As anormalidades hematológicas de origem paraneoplásica são identificadas em diversos tipos de neoplasias que acometem cães e gatos. Nas neoplasias mamárias em cadelas, já foram identificadas anormalidades relacionadas com a coagulação, onde verificou-se que a coagulação intravascular disseminada (CID) clínica e subclínica pode estar presente em 83% das cadelas com carcinoma mamário. Na medicina humana, é dada relevância à investigação de tais alterações uma vez que são fatores indicadores de prognóstico do câncer. Enquanto isso, na medicina veterinária, são escassos os estudos que relacionam as alterações hematológicas com o tipo tumoral, estadiamento e determinação de prognóstico. O objetivo do presente estudo foi realizar a avaliação hematológica, bioquímica e da hemostasia de cadelas acometidas por neoplasia mamária para identificar a alteração mais frequente, além de relacionar as anormalidades com o estadiamento tumoral. Para isso, foram utilizadas 25 cadelas atendidas pelo Grupo de Estudos em Oncologia em pequenos animais (ONCOVET) do Hospital de Clínicas Veterinárias da UFRGS (HCV-UFRGS) durante o período de 4 meses. Foi realizado coleta de sangue para hemograma, contagem de plaquetas, bioquímica sérica (albumina, ALT, cálcio, creatinina, FA, glicose, ureia) e teste de coagulação que constou de TP (tempo de protrombina), TTPa (tempo de tromboplastina parcial ativada), TT (tempo de trombina), fibrinogênio e mensuração do dímero-D. O estadiamento tumoral foi obtido através do exame físico e do resultado da biopsia das mamas. As anormalidades encontradas incluíram anemia, leucocitose neutrofílica, monocitose, eosinofilia, trombocitose, hipoalbuminemia, hipocalcemia, hipoglicemia e diminuição dos níveis de ureia sanguínea. Entretanto, essas alterações não foram relacionadas diretamente com a progressão tumoral, uma vez que não houve diferença entre os grupos avaliados. Apenas as variáveis RDW e ALT apresentaram relação significativa entre os grupos, contudo, sem relevância clínica. No teste de coagulação, houve diferença significativa entre os grupos apenas no TT e fibrinogênio, que foi relacionado com o estadiamento tumoral. / Hematological abnormalities of paraneoplastic origin are identified in several types of cancers that affect dogs and cats. In dogs with mammary neoplasms, abnormalities associated with coagulation have been identified, and verified that disseminated intravascular coagulation (DIC) clinical and subclinical may be present in 83% of dogs with mammary carcinoma. In human medicine, research in this field has been relevant since those factors are indicators of cancer prognosis. Meanwhile, in veterinary medicine, there are few studies that relate hematological changes with tumor type, staging and determination of prognosis. The aim of this study was to evaluate the hematological, biochemical and hemostathic abnormalities in bitches affected by mammary cancer to identify the most frequent alteration and associate with tumor staging. For this, 25 bitches attended by the Oncology Study Group in small animals (ONCOVET) of the Veterinary Hospital of UFRGS (HCV-UFRGS) for the period of 4 months were used. Blood collection for complete blood count, platelet count, serum biochemistry (albumin, ALT, calcium, creatinine, ALP, glucose, urea) and coagulation test that consisted of PT (prothrombin time), aPTT (activated partial thromboplastin time), TT (thrombin time), fibrinogen and D-dimer measurement were performed. Tumor staging was obtained by physical examination and the results of the biopsy of the breast. The abnormalities found included anemia, neutrophilic leukocytosis, monocytosis, eosinophilia, thrombocytosis, hypoalbuminemia, hypocalcemia, hypoglycemia and decreased levels of urea. However, these changes were not associated directly with tumor progression, since there was no difference among the groups. Only the RDW and ALT variables was associated significantly between the groups, however, with no clinical relevance. In the coagulation test, there was significant difference between the groups only in TT and fibrinogen, which was associated with tumor staging.

Resumo: Dietas com níveis energéticos elevados e suplementação com bST a novilhas pré-púberes diminuem a idade ao primeiro mas podem causar alterações de móleculas envolvidas no processo de desenvolvimento mamário. No presente trabalho objetivou-se examinar a expressão diferencial de genes relacionados ao desenvolvimento parenquimal de novilhas em crescimento acelerado e suplementadas com bST. Conduziu-se dois experimentos em fatorial 2x2, com 32 amostras de tecidos coletados de novilhas abatidas na fase pós-púbere inicial. Num experimento, aplicou-se a técnica de microarranjos com uma biblioteca de cDNA específica para bovinos (NBFGC) aonde avaliou-se simultaneamente a expressão de 18.263 diferentes ESTs. O segundo experimento analisou a expressão gênica de leptina e seu receptor no parênquima com a técnica de PCR em tempo real (qRT-PCR). Adicionalmente, foi realizado um experimento in vitro com linhagem de células mamárias MAC-T para verificação de possíveis interações entre a IL-6 e IGF-1 na proliferação celular. A administração de bST estimulou a expressão de diversos genes positivamente relacionados ao desenvolvimento parenquimal e inibiu a expressão de genes negativamente relacionados a esse processo, inclusive da leptina. A dieta causou poucas alterações nesses genes, mas aumentou a expressão de leptina no parênquima. A IL-6 diminuiu a proliferação celular induzida pelo IGF-1. Os resultados obtidos demonstraram alterações na expressão de genes relacionados ao desenvolvimento tecidual em ambos os tratamentos, sendo que o bST atenuou os efeitos da dieta em genes negativamente relacionados a esse processo. / Abstract: High-energy diet and bST administration during pre-pubertal phase decrease age at first calving. However, these managements modify mammary development altering the expression of several genes related to parenchyma development. The objective of the present study was to analyze the effects of a highenergy diet and bST administration in the expression of genes related to tissue development in heifer mammary parenchyma. Two experiments were conducted in a 2X2 factorial, with 32 samples collected from animals killed during early post-pubertal phase. In the first experiment, the microarray technique was performed with a bovine specific cDNA library containing 18.263 ESTs. The second experiment focused in the expression of leptin and leptin-receptor in mammary parenchyma, using the qRT-PCR technique. Additionally, an in vitro experiment was conducted using an immortalized mammary epithelial cell line (MAC-T) to check possible interactions between IL-6 and IGF-1 in cell proliferation. bST administration stimulated the expression of several genes positive related to tissue development and inhibited the expression of genes negative related to this process, including leptin. High-energy diet altered the expression of few genes, but increased the expression of leptin in mammary parenchyma. IL-6 decreased IGF-1 induced cell-proliferation. The results point to alteration in expression of genes related to tissue development in both treatments and suggest that bST administration attenuates the effects of high-energy diet in genes negative related to tissue development. / Orientador: Mauro Dal Secco de Oliveira / Coorientador: Michael J. Vandehaar / Banca: Luís Felipe Prada e Silva / Banca: Maria Lúcia Pereira Lima / Banca: Renato Luis Furlan / Banca: Paulo de Figueiredo Vieira / Doutor

Orientador: João Alberto Negrão / Banca: Izabelle Auxiliadora Molina de Almeida Teixeira / Banca: Marco Aurélio de Felício Porcionato / Resumo: A seleção de animais mais precoces e produtivos, e a melhoria da qualidade do leite são os principais objetivos dos caprinocultores. Com o objetivo de avaliar a interação de dois importantes hormônios, o Cortisol (CORT) e o Fator de Crescimento Análogo à Insulina -1 (IGF-1) na lactação, e suas possíveis influências no desenvolvimento da glândula mamária, produção e qualidade do leite, 38 cabritas (na 1ª fase do experimento) e 24 cabras (na 2ª e 3ª fase do experimento) Saanen divididas em quatro grupos (G1- tratadas com GH e desafiadas com ACTH; G2 - tratadas com GH e desafiadas com placebo; G3 - tratadas com placebo e desafiadas com ACTH; e G4 - tratadas com placebo e desafiadas com placebo). Ao longo de todo experimento foram realizadas coletas de sangue pontuais e durante os desafios, medidas morfométricas internas e externas de úbere e tetos foram realizadas e análises da composição do leite. Não houve diferença no desenvolvimento de úbere e tetos entre animais tratados e não tratados com GH, porém houve maior produção de leite pelos animais tratados, confirmando a eficiência do hormônio em promover maior persistência de lactação. Também houve uma resposta antagônica do IGF-1 ao CORT, durante o desafio das cabras na 3ª fase do experimento, resultado que pode indicar que animais adultos tratados com GH são menos suscetíveis ao estresse que os animais não tratados. O desafio com ACTH e o tratamento com GH não alteraram a composição do leite e a contagem de células somáticas. / Abstract: The selection of early animals and more productives, and improving the quality of milk are the main goals of goat breeders. With the objective of evaluating the interaction of two important hormones, cortisol (CORT) and the Insulin-Like Growth Factor -1 (IGF-1) in milk, and their possible influences on the development of the mammary gland, yield and quality of milk, 38 kid goats (the 1st. phase of the trial) and 24 goats (in the 2nd and 3rd phase of the trial) Saanen divided into four groups (G1- treated with GH (growth hormone) and challenged with ACTH (adrenocorticotropic hormone), G2 - treated with GH and challenged with placebo; G3 - treated with placebo and challenged with ACTH, and G4 - treated with placebo and challenged with placebo). Throughout experiment samples of blood were collected, internal and external morphometric measures of udder and teats reviews were performed and were performed composition of milk. There were not differences in the development of udder and teats of animals treated and not treated with GH, but treated animals showing higher production than non treated goats, confirming the effectiveness of treatment with GH in promoting increase on persistence of lactation. There was also a response of IGF- 1 antagonist to CORT, the challenge of goats during the 3rd phase of the experiment, result that may indicate that adults treated with GH are less susceptible to stress that the animals not treated. The challenge with ACTH and treatment with GH did not alter the composition of milk and somatic cell count. / Mestre

Resumo: Não disponível. / Abstract: Not available. / Orientador: Luís Fernando Barbisan / Coorientador: João Lauro Viana de Camargo / Banca: Antonio Carlos Alessi / Banca: Ione Pellegatti Lemonica / Banca: Danielle Palma de Oliveira / Banca: Helenice de Souza Spinosa / Doutor

Dentre os vários fatores que podem influenciar a polarização das respostas imunológicas nos animais destacam-se o tipo do agente agressor envolvido, as células apresentadoras de antígenos atuantes no processo e o microambiente formado pelas citocinas. Diferentes perfis de citocinas podem determinar o curso da doença, uma vez que distúrbios no equilíbrio da produção e liberação destes polipeptídeos têm papel significativo no desencadeamento e agravamento de diversas enfermidades. Vírus podem interferir com as funções das citocinas em vários níveis, influenciando na natureza da resposta imune do hospedeiro. Este desequilíbrio entre a produção de citocinas do tipo Th1 e Th2 é geralmente considerado como uma das causas da cronificação das infecções por retrovírus. O VLB, assim como outros retrovírus, também possui esta capacidade de manipular a resposta imune do hospedeiro a fim de direcionar os estágios alinfocitóticos (AL) e de linfocitose persistente (LP). No entanto, seu efeito diretamente sobre as citocinas no sangue e leite dos animais infectados ainda não foi completamente elucidado. Desta forma, o presente estudo objetivou a avaliação da resposta imune contra o VLB em vacas naturalmente infectadas por meio da quantificação de um perfil abrangente de citocinas presentes tanto no soro sanguíneo como no lácteo minimizados outros aspectos de estimulação que não fossem a presença do próprio vírus in vivo. Para tanto foram coletadas amostras de sangue e leite de 11 animais divididos em seus respectivos grupos experimentais (4, 3 e 4 animais para os grupos negativo, AL e LP, respectivamente) e determinadas as concentrações séricas e lácteas de IL-1β, IL-4, IL-10, TNF-α e IFN-γ. Para as comparações entre os grupos utilizou-se o teste não paramétrico de Kruskal-Wallis e quando estes apresentaramse significativos aplicou-se \"pós-teste\" para verificar onde tal diferença ocorreu. O nível de significância aplicado nos testes foi de 5 % (P≤0,05), sempre considerando hipóteses alternativas bicaudais. Verificou-se que não houve diferença nas concentrações das citocinas IL-1β, IL-4, IFN-γ e TNF-α no sangue dos animais pertencentes aos diferentes grupos experimentais. Quanto às concentrações das citocinas no soro lácteo dos bovinos testados nos diferentes grupos, os resultados demonstraram comportamentos semelhantes aos habitualmente descritos na literatura científica para o sangue periférico, sugerindo que a atuação do VLB sobre a glândula mamária promova interações análogas às observadas sistemicamente. Os resultados obtidos apontam para um envolvimento do VLB sobre o perfil de citocinas da glândula mamária nos animais sororreagentes. / Among the many factors that can influence the polarization of immune responses in animals include the type of the offending agent involved, the antigen-presenting cells active in the process and the microenvironment formed by the cytokine. Different profiles of cytokines may determine the course of the disease, since disturbances in the balance of production and release of these polypeptides play a significant role in triggering and worsening of many diseases. Viruses can interfere with the functions of cytokines at various levels, influencing the nature of the host immune response. This imbalance in cytokine production from Th1 and Th2 is generally regarded as a cause of chronic infections by retroviruses. The BLV, like other retroviruses, also possess this ability to manipulate the host immune response in order to drive the stages non-lymphocytotic and persistent lymphocytosis. However, its effect directly on cytokines in blood and milk of infected animals has not been fully elucidated. Thus, this study aimed at evaluating the immune response against the BLV in cows naturally infected by quantifying a comprehensive profile of cytokines present in both the serum and in whey minimized others aspects of stimulation that was not the presence of the virus itself in vivo. For this purpose, samples of blood and milk of 11 animals divided into their respective experimental groups (4, 3 and 4 animals for groups negative, PL- and PL+, respectively) and certain whey and serum concentrations of IL-1β, IL- 4, IL-10, TNF-α and IFN-γ. For comparisons between groups used the nonparametric Kruskal-Wallis test and when they showed up was applied to significant \"post test\" to see where this difference occurred. The level of significance in the tests was 5 % (P≤ 0.05), when considering two-tailed alternative hypotheses. There was no difference in the concentrations of IL-1β, IL-4, IFN-γ and TNF-α in the blood of animals belonging to the different experimental groups. The concentrations of cytokines in the whey of cattle in different groups, the results showed behaviors similar to those usually described in scientific literature to the peripheral blood, suggesting that the action of BLV on the mammary gland promotes interactions similar to those seen systemically. The results indicate an involvement of BLV on the cytokine profile of the mammary gland in animals seropositive.

La maîtrise de la conduite d’élevage est essentielle pour produire des chevrettes qui expriment pleinement leur potentiel laitier au cours de leur carrière. Elle implique la détermination de phases clés au cours de la période de croissance, notamment pour la mise en place de la glande mammaire. L’objectif de cette thèse était de déterminer les effets d’un sevrage précoce combiné à différentes conduites alimentaires post-sevrage sur les performances zootechniques de chevrettes de race alpine ainsi que sur le développement de leur glande mammaire. Tout d’abord, nous avons montré qu’un sevrage précoce à 10 kg de poids vif n’affectait ni le développement corporel, ni les performances de reproduction, ni même la production laitière (PL) des chevrettesPar contre, un apport plus élevé de concentré pendant la phase d’élevage a augmenté le développement corporel et le poids de la glande mammaire. Toutefois, cela n’a impacté ni le développement du parenchyme mammaire au cours de la phase pré-pubère ou en début de gestation, ni même la proportion de Cellule Epithéliale Mammaire (CEM). La prolifération des CEM était identique quelle que soit la quantité de concentré ingérée. La PL n’a pas été modifiée par l’augmentation de l’ingestion de concentré. La modification de la concentration en énergie et en protéines du concentré en phase pré-pubère n’a eu d’effet ni sur les performances de croissance ou de reproduction, ni sur le développement de la glande mammaire. La chèvre semble donc s’adapter aux modifications de la conduite alimentaire sans pour autant que l’augmentation de croissanc / Control of husbandry management is essential to produce female goat kids that fully express their milk potential during their career. It involves the determination of key periods during growth, particularly for the mammary gland development. The objective of this thesis was to determinate the effects of early weaning combined with different post-weaning feeding strategies on the performances of alpine goat kids as well as on their mammary gland development. On the one hand, we showed that early weaning at 10 kg of body weight did not affect body development, reproductive parameters or milk production (MP). On the other hand, a high concentrate intake during the rearing period increased body development and the weight of mammary glands.However, this did not impact the development of mammary parenchyma during the pre-pubertal phase or at the beginning of gestation, nor even the proportion of Mammary Epithelial Cells (MEC). The proliferation of MEC was identical regardless of the amount of concentrate ingested. MP was not affected by increasing concentrate intake. Modifying the energy and protein concentration of the concentrate in the pre-pubertal period had no effect on growth, reproductive performance, mammary gland development or mammary parenchyma development. Therefore, goats seem to adapt to changes in feeding strategies without a negative effect of the increase in growth on their ability to produce milk.

In this set of studies, a proteomic approach was used to investigate the protein profile of the mammary epithelial cell (MEC) through different stages of mammary development. The HC11 cell line was used to investigate protein changes between undifferentiated and differentiated MEC, which represent the pregnant and lactating states of the cells. This comparison revealed an interesting differential expression profile underscoring many recognized processes that occur in differentiated MECs, while others unveiled differences between MEC differentiation in vitro and in vivo. Primary MEC were also isolated from virgin, pregnant, and primiparous quiescent mice to compare the virgin state of the cell to the other two stages of development. These comparisons added to a previous dataset of primary isolated MEC and generated data that implied a surprising level of activity in virgin MEC relative to the other stages of development investigated. Differentially expressed proteins in the virgin and primiparous quiescent comparison also added to evidence of persisting changes occurring in the gland after a full term pregnancy that are implicated in the risk for breast cancer development. Data sets generated in the same manner from differentiating MEC were used in the development of a database to help manage the growing list of differentially expressed proteins and aid in the identification of potential interesting patterns of regulation during mammary development and differentiation.

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.

Most members of the PAS (PER-ARNT-SIM) protein family are transcription factors, mediating development and adaptive responses to the environment, such as circadian rhythms and toxin responses. Because the PAS domain mediates protein-protein interactions and functional cross-talk between distinct biological processes, we hypothesized that PAS genes in the circadian clockworks, namely Per1 and Per2, may be involved in development and toxin responses, which are modulated by other PAS members. To explore the possible role of clock genes in development, we examined mammary epithelial cells in vitro and the mouse mammary gland in vivo for evidences of changes in clock gene expression during different stages of development and differentiation. Our results showed that Per1 and Bmal1 expression were up-regulated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. A similar differentiation-dependent profile of clock gene expression was observed in mouse mammary glands; Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. These data suggest that circadian clock genes may play a role in mouse mammary gland development. To examine clock gene function in toxin responses, we evaluated whether disruption or inhibition of Per1 and/or Per2 alters toxin-induced activity of the AhR signaling pathway in the mouse mammary gland and liver. We assessed the activation of the AhR signaling pathway in response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypical AhR agonist, by analyzing the mRNA abundance of its two target genes, cytochrome P450, subfamily I, polypeptide 1 (Cyp1A1) and Cyp1B1. Our results showed that the targeted disruption of Per1, but not Per2, significantly increases the TCDD-induced p450 expression in the mammary gland and liver in vivo. Similar changes in TCDD-mediated p450 expression were observed in vitro using mammary primary cultures of mammary cells derived from from Per1ldc, Per2ldc and Per1ldc/Per2ldc mutant mice and Hepa1c1c7 cells subjected to siRNA-mediated inhibition of Per1 or Per2. These discoveries suggest that the clock gene Per1 may modulate toxin responses perhaps by functioning as a negative regulator for TCDD-mediated activation of the AhR signaling pathway.

The aim of this thesis was to characterize the expression of the peptides ghrelin and obestatin, as well as other neuroendocrine markers in human normal tissues, in invasive breast cancer and a wide panel of neuroendocrine tumors (NETs). In normal tissues the expression of ghrelin and obestatin was mainly localized to the gastric mucosa, and in lesser extent in the remaining gastrointestinal tract, endocrine pancreas and mammary glands. Double immunofluorescence studies demonstrated that ghrelin and obestatin were co-localized in the same cells displaying the same cytoplasmic distribution. In normal breast tissue, ghrelin, obestatin, adrenomedullin, apelin and vesicular monoamine transporter 2 were specifically demonstrated in the luminal epithelial cells. Consecutive sections indicated that mammary epithelial cells could express several of these peptides. Secretogranin II and III were also detected in breast tissue, but their presence was restricted to the outer layer of myoepithelial cells, whereas chromogranin B immunoreactivity was found in both the epithelial and myoepithelial cells. Ghrelin and obestatin immunoreactivity was seen in invasive breast cancer, where the expression could be correlated to factors associated with prognosis. Furthermore, multivariate analysis indicated that ghrelin expression was a possible independent prognostic factor for prolonged recurrence-free and breast cancer-specific survival. In a panel of NETs and endocrine-related disorders it was revealed that ghrelin and obestatin immunoreactivity was primarily found in tumors originating from the respective normal tissues. The two proteins were detected in only a few cases and only occasional tumor cells were immunoreactive. In conclusion, ghrelin and obestatin are localized in the gastrointestinal tract, endocrine pancreas and mammary glands. This thesis has contributed to our understanding of the distribution of ghrelin and obestatin in both normal tissue and tumor cells. A potential role of ghrelin as a prognostic factor in invasive breast cancer has been identified and should be further explored.