Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-13T11:48:23Z No. of bitstreams: 1 texto completo.pdf: 179843 bytes, checksum: 3db2311873e200a06702cfed6db58952 (MD5) / Made available in DSpace on 2017-06-13T11:48:23Z (GMT). No. of bitstreams: 1 texto completo.pdf: 179843 bytes, checksum: 3db2311873e200a06702cfed6db58952 (MD5) Previous issue date: 2002-10-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Foram isoladas 20 leveduras de fezes de leitões recém-nascidos com o objetivo de selecionar espécies com atividade killer e potencial probiótico para serem utilizadas como suplemento na alimentação de suínos. Dentre as leveduras analisadas quanto à atividade killer, incluiu-se 234 leveduras da micoteca do Laboratório de Fisiologia de Microrganismos do BIOAGRO, isoladas de vários laticínios na região da Zona da Mata. Dos 254 isolados, 9 apresentaram atividade killer positiva contra Criptococcus laurentii. Esses isolados killer foram selecionados quanto a resistência a 0,3% de sais biliares e ao ácido clorídrico em pH 2,0 e crescimento à temperatura próxima da retal de suínos (39oC). Três isolados (7, P2 e P3) apresentaram melhor desempenho quanto ao teste de resistência a esses obstáculos fisiológicos e seriam os mais indicados para utilização em probióticos para suínos. O soro de queijo ultrafiltrado fermentado por Kluyveromyces lactis apresentou potencial prebiótico no crescimento dos isolados em concentrações de 6% (v/v). Os isolados killer P2 e P3 associados a 6% (v/v) de soro ultrafiltrado fermentado por Kluyveromyces lactis constituem uma possível estratégia dietética para aumento do desempenho de leitões recém-nascidos. / Yeasts were isolated from three days old piglets faeces in order to select species with killer activity and with probiotic potential for their use as supplement in the swine feeding. Amongst the analyzed yeasts as the killer activity, there were yeasts of the Yeast Collection of the Laboratory of Physiology of Microorganisms of BIOAGRO, isolated from several industries of dairy products. Killer activity against Criptococcus laurentii was screened in 254 yeasts isolate, and nine were selected. Those killer yeasts were then selected by their capacity growth at 0,3% of bile salts, hydrochloric acid (pH 2,0) and at swine rectal temperature. The killer yeast 7, P2 and P3 can be considered as the best among the different strains tested which might be able to tolerate the adverse conditions of the gastrointestinal tract of the pigs and serve as a good agent for use as a microbial feed supplement for swine. The ultra filtered whey fermented by Kluyveromyces lactis presented prebiotic potential in the growth of the killer yeast in concentrations of 6% (v/v). The killer yeasts P2 and P3 associated to 6% (v/v) of whey ultra filtered fermented by Kluyveromyces lactis constitute a possible dietary strategy for increasing the performance of young pigs.

Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto. / Submitted by Maurílio Figueiredo (maurilioafigueiredo@yahoo.com.br) on 2014-10-10T20:16:09Z No. of bitstreams: 2 license_rdf: 22190 bytes, checksum: 19e8a2b57ef43c09f4d7071d2153c97d (MD5) TESE_AdenosineProductionCD39.pdf: 1471044 bytes, checksum: 2af62f7d8c073380843cd9a8ae7ce6be (MD5) / Approved for entry into archive by Gracilene Carvalho (gracilene@sisbin.ufop.br) on 2014-11-07T12:09:27Z (GMT) No. of bitstreams: 2 license_rdf: 22190 bytes, checksum: 19e8a2b57ef43c09f4d7071d2153c97d (MD5) TESE_AdenosineProductionCD39.pdf: 1471044 bytes, checksum: 2af62f7d8c073380843cd9a8ae7ce6be (MD5) / Made available in DSpace on 2014-11-07T12:09:27Z (GMT). No. of bitstreams: 2 license_rdf: 22190 bytes, checksum: 19e8a2b57ef43c09f4d7071d2153c97d (MD5) TESE_AdenosineProductionCD39.pdf: 1471044 bytes, checksum: 2af62f7d8c073380843cd9a8ae7ce6be (MD5) Previous issue date: 2014 / Item withdrawn by Gracilene Carvalho (gracilene@sisbin.ufop.br) on 2014-12-05T12:14:11Z Item was in collections: PPCBIOL - Doutorado (Teses) (ID: 17) No. of bitstreams: 2 license_rdf: 22190 bytes, checksum: 19e8a2b57ef43c09f4d7071d2153c97d (MD5) TESE_AdenosineProductionCD39.pdf: 1471044 bytes, checksum: 2af62f7d8c073380843cd9a8ae7ce6be (MD5) / Item reinstated by Gracilene Carvalho (gracilene@sisbin.ufop.br) on 2015-12-15T16:43:13Z Item was in collections: PPCBIOL - Doutorado (Teses) (ID: 17) No. of bitstreams: 2 license_rdf: 22190 bytes, checksum: 19e8a2b57ef43c09f4d7071d2153c97d (MD5) TESE_AdenosineProductionCD39.pdf: 1471044 bytes, checksum: 2af62f7d8c073380843cd9a8ae7ce6be (MD5) / A leishmaniose cutânea (CL), causada por L. amazonensis, é caracterizada por uma intensa imuno- supressão e multiplicação descontrolada do parasito em modelos experimentais e é geralmente grave em humanos, variando desde a forma cutânea até a cutâneo-difusa. Não existem mecanismos precisos conhecidos sobre como L. amazonensis modula a resposta imunológica para que os macrófagos (MФ) infectados com L. amazonensis se tornem refratários à ativação por células T efetoras. Aqui, nós investigamos o possível mecanismo regulador que Leishmania provavelmente pode induzir em MФ residentes durante a interação precoce, de modo a impedir ativação das células. Neste estudo, analisou-se a expressão de CD39 e CD73, por citometria de fluxo, em MФ peritoneais murinos infectados com promastigotas metacíclicas de L. amazonensis e também a porcentagem dessas células que expressam a CD39 e CD73 foi avaliada. Nossos resultados mostraram que em 72hrs inativos os MФ tiveram baixa expressão de CD73. Curiosamente, no entanto, ao contrário de MФ tratados com LPS os infectados com L. amazonensis expressaram altos níveis de CD73. Esta informação foi posteriormente validada pelos resultados de estudos no contexto ex-vivo que mostrou igualmente que MФ infectados são predominantemente CD73+. Quando as atividades enzimáticas de CD39 e CD73 foram bloqueadas, tal como pelo uso de DIDS e MAD αβ, tanto a infecção quanto o número de amastigotas diminuiu significativamente após 48 horas de incubação. Da mesma forma, a inibição dos receptores de adenosina A2a e A2b de ZM241385 e MRS1754 também apresentou os mesmos efeitos sobre a sobrevivência do parasito e infectividade. Em estudo posterior, em busca de um possível papel da HIF- 1α na infecção por Leishmania, investigamos os efeitos da FM19G11, inibidor do HIF- 1α, na expressão de CD39 e CD73, bem como na infecção parasitária . Observou-se que, apesar de HIF - 1α poder influenciar na sobrevivência do parasito, os seus efeitos sobre a expressão de CD39 e CD73 não eram visíveis. Também foi avaliada, por PCR em tempo real, a expressão de receptores de adenosina em populações infectadas, nas quais não se observou nenhuma mudança significativa na expressão após 24 horas de infecção. Além disso, também foi avaliada a produção de citocinas, tais como TNF- α e IL-10 a partir da produção de NO nos grupos tratados. Surpreendentemente, não houve variação nos níveis destes mediadores, sugerindo a existência de outros mecanismos independentes da mediação por citocina para produção de Óxido Nítrico, tais como a produção de ROS ou efeitos leishmanacidas independentes do triptofano. Concluindo, nossos dados mostram que a infecção por L. amazonensis regula a expressão CD73 durante 24 horas de infecção e sua sobrevivência depende de atividades enzimáticas, bem como de receptores A2a e A2b. __________________________________________________________________________________________ / ABSTRACT:Cutaneous leishmaniasis (CL) caused by L. amazonensis is characterized by intense immune-suppression and uncontrolled parasite multiplication in experimental models and is usually severe in humans ranging from cutaneous to diffuse cutaneous leishmaniasis. There are no precise mechanisms known how L. amazonensis modulates immune response so that macrophages (MФ) infected with L. amazonensis are refractory to activation by effector T cells. Here, we investigated the possible regulatory mechanism that Leishmania can likely induce in host MФ during early interaction so as to prevent their host cells from activation. In this study, we analyzed the expression of CD39 and CD73, by flow cytometry, in murine peritoneal MФ infected with metacyclic promastigotes of L. amazonensis and percentage of those cells expressing CD39 and CD73 was evaluated. Our results showed that 72hrs rested MФ down regulated CD73 expression. Interestingly, however, unlike LPS treated MФ, L. amazonensis infected MФ up regulated CD73 expression. This data was further validated by the findings from in ex-vivo studies which equally support that infected MФ are predominantly CD73 positive. When CD39 and CD73 enzymatic activities were blocked such as by the use of DIDS and αβ MAD, both infection and amastigote number decreased significantly within 48hrs of incubation. Similarly, inhibition of adenosine receptors A2a and A2b by ZM241385 and MRS1754 also had the same effects on the parasite survival and infection. In another study, in search of a possible role of HIF-1α in Leishmania infection, we investigated the effects of FM19G11, inhibitor of HIF-1α, on expression of CD39 and CD73 as well as parasitic infection. We observed that although HIF-1α can influence in the parasite survival, their effects on CD39 and CD73 expression were not visible. We also evaluated the expression of adenosine receptors in infected population by real time PCR in which we observed no significant change in the expression after 24hrs of infection. Moreover, we also evaluated cytokine production such as TNF-alpha, IL-10 and NO production from the treated groups. Surprisingly, there was no alternation in the levels of these mediators suggesting other mechanisms, independent of cytokine mediated nitric oxide production such as ROS production or tryptophan independent oxygen anti-leishmanacidal effects, involved in it. In conclusion, our data show that L. amazonensis infected up regulates CD73 expression during 24hrs of infection and its survival is dependent on enzyme activities as well as A2a and A2b receptors.

Orientadora : Profª. Drª. Karin Braun Prado / Coorientador : Prof. Dr. Danillo G. Augusto / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa: Curitiba, 28/03/2017 / Inclui referências : f. 92-102 / Resumo: A familia de genes KIR (do ingles, killer cell immunoglobulin-like receptors) desempenha um papel central na imunidade inata e adaptativa e seu polimorfismo tem sido associado a suscetibilidade diferencial a diversas doencas. Esses genes exibem extensa variabilidade, tanto em termos de ausencia e presenca de genes, quanto em nivel alelico. Os receptores codificados por esses genes sao expressos na superficie das celulas NK e de alguns subtipos de celulas T e podem transduzir sinais ativadores ou inibidores. Pouco se conhece a respeito dos seus niveis de expressao genica diferencial, tampouco dos mecanismos de regulacao. O gene KIR3DL2 codifica um receptor inibidor e e o segundo gene KIR mais polimorfico e polialelico do complexo, com mais de 80 alelos descritos. O objetivo desse trabalho foi analisar se o polimorfismo alelico de KIR3DL2 impacta na sua expressao genica diferencial. Para isso, caracterizamos a diversidade alelica de KIR3DL2 em uma populacao de euro-descendentes de Curitiba e regiao metropolitana (n = 235), atraves de sequenciamento de DNA. As frequencias genotipicas estavam de acordo com as esperadas pelo equilibrio de Hardy-Weinberg, sendo os genotipos mais comuns: 3DL2*001/007 (11,5%), 3DL2*001/002 (6,8%) e 3DL2*002/007 (6,8%). Os alelos mais frequentes encontrados nessa populacao foram 3DL2*007 (21,7%), 3DL2*001 (21,3%) e 3DL2*002 (20%). Um total de 40 individuos com genotipos especificos e comuns na populacao desse estudo foram selecionados para a analise de expressao diferencial por citometria de fluxo. A analise de expressao diferencial foi realizada com os individuos portadores dos alelos mais frequentes encontrados na populacao, sendo eles: 3DL2*001, 3DL2*002, 3DL2*003, 3DL2*005, 3DL2*007, 3DL2*009, 3DL2*010 e 3DL2*011. Verificamos que o polimorfismo alelico de KIR3DL2 esta associado nao somente com niveis de expressao diferencial, mas tambem com diferentes quantidades de celulas NK que exibem KIR3DL2 em sua superficie. O alelo KIR3DL2*002 foi associado ao maior nivel de expressao de KIR3DL2 e ao maior numero de celulas NK 3DL2 positivas. Ja 3DL2*010 foi associado ao menor nivel de expressao e a menor quantidade de celulas NK com KIR3DL2 presente na superficie celular. Alem disso, demonstramos que um polimorfismo na regiao 3' UTR, na posicao 16545A>G (rs1865095) marca os niveis de expressao de KIR3DL2. Sugerimos que esta expressao diferencial possa estar relacionada a ligacao de miRNAs nesta regiao, especificamente o miR-2114-3p. A presenca de ligantes especificos (HLA-A3, -A11 e -B27) nao esta associada a diferentes niveis de expressao de KIR3DL2 (p = 0,5739) nem a diferentes quantidades de celulas NK KIR3DL2 positivas (p = 0,7772). Alem disso, nenhuma correlacao foi encontrada entre a expressao diferencial dos alelos de KIR3DL2 e a expressao diferencial dos alelos de HLA-A. Como perspectivas futuras pretendemos analisar, atraves de co-cultivo celular, a atividade citotoxica das celulas NK de individuos com diferentes genotipos homozigotos, como KIR3DL2*001/KIR3DL2*001, KIR3DL2*002/KIR3DL2*002, KIR3DL2*007/KIR3DL2*007 e KIR3DL2*010/KIR3DL2*010 a fim de corroborar as hipoteses geradas. Palavras-chave: Celulas NK, KIR3DL2, variabilidade alelica, HLA-A3, HLA-A11, HLA-B27, expressao diferencial. / Abstract: The KIR (killer cell immunoglobulin-like receptors) gene family plays a central role in innate and adaptive immunity and has been associated with differential susceptibility to diseases. Besides the uncommon presence and absence polymorphism that occurs in KIR, these genes also exhibit an extensive allelic variation. The receptors encoded by these genes are expressed on the surface of NK cells and on some subset of T cells. They can transduce either activating or inhibiting signals. The differential expression levels and the mechanisms of genetic regulation of these receptors are poorly known. The KIR3DL2 gene encodes an inhibitory receptor and it is one of the most polymorphic and polyallelic KIR, with more than 80 alleles described so far. This study aimed to analyze if there are a profound impact of KIR3DL2 allelic polymorphism on its differential gene expression. Allelic diversity of KIR3DL2 was characterized in an euro-descendant population from Curitiba and metropolitan region, state of PR (n = 235), by sequencing-based typing. Genotype frequencies were in accordance with Hardy-Weinberg equilibrium and the most frequent genotype was 3DL2*001/007 (11.5%), followed by 3DL2*001/002 (6.8%) and 3DL2*002/007 (6.8%). The most frequent alleles in the population were 3DL2*007 (21.7%), 3DL2*001 (21.3%) and 3DL2*002 (20%). A total of 40 individuals with specific and commom genotypes were selected for differential expression analysis by flow citometry. The differential expression analysis was made for the most common alleles found in the population: 3DL2*001, 3DL2*002, 3DL2*003, 3DL2*005, 3DL2*007, 3DL2*009, 3DL2*010 and 3DL2*011. We verified that the allelic polymorphism of KIR3DL2 was associated not only with the differential expression but also with the different amount of NK cells that display KIR3DL2 on their surface. KIR3DL2*002 allele was associated with higher expression of KIR3DL2 and higher number of KIR3DL2 positive NK cells, while KIR3DL2*010 was related to the lowest expression and lowest level of KIR3DL2 positive NK cells. It has also been found that a polymorphism in the 3' UTR, 16545A>G (rs1865095), was associated with KIR3DL2 differential expression level. We suggest that it may be related to miRNAs binding, specifically miR-2114-3p. The presence of specific HLA class I ligands (HLA-A3, -A11 and -B27) was not associated with KIR3DL2 differential expression levels (p = 0.5739) nor with different amount of NK KIR3DL2+ cells (p = 0.7772). In addition, no correlation was found between the differential expression of the KIR3DL2 alleles and the differential expression of the HLA-A alleles. As future perspectives, it is intended to analyze the citotoxic activity of NK cells from individuals with different genotypes like KIR3DL2*001/KIR3DL2*001, KIR3DL2*002/KIR3DL2*002, KIR3DL2*007/KIR3DL2*007 and KIR3DL2*010/KIR3DL2*010 through co-culture to corroborate the hypotheses generated. Key-words: NK cells, KIR3DL2, allelic variability, HLA-A3, HLA-A11, HLA-B27, differential expression.

Resumo: Receptores de células natural killer semelhantes à imunoglobulina (KIR) são expressos por células natural killer (NK) e subtipos de células T. Os genes KIR são altamente polimórficos e este complexo poligênico exibe variação em número de genes ativadores e inibidores, o que regula a atividade de células NK. Moléculas HLA de classe I servem como ligantes para KIR. As interações dos loci de KIR e HLA são importantes para o reconhecimento de alvos pelas células NK. Vários estudos de associação indicam que estes loci estão envolvidos em doenças infecciosas, desordens autoimunes/inflamatórias, câncer e reprodução. Dados funcionais suportam o mecanismo baseado na ação de receptores inibidores e ativadores através de diferentes combinações de genótipos KIR-HLA em doenças. A doença autoimune pênfigo vulgar (PV) é uma desordem bolhosa que afeta a pele em múltiplas camadas, causada por autoanticorpos contra desmogleína 1 e 3, já foi previamente associada com KIR. Neste estudo nós sugerimos que indivíduos KIR3DL2*001+ KIR3DL1+ e baixa frequência de genes ativadores em seu genótipo, exibem uma maior inibição de citotoxicidade quando na presença dos ligantes HLA-A*03/A*11 e HLA-Bw4. Resultados in vitro demonstraram que o cultivo de células NK de diferentes indivíduos com células T CD3 expressando HLA-A*03/A*11 e HLA-Bw4, levou a uma diminuição de citotoxicidade. Isto foi observado em indivíduos saudáveis e pacientes de pênfigo. O bloqueio com anticorpo anti-KIR3DL2 mostrou um aumento da citotoxicidade, corroborando para o potencial efeito inibidor deste gene nesta situação. Além disso, nós verificamos uma maior frequência de células NK (subtipo CD56bright) no sangue periférico de pacientes de PV quando comparado com indivíduos saudáveis. Estes dados em conjunto com a detecção de níveis aumentados de IL-6 e IFN-? nos ensaios de cultivo, contribuem para o entendimento e o papel de células NK na patogênese de pênfigo.

Orientadores: Ronei Luciano Mamoni, Maria Heloisa Souza Lima Blotta / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T10:37:38Z (GMT). No. of bitstreams: 1 Longhi_LarissaNaraAlegrini_M.pdf: 6631958 bytes, checksum: 45b4fc2e64262b037302e0269ebd8f71 (MD5) Previous issue date: 2010 / Resumo: Tradicionalmente o papel das células NK na resposta imunológica tem sido associado com a resistência à infecção viral e tumores, porém estudos recentes apontam para a participação destas células na resposta imunológica contra outras doenças infecciosas. O objetivo deste estudo foi avaliar a possível participação de células NK (CD56+CD3-) na resposta imunológica ao fungo Paracoccidioides brasiliensis. Foram utilizadas células CD56+ isoladas por meio imunomagnético provenientes de pacientes com paracoccidioidomicose e de indivíduos controle. As células CD56+ foram avaliadas quanto à capacidade citotóxica direta contra leveduras de P. brasiliensis (cepa virulenta - Pb18 e avirulenta - Pb265) e contra células-alvo (monócitos) infectadas com leveduras de ambas as cepas. Também foi avaliada a expressão dos constituintes dos grânulos citotóxicos (granzima A, B, perforina e granulisina), receptores de ativação (NKG2D) e de inibição (KIR2DL2/L3/S2 e KIR3DL1), marcadores de ativação (CD69 e CD25), assim como a produção de citocinas (IFN-g e TNF-a) por meio de PCR em tempo real e por citometria de fluxo. A participação da IL-15 na ativação das células NK foi avaliada pela adição da citocina recombinante em alguns experimentos. Para avaliar a participação dos grânulos na atividade citotóxica direta e contra células-alvo em alguns experimentos as células foram tratadas com inibidores específicos (SrCl, concanamicina A ou EGTA). Os resultados demonstraram que células de pacientes apresentam resposta citotóxica (direta e contra células-alvo) inferior àquela observada nos indivíduos controle. A citotoxicidade direta foi dependente de grânulos, mas independente de perforina, enquanto que a citotoxicidade contra células-alvo foi dependente de perforina. A expressão de granulisina (RNAm e proteína) foi maior nas células de indivíduos controle. Além disso, observamos que células CD56+ apresentam a capacidade de secretar essa proteína após o estímulo com leveduras de P. brasiliensis. Também observamos que as células CD56+ de doadores normais são ativadas (aumentam a expressão de CD69 e CD25) quando estimuladas com o fungo, enquanto as células NK de pacientes não mostraram esse aumento, a não ser quando estimuladas com IL-15. Estes resultados demonstraram que as células CD56+ podem participar ativamente da resposta imunológica contra o P. brasiliensis, podendo contribuir tanto para a morte direta do fungo como de células infectadas, e que a granulisina pode desempenhar um papel preponderante no controle da infecção pelo P. brasiliensis. Outro fato importante observado foi que as células CD56+ quando estimuladas por células leveduriformes de P. brasiliensis produzem e secretam IFN-g, uma citocina com atuação importante na ativação de outras células do sistema imunológico como macrófagos e linfócitos, e dessa forma pode contribuir para o desenvolvimento da resposta imunológica adquirida subseqüente à infecção. / Abstract: Besides their role in viral infection and tumor resistance, recent studies have showed that NK cells also participate in the immune response against other infectious diseases. The aim of this study was to evaluate the possible role for NK cells (CD56+) in the immune response against the fungi Paracoccidioides brasiliensis. CD56+ cells from patients with paracoccidioidomycosis and healthy individuals were isolated by immmunomagnetic columms and antibodies. We evaluated the capacity of the direct killing of P. brasiliensis yeast cells (Pb18 virulent strain and Pb285 avirulent strain), as well as, the ability to kill infected target cells (monocytes infected with either virulent or avirulent strains). The CD56+ cells also were assessed in order to evaluate the expression of citotoxic granules (granzyme A, B, perforin and granulysin) and the expression of the activation receptor NKG2D, the inhibition receptors KIR2DL2/L3/S2 and KIR3DL1 and the activation molecules CD69 and CD25. We also determine the capability of production and release of cytokines (IFN-g and TNF-a) using Real Time PCR, flow cytometry and ELISA. The role of IL-15 was analyzed by the addition of recombinant cytokine in some experiments. To determine if the cytotoxic activity was dependent of granules, cell cultures were supplemented with specific inhibitors which prevent granule release. The results showed that cells from patients present a lower citotoxic response when compared to healthy individuals. The direct citotoxicity seems to be granule-dependent but independent on perforin, whereas the citotoxicity against target cells showed to be perforin dependent. It was observed an augmented expression of granulysin (mRNA and protein) in cells from controls, and that CD56+ cells are able to produce and release granulysin after stimulation with P. brasiliensis yeast cells. Furthermore, the analyses of CD56+ cells from controls showed elevated expression of CD25 and CD69 after the stimulus with yeast cells, while cells from patients are only activated in the presence of IL-15. These results demonstrated that CD56+ cells can participate actively of the immune response against the P. brasiliensis infection either by destroying directly yeast cells or by the recognition and killing of infected cells. Granulysin is the possible mediator of the cytotoxic effect observed in this study, once this protein is produced and released by CD56+ cells. Another important data is the finding that CD56+ cells are able to produce IFN-g after the stimulus, which could influence the subsequent acquired immunological response by stimulating others cells as macrophages and lymphocytes. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

The purpose of this study is to explore the implications of the historical perspective when it is linked to the ecological concept of adaptive management. The vehicle for this exploration is a genetically distinct population of killer whales (Orcinus orca), whose core coastal habitat includes the inland waters of Georgia Strait, Juan de Fuca Strait and Puget Sound; a geographic region referred to as the “Salish Sea.” This stock of killer whales, known as the Southern Resident Community, is unique in having a detailed scientific record that spans over two decades and recently this population was listed as “threatened” by the Committee on the Status of Endangered Wildlife in Canada (April 1999). The goal of this study is to take account of the specific ecological history of this killer whale population, and provide an assessment of the resiliency of this stock to withstand present levels of human impacts. In Chapter 1 the academic concepts of historical ecology and adaptive management are reviewed in preparation for their application as theory. Chapter 2 is an inventory of the ecological domain, in which the focal population is assessed by temporally measurable indicators o f its ecological status: population dynamics, feeding ecology, and habitat use. In Chapter 3 temporally measurable indicators of stress such as predation, disease, food resource depletion, toxic exposure, surface disturbance, and underwater noise are examined for their impact upon the carrying capacity of the environment of the whales. Chapter 4 plots both sets of indicators historically as trends in variation from the Sample Mean at different time scales (months, years, decades, centuries), and indexes them in terms of perturbations from the historical norm. In Chapter 5 four basic types of historical trends in environmental impacts are identified that are directly relevant to evaluating the resilience of the management unit. These are: (1) Relic impacts - potential impacts that are no longer present, but may account for present conditions. (2) Adapted impacts - potential impacts that have been around long enough for the management unit to have adapted to them. (3) Cumulative impacts- potential impacts that accumulate slowly in the environment or life history of the management unit before exerting environmental resistance. (4) New impacts - potential impacts with which the management unit has not had previous experience. These four historical criteria allow the manager to identify the most sensitive impacts for present conditions, and identify scales of management for restorative intervention. This resiliency index should have application for most types of ecological systems, or management units, because it describes very generalized types of temporal outcomes, independent of scale and life history pattern of the management unit. In terms of the focal population of killer whales in this study, the historical assessment suggests that: 1) these whales are presently a remnant population due to killing and capture by European settlers from the turn of the century to the 1970s; 2) they have bio-accumulated toxins during the highest historical periods of environmental pollution in the Salish Sea, and this toxic exposure will continue to increase for the whales over the next few decades; 3) this killer whale population has never previously experienced a lack of salmon, so diminishing salmon stocks are potentially a new stress on them; and 4) these killer whales have adapted to vessel traffic and noise for several decades in relation to vessel-based salmon fishing operations, and that this influence has recently been replaced by record levels of whale watching traffic, which potentially poses more severe impacts than fishing vessels because the boats follow the whales, rather than their prey. This historical assessment facilitates the application of “adaptive management” strategies for these whales by providing the basis for predicting the current “resiliency” of this population to adapt to environmental conditions. / Graduate

Philosophiae Doctor - PhD / The current diagnostic, prognostic, predictive and therapeutic monitoring methods used for breast cancer are limited. Thus, research into more specific, sensitive and effective strategies is required. Breast cancer is the most prevalent form of cancer in women worldwide and accounts for the most common cause of death in women every year. Cancer development is characterized by a wide spread of genetic abnormalities of gene sequences that can be used in detecting and monitoring treatment of the disease as a result of altered gene expression patterns which leave a trail of biomarkers. Seven candidate genes (Gene 1-7) were identified from a previous in silico study and their gene products (BRG 1-7) were annotated to be good candidate breast cancer biomarkers. Differential gene expression analysis using quantitative real-time PCR (qRT-PCR) validated the over-expression of Gene 3, Gene 4 and Gene 7 in a breast cancer cell line (MCF7), of which Gene 7, annotated as a Natural killer group 2, member D (NKG2D) ligand, was observed to be the most over-expressed gene. The innate immune system is the first line of the body's physiological defense against diseases and the natural killer (NK) cells, are central to mediating this type of immunity. NK cells are activated when a specific surface receptor such as the NKG2D receptor binds its ligands expressed by tumor cells. To evade being detected by the immune system, cancer cells are reported to shed off the NKG2D ligands and are expected to be present in the bodily fluids of cancer patients. Also, chemotherapeutics have been reported to suppress the natural anti-tumour immune response, thus should be taken into account when designing optimal therapy for cancer patients. The aim of this research was to validate these candidate genes as effective breast cancer biomarkers using several in silico methods as well as molecular techniques and study the effect of Gene 7 on modulating the effect of several pro-apoptotic compounds. The in silico part of the study investigated the functional, protein interaction, pathways, and tissue expression specificity of the candidate biomarkers using computational software such as DAVID, STRING, KEGG, Genecards and GEA. Also an in silico validation of the prognostic/predictive values of the genes was analysed using SurvExpress, KMplot, and GOBO. Protein expression of selected genes was analysed by Western blot, and immunofluorescence analysis. BRG 7 gene was cloned into pcDNA3.1 vector using recombinant DNA technology while commercial shRNA construct was used to 'knock-down' Gene 7 expression. The two constructs were used to transfect MCF-7 and MCF-12A cells. Over-expression and 'knock down' Gene 7 in transfected cells was confirmed using western blot analysis. Stably transfected cells were then treated with three pro-apoptotic compounds (Camptothecin, Doxorubicin and DMSO) for 24 hours. The apoptotic cells were stained with 3, 4, 5, 6-tetrachloro-2', 4', 5', 7' tetraiodofluorescein (TCTF) and then analysed using flow cytometry. Functional analysis linked Gene 1, Gene 2, Gene 4, Gene 6 and Gene 7 to different cancer related processes. The pathway analysis showed Gene 1, Gene 2, Gene 4 and Gene 7 were involved in pathways that can be linked to cancer modulation. The protein-protein interaction analysis showed only BRG 2 was directly linked to two major hallmarks of cancer (Apoptosis and Autophagy). Breast cancer associated Transcription factors were shown to regulate these genes. Gene 1 and Gene 5 as well as the three genes observed to be highly expressed in the qRT-PCR study were validated to differentially express in breast cancer. An additional protein (BRG 8) was identified and postulated to be a good biomarker candidate for breast cancer based on its direct interaction with BRG 7 and estrogen receptor protein (ESR). The prognostic value of the candidate genes were monitored in two datasets (DATA1 and DATA2) in SurvExpress. DATA1 showed that Gene 6 and Gene 8 while DATA2 showed that Gene 3, Gene 6 and Gene 7 were valuable candidate genes in breast cancer prognosis. The survival curves from the two datasets showed the combined genes could predict the outcome of breast cancer patients undergoing treatments. A plot box output from SurvExpress showed most of the genes were differentially expressed comparing two risk groups. The Kaplan Meier plotter confirmed, Gene 1, Gene 3, Gene 4 and Gene 7 have a significant P-value in predicting the survival outcome based on gene differential expression value. GOBO analysis showed the genes may accurately predict the survival outcome of estrogen positive subtype, ERBB2 subtype of estrogen receptor negative and lymph node negative subtype of ER- tumours, but not all subtype of ER- tumours. Western blot analysis showed BRG 7 may be highly expressed in MCF-7 as compared to MCF-12A, BRG 8 was found to be expressed in all cancer cell types analyzed except for MCF-7 and HT29. BRG 2 was found to be expressed in all cancer types analyzed. immunofluorescence analysis showed BRG 3, BRG 4 and BRG 7 are differentially expressed in breast cancer cell line and are more localized on the cell membrane when compared to the breast non-cancer cell line. Over-expression and gene knock down in cells were successfully confirmed with Western blot analysis. Stably transfected MCF-12A cell for over-expression of BRG7 protein, resulted in cell senescent and the cell stopped growing while stably transfected MCF-7 over-expressing BRG7 did not show any morphological changes. Apoptosis was enhanced in cells treated with camptothecin, doxorubicin and DMSO overexpressing BRG7. Apoptosis was reduced in camptothecin and DMSO treated gene 'knock-down' cells but not doxorucin treated. BRG7 gene 'knock down' in transfected cells showed varying response to all three pro-apoptotic compounds. From this study Gene 3, 5, 7 and 8 and their protein levels were confirmed to be differentially expressed in breast cancer cells and could serve as putative biomarkers for breast cancer. However the variance in the effectiveness of individual genes suggests that the set of genes would perform better than individual gene. The modulating role of BRG7 in drug induced apoptosis, suggest it could probably play an important role in personalised medicine and could serve as a biomarker to monitor the prognosis and/or therapeutic outcome of pro-apoptotic drugs in breast cancer patients. These findings will be further investigated in human breast tissues to validate these data.

Although structurally unrelated, the human killer cell immunoglobulin-like (KIR) and the rodent lectin-like Ly49 receptors serve similar functional roles in natural killer (NK) cells. Moreover, both gene families display variegated and mostly mono-allelic expression patterns established at the transcriptional level. DNA methylation, but not histone modifications, has recently been shown to play an important role in maintenance of the expression patterns of KIR genes but the potential role of DNA methylation in the expression of Ly49 genes was unknown. My thesis focuses on the role of epigenetic modifications, especially DNA methylation, in the maintenance of mouse Ly49 gene expression. I show that hypomethylation of the region encompassing the main promoter of Ly49a and Ly49c in primary C57BL/6 (B6) mouse NK cells correlates with expression of these genes. Using B6 x BALB/c Fl hybrid mice, I demonstrate that the expressed allele of Ly49a is hypomethylated while the non-expressed allele is heavily methylated, indicating a role for epigenetics in maintaining mono-allelic Ly49 gene expression. Furthermore, the Ly49a promoter region is heavily methylated in fetal NK cells but variably methylated in non-lymphoid tissues. In apparent contrast to the KIR genes, I show that histone acetylation state of the promoter region strictly correlate with Ly49A and Ly49G expression status. Also, the instability of Ly49G expression on some lymphoid cell lines is at least in part due to changes in the level of histone acetylation of the promoter region. As for the activating Ly49 receptors, it seems that although DNA methylation levels of the promoter regions do correlate with the state of expression of these receptors, the pattern of DNA methylation is different from that of the inhibitory Ly49a and c genes. In conclusion, my results support a role for epigenetic mechanisms in the maintenance of Ly49 expression. Moreover, these epigenetic mechanisms appear to vary among the Ly49 genes and also differ from those governing KIR expression. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Natural killer (NK) cells, lymphocytes of the innate immune system, can recognize a wide array of cells via several receptors families such as Ly49 and NKR-P1. The Nkrp1 gene family encode for C-type lectin-like receptors which can recognize their ligands, Clr, on target cells. Nkrp1 and Clr genes are intertwined in the NK gene complex and are thus inherited together. The Nkrp1-Clr genes in 129S6 and BALB/c mouse strains show significant sequence polymorphism compared to those of C57BL/6 mice while the overall gene organization and gene number are conserved. RT-PCR was utilized to study the expression of individual Nkrp1-Clr genes. In situ hybridization was performed to validate expression results from RT-PCR, as well as to verify the cell types in which Nkrp1-Clr genes are expressed. Surprisingly, our expression studies reveal an interesting pattern of expression of Nkrp1 and Clr genes not only in lymphoid tissues but also in the epithelial cells of the intestine, kidney, eye and lung, the myocytes of the heart and skeletal muscle, and possibly some endothelial cells, indicating novel functions of NK cells in these tissues.

The Tumor Necrosis Factor Receptor Superfamily (TNFR) is responsible in regulating a myriad of physiological function including the regulation of the immune system. Among the members include CD137 (4-1BB), an inducible costimulatory receptor known for its potent activation, proliferation, and survival effects on T cells. Stimulation of NK cells with agonistic α-CD137 antibodies are known to increase IFN-γ production and proliferation in NK cells as well as increase efficacy of anti-tumor responses. However, NK cell death has also been seen in certain circumstances, although the mechanism remains to be determined. In vitro stimulation of NK cells revealed that α-CD137 induced NK cell death occurs through both TNFR1 and TNFR2, although the action of TNF-α and TNF-ß remain uncertain. Death was independent of other cytotoxic mechanisms such as granzyme/perforin, Fas-Fas ligand, and TRAIL. During MCMV infection, α-CD137 induces NK cell death during the early phase of infection reducing viral resistance. This causes increased viral proliferation which drives NK cell proliferation, likely through Ly49H-m157 interactions, to high levels by day 4 of infection. The use of α-CD137 as a tumor therapeutic is promising with several applications undergoing clinical trials. However, my results raise concern of other effects including the depletion of NK cells. This may cause a temporary impairment in immune function against pathogenic infections and a compensatory reaction of NK cell proliferation, both of which may cause damage to the host. However, with proper co-stimulation or co-treatments, this impairment may be overcome and prevent adverse effects in patients.