The problem with which this investigation is concerned is that of determining the nature of events leading to the change. of normal cells into malignant cells. The design of the study is multi-phasic: (A) to establish the presence or absence of an oncogenic virion, (B) to demonstrate by use of the electron microscopy any ultracellular alteration in malignant or transformed tissues, (C) to investigate the nature of the transforming agent in the murine lymphosarcoma, and (D) to employ various methods to demonstrate cellular transformations in vivo and in vitro. It is concluded that the transforming and tumorinducing agent in this' investigation was not a virion, but an infectious ribonucleic acid genome or a segment of a viral genome which had become integrated into the genome of the mouse cells. The vision has lost its ability to form a protein coat; therefore it is not demonstrable as a virion. But the ribonucleic acid is able to infect other cells and transform them from normal to neoplastic tissues.

Thesis advisor: Thomas Seyfried / Lipids, in all their forms from structural components of the membranes (phosphoglycerides, glycolglycerolipids) to signaling molecules (IP3, DAG, prostaglandins, etc.,) post-translational modification of proteins (palmitoylated, farnesylated, prenylated, and GPI anchoring) play an essential role in cancer cell survival, proliferation, and metastasis. Alteration in structural lipids can impair transport, and signaling cascades. Abnormalities in lipids, such as cardiolipin (Ptd2Gro), impair mitochondrial function, bioenergetics, and could play a role in precipitatting the high incidence of spontaneous tumors in VM/Dk mice. This thesis explores the role of glucose and glutamine in their incorporation into lipids in the VM-M3 murine glioblastoma cell line as well as the inheritance of brain cardiolipin fatty acids abnormalities in VM/Dk mice. I used labeled [14C]-U-D-glucose and [14C]-U-L-glutamine to examine the profile of de novo lipid biosynthesis in the VM-M3 cell line. The major lipids synthesized included phosphatidylcholine (PtdCho), phosphatidylethanolamine (EtnGpl), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), sphingomyelin (CerPCho), bis(monoacylglycero)phosphate (BMP) / phosphatidic acid (PtdOH), cholesterol (C), Ptd2Gro, and the gangliosides. The data show that the incorporation of labeled glucose and glutamine into synthesized lipids was dependent on the type of growth environment, and that the VM-M3 glioblastoma cells could acquire lipids, especially cholesterol, from the external environment for growth and proliferation. In addition, this thesis also explores and evaluates the abnormality of Ptd2Gro fatty acid composition in VM mice in comparison to B6 mice. Although previously reported, I confirmed the finding in the abnormal cardiolipin fatty acid composition in the VM mice. The abnormal brain cardiolipin fatty acid composition was found to be inherited as an autosomal dominant trait in reciprocal B6 x VM F1 hybrids for both male and female. Impaired cognitive awareness under hypoxia observed for the VM mice and reciprocal F1 hybrids is associated with abnormalities in neural lipid composition. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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No. of bitstreams: 2 Dissertação - Rosidete Pereira de Bastos - 2008.pdf: 938423 bytes, checksum: 1e65715cda59aae81ec161a84d517cb6 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2008-03-14 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The knowledge about immunology of Leishmania (Viannia) braziliensis-murine infection is poorly known, especially concerning innate immune response and the involvement of leukotrienes in the resistance mechanisms. Leukotrienes are lipid mediators of inflammation that activate microbicidal mechanisms in leukocytes. The present study aimed to evaluate the BALB/c and C57Bl/6 murine infection with two L. (V.) braziliensis isolates obtained from cutaneous leishmaniasis patients and the involvement of leukotrienes in the resistance of infection. Thus, IMG3 and RPL5 isolates were identified as L. (V.) braziliensis by using molecular techniques and the in vitro growth of parasites in Grace´s medium (26°C) was evaluated. The time course of lesion after footpad infection was followed in BALB/c and C57Bl/6 mice. C57Bl/6 mice genetically deficient in interferon gamma (IFNγ) were infected to evaluate the relevance of this cytokine in resistance. The parasite burden in draining lymph nodes and spleens was detected by limiting dilution assay. BALB/c- and C57Bl/6-infected footpads were processed after 12 weeks of infection and those from IFNγ-defecient mice after 4 weeks for histopathological analyses. Tissue sections were stained by hematoxylin-eosin. Parasite capacity to induce nitric oxide (NO) was analyzed in RAW 264.7 cell cultures treated or not with IFNγ and lipopolysaccharide (LPS). Nitrites were detected by using Griess reaction. The NO modulation by endogenous leukotrienes was evaluated through treatment of the cultures with a 5-lipoxygenase (5-LO) inhibitor and a leukotriene B4 (LTB4) antagonist. Macrophage leishmanicidal activity against IMG3 isolate was evaluated in thioglycolateelicited peritoneal macrophages of C57Bl/6 mice. In these cultures, NO was inhibited by aminoguanidine. In vivo leukotriene inhibition was achieved by using a 5-LO inhibitor. In vitro-parasite growth profiles were similar and parasites at the 5th day of culture were used to infection. The lesion course was also similar between isolates in both two mouse stains used, but C57Bl/6 mice presented healing after 12 weeks of infection whereas in BALB/c mice the lesion was persistent. In IFNγ-deficient mice there progressive lesions and visceralization in IMG3- as well as in RPL5-infected mice. Corroborating these data, the parasite burden in draining lymph nodes of BALB/c mice was higher than in C57Bl/6 mice after 12 weeks of infection with IMG3, and parasites (IMG3 and RPL5) were identified in about 50% of the IFNγ-deficient mouse spleens. The histopathological analyses showed an intense dermal infiltrate with vacuolated macrophages heavily parasitized in BALB/c mice (12 weeks) an in IFNγ-deficient mice (4 weeks), but not in C57Bl/6 mice (12 weeks), similarly for IMG3 and RPL5. Both isolates IMG3 an RPL5 induced NO production in RAW 264.7 celll cultures presenting synergism with IFNγ. Endogenous leukotrienes did not affect NO production in these cultures. C57Bl/6 peritoneal macrophages activated with IFNγ/LPS killed IMG3 parasites depending on NO release. In vivo inhibition of leukotriene synthesis did not change the course of infection in C57Bl/6 mice infected with IMG3 isolate. The relevant findings are: BALB/c mouse is susceptible to infection with IMG3 an RPL5 isolates whereas C57Bl/6 is resistant; IFNγ is crucial to the control of the infection; the isolates induce NO and this molecule contributes to macrophage leishmanicidal activity; and also the data suggest that endogenous leukotrienes are not involved in the control of L. (V.) braziliensis in C576Bl/6 mouse. / A imunologia da infecção murina por Leishmaia (Viannia) braziliensis é pouco conhecida, especialmente considerando a imunidade inata e a participação dos leucotrienos nos mecanismos de resistência à infecção. Os leucotrienos são mediadores lipídicos da inflamação que ativam mecanismos microbicidas dos leucócitos. O presente trabalho teve como objetivo avaliar o perfil da infecção de camundongos BALB/c e C57Bl/6 por dois isolados de L. (V.) braziliensis obtidos de pacientes com leishmaniose cutânea e o envolvimento dos leucotrienos endógenos na resistência à infecção. Para isto, os isolados denominados IMG3 e RPL5 foram identificados como L. (V.) braziliensis por meio de técnicas moleculares e foram avaliados quanto ao crescimento in vitro em meio Grace (26°C), e quanto ao curso da evolução da lesão, em camundongos BALB/c e em C57Bl/6. Camundongos C57Bl/6 geneticamente deficientes em interferon gama (IFNγ) foram infectados para avaliar a importância desta citocina no controle da infecção. A carga parasitária foi obtida pelo ensaio da diluição limitante em linfonodos drenantes da lesão e nos baços. As patas dos camundongos BALB/c e C57Bl/6 foram colhidas após 12 semanas de infecção, e dos camundongos deficientes em IFNγ, na 4ª semana, e foram processadas para análises hitopatológicas. A capacidade de indução de óxido nítrico (NO) pelos isolados foi avaliada em culturas de células RAW 264.7 tratadas ou não com IFNγ e lipopolissacarídeo (LPS), sendo os nitritos detectados por reação de Griess. A regulação da produção de NO pelos leucotrienos endógenos foi avaliada por tratamento das culturas de macrófagos com um inibidor de 5-lipoxigenase (5-LO) e um antagonista de receptor de leucotrieno B4 (LTB4). A atividade microbicida dos macrófagos foi avaliada em macrófagos peritoneais (C57Bl/6) infectados com o isolado IMG3, sendo o NO inibido por aminoguanidina. O efeito da inibição de leucotrienos, in vivo, foi avaliado em camundongos C57Bl/6 infectados com o isolado IMG3 e tratados com um inibidor de 5- LO. As curvas de crescimento in vitro foram similares para os dois isolados e os parasitos no 5° dia de cultivo foram usados nos experimentos de infecção. O curso da lesão foi similar entre os dois isolados nos camundongos BALB/c e C57Bl/6, porém enquanto a lesão regrediu nos C57Bl/6, nos camundongos BALB/c, a lesão foi persistente até a 12ª semana de infecção. Em camundongos deficientes em IFNγ houve crescimento progressivo das lesões e visceralização, tanto com o isolado IMG3 quanto com o RPL5. Confirmando estes dados, a carga parasitária nos linfonodos drenantes da lesão nos camundongos BALB/c foi maior do que a encontrada nos C57Bl/6 após 12 semanas de infecção com IMG3 e os parasitos (IMG3 e RPL5) foram encontrados em cerca de 50% dos baços dos camundongos deficientes em IFNγ. As análises histopatológicas mostraram um acentuado infiltrado inflamatório na derme, com macrófagos vacuolizados repletos de parasitos nos camundongos BALB/c (12 semanas), e nos deficientes de IFNγ (4 semanas), mas não nos camundongos C57Bl/6 (12 semanas), similarmente para IMG3 e RPL5. Os isolados IMG3 e RPL5 induziram NO em células RAW 264.7 em sinergismo com IFNγ e os leucotrienos endógenos não alteraram a produção de NO destas células. Macrófagos peritoneais murinos mostraram atividade microbicida de maneira dependente de NO. A inibição in vivo da síntese de leucotrienos não alterou o curso da infecção pelo isolado IMG3 em camundongos C57Bl/6. Coletivamente, os dados mostram que o camundongo BALB/c é suscetível à infecção pelos dois isolados, enquanto o C57Bl/6 é resistente; o IFNγ é essencial para o controle da infecção; os isolados induzem a produção de NO, o qual contribui para a eliminação dos parasitos; e os dados sugerem que os leucotrienos endógenos não estão envolvidos nos mecanismos de resistência dos camundongos C57Bl/6 a L. (V.) braziliensis.

Malignant mesothelioma (MM) is an aggressive tumour, which is highly associated with previous asbestos exposure and is resistant to most conventional anticancer therapies. Previous studies have used a mouse model of to 01 p effective approaches to induction of anti-tumour immunity using modification of tumour cells by the introduction of genetic constructs expressing genes such as that for B7-1 so that tumour growth can be inhibited in vivo. Transfectant clones, AC29 B7-7 and AC29 B7-6, which showed equal levels of expression of B7-1 but were markedly different in tumorigenicity were assessed using suppression subtractive hybridization (SSH) in order to isolate transcripts which may have been differentially expressed in the two clones. SSH allowed isolation of a number of cDNAs which were apparently differentially expressed in the cell lines. These required characterisation in order to determine their possible relevance to tumorigenicity. Two cDNAs designated as 7-7-76 and 7-7-43 had been isolated previously and the aim of this project was to characterise these cDNAs by sequencing, searching for their homology relationships and investigating gene expression profiles. Preliminary searches revealed that clone 7-7-43 had homology to cyclin-dependent kinase regulatory subunit 1 which plays a role in the cell cycle. On the other hand, clone 77-76 showed only homology to an EST of hypertension related protein and therefore, further investigation was required to obtain the identity of clone 7-7-76. The first part of this project was to in investigate and evaluate gene expression on clone 7-7-43, using both relative RT-PCR and Northern blotting.' In the second part of this project, a more intense study of clone 7-7-76 was conducted. Clone 7-7-76 was investigated for its homology relationships and its gene expression profile. / Results obtained from relative RT-PCR suggested no difference in the expression of the either eDNA clone (7-7-43 and 7-7-76) between the MM clones AC29 B7-6 and AC29 B7-7, the cells used to derive these clones by SSH. Therefore, it was concluded that neither clone 7-7-43 nor 7-7-76 was differentially expressed in MM cells of differing immuno enicit RACE was employed in order to derive a longer sequence of clone 7-7-76 and the newly derived sequence of 7-7-76 was again used to search for homologies using a wider range of sequences for human and other species. These investigations on clone 7-7-76 showed it to correspond to the sequence of human mitofusin 2 which is involved in determining mitochondrial morphology The results determined in this project suggest that clones 7-7-43 and 7-7-76 are not differentially expressed in the range of MM cell lines tested. The data have however highlighted the potential of the SSH technique to easily derive cDNA clones worthy of investigation, but underline the possibility of false positive clones being isolated. The need for an efficient, accurate screening procedure such as real-time PCR is acknowledged.

The Langendorff perfused murine heart has become an increasingly important research model in cardiovascular physiology and pharmacology. However, the model remains relatively poorly characterised when compared with the widely employed rat preparation. The purpose of the research within this thesis was initially two-fold: 1) to characterise the functional and substrate-dependent properties of the murine model; and 2) to characterise the relationships between glycolysis, ischaemic tolerance and adenosine-mediated cardioprotection in the mouse. Initial studies, confirmed by simultaneous/subsequent work in other laboratories, revealed the frequent occurrence of regular cyclic oscillations in contractile function and coronary flow in glucose-perfused isovolumically contracting hearts. This phenomenon (labelled 'cycling') was unaltered by inhibition of ?-adrenergic receptors, prostaglandins, and nitric oxide synthase. However, A1/A2 adenosine receptor agonism did abolish the oscillations in flow and reduced contractile oscillations by 50%. Importantly, cycling was eliminated by addition of 50 IU/l insulin to perfusion fluid, or provision of 5 mM pyruvate as a co-substrate with glucose. These data suggest that functional 'cycling' in glucose-perfused murine hearts likely occurs as a result of a mismatch between substrate metabolism (energy supply) and myocardial energy demand. It may be that glycolysis with exogenous glucose is insufficient to ensure appropriate matching of myocardial energy supply and demand. For this reason, it is advisable to employ a co-substrate such as pyruvate in studies of murine hearts. Further studies performed within this thesis generally employ this co-substrate addition. Addition of pyruvate as co-substrate removes 'cycling' but is also known to inhibit/modify glycolysis, which may affect ischaemic tolerance and/or cardioprotection mediated by adenosine. Experiments throughout this thesis demonstrated that pyruvate-perfusion improved tolerance to both ischaemia (delayed time to onset of ischaemic contracture; TOC) and reperfusion (reduced diastolic dysfunction and cell death). The delay in TOC as a result of pyruvate-perfusion also suggests that contracture is not solely influenced by anaerobic glycolysis (as outlined in current paradigms). To test the relevance of glycolysis to ischaemic injury hearts were subjected to various forms of glycolytic inhibition. Glycolysis was inhibited by use of 10 mM pyruvate, (iodoacetic acid) IAA treatment, and glycogen depletion by pre-ischaemic substrate-free perfusion (all groups employing pyruvate as sole-substrate). Each form of glycolytic modification resulted in significant delays in TOC, in complete contrast to findings from other models and species. Glycogen depletion also reduced the peak level of contracture. These findings indicate that the mouse is either unique in terms of substrate metabolism and mechanisms of contracture (an unlikely possibility), or raise serious questions regarding current models of contracture development during ischaemia (theorised to be delayed by prolonging anaerobic glycolysis). Modification of glycolysis also altered post-ischaemic outcome, with pyruvate perfusion and glycogen depletion both enhancing functional recoveries. However, IAA treated hearts, despite near-identical ischaemic tolerance (ie contracture development) to pyruvate-perfused hearts, displayed very poor functional recovery, which was below that for all other groups. These data clearly reveal that blocking glycolysis improves tolerance to ischaemia (as evidenced by reduced contracture), provide evidence of dissociation of ischaemic injury or contracture from post-ischaemic recovery, and confirm the key importance of glycolysis in enhancing recovery from ischaemia. Since tolerance to ischaemia/reperfusion was shown to be glycolysis dependent, and since it has been theorised that adenosine protects hearts through modulating glycolysis, the relationships between glycolytic inhibition and adenosine-mediated cardioprotection was tested. In a number of studies, exogenously applied adenosine was shown to protect both glucose- and pyruvate-perfused hearts (supporting no dependence of adenosinergic protection on glycolysis). However, to more equivocally test the role of glycolysis effects of IAA were studied and were shown to markedly limit protection with adenosine. The effects of adenosine during ischaemia were abolished by IAA treatment, and effects on post-ischaemic recovery were reduced (but not eliminated). Similar results were acquired for protection with endogenous adenosine (using iodotubercidin to block adenosine phosphorylation). Collectively, these data reveal that adenosinergic protection during ischaemia depends entirely upon glycolysis while protection during reperfusion likely involves glycolysis dependent and independent processes. However, glycolysis is required for full recovery of function during reperfusion. Further studies assessed the involvement of glycolysis in cardioprotection afforded by transgenic A1 adenosine receptor (A1AR) overexpression. It was found that pyruvate-perfusion provided the same protection as A1AR overexpression, and the two responses (to pyruvate and A1AR overexpression) were not additive. Thus, it is probable that common mechanisms are targeted in both responses (likely glycolysis). Finally, the effects of adenosine and pyruvate on oxidant injury were studied, testing whether interactions between adenosine and pyruvate observed in prior work within this thesis could be explained by alterations in anti-oxidant responses. It was found that adenosine has quite profound anti-oxidant responses in glucose-perfused hearts, with very selective effects on markers of damage. Pyruvate also had some anti-oxidant effects but interestingly it reduced the anti-oxidant effects of adenosine. In conclusion, the work entailed within this thesis demonstrates that the isolated mouse heart model may possess unique properties and should be further characterised by potential users in order to improve its utility, and the reliability of experimental findings (chiefly when studying ischaemia-reperfusion). Other work within thesis demonstrates that modification of glycolysis is important in dictating recovery from ischaemia-reperfusion, and also impacts on adenosine-mediated protection (principally but not exclusively during ischaemia itself). The manner in which glycolysis is modified and contributes to protection remains unclear.

Surgery precipitates a hypercoagulable state and has been shown to increase the development of cancer metastases in animal models, however mechanism(s) responsible for this are largely unknown. We hypothesize that the prometastatic effect of surgery may be secondary to postoperative hypercoagulable state. Surgical stress was induced in mice by partial hepatectomy or nephrectomy, preceded by intravenous injection of CT26-LacZ or B16F10-LacZ cells to establish pulmonary metastases with or without perioperative anticoagulation and their lung tumor cell emboli (TCE) were quantified. Fibrinogen and platelets were fluorescently labeled prior to surgical stress to evaluate TCE-associated fibrin and platelet clots. Surgery significantly increased metastases while anticoagulation with five different agents attenuated this effect. Fibrin and platelet clots were associated with TCE significantly more frequently in surgically stressed mice. Surgery promotes the formation of fibrin and platelet clots around TCE and this appears to be the mechanism for the increase in metastases seen following surgery.

Surgery precipitates a hypercoagulable state and has been shown to increase the development of cancer metastases in animal models, however mechanism(s) responsible for this are largely unknown. We hypothesize that the prometastatic effect of surgery may be secondary to postoperative hypercoagulable state. Surgical stress was induced in mice by partial hepatectomy or nephrectomy, preceded by intravenous injection of CT26-LacZ or B16F10-LacZ cells to establish pulmonary metastases with or without perioperative anticoagulation and their lung tumor cell emboli (TCE) were quantified. Fibrinogen and platelets were fluorescently labeled prior to surgical stress to evaluate TCE-associated fibrin and platelet clots. Surgery significantly increased metastases while anticoagulation with five different agents attenuated this effect. Fibrin and platelet clots were associated with TCE significantly more frequently in surgically stressed mice. Surgery promotes the formation of fibrin and platelet clots around TCE and this appears to be the mechanism for the increase in metastases seen following surgery.

Le RadLV/VL₃ clone V-13 est un rétrovirus murin thymotrope, non défectif, fortement leucémogène et écotrope. Il induit des lymphomes des cellules T en s'intégrant dans le génome de l'hôte. Le virus peut causer une tumeur en modifiant le fonctionnement des gènes près desquels il s'est intégré. Ces intégrations, si retrouvées au même endroit dans plus d'une tumeur, définissent un site commun d'intégration. Ce projet a pour but de trouver un ou des nouveaux sites d'intégration du rétrovirus RadLV/VL₃ clone V-13. Pour ce faire, un oligonucléotide de séquence connue, nommé splinkerette, a été utilisé. L'ADN d'une souris tumorale est coupée puis une splinkerette y est ligasée. Ceci permet d'amplifier, par des réactions de PCR, les régions flanquantes aux intégrations rétrovirales. Une fois clonées, les produits des PCR sont séquencés, permettant de situer les intégrations dans le génome de la souris, qui est disponible dans les banques de données. Il est ainsi possible de repérer les gènes à proximité de l'intégration rétrovirale. Certains gènes d'intérêt ont déjà été retrouvés grâce à cette technique pour d'autres rétrovirus. Dans le cas du RadLV/VL₃, parmi une vingtaine de tumeurs et environ 70 bandes analysées, les gènes Notch l, Rasgrp1, Rorc, Lef1, Gfi1, Ncor2, Scarb1, Lfng, Mad, Myb, Ahi1, Supt4h, Bzrap1, Sept9, Fos, Jundm2, Myc, Pim1, Ccnd3, Bcl211 et Gpc3 ont été trouvés. Plusieurs de ces oncogènes n'ont jamais été associés au rétrovirus RadLV/VL₃. Deux régions potentiellement oncogéniques sur les chromosomes 7 et 11 ont été identifiées. La compréhension du fonctionnement des oncogènes permettra d'empêcher leur expression aberrante, ou d'utiliser des rétrovirus comme vecteurs dans la thérapie génique afin de corriger l'expression de gènes mutés. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : ADN, Clonage, Leucémie, Oncogène, PCR, RadLV/VL₃, Rétrovirus, Séquençage, Site commun d'intégration, Sonde radioactive, Splinkerette, Tumeur.

Ph.D.

The lower esophageal sphincter (LES) is characterized by basal tone and appropriately timed neurogenic relaxation. The physiological mechanisms underlying these crucial LES functions remain poorly understood. The current studies were designed to characterize the electrophysiological properties and neural regulation of LES circular smooth muscle (CSM), and to determine whether interstitial cells of Cajal (ICC) play a role in neurotransmission. Conventional intracellular recordings were performed in CD1, nNOS knock-out, eNOS knock-out and W/Wv mutant mice. Mouse LES consists of “sling” and “clasp” smooth muscle, which were studied separately in CD1 mice. In subsequent studies of mutant mice and respective controls, only the clasp muscle was examined, Immunohistochemical c-Kit staining of ICC was performed in wild-type and W/Wv mutant mice that were first characterized electrophysiologically. The smooth muscle of the LES clasp and sling displayed unitary membrane potentials with a resting membrane potential (RMP) of ~ -43 mV. Spontaneous nifedipine-sensitive action potentials superimposed on the unitary potentials were usually recorded in the LES clasp, but not sling muscle. A monophasic inhibitory junction potential (IJP) was recorded in sling CSM, whereas a biphasic IJP consisting of an initial IJP, followed by long-lasting slow IJP (LSIJP) was recorded in clasp. Further pharmacological studies using control and various knockout mice suggest that: 1. the CSM of the mouse LES is innervated by cholinergic, nitrergic and purinergic nerves; 2. the LSIJP is mediated entirely by nitrergic nerves, whereas purinergic and nitrergic nerves produce the monophasic IJP in the LES sling and initial phase of biphasic IJP in the LES clasp; 3. Ca2+/CaM-kinase II is involved in the regulation of the nitrergic IJPs; 4. TREK-1 K+ channels are not involved in the nitrergic IJP; 5. purinergic and cholinergic neurotransmission is intact in LES CSM of W/Wv mutant mice, whereas nitrergic neurotransmission is impaired in about half of the animals. In animals in which nitrergic neurotransmission was intact, ICC-IM were markedly deficient immunohistologically, suggesting that ICC are not required for nitrergic neurotransmission; 6. impaired nitrergic neurotransmission in W/Wv mutant mice is associated with dysfunction of a Ca2+-dependent signaling cascade primed by spontaneous Ca2+ release from the sarcoplasmic reticulum. / Thesis (Ph.D, Physiology) -- Queen's University, 2008-01-24 15:54:52.175