Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Iron is an essential element that plays a role in the process of respiration, oxygen transport and as a principle cofactor to several enzymes. Iron homeostasis is a finely regulated process since excess levels become toxic to healthy cells via the production of reactive oxygen species. A plethora of genes that control several key points throughout this regulatory process have been identified. Research focusing on changes in expression levels and downstream functional effects of these genes has become increasingly important over the past decade. One area of particular interest has emerged since a link between iron status and host response to Mycobacterium tuberculosis infection was discovered. Although the prevalence of Tuberculosis has decreased across the globe with the exception of Africa and parts of Europe, the mortality rate remains high. Therefore, research that focuses on understanding an individual’s predetermined susceptibility to TB infection at the genetic level could provide health care practitioners with the tools required to identify and educate at-risk individuals prior to TB infection. RT-qPCR was utilised to determine expression profiles for eight iron genes (CP, CYBRD1, FTH, FTL, LTF, HFE, HMOX1, and SCL40A1) normalised to three reference genes (ACTB, GUSB, and RPL37A1). Up-regulation is demonstrated in the TB group for transcript levels recorded for CYBRD1, HFE, HMOX1, and SLC40A1. Several measured serum parameters including conjugated, unconjugated, total bilirubin, and total protein were increased in the TB group while albumin was significantly lower in this group. Correlation analysis demonstrated that a positive correlation exists between transferrin saturation and iron and a negative correlation exists between transferrin and ferritin levels. Individuals categorised with low serum iron levels demonstrated lower CP/GUSB levels and higher HMOX1/GUSB levels. Individuals categorised with low transferrin saturation levels demonstrated higher FTL/GUSB and SLC40A1/GUSB levels and lower CP/GUSB. Results from this study provide further evidence for the relationship between iron status and TB infection rates, although protein studies are required to confirm these results. The data obtained illustrate the important role that these profiles and iron parameters may play in the clinical field when identifying at-risk individuals. Further investigation that focuses on which gene profile and parameter combinations show the most distinctive utility in the clinical setting is warranted. / AFRIKAANSE OPSOMMING: Yster is ‘n noodsaaklike element wat ‘n rol speel in die proses van respirasie en die vervoer van suurstof en ook ‘n belangrike ko-faktor vir verskeie ensieme is. Yster homeostase is op ‘n fyn manier gereguleer omdat oormatige vlakke toksies kan wees vir gesonde selle wanneer reaktiewe suurstofspesies geproduseer word. ‘n Magdom gene wat verskeie sleutelpunte in hierdie proses kontroleer is voorheen identifiseer. Navorsing wat fokus op die veranderinge in geenuitdrukkingsvlakke en die funksionele gevolge daarvan het oor die afgelope dekade toenemend belangrik geword. Een gebied van spesifieke belang het na vore gekom nadat ‘n verband tussen ystervlakke en die manier waarop die immuunstelsel reageer op Mycobacterium tuberculosis infeksie, ontdek is. Alhoewel die voorkoms van Tuberkulose wêreldwyd, behalwe in Afrika en sekere dele van Europa, afgeneem het, bly die sterftesyfer hoog. Daarom kan navorsing wat daarop fokus om ‘n individu se voorafbepaalde vatbaarheid vir TB-infeksie op die genetiese vlak te verstaan dalk aan gesondheidswerkers die regte instrumente verskaf om hoë-risiko individue te identifiseer en op te voed voordat hulle TB ontwikkel. RT-qPKR is gebruik om die geenuitdrukkingsvlakke van agt ystergene, wat met drie verwysings-gene (ACTB, GUSB, en RPL37A1) genormaliseer is, te bepaal. ‘n Toename in die uitdrukkingsvlakke van CYBRD1, HFE, HMOX1, en SLC40A1 is in die TB-groep waargeneem. Die bloedvlakke van verskeie parameters insluitend gekonjugeerde, ongekonjugeerde, totale bilirubin, en totale proteïen was hoër in die TB-groep, terwyl albuminvlakke laer was in hierdie groep. Korrelasie-analise het ‘n positiewe korrelasie tussen transferrin-versadiging en yster getoon, terwyl daar ‘n negatiewe korrelasie tussen transferrin- en ferritinvlakke gevind is. Individue met lae ystervlakke het laer CP/GUSB-vlakke en hoër HMOX1/GUSB-vlakke getoon. Individue met lae transferrin-versadiging het hoër FTL/GUSB- en SLC40A1/GUSB-vlakke en laer CP/GUSB-vlakke getoon. Resultate uit hierdie studie verskaf verdere getuienis dat daar ‘n verwantskap tussen ystervlakke en TB-infeksiekoerse bestaan, alhoewel proteïenstudies nodig is om hierdie resultate te bevestig. Die data dui op die belangrike rol wat hierdie profiele en ystervlakke in die kliniese veld mag speel in die identifisering van hoë-risiko individue. Verdere ondersoek, gefokus op watter geenprofiel en parameterkombinasies die grootste nut in die kliniese omgewing bied, is geregverdig.

Made available in DSpace on 2014-12-17T14:16:23Z (GMT). No. of bitstreams: 1 ValdjaneS.pdf: 2387721 bytes, checksum: d21580a2388ac377d21ffb9abbbd4392 (MD5) Previous issue date: 2009-03-27 / Ferritin is a protein composed of heavy and light chains, non-covalently linked and which accommodates, in its core, thousands of atoms of iron. Furthermore, this protein represents the stock of iron in the body and it is characterized as an acute marker and predictor of diseases, such as iron deficiency anemia, hereditary hemochromatosis and others. Considering the variability of reference values and the analytical methods currently available, the aim of this work was to propose 95% confidence intervals for adults in the State of Rio Grande do Norte, Brazil, after determining the average concentration of serum ferritin for both sexes, beyond its correlation with the age. We analyzed 385 blood samples, collected by venipuncture from individuals residing in the State, after 12-14 hours of fast. The populational sample had 169 men and 216 women between 18-59 years old, which filled a questionnaire on socioeconomic, food habits and accounts about previous and current diseases. The sample collections were itinerant and the results of erythrogram, fasting glucose, alanine aminotransferase, aspartate aminotransferase, &#947;-glutamyl transferase, urea, creatinine, leukocyte count and platelets, beyond C-reactive protein, were issued to each participant, so that, after selection of the apparently healthy individuals, the dosage of serum ferritin was carried out. Statistical analysis was performed using the softwares SPSS 11.0 Windows version, Epi Info 3.3.2 and Graf instant pad (version 3.02), and the random population sample was single (finite population), for which the test of linear correlation and diagram of dispersion were also made. After selection of individuals and determination of serum ferritin, the most discrepant outliers were disregarded (N = 358, Men = 154/Women = 207) and the average value determined for the masculine sex individuals was 167,18 ng / dL; for the feminine sex individuals, the average value obtained was 81,55 ng / dL. Moreover, we found that 25% of men had values < 90,30 ng / dL; 50% &#8804; 156,25 ng / dL and 75% &#8804; 229,00 ng / dL. In the group of women, 25% had values < 38,80 ng / dL; 50% &#8804; 65,00 ng / dL and 75% &#8804; 119,00 ng / dL. Through the correlation coefficient (r = 0,23 with p = 0,003), it is possible to suggest the existence of positive linear correlation between age and serum ferritin for men. The correlation coefficient for women (r = 0,16 with p = 0,025) also confirms the existence of positive linear correlation between serum ferritin and age. Considering the analysis carried out and specific methods corroborating with the proposed benchmarks, we concluded that the average value found for men is higher than that found for women. Furthermore, this scenario rises with age for both sexes, and the 95% confidence intervals obtained were 74 ng/dL &#8804; &#956; &#8804; 89 ng/dL and 152ng/dL &#8804; &#956; &#8804;183ng/dL for the feminine and masculine sex individuals respectively / A ferritina ? uma prote?na composta por cadeias leves e pesadas ligadas n?o- covalentemente e que acomoda, em seu n?cleo, milhares de ?tomos de ferro. Al?m disso, esta prote?na representa os estoques de ferro no organismo e caracteriza-se como marcador de fase aguda e preditor de doen?as como anemia por defici?ncia de ferro, hemocromatose heredit?ria, entre outras. Diante da variabilidade de valores de refer?ncia e m?todos anal?ticos dispon?veis atualmente, a presente pesquisa objetivou propor intervalos de refer?ncia com 95% de confian?a para adultos do Estado do Rio Grande do Norte, ap?s determina??o da concentra??o m?dia de ferritina s?rica para ambos os sexos, correlacionando-as tamb?m com a idade. Foram analisadas 385 amostras de sangue, coletadas ap?s 12-14 horas de jejum por venopun??o, de indiv?duos residentes no Estado, sendo 169 homens e 216 mulheres entre 18-59 anos, os quais responderam a um question?rio relacionado a aspectos s?cio-econ?micos, alimentares, hist?rico de doen?as anteriores e queixas atuais. A coleta teve car?ter itinerante, tendo sido emitidos a cada participante os resultados de eritrograma, glicose em jejum, alanina aminotransferase, aspartato aminotransferase, &#947;- glutamil transferase, ureia, creatinina, contagem de leuc?citos e plaquetas, al?m de prote?na C reativa de modo que, ap?s sele??o dos indiv?duos aparentemente saud?veis, foi feita a dosagem de ferritina s?rica. As an?lises estat?sticas foram realizadas utilizando-se os softwares SPSS Windows vers?o 11.0, Epi Info 3.3.2 e Graf pad instant (vers?o 3.02), sendo que a amostra populacional foi aleat?ria simples (popula??o finita) para a qual foi feito o teste de correla??o linear e diagrama de dispers?o. Ap?s a sele??o dos indiv?duos e determina??o da ferritina s?rica, os outliers mais discrepantes foram desconsiderados, obtendo-se um valor m?dio para os indiv?duos do sexo masculino de 167,18 ng/dL e, para os indiv?duos do sexo feminino de 81,55 ng/dL. Analisando os valores obtidos, temos que 25% dos homens apresentaram valores < 90,30 ng/dL; 50% &#8804; 156,25 ng/dL e 75% &#8804; 229,00 ng/dL. No grupo das mulheres, 25% apresentam valores < 38,80 ng/dL; 50% &#8804; 65,00 ng/dL e 75% &#8804;119,00 ng/dL. Por meio do coeficiente de correla??o, r = 0,23, e p = 0,003, ? poss?vel confirmar a exist?ncia de correla??o linear positiva entre idade e ferritina s?rica dos indiv?duos do sexo masculino, assim como o coeficiente de correla??o r = 0,16 e p = 0,025 confirma a exist?ncia de correla??o linear positiva entre a ferritina s?rica e a idade das mulheres. Diante das an?lises realizadas e corroborando com m?todos espec?ficos para proposi??o de valores de refer?ncia, conclu?mos que o valor m?dio encontrado para homens ? superior ao valor m?dio encontrado para mulheres, elevando-se com a idade para os indiv?duos de ambos os sexos. Al?m disso, os intervalos de refer?ncia determinados com 95% de confian?a, foram de 74 ng/dL &#8804; &#956; &#8804; 89 ng/dL e de 152 ng/dL &#8804; &#956; &#8804; 183 ng/dL, para os indiv?duos dos sexos feminino e masculino, respectivamente

The thesis "The proteomic analysis in hematology: Identification of alfa2- macroglobulin as a specific carrier for the hormone hepcidin and proteomic analysis of the leukemic K562 cell differentiation induced by sodium butyrate" describes proteomic approaches, used for the identification and functional characterisation of proteins, which are binding and transporting the iron metabolism regulating hormone hepcidin. Proteomic techniques are also exploited for the identification of proteins, participating in erythroid differentiation of the model cell line K562. In the first section of the thesis, non-denaturing, native techniques, such as chromatography and native electrophoresis are used, in the second section, the control and butyrate - induced K562 cell proteomes are compared using the classical 2D - SDS polyacrylamide gel electrophoresis approach. The methods, described in the thesis are broadening the spectrum of available techniques in experimental hematology. The results, described in this thesis together with the accompanying published manuscripts broaden our knowledge in the function of proteins of iron metabolism and proteins, functioning in erythroid differentiation. Key words: proteomic analysis, hepcidin, alfa2-macroglobulin, iron metabolism, CML, K562, sodium butyrate

Orientador: Helena Zerlotti Wolf Grotto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T08:24:50Z (GMT). No. of bitstreams: 1 Alvarenga_ElianadaCosta.pdf: 3519502 bytes, checksum: cc024f9bd1176e4c4f56d989bba5a487 (MD5) Previous issue date: 2009 / Resumo: A deficiência de ferro é provavelmente o distúrbio nutricional mais freqüente no mundo. O ferro é um componente essencial da molécula de hemoglobina, da mioglobina e de diversas enzimas. Tem papel fundamental no transporte de oxigênio, na transferência de elétrons e atua como cofator em muitos processos enzimáticos, incluindo a síntese de DNA. Diversos estudos têm mostrado a contribuição da infecção pelo Helicobacter pylori (H. pylori) no desenvolvimento da anemia ferropriva e a associação entre o H. pylori e a diminuição do estoque de ferro. O objetivo do presente trabalho foi verificar a possível associação entre a infecção pelo H. pylori e a deficiência de ferro em um grupo de pacientes adultos. Desse modo pretendeu-se conhecer melhor as alterações hematológicas presentes nos pacientes infectados pelo H. pylori, principalmente as relacionadas ao metabolismo do ferro. Foram estudados 156 pacientes adultos de ambos os sexos que foram submetidos à endoscopia digestiva alta para esclarecimento diagnóstico. Desses 156 pacientes, 125 apresentaram alterações à endoscopia, que justificaram a realização da biópsia gástrica. A avaliação da presença de anemia foi feita pelos dados hematimétricos e pelo conteúdo de hemoglobina dos reticulócitos (Ret-He) e o estado do ferro foi avaliado pelas dosagens de ferro sérico, capacidade de ligação do ferro à transferrina e ferritina sérica. A possível participação da atividade inflamatória na eritropoiese foi verificada pela correlação entre os parâmetros hematimétricos e do estado do ferro com os níveis de proteína C reativa (PC-R). O diagnóstico do H. pylori foi realizado através da análise histológica, teste da urease e teste sorológico (IgG anti- H. pylori). Os 125 pacientes foram separados em H. pylori positivo (n= 75) e negativo (n= 50). Não houve diferença significativa nos valores dos parâmetros hematológicos e bioquímicos, tendo sido diferentes somente os valores de IgG anti-H. pylori e de gastrina sérica, significativamente superiores no grupo H. pylori positivo. Foi observada uma correlação inversa fraca, mas significativa entre os níveis de PC-R e Ret-He e entre gastrina sérica e Ret-He no grupo H. pylori positivo. De acordo com os critérios laboratoriais, dos 125 pacientes apenas 17 (13,6%) mostraram níveis de Hb indicativos de anemia, sendo que desses, 7 (5,6%) mostraram ser positivos para a infecção pelo H. pylori. Não foi observada diferença na freqüência da deficiência de ferro entre os grupos de pacientes infectados pelo H. pylori e os não infectados pelo H. pylori. Dentre os pacientes estudados a gastrite foi confirmada em 110 pacientes, sendo que 74 (67,2%) eram H. pylori positivo. A gastrite foi classificada de acordo com o sistema de Sidney em: moderada/intensa (36.63%), leve (63,37%) e inespecífica (8,1%). Quando os pacientes com gastrite moderada/intensa foram comparados com os com gastrite leve, não houve diferença entre os parâmetros estudados, exceto os valores da titulação de IgG, que foram significativamente superiores no grupo com gastrite moderada/intensa. Portanto, podemos concluir que no grupo de indivíduos estudados a infecção pelo H. pylori e a intensidade da gastrite não foram fatores determinantes ao desenvolvimento da deficiência de ferro. / Abstract: Iron deficiency is probably the most common nutritional disorder in the world. Iron is an essential component of the molecule of hemoglobin, myoglobin and various enzymes. It has a fundamental role in oxygen transport, in electrons transference and acts as a cofactor in many enzymatic processes, including synthesis of DNA. Several studies have shown the contribution of Helicobacter pylori (H. pylori) infection in the development of anemia and the association between H. pylori and reduction of iron store. The objective of this study was to investigate the association between H. pylori infection and iron deficiency in a group of adult patients. Thus we intend to study the hematological alterations in H. pylori infected patients, mainly those related to iron metabolism. We studied 156 adult patients of both sex undergoing routine upper endoscopy. Of these 156 patients, 125 had gastric biopsies stained for H. pylori identification. The evaluation of the presence of anemia was performed by erythrocyte indeces and reticulocyte hemoglobin content (RET-He) and the iron status was assessed by measurements of serum iron, iron binding capacity of the transferrin and serum ferritin levels. Inflammatory activity influence on erythropoiesis was verified by the correlation among erythrocyte parameters and the state of the iron with C-reactive protein (CR-P) levels. The diagnosis of H. pylori was performed by histological, urease and serological (IgG anti-H. pylori) methods. Patients were divided into H. pylori positive (n = 75) and negative (n = 50). There was no significant difference in hematological and biochemical parameters, except for IgG anti-H. pylori values and serum gastrin, significantly higher in H. pylori positive group. There was a weak but significant inverse correlation between CR-P and Ret-He levels and between serum gastrin and Ret-He in H. pylori positive group. According to laboratory criteria, only 17 of 125 patients (13.6%) showed levels of hemoglobin indicative of anemia, 7 of 17 patients were positive for H. pylori infection. There was no difference in the frequency of iron deficiency anemia between H. pylori positive and negative groups. Gastritis was confirmed in 110 patients, of these 74 (67.2%) were H. pylori positive. The gastritis was classified according to the Sydney system as: moderate / severe (36.63%), mild (63.37%) and nonspecific (8.1%). There was no difference between groups with moderate/ severe gastritis and mild gastritis concerning hematologic and biochemical parameters, except the values of the titers of IgG, which were significantly higher in the group with moderate / severe gastritis. Therefore, we conclude that infection by H. pylori and the intensity of gastritis were not determining factors for the development of iron deficiency in the studied group. / Universidade Estadual de Campi / Ciencias Biomedicas / Mestre em Ciências Médicas

A presente pesquisa teve a finalidade de estudar a hematologia de bezerros clonados da raça Nelore, originados por meio da técnica de transferência nuclear de células somáticas (TNCS) por meio da avaliação do quadro eritrocitário, da dinâmica dos tipos de hemoglobina e do metabolismo do ferro destes animais durante o primeiro mês de vida. O delineamento experimental envolveu a colheita de 260 amostras de sangue e soro sanguíneo de 20 bezerros distribuídas nos seguintes momentos: imediatamente após o nascimento, 12 horas após o nascimento, 1 dia de vida, 2 , 3 , 4, 5, 7, 10, 15, 20 e 30 dias de vida. Os animais foram divididos em quatro grupos experimentais: 12 bezerros obtidos por meio da técnica de TNCS pelos laboratórios A e 8, 4 por meio de fertilização in vitro (FIV) e 4 por monta natural (MN). A ocorrência de anemia de grau moderado a grave, do tipo normocítico e normocrômico, foi observada em 100 % (5/5) dos 5 bezerros clonados pelo Laboratório A, enquanto a incidência nos bezerros clonados pelo Laboratório B foi igual a 14,2 % (1/7), nos bezerros obtidos por fertilização in vitro foi igual 50,0 % (2/4) e em bezerros obtidos por monta natural foi igual a 50,0 % (2/4). A avaliação do eritrograma dos bezerros cio nados pelo Laboratório A evidenciou que a anemia instalou-se gradualmente a partir das 12 horas de vida atingindo ao final da primeira semana, a, sua intensidade máxima, sendo observada a recuperação gradativa dos valores eritrograma a partir do 15°dia de vida. Os valores médios obtidos para o eritrograma dos bezerros clonados do Laboratório A no 7º dia de vida foram os seguintes: Hemácias - 4,33X106/mm3-; Volume Globular - 23 %, Taxa de Hemoglobina - 7,25 g/dL; VCM - 52,89 µ3-; HCM - 16,65 pg; CHCM - 31,47%. A anemia observada nos bezerros clonados pelo Laboratório A era de origem ferropriva, pois se evidenciou nesses animais uma significativa diminuição dos teores séricos de ferro associada à diminuição do índice de saturação da transferrina (1ST), enquanto os valores da capacidade total de ligação do ferro (CTLF) não sofreram influência durante o período. Os valores médios obtidos para o metabolismo de ferro dos bezerros clonados pelo Laboratório A no 7º dia de vida foram os seguintes: teores séricos de ferro - 47,35 mg/dL; capacidade total de ligação de ferro - 455,90 mg/dL, índice de saturação da transferrina - 9,64%. Durante o estudo dos tipos de hemoglobinas, utilizando-se técnica de eletroforese foram identificados três fenótipos de hemoblogina adulta (Hb-A; Hb-B e Hb-AB) e a presença de hemoglobina fetal (Hb-F), não sendo observadas anomalias que pudessem sugerir a ocorrência de hemoglobinopatias hereditárias e/ ou congênitas. Verificou-se que as taxas de Hb-A, nos clones com fenótipo Hb-AB e Hb-A, permaneceram estáveis durante todo o período experimental, enquanto nos bezerros obtidos por fertilização in vitro ou monta natural com os mesmos fenótipos (Hb-A e Hb-AB) observou-se a partir de 120 horas de vida um gradativo aumento das taxas de Hb-A. Durante a avaliação da dinâmica da hemoglobina do tipo fetal (Hb-F) no primeiro mês de vida observou-se, que todos os grupos animais apresentaram comportamento similar, caracterizado por sua diminuição com o desenvolvimento etário. / The present work aimed to study the hematology of cloned Nelore calves produced using the technique of somatic cell nuclear transfer (SCNT), by evaluating erythrocyte parameters, hemoglobin dynamics, and iron metabolism in the animals during the first month after birth. The experimental design included the collection of 260 blood and blood serum samples from 20 calves in the following times: immediately after birth, 12 hours after birth, 1st of life, 2nd, 3rd, 4th, 5th, 7th, 20th and 30th day of life. The animals were classified in four experimental groups: 12 calves produced, using SCNT for two commercial laboratories (laboratory A=5 calves and laboratory B= 7 calves), four calves produced by in vitro fertilization (IVF) and four calves produced by natural mating (NM). Mild to severe normocytic and normochromic anemia was observed in 100% (5/5) cloned calves from laboratory A, and 14.2% (117) cloned calves from laboratory B. In both IVF and NM calves, anemia was observed in 50% (214) of the calves. Erytrogram evaluation of cloned calves from laboratory A showed that anemia developed gradually from 12 hours after birth, was most intense at the end of the first week, and then erytrogram normal values were recovered after the 15th day of life. Mean values for the laboratory A cloned calves erytrogram in the 7th day of life were the following: Red cells 4033X106/mm3, hematocrit 23%, hemoglobin 7.25g/dL, MCV 52.89µ3-; MCH 16.65, MCHC 31.47%. Anemia observed in cloned calves from laboratory A was caused by iron deficiency, since a significant decrease in iron se rum levels together with a decrease in transferrin saturation index (TSI) was confirmed. At the same time, Total iron-binding capacity (TIBC) was not changed in this period of time. Mean iron metabolism values for cloned calves from laboratory A were the following: serum iron amount: 47,35mg/dL, TIBC 455,90 mg/dL and TSI 9,64%. Hemoglobin identification by eletrophoresis identified three adult hemolglobin phenotypes (A-Hb; B-Hb e AB-Hb) and the fetal hemoglobin (F-HB), and there was no sign of hereditary hemoglobin disorders were observed. The rate of A-Hb in cloned calves with A-Hb and AB-Hb phenotypes was maintained during the experimental period. Nonetheless, for the IVF calves with the same phenotypes (A-Hb and AB-Hb) a constant increase in the A-Hb was noticed. For the evaluation of F-Hb dynamics in the first month of life, results showed similar pattern, characterized by its decrease with age.

The resistance to tamoxifen, a drug used in the adjuvant therapy for hormone sensitive breast cancer, represents a major clinical obstacle. Although various mechanisms leading to tamoxifen resistance have been described and intensively studied, a significant number of patients still become resistant to the treatment and eventually relapse. Tamoxifen therapy has been shown to enrich tumors with cancer stem cells (CSCs), which are naturally resistant, and have self-renewal ability and the potential to form secondary tumors. Metabolic rewiring, altered iron metabolism and upregulation of ATP-binding cassette (ABC) transporters have been shown to be important in the maintenance of CSC phenotype. Therefore, we investigated these mechanisms as possible contributors to tamoxifen resistance in vitro in two tamoxifen resistant (Tam5R) cell lines that we established. We show that Tam5R cells have dramatically disassembled and less active mitochondrial supercomplexes (SCs) and higher level of mitochondrial superoxide, together with a fragmented mitochondrial network. Such dysfunction of mitochondria results in the AMP-activated protein kinase (AMPK) activation and metabolic rewiring towards glycolysis. Importantly, cells lacking functional mitochondria are significantly more resistant to tamoxifen, supporting...

The resistance to tamoxifen, a drug used in the adjuvant therapy for hormone sensitive breast cancer, represents a major clinical obstacle. Although various mechanisms leading to tamoxifen resistance have been described and intensively studied, a significant number of patients still become resistant to the treatment and eventually relapse. Tamoxifen therapy has been shown to enrich tumors with cancer stem cells (CSCs), which are naturally resistant, and have self-renewal ability and the potential to form secondary tumors. Metabolic rewiring, altered iron metabolism and upregulation of ATP-binding cassette (ABC) transporters have been shown to be important in the maintenance of CSC phenotype. Therefore, we investigated these mechanisms as possible contributors to tamoxifen resistance in vitro in two tamoxifen resistant (Tam5R) cell lines that we established. We show that Tam5R cells have dramatically disassembled and less active mitochondrial supercomplexes (SCs) and higher level of mitochondrial superoxide, together with a fragmented mitochondrial network. Such dysfunction of mitochondria results in the AMP-activated protein kinase (AMPK) activation and metabolic rewiring towards glycolysis. Importantly, cells lacking functional mitochondria are significantly more resistant to tamoxifen, supporting...

The effect of two levels of dietary protein and exercise on iron metabolism in pregnant Sprague-Dawley rats was studied. Animals were assigned to the following diet and exercise groups on the first day of gestation: high protein sedentary (HS), high protein exercise (HEx), low protein sedentary (LS), low protien exercise (LEx). Animals in the exercise groups swam continuously for 75 minuites the first day and 90 minutes daily thereafter, throughout the study. Hemoglobin, hematocrit, liver iron concentration and spleen iron concentration were measured at day 0, 3, 6, 9, 12, 15, 18, and 21 of gestation. Mean hemoglobin, hematocrit and liver iron concentration values were lower at day 21 than at day 0 of gestation. Mean hematocrit and hemoglobin for the LEx group were the lowest for days 9 through 15. At day 15 the mean hematocrit for the LEx group was significantly (p < 0.05) lower than that of the other groups. The HEx group had the highest hematocrit and hemoglobin values at day 21 of gestation; hemoglobin was significantly (p < 0.05) higher. No difference in mean spleen iron concentration from day 0 to day 21 was found, however, the low protein groups had higher spleen iron values early in pregnancy that the high protein groups. The mean spleen iron concentration of the LEx group was significantly (p < 0.05) higher than those of the HS and HEx groups at day 6. A trend for higher liver iron concentration values of the low protein groups than high protein groups was also observed. The LEx group had a significantly (p <0.05) higher mean liver iron concentration at day 18 than the other groups. Both protein nutriture and exercise appear to affect iron metabolism in pregnant rats. / M.S.

Pseudomonas is a diverse genus of Gram-negative bacteria that includes pathogens of plants, insects, and humans as well as environmental strains with no known pathogenicity. Pseudomonas fluorescens itself encompasses a heterologous group of bacteria that are prevalent in soil and on foliar and root surfaces of plants. Some strains of P. fluorescens suppress plant diseases and the genomic sequences of many biological control strains are now available. I used a combination of bioinformatic and phylogenetic analyses along with mutagenesis and biological assays to identify and compare the TonB-dependent outer-membrane proteins (TBDPs) of ten plant-associated strains of P. fluorescens and related species. TBDPs are common in Gram-negative bacteria, functioning in the uptake of ferric-siderophore complexes and other substrates into the cell. I identified 14 to 45 TBDRs in each strain of P. fluorescens or P. chlororaphis. Collectively, the ten strains have 317 TBDPs, which were grouped into 84 types based upon sequence similarity and phylogeny. As many as 13 TBDPs are unique to a single strain and some show evidence of horizontal gene transfer. Putative functions in the uptake of diverse groups of microbial siderophores, sulfur-esters, and other substrates were assigned to 28 of these TBDP types based on similarity to characterized orthologs from other Pseudomonas species. Redundancy of TBDP function was evident in certain strains of P. fluorescens, especially Pf-5, which has three TBDPs for ferrichrome/ferrioxamine uptake, two for ferric-citrate uptake and three for heme uptake. Five TBDP types are present in all ten strains, and putative functions in heme, ferrichrome, cobalamin, and copper/zinc uptake were assigned to four of the conserved TBDPs. The fluorescent pseudomonads are characterized by the production of pyoverdine siderophores, which are responsible for the diffusible UV fluorescence of these bacteria. Each of the ten plant-associated strains of P. fluorescens or P. chlororaphis has three to six TBDPs with putative roles in ferric-pyoverdine uptake (Fpv). To confirm the roles of the six Fpv outer membrane proteins in P. fluorescens Pf-5, I introduced deletions into each of the six fpv genes in this strain and evaluated the mutants and the parental strain for heterologous pyoverdine uptake. I identified at least one ferric-pyoverdine that was taken up by each of the six Fpv outer-membrane proteins of Pf-5. By comparing the ferric-pyoverdine uptake assay results to a phylogenetic analysis of the Fpv outer-membrane proteins, I observed that phylogenetically-related Fpv outer-membrane proteins take up structurally-related pyoverdines. I then expanded the phylogenetic analysis to include nine other strains within the P. fluorescens group, and identified five additional types of Fpv outer-membrane proteins. Using the characterized Fpv outer-membrane proteins of Pf-5 as a reference, pyoverdine substrates were predicted for many of the Fpv outer-membrane proteins in the nine other strains. Redundancy of Fpv function was evident in Pf-5, as some pyoverdines were recognized by more than one Fpv. It is apparent that heterologous pyoverdine recognition is a conserved feature, giving these ten strains flexibility in acquiring iron from the environment. Overall, the TBDPs of the P. fluorescens group are a functionally diverse set of structurally-related proteins present in high numbers in many strains. While putative functions have been assigned to a subset of the proteins, the functions of most TBDPs remain unknown, providing targets for further investigations into nutrient uptake by P. fluorescens spp.. The work presented here provides a template for future studies using a combination of bioinformatic, phylogenetic, and molecular genetic approaches to predict and analyze the function of these TBDPs. / Graduation date: 2012

Jede Zelle des Säugetierorganismus benötigt Eisen als Spurenelement für zahlreiche oxidativ-reduktive Elektronentransfer-Reaktionen und für Transport und Speicherung von Sauerstoff. Der Organismus unterhält daher ein komplexes Regulationsnetzwerk für die Aufnahme, Verteilung und Ausscheidung von Eisen. Die intrazelluläre Regulation in den verschiedenen Zelltypen des Körpers ist mit einer globalen hormonellen Signalstruktur verzahnt. Sowohl Eisenmangel wie Eisenüberschuss sind häufige und ernste menschliche Krankheitsbilder. Sie betreffen jede Zelle, aber auch den Organismus als Ganzes. In dieser Dissertation wird ein mathematisches Modell des Eisenstoffwechsels der erwachsenen Maus vorgestellt. In ihm wird die Flussbilanz des Eisens in den wichtigsten Zelltypen in Form von transmembranalen und intrazellulären kinetischen Gleichungen dargestellt, und es werden diese Zellmodelle mit dem zentralen Eisenaustausch-Kompartiment (Blutplasma) des Körpers integriert. Der Eisenstatus wird charakterisiert als Gehalt an labil gebundenen Eisen und an ferritin-gebundenen Eisen für jede Zelle. Der Stoffwechsel wird als Netzwerk von Flussdynamik formuliert. Der experimentelle Input in dieses Modells stammt von verschiedenen Quellen. Radioaktive Tracerdaten, gemessen am intakten Tier (Mausstamm C57BL6 – das am intensivsten studierte Tiermodell) unter varrierten physiologischen Bedingungen lieferten den experimentellen Hintergrund, von dem aus Clearance-Parameter durch numerisches Fitting ermittelt wurden. Es wird gezeigt, dass das Modell mit entsprechend adaptierten Parametersätzen die wichtigsten metabolischen und regulatorischen Ereignisse in Übereinstimmung mit den Messungen darstellen kann. In Zukunft soll die quantitative Übereinstimmung mit Daten aus weiteren genetischen Rekonstruktionen (globale und zell-spezifische knock-outs und konstitutive Expression relevanter Gene des Modellorganismus Maus) hergestellt werden. / Every cell of the mammalian organism needs iron as trace element in numerous oxido-reductive processes as well as for transport and storage of oxygen. The mammalian organism maintains therefore a complex regulatory network of iron uptake, excretion and intra-body distribution. Here a mathematical model of iron metabolism of the adult mouse is presented. It formulates the iron flux balance of the most important cell types of the organism in the form of transmembraneous and intracellular kinetic equations and integrates these cell models with the central exchange compartment (blood plasma) of the body. The iron status is represented as content of labile iron and of ferritin-bound iron in every cell type, and the metabolism is formulated as a network of flux dynamics. The experimental input into the model stems from different sources. Radioactive tracer data measured in the intact animal (mouse strain C57BL6 - the most intensively studied animal model) under various physiological conditions provided the experimental background from which clearance parameters could be obtained by numerical parameter fitting. Future research should render more precise the quantitative representation of genetic reconstructions (global and cell-type-addressed knock-out and constitutive expression of relevant genes of the model mouse strain).