E2F1, un régulateur crucial du métabolisme dans les cellules normales et cancéreuses.Résumé: Le facteur de transcription E2F1 est largement décrit pour son implication dans le contrôle du cycle cellulaire. Notre laboratoire et d'autres ont montré qu'il jouait également un rôle majeur dans le contrôle de l'homéostasie du glucose et des lipides. Dans les travaux présentés dans cette thèse, nous avons démontré ici en utilisant les souris invalidées pour E2F1 (souris E2f1-/-), qu'il joue un rôle dans le mécanisme de sécrétion d'insuline, dans la lipogenèse et la gluconéogenèse hépatique et dans le contrôle du métabolisme oxydatif. Dans la cellules β pancréatique, E2F1 contrôle la sécrétion d'insuline via le contrôle de l'expression de Kir6.2. Nous avons également montré l'implication de E2F1 dans la régulation de l'expression de gènes oxydatifs dans le TAB et le muscle. De plus, l'étude du foie de ces souris a permis de montrer le rôle de E2F1 dans le contrôle de la lipogenèse et la néoglucogenèse. E2F1 semble en effet capable de réguler l'expression d'un gène clé de la lipogenèse, Fas et de la G6Pase, un gène impliqué dans la production hépatique de glucose, en coopérant avec le facteur de transcription foxo-1. Enfin, nous avons observé que la diminution de la néoglucogenèse en absence de E2F1 empêche la formation de métastases pulmonaires. Ces différents résultats démontrent que E2F1 est un régulateur clé du métabolisme, et que la modulation de son activité pourrait avoir des conséquences majeures dans le développement de maladies comme le diabète, l'obésité, les myopathies et le cancer.Mots clés: E2F1, sécrétion d'insuline, métabolisme oxydatif, lipogenèse, gluconéogenèse, cancer. / E2F1, a crucial regulator of metabolism in normal and cancer cells. Abstract: E2F1 is a key transcription factor involved in the control of the cell cycle. We and others have previously demonstrated a a major role for E2F1 in the control of glucose and lipid homeostasis. In this thesis, we showed bu using E2F1 null mice, that E2F1 plays a major role in the control of insulin secretion, oxidative metabolism, lipogenesis and gluconeogenesis. E2F1 controls insulin secretion through the modulation of Kir6.2 expression. Moreover, we demonstrated that E2F1 controls the expression of oxidative genes in BAT and muscle. In addition, we observed that E2F1 is involved in the control of lipogenesis and gluconeogenesis in the liver. E2F1regulates the expression of key lipogenic genes, such as Fas, and G6Pase, a gene involved in hepatic glucose production, through cooperation with foxo-1. Finally, we observed that the inhibition of gluconeogenesis upon E2f1 genetic ablation impaired the formation of lung metastases. These different results show that E2F1 is a key regulator of metabolism, and that modulating its activity could have High outcomes on diseases such as diabetes, obesity, muscular distrophies or cancers.Key words: E2F1, insulin secretion, oxidative metabolism, lipogenesis, gluocneogenesis, cancer.

A galectina-1 (Gal-1) é uma lectina que reconhece ?-galactosídeos e participa de vários processos biológicos, incluindo a modulação da resposta inflamatória. Dados da literatura mostram a participação desta lectina na indução da exposição de fosfatidilserina (FS - um marcador de apoptose), na geração de espécies reativas do oxigênio (EROs) e na modulação quimiotática de neutrófilos. Entretanto, ainda são escassos os dados relacionados ao impacto biológico da Gal-1, exógena e endógena, sobre a biologia destas células. Neste trabalho foram avaliados, in vitro, alguns aspectos funcionais da interação Gal-1/neutrófilo. Determinou-se o nível de expressão da Gal-1 (Western Blotting) e de seu mRNA (PCR real time), em leucócitos humanos obtidos do sangue periférico de doadores sadios e em células da linhagem promielocítica humana (HL-60). Leucócitos do sangue periférico e células HL-60 não expressam níveis detectáveis da proteína e também do mRNA para Gal-1. Por meio de ensaios de quimiluminescência (QL) foi possível analisar a capacidade da Gal-1 recombinante humana de induzir e modular a produção de EROs em neutrófilos humanos não ativados e ativados com fMLP (n-Formil-Methionyl-Leucyl-Phenylalanine). A Gal-1 induz a produção de EROs de modo dose-dependente em neutrófilos ativados com fMLP. Entretanto, em neutrófilos não ativados esta lectina não induz o metabolismo oxidativo e, além disso, é capaz de modular negativamente a produção de EROs em resposta ao fMLP. Tanto nas células não ativadas quanto ativadas com fMLP, os efeitos da Gal-1 na produção de EROs estão parcialmente associados a sua propriedade lectínica. Na literatura ainda não há relatos sobre a interferência da Gal-1 no metabolismo oxidativo em neutrófilos ativados com repetidas doses de fMLP. Sabe-se que o tratamento sucessivo com fMLP reduz os níveis de produção de EROs por neutrófilos, no entanto, a presença de Gal-1 não interferiu neste processo. Interessantemente, neutrófilos recuperados do peritônio de camundongos Gal-1-/- liberam mais EROs em resposta ao fMLP e a Gal-1 exógena quando comparado aos neutrófilos de animais selvagens. Com base nos achados in vitro e sabendo que na sepse polimicrobiana o neutrófilo desempenha um papel importante, o próximo passo foi utilizar o modelo de M-CLP (sepse moderada) em camundongos destituídos (Gal-1-/-) ou não (Gal-1+/+) do gene da Gal-1. Animais Gal-1-/-, apresentam menor taxa de sobrevivência. O influxo de neutrófilos e a carga bacteriana no peritônio são maiores nos animais Gal-1-/- apesar da menor quantidade de bactérias detectadas no sangue, em relação aos animais selvagens. No pulmão, o influxo de neutrófilos é semelhante para ambos os grupos. No entanto, após a injeção intraperitoneal de 107 Unidades Formadoras de Colônias (UFC) de bactérias, camundongos Gal-1-/- apresentam maior atividade bactericida no lavado peritoneal e sangue, em relação aos selvagens. A participação da Gal-1 na homeostase de neutrófilos foi demonstrada in vitro, por citometria de fluxo (anexina-V-FITC), onde a indução de FS nos neutrófilos tratados com Gal-1 favoreceu a fagocitose destas células por macrófagos. Portanto, este conjunto de resultados sugere que a Gal-1, exógena ou endógena, pode modular funções imunológicas de neutrófilos e participar da regulação do processo inflamatório/infeccioso sistêmico. / Galectin-1 (Gal-1) is a lectin that recognizes ?-galactosides and participates in biological processes, including modulation of the inflammatory response. Literature data show the involvement of this lectin to induce exposure of phosphatidylserine (PS - a marker of apoptosis), in the generation of reactive oxygen species (ROS) and in modulation of neutrophil chemotaxis. However, there are few data related to the biological impact of exogenous and endogenous Gal-1 on the biology of these cells. This study evaluated, in vitro, some functional aspects of the interaction of Gal-1 and neutrophil. It was determined the expression level of Gal-1 (Western blotting) and its mRNA (real time PCR) on human leukocytes obtained from peripheral blood of healthy donors and human promyelocytic cell line (HL-60). Peripheral blood leukocytes and HL-60 cells do not express detectable levels of this protein as well as the Gal-1 mRNA. Through chemiluminescence testing (CL) it was possible to analyze the ability of recombinant human Gal-1 to induce and modulate the production of ROS in naïve and activated (n-Formyl-Methionyl-Leucyl-Phenylalanine-fMLP) human neutrophils. Gal-1 induces ROS production in a dose-dependent way in fMLP activated neutrophils. However, in naive neutrophils this lectin does not induce oxidative stress and can negatively modulate ROS production in response to fMLP. The effects of Gal-1 on ROS production in both non-activated cells and activated cells are partially associated with their lectin property. In the literature there are no data about the interference of Gal-1 on ROS production in activated neutrophils with repeated doses of fMLP. It is known that the subsequent treatment with fMLP reduced levels of ROS production by neutrophils; however, the presence of Gal-1 did not affect this process. Interestingly, peritoneum neutrophils from Gal-1-/- mice release more ROS in response to fMLP and exogenous Gal-1 when compared to neutrophils from wild type animals. Based on the in vitro findings and considering that in polymicrobial sepsis, neutrophils play an important role, the next step was to use the M-CLP model (moderate sepsis) in mice lacking (Gal-1-/-) or not (Gal-1+/+) Gal-1 gene. Gal-1-/- animals present lower survival rate and fewer bacteria in the blood despite having higher bacterial load in infectious focus in relation to wild type mice. The rates of neutrophils influx into the peritoneum and lungs are similar for both groups. The participation of Gal-1 in the homeostasis of neutrophils was demonstrated in vitro by flow cytometry (annexin V-FITC), where induced PS by Gal-1 in the neutrophils enhanced the phagocytosis of these cells by macrophages. Therefore, this set of results suggests that Gal-1, exogenous or endogenous, can modulate immune functions of neutrophils and participate in the regulation of inflammatory/infectious disorders.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第21845号 / 人博第874号 / 新制||人||210(附属図書館) / 2018||人博||874(吉田南総合図書館) / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 石原 昭彦, 教授 久代 恵介, 教授 神﨑 素樹 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

A tolerância ao Cádmio (Cd) é um assunto de relevada importância, devido aos vários problemas que este metal pode causar para a agricultura, levando à queda de produção e perda da qualidade dos alimentos, representando, também, riscos à saúde humana pelo consumo de produtos contaminados com esse metal. Neste trabalho, fizemos um estudo geral sobre a tolerância ao Cd em plantas de tomateiro, partindo de 3 abordagens complementares, onde geramos variabilidade genética por meio de mutagênese usando um tomateiro modelo (cultivar Micro-Tom); exploramos uma pequena fração da variabilidade genética da espécie para identificar genótipos com diferentes graus de tolerância ao Cd e, finalmente, realizamos estudos ligados ao metabolismo oxidativo de duas cultivares selecionadas, sendo uma sensível e outra mais tolerante a este metal. Este trabalho nos conduziu também ao desenvolvimento e adaptação de metodologias para a avaliação e seleção de plantas tolerantes a metais pesados, dentre as quais propomos a utilização de um Índice de Tolerância adequado para avaliar cultivares morfologicamente diferentes. Por fim, os resultados obtidos neste trabalho possibilitaram uma visão geral sobre os parâmetros de tolerância ao Cd em plantas de tomateiro, bem como o estudo dos principais padrões de resposta no metabolismo oxidativo de genótipos mais sensíveis e mais tolerantes a este metal. / Tolerance to heavy metal Cadmium (Cd) is an important subject because this metal can cause several problems in agriculture, such as decrease in production and loss of food quality, representing risks to human health by consumption of vegetables contaminated with this metal. In this research, we studied Cd-tolerance in tomato plants, using three complementary approaches, generating genetic variability by mutagenesis using a tomato model (cultivar Micro-Tom), exploring a small fraction of the genetic variability of species to identify genotypes with different degrees of Cd-tolerance and, finally, we conducted studies related to the oxidative metabolism of two cultivars, with low and high tolerance to this metal. Additionally, this work results in the development and adaptation of methodologies for the evaluation and selection of tolerant plants to heavy metals. Then, we propose an appropriate Tolerance Index to evaluate morphologically different cultivars. In conclusion, the results of this study are an overview of the parameters of Cd-tolerance in tomato plants, as well as the study of the some patterns of response in the oxidative metabolism of genotypes more sensitive and more tolerant to this metal.

A relação entre nutrição e fenótipo é uma questão especialmente desafiadora em casos de polifenismo facultativo, como no caso das castas de insetos sociais, por exemplo, a abelha melífera Apis mellifera. Após estudos de vias de sensoriamento de nutrientes, modificações inesperadas nestas vias de sinalização revelaram a resposta à hipóxia como um possível mecanismo subjacente à regulação do tamanho corporal e crescimento de órgãos. Uma vez que essa resposta está intimamente ligada a condições metabólicas das células, o presente estudo foi concebido para investigar possíveis alterações no metabolismo mitocondrial e oxidativo no processo de diferenciação de castas em A. melífera na fase larval, com foco no corpo gorduroso, que é o centro metabólico dos insetos. Partindo da hipótese de que rainhas e operárias são criadas em células de cria abertas sob condições iguais de disponibilidade de oxigênio, nós investigamos o número e a distribuição mitocondrial, bem como as taxas de consumo de oxigênio em mitocondrias de células de corpo gorduroso durante estágios larvais críticos. Por meio de análises de imunofluorescência e microscopia eletrônica encontramos maior densidade de mitocôndrias no corpo gorduroso larval da rainha, dado corroborado pela quantificação de unidades funcionais mitocondriais por ensaio de citrato sintase. Medições de consumo de oxigênio por respirometria de alta resolução revelaram que as larvas de rainha têm capacidades máximas mais altas de produção de ATP, com menor demanda fisiológica, mas com mesma eficiência mitocondrial que operárias. A análise da expressão de fatores relacionados à mitogênese mostrou que os homólogos dos genes codificadores dos fatores de transcrição TFB1 e TFB2 e de um regulador nutricional, ERR, estão mais expressos em larvas de rainha. Apesar das diferenças encontradas na respiração mitocondrial entre as duas castas, as mesmas apresentaram níveis similares de produção de lactato e peróxido de hidrogênio, sem grandes alterações referentes à condições de estresse oxidativo e ao estado redox das célula. Encontramos altos níveis de expressão de genes codificadores das enzimas do sistema antioxidante MnSOD e catalase em células do corpo gorduroso larval de rainha, que garantem a redução de eventuais danos oxidativos. Estes resultados são fortes evidências de que a nutrição diferencial das larvas pelas operárias adultas, como estímulo externo da indução do desenvolvimento das castas, diferencialmente afeta a dinâmica e funcionalidade mitocondrial como elemento intrínseca da plasticidade fenotípica neste inseto social. / The connection between nutrition and phenotype is a particularly challenging issue in cases of facultative polyphenism, as for instance in the honeybee Apis mellifera. After studies on nutrient sensing pathways found unexpected modifications in these signaling pathways, a response to hypoxia was revealed as a possible mechanism underlying regulation of body size and organ growth. Since this response is closely linked to the metabolic conditions of the cells, the present study was designed to investigate the role of the mitochondrial and oxidative metabolism in the fat body of honeybee larvae, which is the metabolic center in insects, in the context of the caste differentiation process in A. mellifera. Based on the fact that honey bee larvae are reared in open brood cells, the queen and worker larvae should be exposed to equal oxygen diffusion conditions, and hence we investigated mitochondrial number and intracellular distribution, as well as rates of mitochondrial oxygen consumption in fat body cells during the larval stages critical for caste differentiation. By means of immunofluorescence and electron microscopy we found a higher density of mitochondria in the fat body of queen larvae. This result was corroborated by the quantification of mitochondrial functional units using a citrate synthase assay. Measurements of oxygen consumption obtained by high resolution respirometry revealed that queen larvae have higher maximum capacities of ATP production, with less physiological demand and higher mitochondrial efficiency than workers. Analysis of the expression of genes related to mitogenesis showed that Apis homologs of the transcription factors TFB1 and TFB2 and of the nutritional regulator, ERR are higher expressed in queen larvae. Despite differences in mitochondrial respiration, the two castes presented similar levels of lactate and hydrogen peroxide production, and without major chang in oxidative stress and cellular redox status. The high transcriptional levels of genes encoding enzymes of the antioxidant system, MnSOD and catalase observed in fat body cells of queen larvae guarantee that oxidative damage is reduced during larval development. These results are strong evidence that the differential nutrition of honey bee larvae by the adult worker, as the external stimulus for caste induction, differentially affects mitochondrial dynamics and functionality as an intrinsic element of phenotypic plasticity in this social insect.

<p>In this thesis the oxidative metabolism by blood eosinophils from birch pollen allergic subjects was studied and compared to that by eosinophils from healthy controls, during and out of the pollen season. The effects and mechanisms of <i>in vitro</i> IL-5 priming on blood eosinophils were investigated and compared to the effects of <i>in vivo</i> priming during pollen exposure.</p><p>The main findings of this work were that the oxidative metabolism by blood eosinophils taken from pollen allergic subjects is reduced during the pollen season. The eosinophils taken from asymptomatic allergics have a reduced capacity to produce oxygen free radicals as compared to non-allergic controls. The oxidative metabolism by blood eosinophils from allergic subjects is primed <i>in vivo</i> during the pollen season, as compared to the healthy controls and as compared to out of season. IL-5 primed the oxidative metabolism by eosinophils from allergic subjects in a similar way as eosinophils from healthy controls, both during and out of pollen exposure. The total and tyrosine phosphorylation patterns obtained were identical in eosinophils from allergic subjects and non-allergic controls during the pollen season. Spontaneous phosphorylation was the same in both groups and different from that after IL-5 priming. The oxidative metabolism of blood eosinophils is composed of different stages. The initial stage, measured as the t_½rises of the CL curves, is an indication of the state of priming of the cell, while the end stage, measured as the peaks of the CL curves, is an estimate of the total radical production by the cells. IL-5 priming affected these two stages differently and the two stages are regulated by different signal transduction pathways and IL-5 priming causes a by-passing of MEK.</p><p>In conclusion, in this thesis it is shown that blood eosinophils from allergic subjects are primed <i>in vivo</i> during exposure to their allergen. This <i>in vivo</i> priming leads on one hand to a reduced oxidative metabolism during the pollen season, but also to a faster onset of radical production as a response to certain stimuli. Our data do not provide any evidence of IL-5 involvement in the <i>in vivo</i> priming of blood eosinophils from allergic patients during pollen exposure.</p>

In this thesis the oxidative metabolism by blood eosinophils from birch pollen allergic subjects was studied and compared to that by eosinophils from healthy controls, during and out of the pollen season. The effects and mechanisms of in vitro IL-5 priming on blood eosinophils were investigated and compared to the effects of in vivo priming during pollen exposure. The main findings of this work were that the oxidative metabolism by blood eosinophils taken from pollen allergic subjects is reduced during the pollen season. The eosinophils taken from asymptomatic allergics have a reduced capacity to produce oxygen free radicals as compared to non-allergic controls. The oxidative metabolism by blood eosinophils from allergic subjects is primed in vivo during the pollen season, as compared to the healthy controls and as compared to out of season. IL-5 primed the oxidative metabolism by eosinophils from allergic subjects in a similar way as eosinophils from healthy controls, both during and out of pollen exposure. The total and tyrosine phosphorylation patterns obtained were identical in eosinophils from allergic subjects and non-allergic controls during the pollen season. Spontaneous phosphorylation was the same in both groups and different from that after IL-5 priming. The oxidative metabolism of blood eosinophils is composed of different stages. The initial stage, measured as the t½rises of the CL curves, is an indication of the state of priming of the cell, while the end stage, measured as the peaks of the CL curves, is an estimate of the total radical production by the cells. IL-5 priming affected these two stages differently and the two stages are regulated by different signal transduction pathways and IL-5 priming causes a by-passing of MEK. In conclusion, in this thesis it is shown that blood eosinophils from allergic subjects are primed in vivo during exposure to their allergen. This in vivo priming leads on one hand to a reduced oxidative metabolism during the pollen season, but also to a faster onset of radical production as a response to certain stimuli. Our data do not provide any evidence of IL-5 involvement in the in vivo priming of blood eosinophils from allergic patients during pollen exposure.

Although androgens are known to exert anabolic effects in skeletal muscle, overexpression of androgen receptor (AR) selectively in this tissue causes androgen dependent motor deficits and muscular atrophy. The cellular and subcellular changes underlying this phenotype are unknown. Therefore, this study aimed to elucidate the ultrastructural and histologic changes accompanying myopathy and to determine the importance of androgens and overexpression level for myopathic features. Transmission electron microscopy revealed augmented mitochondrial content and reduced myofibril width in androgen exposed transgenics. Additionally, male transgenics demonstrated increased glycogen content. Histochemical analyses confirmed sex-specific changes in glycogen content and revealed a surprising loss in the proportion of oxidative fibers in symptomatic animals. However, increased mitochondrial content was confirmed by the presence of ragged red fibers. Overexpression of AR in muscle fiber results in mitochondrial pathology and dysregulation of glycogen metabolism, possibly reflecting normal but exaggerated function of androgens in skeletal muscle fibers.

Although androgens are known to exert anabolic effects in skeletal muscle, overexpression of androgen receptor (AR) selectively in this tissue causes androgen dependent motor deficits and muscular atrophy. The cellular and subcellular changes underlying this phenotype are unknown. Therefore, this study aimed to elucidate the ultrastructural and histologic changes accompanying myopathy and to determine the importance of androgens and overexpression level for myopathic features. Transmission electron microscopy revealed augmented mitochondrial content and reduced myofibril width in androgen exposed transgenics. Additionally, male transgenics demonstrated increased glycogen content. Histochemical analyses confirmed sex-specific changes in glycogen content and revealed a surprising loss in the proportion of oxidative fibers in symptomatic animals. However, increased mitochondrial content was confirmed by the presence of ragged red fibers. Overexpression of AR in muscle fiber results in mitochondrial pathology and dysregulation of glycogen metabolism, possibly reflecting normal but exaggerated function of androgens in skeletal muscle fibers.

Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-14Bitstream added on 2014-06-13T19:12:34Z : No. of bitstreams: 1 soeiro_cs_me_araca.pdf: 414870 bytes, checksum: 7e39d30ed89b5dd3fa716cdd85d43676 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O metabolismo oxidativo e apoptose dos neutrófilos de pacientes humanos nefropatas e sua relação com as toxinas urêmicas tem sido, nos últimos anos, amplamente investigados devido sua importância como elemento imunossupressor. Recentemente surgiram evidências de que a uremia causa disfunção neutrofílica em cães nefropatas, porém não se sabe se o acúmulo de compostos nitrogenados, que também ocorrem nas azotemias não renais, igualmente afeta a função dos neutrófilos. O objetivo do presente estudo foi testar ex vivo a hipótese de que plasmas urêmicos e azotêmicos igualmente afetam o metabolismo oxidativo e a apoptose dos neutrófilos de cães. Para tal, neutrófilos de cães sadios foram isolados e incubados com plasma autólogo, plasma de cão azotêmico e urêmico. A produção de superóxido, com e sem o estimulo com PMA, foi estimada pelo método de redução do nitroazul tetrazólio (NBT) e por citometria de fluxo capilar utilizando-se a sonda hidroetidina (HE). A taxa de neutrófilos viáveis, em apoptose inicial e final, foi quantificada por citometria utilizando-se Anexina V-PE e o índice apoptótico mensurado pelo método morfométrico. A produção de superóxido gerada pelos neutrófilos isolados, em ambos os tratamentos (plasma urêmico e azotêmico) apresentou significante redução (p<0,05). Já a apoptose dos neutrófilos de cães sadios foi acelerada, quando incubados com plasmas urêmico e azotêmico. Pode-se concluir que os componentes presentes nos plasmas urêmicos e azotêmicos alteram ex vivo o metabolismo oxidativo e a apoptose dos neutrófilos, fortalecendo a hipótese de que in vivo ambas condições podem comprometer a imunidade inata de cães / The oxidative metabolism and apoptosis of neutrophils from human patients with nephropathy and its relation with uremic toxins has been widely investigated in the last years because of its importance as an immunosuppressive element. Recently evidences suggests that uremia causes neutrophil dysfunction in dogs with renal disease, but it is unclear whether the accumulation of nitrogen compounds, which also occur in non-renal azotemia, can as well affect the role of neutrophils. The objective of this study was to test ex vivo the hypothesis that uremic and azotemic plasma also affects the oxidative metabolism and apoptosis of neutrophils in dogs. To this end, neutrophils from healthy dogs were isolated and incubated with autologous plasma, plasma of azotemic and uremic dog. The production of superoxide, with and without PMA stimulation was estimated by the method of nitroblue tetrazolium reduction (NBT) and by capillary flow cytometry using the hydroethidine probe (HE). The rate of viable, in early and late apoptosis neutrophils was quantified by flow cytometry using Annexin V-PE and theapoptotic index was measured by morphometric method. The production of superoxide generated by isolated neutrophils in both treatments (azotemic and uremic plasma) showed a significant reduction (p <0.05). Neutrophil apoptosis of healthy dogs was accelerated when incubated with uremic and azotemic plasma. In conclusion, the components present in uremic and azotemic plasma change ex vivo the oxidative metabolism and apoptosis of neutrophils, emphasizing the hypothesis that in vivo both conditions can compromise the innate immunity of dogs