaNAs) through simple heating-drying reactions from neutral aqueous solutions is demonstrated, and these linkages are shown to be hydrolytically stable under a considerable range of solution conditions. Computational models demonstrate that the glyoxylate linkage is an excellent electronic and isosteric replacement for phosphate. Molecular dynamics simulations also indicate that a gaNA duplex would have structural properties that closely match a phosphate-linked RNA helix, suggesting the possibility for cross-pairing between gaNAs and RNAs, allowing for sequence transfer and genetic continuity through the evolution from proto-RNAs to RNA. The principles illustrated in this thesis by 2-pyrimidinone nucleoside and gaNA synthesis can be extended to other prebiotic condensation reactions. Factors affecting condensation yield, such as thermodynamics, kinetics, reactant solubility, and salt effects, are summarized herein.

The Snail protein functions as a transcriptional regulator to establish early mesodermal cell fate in Drosophila. Later, in germ band-extended embryos, Snail is considered a pan-neural protein based on its extensive expression in neuroblasts. The evidence presented in thesis links snail expression and function in CNS. Cloning and functional characterization of a novel snail homologue, in Drosophila, are also described here. Cloning of this gene, worniu (Chinese for snail), revealed that the neural function of snail is masked by this and another closely related gene escargot. Both Escargot and Worniu contain zinc finger domains that are highly homologous to that of Snail. These three members of Snail protein family are redundantly required for CNS development. Although not affecting formation of neuroblasts, the loss of expression of these three members correlates with disruption of Nb asymmetry and division. Downstream targets of Snail protein family, in these processes, are inscuteable and string. In mutant embryos, which have the three genes deleted, the RNA expression of inscuteable and string is significantly lowered. Consistent with the gene expression defects, the mutant embryos have loss of asymmetric localization of prospero RNA in neuroblasts and nuclear localization of Prospero protein in ganglion mother cells. Transgenic expression of inscuteable and string together, in the snail family deletion mutant, efficiently restores the Prospero expression in GMC, demonstrating that the two genes are key targets of Snail in Nbs. Like in the mesoderm, in CNS Snail function depends on interaction with dCtBP co-repressor. These results suggest that Sna [Snail] family of proteins control both asymmetry and cell division of neuroblasts by activating, perhaps indirectly, the expression of inscuteable and string.

Calcium ions (Ca2+) are involved in almost all neuronal functions, providing the link between electrical signals and cellular activity. This work examines the mechanisms by which a neuron can regulate the movement and sequestration of Ca2+ through specific channels such that this ubiquitous ion can encode specific functions. My initial focus was using intracellular calcium ([Ca2+]i) imaging techniques to study the influence of the inhibition of specific voltage gated calcium channels (VGCC) by ethanol on a depolarization induced rise in [Ca2+]i in neurohypophysial nerve terminals. This research took an unexpected turn when I observed an elevation of [Ca2+]i during perfusion with ethanol containing solutions. Control experiments showed this to be an artifactual result not directly attributable to ethanol. It was necessary to track down the source of this artifact in order to proceed with future ethanol experiments. The source of the artifact turned out to be a contaminant leaching from I.V. drip chambers. Due to potential health implications stemming from the use of these drip chambers in a clinical setting as well as potential artifactual results in the ethanol field where these chambers are commonly used, I choose to investigate this phenomenon more rigorously. The agent responsible for this effect was shown to be di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer that has been shown to be carcinogenic in rats and mice. The extraction of this contaminant from the I.V. drip chamber, as measured by spectrophotometry, was time-dependent, and was markedly accelerated by the presence of ethanol in the solution. DEHP added to saline solution caused a rise in [Ca2+]i similar to that elicited by the contaminant containing solution. The rise in calcium required transmembrane flux through membrane channels. Blood levels of DEHP in clinical settings have been shown to exceed the levels which we found to alter [Ca2+]i. This suggests that acute alterations in intracellular calcium should be considered in addition to long-term effects when determining the safety of phthalate-containing plastics. As part of a collaboration between Steven Treistman and Robert Messing's laboratory at UCSF, I participated in a study of how ethanol regulates N-type calcium channels which are known to be inhibited acutely, and upregulated in the chronic presence of ethanol. Specific mRNA splice variants encoding N-type channels were investigated using ribonuclease protection assays and real-time PCR. Three pairs of N-type specific α-subunit Cav2.2 splice variants were examined, with exposure to ethanol observed to increase expression of one alternative splice form in a linker that lacks six bases encoding the amino acids glutamate and threonine (ΔET). Whole cell electrophysiological recordings that I carried out demonstrated a faster rate of channel activation and a shift in the voltage dependence of activation to more negative potentials after chronic alcohol exposure, consistent with increased expression of ΔET variants. These results demonstrate that chronic ethanol exposure not only increases the abundance of N-type calcium channels, but also increases the expression of a Cav2.2 splice variant with kinetics predicted to support a larger and faster rising intracellular calcium signal. This is the first demonstration that ethanol can up-regulate ion channel function through expression of a specific mRNA splice variant, defining a new mechanism underlying the development of drug addiction. Depolarizing a neuron opens voltage gated Ca2+ channels (VGCC), leading to an influx of Ca2+ ions into the cytoplasm, where Ca2+ sensitive signaling cascades are stimulated. How does the ubiquitous calcium ion selectively modulate a large array of neuronal functions? Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca2+ current and the resulting rise and decay of [Ca2+]i, showing that equal amounts of Ca2+ entering through N-type and L-type voltage gated Ca2+ channels result in significantly different [Ca2+]i temporal profiles. When the contribution of N-type channels was reduced, a faster [Ca2+]i decay was observed. Conversely, when the contribution of L-type channels was reduced, [Ca2+]i decay was slower. Potentiating L-type current or inactivating N-type channels both resulted in a more rapid decay of [Ca2+]i. Channel-specific differences in [Ca2+]i decay rates were abolished by depleting intracellular Ca2+ stores suggesting the involvement of Ca2+-induced Ca2+ release (CICR). I was able to conclude that Ca2+ entering through N-type, but not L-type channels, is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR generates a unique intracellular Ca2+ signal depending on the route of entry, potentially encoding the selective activation of a subset of Ca2+ -sensitive processes within the neuron.