Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Thu Annelise Nguyen / Preclinical animal models of mammary carcinoma formation are vital for the advancement of cancer research, specifically in drug development. Two different types of animal models were utilized to determine the efficacy of combinational treatment of common antineoplastic drugs and the new class of primaquines that act as gap junction enhancers (PQs) at attenuating mammary tumor growth. The classic xenograft mouse model was used to show that PQs could increase the efficacy of cisplatin and paclitaxel. Combinational treatment induced an upregulation of connexin and caspase expression in the isolated tumor. Next the transgenic PyVT mouse model was characterized by multiple factors, including hormone receptor status, molecular markers for survival and proliferation, tissue histopathology, and secondary metastases during multiple stages of tumor development. This model showed limited therapeutic response to the antineoplastic drugs tested. PQ1 effectively attenuated tumor growth at all stages of tumorigenesis in the PyVT model, while PQ7 was determined to be an effective chemopreventive compound rather than chemotherapeutic. The PQs altered the expression profiles of connexins during tumorigenesis. Together the results indicate that PQs have an anticancer effect that is more efficient at attenuating tumor growth than the common antineoplastic compounds. Lastly the PyVT mouse model was used to determine the efficacy of antineoplastic compounds on male mammary carcinoma development. Interestingly, the antineoplastic compound that attenuated female mammary carcinoma growth did not produce a therapeutic response in the males and vice versa, suggesting a need for further studies into the male response to therapy.

As junções GAP são canais intercelulares responsáveis pela comunicação de células vizinhas, por onde passam pequenas moléculas e íons que mantêm a homeostasia celular. A junção GAP é formada seis proteínas, as conexinas. Na célula endotelial encontram-se as conexinas 37, 40 e 43. Nesse estudo, estimulamos a angiogênese em córnea de camundongos, através da cauterização com cristal de nitrato de prata. Foram utilizados camundongos heterozigotos para o gene da conexina 43 (Cx43+/-) e camundongos selvagens (Cx43+/+). As córneas foram analisadas 2 e 6 dias após a cauterização atravéspor meio da morfologia vascular, detecção das Cx37, Cx40, Cx43, PCNA por meio de Western Blot e avaliação ultraestrutural das células endoteliais. Como resultado obtivemos uma menor área de preenchimento vascular nos animais Cx43+/- em 2 e 6 dias após a lesão corneal, porém, em relação a extensão dos vasos não foi observado diferenças entre os grupos. Uma menor proliferação celular foi verificada através da detecção do PCNA, nos animais heterozigotos, somente após 2 dias da lesão corneal. Não houve alteração da Cx37 e Cx40 entres os grupos. A Cx43 parece ser uma conexina importante para a célula endotelial durante o processo de angiogênese. / The GAP junctions are intercellular streams responsible for the communication between close cells, which allow small molecules and ions to pass through them maintaining the cellular homeostasis. The GAP junction is formed of six proteins, the connexin. In the endothelial cell, there are the connexin 37, 40 and 43. In this study, we stimulated the angiogenesis in the mice\'s cornea through its cauterization using silver\'s crystal glass. It was used heterozygote mice to the gene of connexin 43 (Cx43+/-) and wild mice (Cx43+/+). The corneas were analyzed 2 and 6 days after the cauterization through the vascular morphology, detection of Cx37, Cx40, Cx43, PCNA through Western Blot and ultrastructural evaluation of the endothelial cells. As a result, we obtained a smaller area of vascular fillness in the animals Cx43+/- with 2 and 6 days of corneal injury, however, in regard to the extensions of the vessels, it wasn\'t observed any changes between the groups. A smaller proliferation of cells was verified, through the detection of PCNA, in the heterozygote animals only 2 days after the corneal injury. There wasn\'t any modification of the Cx37 and Cx40 between groups. The Cx43 seems to be an important connexin to the endothelial cell during the process of angiogenesis.

A conexina 32 (Cx32) é uma estrutura protéica que constitui os canais que promovem as comunicações intercelulares via junções comunicantes (GJIC), permitindo difusão de pequenas moléculas citoplasmáticas de uma célula à outra. Este trabalho objetivou os estudos destas estruturas devido a sua importância em processos hepáticos, mais especificamente, a fibrose hepática. O presente estudo foi realizado através da administração oral da droga hepatotoxica dimetilnitrosamina (DMN) em ratas Wistar duas vezes por semana em dias consecutivos no prazo de cinco semanas. A necropsia destes animais foi realizada após cinco semanas da última administração da droga e revelou um quadro de fibrose hepática, em contra partida aos resultados obtidos em um grupo controle com a mesma quantidade de animais. O material fibrótico foi submetido à análise imunohistoquímica que revelou uma presença preferencial de Cx32 dispersa no citoplasma, o que pode levar à hipótese de problemas no mecanismo de transporte citoplasmático destas estruturas, em contrapartida ao material pertencente ao grupo controle que evidenciou a presença das Cx32 na membrana plasmática formando placas juncionais. Quando submetido à análises moleculares o fígado fibrótico revelou uma diminuição da expressão gênica embora o produto protéico deste material quando comparado ao grupo controle não tenha se mostrado diminuído. / The connexin 32 (Cx32) is a proteic structure that constitute the channels that promote the cell communication by means of the gap junction (GJIC), allowing the diffusion of short cytoplasmic molecules from a cell to another. This work aimed to study these structures due to their importance in the hepatic metabolic processes. The hepatic fibrosis was triggered by the oral administration of dimethylnitrosamine (DMN) in the female rat Wistars twice a week in consecutive days during five weeks. The necropsy of these animals was carried out after the last drug administration. They presented a hepatic fibrosis state. The fibrotic material was submitted to the imunohistochemical analysis, which showed a preferencial presence of Cx32 in the cytoplasm, whereas in the control group the Cx32 was located at the membranes, in the junctional plaques. The molecular analysis showed a decrease of the genic expresson of the fibrotic material, however the proteic product wasn? t reduced in comparison with the control group as it was shown by western blot. We concluded that the fibrotic state introduced a disturbance in the intracellular distribution and genic expression of the connexin 32.

This study investigated how gastrointestinal and skin bacterial infections were affected by differential expression of connexin (Cx) 26 and a disintegrin and metalloprotease (ADAM) 17 in vitro. Cx26 is a component of gap junctions, which facilitate the transfer of small molecules between two cells. Recessive mutations in Cx26 cause non syndromic hearing loss (NSHL), and in certain populations, specific mutations account for the majority of Cx26 related NSHL. Their common occurrence suggests that they may provide a heterozygous, protective advantage to carriers. In this study adherence by the attaching and effacing pathogen Enteropathogenic Escherichia coli (EPEC) was significantly reduced in cells expressing mutant Cx26 compared to wild type Cx26. Furthermore, EPEC adherence and invasion of an alternative enteric pathogen, Shigella flexneri were reduced following treatment with Cx26 short-interfering-RNA in intestinal cells. These findings suggest that the loss of functional Cx26 expression improves protection against enteric bacteria. ADAM17 releases substrates including tumour necrosis factor alpha and ligands of the epidermal growth factor receptor and therefore is involved in the induction of immune responses and maintenance of the epidermal barrier. This study demonstrated that ADAM17 provides protection during Staphylococcus aureus infection of keratinocytes. Subsequently the protective effects of ADAM17 mediated protection were explored. Secretion of the proinflammatory cytokines Interleukins 6 and 8 correlated with ADAM17 activity. Additionally gene expression profiling was performed which identified the IL-17 signalling pathway, which is known to be important during S. aureus infection, as a potential downstream target of ADAM17. In summary, based on these findings, Cx26 and ADAM17 may represent potential therapeutic targets for gastrointestinal and skin bacterial pathogens.

As junções GAP são canais intercelulares responsáveis pela comunicação de células vizinhas, por onde passam pequenas moléculas e íons que mantêm a homeostasia celular. A junção GAP é formada seis proteínas, as conexinas. Na célula endotelial encontram-se as conexinas 37, 40 e 43. Nesse estudo, estimulamos a angiogênese em córnea de camundongos, através da cauterização com cristal de nitrato de prata. Foram utilizados camundongos heterozigotos para o gene da conexina 43 (Cx43+/-) e camundongos selvagens (Cx43+/+). As córneas foram analisadas 2 e 6 dias após a cauterização atravéspor meio da morfologia vascular, detecção das Cx37, Cx40, Cx43, PCNA por meio de Western Blot e avaliação ultraestrutural das células endoteliais. Como resultado obtivemos uma menor área de preenchimento vascular nos animais Cx43+/- em 2 e 6 dias após a lesão corneal, porém, em relação a extensão dos vasos não foi observado diferenças entre os grupos. Uma menor proliferação celular foi verificada através da detecção do PCNA, nos animais heterozigotos, somente após 2 dias da lesão corneal. Não houve alteração da Cx37 e Cx40 entres os grupos. A Cx43 parece ser uma conexina importante para a célula endotelial durante o processo de angiogênese. / The GAP junctions are intercellular streams responsible for the communication between close cells, which allow small molecules and ions to pass through them maintaining the cellular homeostasis. The GAP junction is formed of six proteins, the connexin. In the endothelial cell, there are the connexin 37, 40 and 43. In this study, we stimulated the angiogenesis in the mice\'s cornea through its cauterization using silver\'s crystal glass. It was used heterozygote mice to the gene of connexin 43 (Cx43+/-) and wild mice (Cx43+/+). The corneas were analyzed 2 and 6 days after the cauterization through the vascular morphology, detection of Cx37, Cx40, Cx43, PCNA through Western Blot and ultrastructural evaluation of the endothelial cells. As a result, we obtained a smaller area of vascular fillness in the animals Cx43+/- with 2 and 6 days of corneal injury, however, in regard to the extensions of the vessels, it wasn\'t observed any changes between the groups. A smaller proliferation of cells was verified, through the detection of PCNA, in the heterozygote animals only 2 days after the corneal injury. There wasn\'t any modification of the Cx37 and Cx40 between groups. The Cx43 seems to be an important connexin to the endothelial cell during the process of angiogenesis.

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system leads to the rewarding properties of alcohol. The mesolimbic DA system, which plays an important role in regulating reward and addiction, consists of DA neurons in the midbrain ventral tegmental area (VTA) that innervate the nucleus accumbens (NAc). It is believed that VTA DA neurons are inhibited by local gamma-aminobutyric acid (GABA) interneurons that express connexin-36 (Cx36) gap junctions (GJs). We have previously demonstrated that blocking Cx36 GJs suppresses electrical coupling between VTA GABA neurons and reduces ethanol intoxication and consumption suggesting that electrical coupling between mature VTA GABA neurons underlies the rewarding properties of ethanol. The aim of this study was to further investigate the role of VTA GABA neurons expressing Cx36 GJs in regulating DA neuron activity and release and mediating ethanol effects on VTA GABA neurons. To this end, we customized a Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) viral vector to target VTA GABA neurons expressing Cx36 GJs in order to chemogenetically modulate their activity. In order to more conclusively demonstrate the role of this sub population of VTA GABA neurons in regulating DA neural activity and release we used electrophysiology to characterize the electrical changes that occur in VTA DA and GABA neurons when Cx36-expressing VTA GABA cells were selectively activated. In addition, we evaluated the effects of activation of VTA GABA neurons on brain stimulation reward and alcohol consumption in ethanol naive and dependent mice. Results indicate that there are two populations of GABA neurons in the VTA, one that is GAD65+/Cx36+ and one that is GAD67+/Cx36-. Activation of Cx36+ VTA GABA neurons by clozapine-n-oxide (CNO) in mice injected with Gq DREADD activated VTA DA neurons and subsequent DA release in the NAc, suggesting that Cx36-containing GABA neurons are inhibiting non-Cx36 GABA neurons to disinhibit DA neurons. In hM3Dq animals, CNO administration provided a rewarding stimulus in the conditioned pace preference paradigm, and reduced consumption in the drink-in-the-dark ethanol consumption paradigm in dependent and naïve mice. A better understanding of the circuitry of the mesolimbic DA system is key to understanding the mechanisms that lead to addiction and may ultimately lead to improved therapies for substance abuse.

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system leads to the rewarding properties of alcohol. The mesolimbic DA system, which plays an important role in regulating reward and addiction, consists of DA neurons in the midbrain ventral tegmental area (VTA) that innervate the nucleus accumbens (NAc). It is believed that VTA DA neurons are inhibited by local gamma-aminobutyric acid (GABA) interneurons that express connexin-36 (Cx36) gap junctions (GJs). We have previously demonstrated that blocking Cx36 GJs suppresses electrical coupling between VTA GABA neurons and reduces ethanol intoxication and consumption suggesting that electrical coupling between mature VTA GABA neurons underlies the rewarding properties of ethanol. The aim of this study was to further investigate the role of VTA GABA neurons expressing Cx36 GJs in regulating DA neuron activity and release and mediating ethanol effects on VTA GABA neurons. To this end, we customized a Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) viral vector to target VTA GABA neurons expressing Cx36 GJs in order to chemogenetically modulate their activity. In order to more conclusively demonstrate the role of this sub population of VTA GABA neurons in regulating DA neural activity and release we used electrophysiology to characterize the electrical changes that occur in VTA DA and GABA neurons when Cx36-expressing VTA GABA cells were selectively activated. In addition, we evaluated the effects of activation of VTA GABA neurons on brain stimulation reward and alcohol consumption in ethanol naive and dependent mice. Results indicate that there are two populations of GABA neurons in the VTA, one that is GAD65+/Cx36+ and one that is GAD67+/Cx36-. Activation of Cx36+ VTA GABA neurons by clozapine-n-oxide (CNO) in mice injected with Gq DREADD activated VTA DA neurons and subsequent DA release in the NAc, suggesting that Cx36-containing GABA neurons are inhibiting non-Cx36 GABA neurons to disinhibit DA neurons. In hM3Dq animals, CNO administration provided a rewarding stimulus in the conditioned pace preference paradigm, and reduced consumption in the drink-in-the-dark ethanol consumption paradigm in dependent and naïve mice. A better understanding of the circuitry of the mesolimbic DA system is key to understanding the mechanisms that lead to addiction and may ultimately lead to improved therapies for substance abuse.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 109-118).

To maintain human health, the lymphatic system requires a structurally and functionally sound network of lymph vessels to absorb lipid-based nutrients, preserve extracellular fluid homeostasis, and mediate immune responses. Aside from lymphedema, investigations in the past few decades have found that impairment of the lymphatic vasculature is also involved in processes such as inflammation, tumor metastasis, fat metabolism, and obesity. However, despite a long history of study and rekindled vigor in the field of lymphatic vascular research, our knowledge of lymph vessel development and physiology is still quite limited. Recently, mutations in a protein family known as connexins (Cxs) were identified as the cause of lymphatic dysfunction in some cases of inherited lymphedema. This dissertation explores the role of primarily two specific connexins, Cx37 and Cx43, and the transcription factor Foxc2 in the morphogenesis and function of the lymphatic vasculature in mice. To accomplish this, phenotypic characterization of mice with genetic deficiencies (knockout mice) in Cx37, Cx43, and/or Foxc2 was performed principally via necropsy, histological techniques (immuno-fluorescence microscopy and H&E staining), and Evans blue dye (EBD) injections. Developmental abnormalities were found in lymphatic vascular growth, patterning, and remodeling in mice lacking Cx37, Cx43, Foxc2 or a combined deficiency of these proteins. Reductions or complete loss of lymphatic valves were a common finding in mice lacking one or more of these proteins. These valve deficits underlay lymphatic insufficiencies that resulted in lymphedema and chylothorax in some genotypes. Foxc2 was found to be a regulator of Cx37 expression. Moreover, Foxc2 was also dependent on Cx37 function for proper morphogenesis of lymph vessels. These findings pertaining to the expression of connexins in the lymphatic vasculature, their role in lymphatic valvulogenesis, and the interdependence of Cx37 and Foxc2 during lymph-vascular development represent my original contributions to human knowledge.

Vasomotion is the spontaneous oscillation of vascular tone, occurring due to synchronization of internal calcium fluctuations between multiple vascular smooth muscle cells by gap junction and electrical communication. Although altered vasomotion has been observed in a variety of pathological situations, characterization of these alterations has been lacking. Using a novel method of spectral quantification, and two experimental models known to have altered vascular structure, the present thesis was designed to evaluate whether vasomotion characteristics could be correlated with altered vascular structure. Rats with perinatal iron deficiency (PID) have previously been shown to possess altered vascular structure. When phenylephrine-mediated contractile and acetylcholine-mediated dilatory responses were investigated in PID animals, they both displayed blunted relaxation as compared to control vessels. When vasomotion characteristics were quantified, vessels taken from PID animals exhibited a decreased power in the very low frequency window (VLF <0.2 Hz). Changing vessel oxygenation to 10% O2 from 95% O2 did not result in significant alterations of vasomotion characteristics. The primary frequency of oscillation was investigated with a peak finder, and found to be significantly different compared to control in both the aorta and renal arteries obtained from PID animals. To investigate the effect of antihypertensive treatment (enalapril and hydrochlorothiazide) on gap junction communication, spontaneously hypertensive rats (SHR) were subject to a 2-week intensive angiotensin converting enzyme inhibitor treatment. This treatment resulted in significant vascular structural regression. All vessels (aorta, renal, mesenteric) from treated animals had greater proportions of power in the VLF window, with both the mesenteric and renal vessels exhibiting a primary peak of oscillation around 0.2 Hz; whereas the aorta had a primary peak at 0.12 Hz. Investigating altered gap junction communication with the gap junction blocker 18-α glycyrrhetinic acid, revealed that vascular bed location was the determining factor of vasomotion response. Immunoblotting did not indicate differences in connexin 43, a major gap junction protein in the vascular smooth muscle. These studies suggest that vasomotion characteristics can be used as a method of vascular phenotype investigation; vasomotion characteristics were significantly different in vessels taken from PID and hypertensive animals as compared to control and antihypertensive-treated animals, respectively. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-09-26 11:39:44.043