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Submitted by WALTER HEINZ FERINGER JUNIOR null (walterferinger@gmail.com) on 2018-03-22T22:47:37Z No. of bitstreams: 1 TESE_WALTER_HEINZ_FERINGER_JUNIOR.pdf: 2433033 bytes, checksum: 618fd780a0ad05e04e544c2769f96c3d (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-03-23T10:43:56Z (GMT) No. of bitstreams: 1 feringerjunior_wh_dr_jabo.pdf: 2433033 bytes, checksum: 618fd780a0ad05e04e544c2769f96c3d (MD5) / Made available in DSpace on 2018-03-23T10:43:56Z (GMT). No. of bitstreams: 1 feringerjunior_wh_dr_jabo.pdf: 2433033 bytes, checksum: 618fd780a0ad05e04e544c2769f96c3d (MD5) Previous issue date: 2017-11-13 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O principal mecanismo de transporte dos íons lactato e H+ em equinos e cães é o complexo transportador formado pelos transportadores de monocarboxilatos, isoformas 1 (MCT1) e 4 (MCT4) juntamente com a proteína auxiliar CD147. Objetivando identificar diferenças entre equinos com desempenho distinto, 16 equinos da raça Brasileiro de Hipismo (BH) foram distribuídos em dois grupos, desempenho inferior (DI, n=8) e desempenho superior (DS, n=8) que foram submetidos a teste de salto incrementai (TSI). Realizou-se biópsia do músculo Gluteus medius para tipificação e análise das expressões das isoformas MCT1, MCT4 e CD147. Amostras sanguíneas foram colhidas para avaliar as expressões MCT1 e CD147 das hemácias. Aplicaram-se testes de normalidade de Shapiro Wilk e homogeneidade de Levene. As medidas morfométricas foram submetidas ao teste de Tukey. Teste “t” de Student não pareado para a comparação das médias dos grupos DI e DS. Aplicou-se correlação de Spearman para as expressões dos transportadores. Para todas as análises utilizou-se p≤0,05. Não houve diferença entre os grupos quanto à frequência de cada tipo de fibra e constatou-se maior quantidade das fibras tipo I em relação às fibras IIA e IIX em todos os equinos avaliados. Não houve diferença entre os pesos moleculares e a expressão das proteínas MCT1, MCT4, e CD147 musculares ou sanguíneas. Houve correlações positivas entre MCT1 vs. CD147 e MCT4 vs. CD147 musculares dos grupos DI e DS. As correlações encontradas foram esperadas uma vez que as isoformas estudadas dependem intimamente da proteína auxiliar CD147 para o transporte. Os equinos BH não apresentaram diferenças nas expressões dos MCT1,4 e CD147, musculares ou sanguíneos, mesmo com níveis de condicionamento diferentes. Com o objetivo de investigar as concentrações de lactato plasmático e das hemácias e avaliar as expressões eritrocitáras do complexo transportador MT1/CD147, 6 cães da raça American Pitbull Terrier (APBT) foram submetidos ao teste de esforço incremental (TEI) em esteira. No final de cada incremento de velocidade foi coletado sangue da veia cefálica. Foram mensuradas concentrações de lactato sanguíneo (LS), plasmático (LP), pH e hematócrito (Ht). A concentração do lactato dentro das hemácias (LH) foi estimada e estabeleceu-se a relação LH:LP. As expressões sanguíneas do complexo MCT1/CD147 foram avaliadas por Western Bloting. Aplicou-se análise de variância de uma via seguido pelo teste de Dunn’s. Para pH e Ht aplicou-se teste t de student para amostras pareadas e a correlação de Pearson foi utilizada para MCT1 e CD147, estabeleceu-se nível de significância P≤0,05. LS, LP e LH e pH não apresentaram diferenças entre si, a relação LH:LP foi próxima de 1 com tendência de aumento. MCT1 e CD147 apresentaram 48 e 59 kDa de peso molecular e 1,27 e 1,05 de unidades ópticas arbitrárias (UOA). Não foram encontradas correlações entre MCT1 e CD147. A grande velocidade de transporte do MCT1/CD147 explica a relação LP:LH próxima de 1, esta velocidade e o mecanismo de arquejo podem explicar os valores de pH constantes. A raça APBT, quando submetidos à atividade física apresentaram tendência de aumento da relação LH:LP e expressam de maneira homogênea o complexo MCT1/CD147. / The central transport mechanism of lactate and H+ ions in horses and dogs is the carrier complex formed by the monocarboxylate, isoform 1 (MCT1) and 4 (MCT4) associated with the ancillary protein CD147. This study aimed to identify possible differences between horses with different performances levels, 16 horses of the Brazilian Sport Horse breed (BH) were distributed in two groups, inferior performance (IP, n = 8) and superior performance (SP, n = 8). A Gluteus medius muscle biopsy was performed for cellular typing and analysis of MCT1, MCT4, and CD147 muscle expressions. By jugular venipuncture, blood samples were collected to evaluate MCT1 and CD147 expressions in the red blood cells (RBC). Normality Shapiro Wilk test and homogeneity of Levene were applied. The morphometric measurements were submitted to the Tukey test, and not paired Student's t-test were applied to compare the mean of the IP and SP groups for all variables and was used Spearman's correlation for isoform expressions, for all analyzes, p≤0.05. There were no differences between the groups regarding the frequency of each type of fiber and a higher number of type I fibers were observed about the IIA and IIX fibers in all groups. There was no difference between molecular weights and expressions of MCT1, MCT4, and CD147 in muscle or blood. There were positive correlations between muscles MCT1 vs CD147 and MCT4 vs CD147 in both groups. The relationships found were expected since the MCT1 and 4 depended on the CD147 ancillary protein for correct functioning. The BH horses do not present differences in the muscle or RBC expressions of MCT1, 4 and CD147, even with different conditioning levels. To investigate plasma and erythrocyte lactate concentrations and to evaluate erythrocyte expression of the MT1/CD147 transporter complex, six dogs of the American Pit Bull Terrier breed (APBT) were submitted to a treadmill incremental effort test (IET). At the end of each increment of speed, blood was collected from the cephalic vein. Concentrations of blood (BL) and plasma lactate (PL), pH and hematocrit (Ht) were measured. The concentration of lactate inside the red blood cells (LC) was estimated and the LC: PL ratio was established, the blood expressions of the MCT1/CD147 transporter complex were evaluated by western blot. Data were submitted to the Shapiro-Wilks normality test, one-way ANOVA and Dunn's test. For pH and Ht, paired Student's t-test was applied, and Pearson's correlation was used for MCT1 and CD147 analysis, for all analyzes, p≤0.05. BL, PL, LC, pH showed no differences, the LC: PL ratio was close to 1 with an increasing tendency. MCT1 and CD147 presented 48 and 59 kDa of molecular weight and 1.27 and 1.05 of arbitrary optical units (AOU). No correlations were found between MCT1 and CD147. The high transport velocity of the MCT1/CD147 could explain the LC: PL ratio close to 1, this velocity plus the grasping mechanism may explain the constant of pH values. The APBT submitted to intense physical activity showed a tendency to increase the LC: PL ratio, and homogeneously express the MCT1/CD147 complex / FAPESP: 11/11080-0

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Orientador: Guilherme de Camargo Ferraz / Resumo: O principal mecanismo de transporte dos íons lactato e H+ em equinos e cães é o complexo transportador formado pelos transportadores de monocarboxilatos, isoformas 1 (MCT1) e 4 (MCT4) juntamente com a proteína auxiliar CD147. Objetivando identificar diferenças entre equinos com desempenho distinto, 16 equinos da raça Brasileiro de Hipismo (BH) foram distribuídos em dois grupos, desempenho inferior (DI, n=8) e desempenho superior (DS, n=8) que foram submetidos a teste de salto incrementai (TSI). Realizou-se biópsia do músculo Gluteus medius para tipificação e análise das expressões das isoformas MCT1, MCT4 e CD147. Amostras sanguíneas foram colhidas para avaliar as expressões MCT1 e CD147 das hemácias. Aplicaram-se testes de normalidade de Shapiro Wilk e homogeneidade de Levene. As medidas morfométricas foram submetidas ao teste de Tukey. Teste “t” de Student não pareado para a comparação das médias dos grupos DI e DS. Aplicou-se correlação de Spearman para as expressões dos transportadores. Para todas as análises utilizou-se p≤0,05. Não houve diferença entre os grupos quanto à frequência de cada tipo de fibra e constatou-se maior quantidade das fibras tipo I em relação às fibras IIA e IIX em todos os equinos avaliados. Não houve diferença entre os pesos moleculares e a expressão das proteínas MCT1, MCT4, e CD147 musculares ou sanguíneas. Houve correlações positivas entre MCT1 vs. CD147 e MCT4 vs. CD147 musculares dos grupos DI e DS. As correlações encontradas foram esperadas ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The central transport mechanism of lactate and H+ ions in horses and dogs is the carrier complex formed by the monocarboxylate, isoform 1 (MCT1) and 4 (MCT4) associated with the ancillary protein CD147. This study aimed to identify possible differences between horses with different performances levels, 16 horses of the Brazilian Sport Horse breed (BH) were distributed in two groups, inferior performance (IP, n = 8) and superior performance (SP, n = 8). A Gluteus medius muscle biopsy was performed for cellular typing and analysis of MCT1, MCT4, and CD147 muscle expressions. By jugular venipuncture, blood samples were collected to evaluate MCT1 and CD147 expressions in the red blood cells (RBC). Normality Shapiro Wilk test and homogeneity of Levene were applied. The morphometric measurements were submitted to the Tukey test, and not paired Student's t-test were applied to compare the mean of the IP and SP groups for all variables and was used Spearman's correlation for isoform expressions, for all analyzes, p≤0.05. There were no differences between the groups regarding the frequency of each type of fiber and a higher number of type I fibers were observed about the IIA and IIX fibers in all groups. There was no difference between molecular weights and expressions of MCT1, MCT4, and CD147 in muscle or blood. There were positive correlations between muscles MCT1 vs CD147 and MCT4 vs CD147 in both groups. The relationships found were expected since the MCT1 and 4 depended on the CD... (Complete abstract click electronic access below) / Doutor

It has been shown that tumour cells are capable of switching to glycolytic metabolism for the production of ATP even in the presence of oxygen, this is known as aerobic glycolysis or the 'Warburg effect'. The glycolytic phenotype has been associated with tumour aggressiveness and poor outcome in several cancer types. This makes the area of cancer metabolism an attractive area for the potential identification of new therapeutic targets. One key component, required for cells to maintain the glycolytic phenotype, is the presence of monocarboxylate transporters that are capable of exporting lactate. These transporters are vital for the maintenance of the intracellular pH of cells under these conditions. This study was centred around the hypothesis that altering expression of MCTs would impact on the metabolism of tumour cells and, therefore, other key characteristics of cells relating to metastatic capabilities and survival following treatment. For the purpose of this work, prostate cancer cell lines were transfected with lentiviral particles targeting overexpression of MCT1 or MCT4, or knockdown of MCT4. Following transfection, cellular metabolic profiles were assessed under normoxic and hypoxic conditions and the metastatic phenotype of each cell line was investigated. Additionally, the effect of MCT expression on response to chemotherapy and radiation therapy was explored, and a siRNA metabolome screen was performed to identify combinations of targets that may produce synthetic lethality in prostate cancer cell lines. It was shown that changes in the expression of MCT1 or MCT4 did not cause significant changes in the metastatic phenotypes of the prostate cancer cell lines investigated. Some differences were observed in the metabolic pathways used by these prostate cancer cells following alterations in MCT expression. For example, overexpression of MCT1 in DU145 cells resulted in an increase in intracellular lactate. Additionally, MCT4 knockdown in PC3 cells was able to reduce OXPHOS under reduced oxygen. MCT1 overexpression was able to sensitise androgen-independent prostate cancer cells to treatment with chemotherapy and radiation therapy. Furthermore, combinations of siRNA treatments were identified that may be capable of producing synthetic lethality. In summary, findings in this study indicated that targeting MCT1 and MCT4 expression could offer therapeutic benefit in prostate cancer. However, it was also highlighted that the roles of these transporters are specific to cancer type, and even cell line.

Upon activation, T cells shift towards a metabolic program characterized by increased glucose metabolism in order to sustain proliferation and effector function. Surprisingly, while resting T lymphocytes degrade glucose aerobically to CO2, proliferating T cells metabolize glucose almostentirely to lactate in the presence of oxygen through aerobic glycolysis (the Warburg effect). This metabolic switch comprises the upregulation of glycolytic enzymes and glucose transporters to the cell membrane, leading to an increase of glycolytic flux and the concomitant production of lactate. Despite many decades of research, we still do not fully understand the mechanisms that make proliferating T cells choose glycolysis rather than oxidation of glucose to produce energy. Since activated T lymphocytes depend on a glycolytic metabolism, they must release lactate, which inthese cells is facilitated by the proton-linked monocarboxylate transporter MCT1. The transporter is part of a protein family of 14 members among which MCT1–4 facilitate the passive transmembrane transport of monocarboxylates such as lactate, pyruvate and ketone bodies. The observation that pharmacological MCT1 inhibition has shown anti-proliferative effect on T cells suggests that lactate transport is essential to T cell expansion triggered after antigen recognition. The aim of our research is to investigate the importance of MCT1-dependent regulation in T cellmetabolism. Following TCR stimulation, MCT1 was expressed early in T cells unlike MCT4 whose significant expression was detected at later time point. To investigate the role played by MCT1 in the early steps of T cell activation, we generated a transgenic mouse model where conditional deletion of the MCT1 gene was achieved specifically in T cells. Phenotype and T cell distribution in thymus and peripheral organs were normal in MCT1fl/fl CD4Cre mice. However, lack of MCT1 expression decreased the proliferative capacity of in vitro activated CD4+ or CD8+ T cells without altering their viability. We observed that the IL-2 production was also affected by the lack of MCT1 expression, in line with decreased proliferative ability. Moreover, in vivo, T cell expansion that followed antigenic stimulation as well as T cell-mediated immune response to infection were deficient in MCT1fl/flCD4Cre mice. Our data indicate that this situation resulted from a cellular energy shortage caused by reduced glycolytic activity soon after activation. Moreover, energy crisis was amplified by the necessity to use ATP-consuming mechanisms for excluding H+ protons from the cytosol of activated MCT1-deficient T cells. Thus, in T cells, early MCT1 expression after activation ensures an energy saving mechanism for regulating cytoplasm acidification. Our observations also indicate that a high glycolytic flux is required in dividing T cells to maintain pH homeostasis. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Einleitung: Monocarboxylattransporter (MCT) 1 & 4 sind in zahlreichen Geweben als Kotransporter für Monocarboxylate und Protonen beschrieben. Auch im Pansenepithel werden MCT benötigt, um kurzkettige Fettsäuren (SCFA) aus dem Pansenlumen in die Pansenepithelzelle aufzunehmen (MCT4) und um SCFA und deren Metabolite aus der Pansenepithelzelle in das Blut auszuschleusen (MCT1). Die transepitheliale Permeation von SCFA über die Pansenwand ist von enormer Bedeutung, da sie die wichtigste Energiequelle der Wiederkäuer darstellen. Die beteiligten Transportprozesse müssen dementsprechend einer Anpassung an variierende Mengen von SCFA unterliegen. Bisherige Studien bei anderen Spezies deuten auf eine Regulation des MCT1 auf mRNA Ebene über den Peroxisom-Proliferator-aktivierten Rezeptor α (PPARα) hin. Ziele der Untersuchung: Das Ziel dieser Arbeit war herauszufinden, ob MCT1 in ovinen Pansenepithelzellen über PPARα reguliert wird und ob auch MCT4 dieser Regulation unterliegt. Eine gleichzeitige Regulation beider Transporter läge nahe, da sie gemeinsam an der transepithelialen Permeation beteiligt sind. Die Auswirkungen solch einer Regulation auf die Proteinexpression und die Transportleistung der MCT sollte charakterisiert werden. Ebenfalls war das Potenzial der bei erhöhter Kraftfutterfütterung vermehrt anfallenden SCFA Butyrat auf die MCT1 Expression zu untersuchen. Material & Methoden: Aus dem Vorhof von Schafen wurden Pansenepithelzellen gewonnen und entsprechend einer bereits etablierten Methode kultiviert. Nach einer Subkultivierung wurden die Zellen immunzytochemisch mit Antikörpern gegen MCT1, MCT4 und Na+/K+-ATPase untersucht, um deren Lokalisation in den kultivierten Pansenepithelzellen zu bestimmen. Weiterhin erfolgte eine Behandlung mit WY 14.643, einem spezifischen, synthetischen PPARα Agonisten, sowie mit GW 6471, einem Antagonisten des PPARα. Mittels qPCR wurden die relativen mRNA Mengen von MCT1, MCT4, ACO, CPT1A und CACT bestimmt und auf die Referenzgene GAPDH und Na+/K+-ATPase normalisiert. Die Proteinexpression von MCT1 und MCT4 wurde mittels Western Blot bestimmt. Zur funktionellen Quantifizierung wurde der intrazelluläre pH-Wert der Zellen mittels Spektrofluorometrie gemessen und der laktatabhängige Protonentransport als Vergleichswert zwischen den Behandlungen genutzt. Um den MCT-abhängigen Teil des Transportes zu bestimmen, wurde ein spezifischer MCT1 & 4 Inhibitor, die p-Hydroxymercuribenzensulfonsäure (pHMB) eingesetzt. Die Zellen wurden mit Butyrat über einen Zeitraum von 6 und 48 h induziert. Die Erfassung der MCT1 Expression erfolgte mittels semiquantitativer PCR. Ergebnisse: MCT1 & 4 sind sowohl in der Zellmembran als auch intrazellulär in den Pansenepithelzellen lokalisiert. Die mRNA Expressionsdaten konnten zeigen, dass MCT1 und die PPARα Zielgene durch WY 14.643 hochreguliert werden konnten, wohingegen die MCT4 Expression keine eindeutige Antwort auf die Stimulation zeigt. Die Behandlung mit den Antagonisten zeigt eine Abhängigkeit der MCT1 Expression von PPARα, die MCT4 Expression konnte dagegen nicht beeinflusst werden. Mittels pHMB gelang es, den laktatabhängigen Protonenexport fast vollständig zu blocken. Sowohl laktatabhängiger Protonenexport als auch die Proteinexpression zeigten keine Änderung durch WY 14.643 Stimulation. Die Butyratexposition veränderte die Morphologie der Pansenepithelzellen und schien nicht geeignet für Untersuchungen der mRNA Expression zu sein. Schlussfolgerungen: Es konnte in dieser Arbeit erstmals gezeigt werden, dass MCT1 in Pansenepithelzellen über PPARα reguliert wird, nicht aber MCT4. PPARα scheint demnach einer der entscheidenden Angriffspunkte für die Regulation des SCFA Transportes zu sein, dessen natürliche Liganden im Pansen aber noch nicht bekannt sind. Damit legt diese Arbeit den Grundstein für regulative Studien am intakten Pansenepithel. / Introduction: Monocarboxylate transporters (MCT) 1 & 4 are cotransporters of monocarboxylates and protons in a variety of mammalian cell types. In the ruminal epithelium MCT are necessary to transport short-chain fatty acids (SCFA) from the lumen into the ruminal epithelial cell (MCT4) and to discharge SCFA and their metabolites from the cell into the blood (MCT1). Transepithelial permeation of SCFA is of great importance, because they are the main source of energy for ruminants. The regulation of appropriate transport proteins should thus be subject to the adaptation to varying SCFA amounts. Previous studies in other species suggested that gene expression of MCT1 is regulated by peroxisome proliferator-activated receptor α (PPARα), a ligand-activated nuclear receptor. Aims: The aim of the study was to examine if MCT1 in ruminal epithelial cells is regulated by PPARα and furthermore if MCT4 can be regulated by PPARα, as well. A simultaneous regulation seems likely, because both are acting jointly in the transepithelial transporting of SCFA. The implications of such a regulation on protein expression and transport capacity of MCT should be characterized. The effect of butyrate, a SCFA which increases under concentrate feeding, on MCT1 expression was determined. Materials & Methods: Ruminal epithelial cells of sheep were cultivated according to methods previously established. After subcultivation, immunocytochemistry with antibodies against MCT1, MCT4 and Na+/K+-ATPase was performed to determine their localization in ruminal epithelial cells. For studying the influence of PPARα, WY 14.643, a synthetic and selective ligand of PPARα, and GW 6471, a synthetic antagonist of PPARα, were applied to the culture medium of the cells. After processing the specimens, the relative amount of mRNA of MCT1, MCT4 and the target genes ACO, CPT1A and CACT were analyzed by qPCR and normalized on the reference genes GAPDH and Na+/K+-ATPase. Protein abundance of MCT1 & 4 was measured by using the Western Blot method. Functional quantification was measured by the intracellular pH (pHi) of cells using spectrofluorometry as well as comparing the effect of WY 14.643 treatment on lactate-dependent proton export. To determine the MCT-dependent part of the pHi recovery, p-hydroxymercuribenzoic acid (pHMB), a specific inhibitor of MCT1 & 4, was applied. Cells were also treated with butyrate for 6 h and 48 h and the mRNA abundance of MCT1 was analyzed by semiquantitative PCR. Results: Both MCT1 and MCT4 were localized in the cell membrane as well as in the cytoplasm of ruminal epithelial cells. By qPCR it could be demonstrated that the mRNA abundance of MCT1 and PPARα target genes in the ruminal epithelial cells was increased by WY 14.643 in comparison to untreated cells, whereas the response of MCT4 did not yield distinct results. Treatment with the PPARα antagonist pointed out, that MCT1 is influenced by PPARα, but not MCT4. Lactate-dependent proton export was blocked almost completely by pHMB. Both lactate-dependent proton export and protein expression were not altered by WY 14.643 treatment. Butyrate exposure changed the morphology of ruminal epithelial cells and seemed unsuitable for the analysis of mRNA expression. Conclusion: For the first time, it could be demonstrated, that MCT1 in ruminal epithelial cells is regulated by PPARα, but not MCT4. PPARα seems to be a vital target in the rumen for SCFA transport regulation, whose natural triggers have yet to be identified. Furthermore, this study provides the basis for regulative studies on intact ruminal epithelium.

Les carcinomes rénaux (RCC) sont subdivisés en plusieurs sous-types, définis selon des critères histologiques, phénotypiques et génétiques. Le diagnostic différentiel de ces tumeurs est primordial avec des conséquences pronostiques et thérapeutiques. Génétique et diagnostic : Nous avons étudié les données cliniques, histologiques, immunohistochimiques et génétiques d'une série de RCC papillaires (PRCC) de type 1 et 2. Une caractérisation génomique exhaustive complétée par NGS nous a permis de classer les PRCC de type 2 dans plusieurs groupes d'évolution variable. Nos résultats fournissent des informations inédites sur la pathogenèse des PRCC qui donnent des pistes pour un traitement personnalisé. Métabolisme, grade tumoral et phénotype : Dans une série de RCC à cellules claires (ccRCC), nous avons analysé les caractéristiques de ces tumeurs et l'expression des protéines impliquées dans le métabolisme et les isoformes de HIF. Cette étude nous a permis de mettre en évidence quantitativement une corrélation entre l'expression de MCT1, GLUT1 et CAXII et le grade de Fuhrman, et qualitativement une localisation périphérique de HIF2alpha et la co-localisation des protéines HIF2alpha et HAF. Stratégies théranostiques : Dans l’optique de définir les traitements les plus appropriés pour les patients atteints de RCC, nous avons fait un parallèle entre la sensibilité aux thérapies ciblées des patients (in vivo) et de cellules dérivées de la tumeur initiale (in vitro). Nous avons démontré que la réponse chez les patients et dans les cellules était équivalente et donc que des tests in vitro sont une piste pour définir des traitements personnalisés des patients atteints de ccRCC. / Renal carcinomas (RCC) are divided into several subtypes, defined by histological, genetic and phenotypic criteria. The differential diagnosis of these tumors is important with prognostic and therapeutic implications. Genetics and diagnosis: We studied the clinical, histological, immunohistochemical and genetic of papillary RCC (PRCC) type 1 and 2 cohort. An extensive genomic characterization completed by NGS has allowed us to classify type 2 PRCC in several groups of variable clinical evolution. Our results provide new information on the pathogenesis of PRCC that provide perspectives for personalized treatment. Metabolism, tumor grade and phenotype: In a series of clear cell RCC (ccRCC), we analyzed the characteristics of these tumors and the expression of proteins involved in the metabolism and isoforms of HIF. This study allowed us to demonstrate quantitative correlation between the expression of MCT1, GLUT1 and CA XII and Fuhrman grade, and qualitatively peripheral HIF2alpha localization and co-localization of proteins HIF2alpha and HAF. Theranostic strategies: In order to define the most appropriate treatment for patients with RCC, we made a parallel between sensitivity to targeted therapies of patients (in vivo), and cells derived from the original tumor (in vitro). We have demonstrated that the response in patients and in cells and was similar, thus in vitro assays are a way to define personalized treatment for ccRCC.

Einleitung: Monocarboxylattransporter (MCT) 1 & 4 sind in zahlreichen Geweben als Kotransporter für Monocarboxylate und Protonen beschrieben. Auch im Pansenepithel werden MCT benötigt, um kurzkettige Fettsäuren (SCFA) aus dem Pansenlumen in die Pansenepithelzelle aufzunehmen (MCT4) und um SCFA und deren Metabolite aus der Pansenepithelzelle in das Blut auszuschleusen (MCT1). Die transepitheliale Permeation von SCFA über die Pansenwand ist von enormer Bedeutung, da sie die wichtigste Energiequelle der Wiederkäuer darstellen. Die beteiligten Transportprozesse müssen dementsprechend einer Anpassung an variierende Mengen von SCFA unterliegen. Bisherige Studien bei anderen Spezies deuten auf eine Regulation des MCT1 auf mRNA Ebene über den Peroxisom-Proliferator-aktivierten Rezeptor α (PPARα) hin. Ziele der Untersuchung: Das Ziel dieser Arbeit war herauszufinden, ob MCT1 in ovinen Pansenepithelzellen über PPARα reguliert wird und ob auch MCT4 dieser Regulation unterliegt. Eine gleichzeitige Regulation beider Transporter läge nahe, da sie gemeinsam an der transepithelialen Permeation beteiligt sind. Die Auswirkungen solch einer Regulation auf die Proteinexpression und die Transportleistung der MCT sollte charakterisiert werden. Ebenfalls war das Potenzial der bei erhöhter Kraftfutterfütterung vermehrt anfallenden SCFA Butyrat auf die MCT1 Expression zu untersuchen. Material & Methoden: Aus dem Vorhof von Schafen wurden Pansenepithelzellen gewonnen und entsprechend einer bereits etablierten Methode kultiviert. Nach einer Subkultivierung wurden die Zellen immunzytochemisch mit Antikörpern gegen MCT1, MCT4 und Na+/K+-ATPase untersucht, um deren Lokalisation in den kultivierten Pansenepithelzellen zu bestimmen. Weiterhin erfolgte eine Behandlung mit WY 14.643, einem spezifischen, synthetischen PPARα Agonisten, sowie mit GW 6471, einem Antagonisten des PPARα. Mittels qPCR wurden die relativen mRNA Mengen von MCT1, MCT4, ACO, CPT1A und CACT bestimmt und auf die Referenzgene GAPDH und Na+/K+-ATPase normalisiert. Die Proteinexpression von MCT1 und MCT4 wurde mittels Western Blot bestimmt. Zur funktionellen Quantifizierung wurde der intrazelluläre pH-Wert der Zellen mittels Spektrofluorometrie gemessen und der laktatabhängige Protonentransport als Vergleichswert zwischen den Behandlungen genutzt. Um den MCT-abhängigen Teil des Transportes zu bestimmen, wurde ein spezifischer MCT1 & 4 Inhibitor, die p-Hydroxymercuribenzensulfonsäure (pHMB) eingesetzt. Die Zellen wurden mit Butyrat über einen Zeitraum von 6 und 48 h induziert. Die Erfassung der MCT1 Expression erfolgte mittels semiquantitativer PCR. Ergebnisse: MCT1 & 4 sind sowohl in der Zellmembran als auch intrazellulär in den Pansenepithelzellen lokalisiert. Die mRNA Expressionsdaten konnten zeigen, dass MCT1 und die PPARα Zielgene durch WY 14.643 hochreguliert werden konnten, wohingegen die MCT4 Expression keine eindeutige Antwort auf die Stimulation zeigt. Die Behandlung mit den Antagonisten zeigt eine Abhängigkeit der MCT1 Expression von PPARα, die MCT4 Expression konnte dagegen nicht beeinflusst werden. Mittels pHMB gelang es, den laktatabhängigen Protonenexport fast vollständig zu blocken. Sowohl laktatabhängiger Protonenexport als auch die Proteinexpression zeigten keine Änderung durch WY 14.643 Stimulation. Die Butyratexposition veränderte die Morphologie der Pansenepithelzellen und schien nicht geeignet für Untersuchungen der mRNA Expression zu sein. Schlussfolgerungen: Es konnte in dieser Arbeit erstmals gezeigt werden, dass MCT1 in Pansenepithelzellen über PPARα reguliert wird, nicht aber MCT4. PPARα scheint demnach einer der entscheidenden Angriffspunkte für die Regulation des SCFA Transportes zu sein, dessen natürliche Liganden im Pansen aber noch nicht bekannt sind. Damit legt diese Arbeit den Grundstein für regulative Studien am intakten Pansenepithel. / Introduction: Monocarboxylate transporters (MCT) 1 & 4 are cotransporters of monocarboxylates and protons in a variety of mammalian cell types. In the ruminal epithelium MCT are necessary to transport short-chain fatty acids (SCFA) from the lumen into the ruminal epithelial cell (MCT4) and to discharge SCFA and their metabolites from the cell into the blood (MCT1). Transepithelial permeation of SCFA is of great importance, because they are the main source of energy for ruminants. The regulation of appropriate transport proteins should thus be subject to the adaptation to varying SCFA amounts. Previous studies in other species suggested that gene expression of MCT1 is regulated by peroxisome proliferator-activated receptor α (PPARα), a ligand-activated nuclear receptor. Aims: The aim of the study was to examine if MCT1 in ruminal epithelial cells is regulated by PPARα and furthermore if MCT4 can be regulated by PPARα, as well. A simultaneous regulation seems likely, because both are acting jointly in the transepithelial transporting of SCFA. The implications of such a regulation on protein expression and transport capacity of MCT should be characterized. The effect of butyrate, a SCFA which increases under concentrate feeding, on MCT1 expression was determined. Materials & Methods: Ruminal epithelial cells of sheep were cultivated according to methods previously established. After subcultivation, immunocytochemistry with antibodies against MCT1, MCT4 and Na+/K+-ATPase was performed to determine their localization in ruminal epithelial cells. For studying the influence of PPARα, WY 14.643, a synthetic and selective ligand of PPARα, and GW 6471, a synthetic antagonist of PPARα, were applied to the culture medium of the cells. After processing the specimens, the relative amount of mRNA of MCT1, MCT4 and the target genes ACO, CPT1A and CACT were analyzed by qPCR and normalized on the reference genes GAPDH and Na+/K+-ATPase. Protein abundance of MCT1 & 4 was measured by using the Western Blot method. Functional quantification was measured by the intracellular pH (pHi) of cells using spectrofluorometry as well as comparing the effect of WY 14.643 treatment on lactate-dependent proton export. To determine the MCT-dependent part of the pHi recovery, p-hydroxymercuribenzoic acid (pHMB), a specific inhibitor of MCT1 & 4, was applied. Cells were also treated with butyrate for 6 h and 48 h and the mRNA abundance of MCT1 was analyzed by semiquantitative PCR. Results: Both MCT1 and MCT4 were localized in the cell membrane as well as in the cytoplasm of ruminal epithelial cells. By qPCR it could be demonstrated that the mRNA abundance of MCT1 and PPARα target genes in the ruminal epithelial cells was increased by WY 14.643 in comparison to untreated cells, whereas the response of MCT4 did not yield distinct results. Treatment with the PPARα antagonist pointed out, that MCT1 is influenced by PPARα, but not MCT4. Lactate-dependent proton export was blocked almost completely by pHMB. Both lactate-dependent proton export and protein expression were not altered by WY 14.643 treatment. Butyrate exposure changed the morphology of ruminal epithelial cells and seemed unsuitable for the analysis of mRNA expression. Conclusion: For the first time, it could be demonstrated, that MCT1 in ruminal epithelial cells is regulated by PPARα, but not MCT4. PPARα seems to be a vital target in the rumen for SCFA transport regulation, whose natural triggers have yet to be identified. Furthermore, this study provides the basis for regulative studies on intact ruminal epithelium.

<p>Pathophysiologic lactate accumulation is characteristic of solid tumors and has been associated with metastases and poor overall survival in cancer patients. In recent years, there has been a resurgence of interest in tumor lactate metabolism. In the past, our group has shown that lactate can be used as a fuel in some cancer cell lines; however, survival responses to exogenous lactate alone are not well-described. We hypothesized that lactate utilization and cellular responses to exogenous lactate were varied and dynamic, dependent upon factors such as lactate concentration, duration of lactate exposure, and of expression of the lactate transporter, monocarboxylate transporter 1 (MCT1). We hypothesized that pharmacological inhibition of MCT1 with a small molecule, competitive MCT1 inhibitor, &#945;-cyano-4-hydroxycinnamic acid (CHC), could elicit cancer cell death in high lactate conditions typical of that seen in breast cancer. </p><p>My work focused on defining: 1. Lactate levels in locally advanced breast cancer (LABC); 2. Lactate uptake and catabolism in a variety of cancer cell lines; 3. The effect of exogenous lactate on cancer cell survival; 4. Whether the lactate-transporters, MCT1 and MCT4 can be used as markers of cycling hypoxia. </p><p>Lactate levels in LABC biopsies were assessed ex vivo by bioluminescence. NMR techniques were employed extensively to determine metabolites generated from 13C-labeled lactate. Cell viability in response to extracellular lactate ( ± glucose and ± CHC) was measured with Annexin V / 7-AAD staining to assess acute survival responses and clonogenic assays to evaluate long-term colony forming ability after lactate treatment. MCT1 and MCT4 protein expression was evaluated in cancer cell lines with Western blots after exposure to chronic or cycling hypoxia. Immunofluorescence was employed to assess MCT1 and MCT4 expression in head and neck cancer biopsies, and the expression patterns of the transporters were correlated to areas of hypoxia, as indicated by hypoxia marker EF5. </p><p>Lactate concentrations in LABC biopsied ranged from 0 - 12.3 µmol/g of tissue. The LABC dataset was too small to derive statistical power to test if lactate accumulation in LABC biopsies was associated with poor patient outcome or other clinical parameters of known prognostic significance. All cell lines tested (normal and cancer) showed uptake and metabolism of labeled lactate, with dominant generation of alanine and glutamate; however, relative rates and the diversity of metabolites generated was different among cell lines. MCF7 cells showed greater overall lactate uptake (mean = 18mM) over five days than MDA-MB-231 cells (mean = 5.5mM). CHC treatment effectively prevented lactate uptake in cancer cells when lactate concentrations were &#8804;20mM. </p><p>Cell survival was dependent upon lactate concentration and glucose availability. Acute responses to exogenous lactate did not reflect the long-term consequences of lactate exposure. Acutely, HMEC and R3230Ac cells were tolerant of all lactate concentrations tested (0-40mM) regardless of presence or absence of glucose. MCF7 and MDA-MB-231 cells were tolerant of lactate within the concentration ranges seen in biopsies. Cytotoxicity was seen after 24 hr incubation with 40mM lactate (-glucose), but this concentration is three times higher than any measurement made in human biopsies of LABC. Similarly, HMEC and MCF7 cells showed significantly decreased colony formation in response to 40mM exogenous lactate (+ glucose) while R3230Ac and MDA-MB-231 cells showed no impairment in colony-forming abilities with any lactate concentration (+ glucose). 5mM CHC significantly increased cell death responses independent of lactate treatment, indicating off-target effects at high concentrations. </p><p>MCT1 was found to be expressed in a majority of the cell lines tested, except for MDA-MB-231 cells. Cancer cells exposed to exogenous lactate showed upregulation of MCT1 but not MCT4. Chronic hypoxia resulted in an increase in protein expression of MCT4 but a decrease in MCT1 expression in cancer cell lines. The time course of regulation of protein levels of each transporter suggested the possibility of expression of both transporters during cycling hypoxia. When cancer cells were exposed to cycling hypoxia, both transporters showed upregulation. In head and neck tumor biopsies, MCT1 expression was significantly positively correlated to aerobic tumor regions and inversely correlated to hypoxic tumor regions. </p><p>Cancer cell responses to exogenous lactate were not uniform. Some cell lines demonstrated a lactate-tolerant and/or a lactate-consuming phenotype while other cell lines demonstrated lactate-intolerant and/or non-lactate-consuming phenotype. My work indicates that exogenous lactate was well-tolerated at clinically relevant concentrations , especially in the presence of glucose. Evidence of glutamate metabolism from lactate indicated that exogenous lactate partially progresses through the TCA cycle, suggesting that lactate may be utilized for fuel. The cell death elicited from 5mM CHC treatment was not dependent upon presence of lactate, indicating that manipulation of lactate metabolism may not be the best option for targeting cancer metabolism. When attempting to manipulate lactate metabolism in tumors, microenvironmental factors, such as hypoxia and glucose, must be taken into account in order to ensure a predictable and favorable outcome. Together, these results illustrate the importance of characterizing tumor metabolism before therapeutic intervention.</p> / Dissertation