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Utilization of iron by hemoglobin-producing cells is highly efficient. The acquisition of iron from plasma requires the binding of diferric transferrin (Tf) to its cognate receptor (Tf-R) on the erythroid cell membrane, followed by internalization of the Tf - Tf-R complexes via receptor-mediated endocytosis. Through a poorly understood mechanism, iron is targeted to mitochondria, the site of heme biosynthesis. We believe that a direct interaction between iron-containing endosomes and mitochondria is essential for iron transfer to mitochondria and its efficient incorporation into heme. / In order to illustrate the interaction between endosomes and mitochondria, we have employed flow cytometry. Flow cytometry analysis of reticulocytes (erythrocyte precursors which still synthesize hemoglobin) stained with fluorescent dyes specific to mitochondria and endosomes revealed three distinct populations: mitochondria, endosomes and a population labeled with both dyes. This double-labeled population suggests a population composed of endosomes associated with mitochondria. Using non-fluorescent diferric-Tf, we were able to remove the double population, leaving only the endosomal and the mitochondrial population. This finding has confirmed that the double population is the result of the interaction between the two organelles. / Additionally, we established a cell-free assay consisting of fluorescent mitochondria and endosomes isolated from erythroid cells. Using confocal microscopy, we demonstrated a colocalization between the two organelles. We repeated the assay using fluorescent mitochondria and endosomes isolated from HeLa spinner cells. Using the mitochondrial uncoupler CCCP, we were able to significantly reduce the colocalization between the two organelles, indicating that the interaction between the organelles is specific and that the mitochondrial potential is a requirement for organellar interaction. / Based on our results from flow cytometry and confocal microscopy, we conclude that a specific and direct interaction exists between the two organelles.

Iron is recognized as a key element regulating primary production in large regions of the ocean, but nothing is known of its direct effect on higher trophic levels. Growth and metabolism of two species of heterotrophic protozoans fed iron-rich and iron-poor prey were thus examined. Maximum growth rates of Paraphysomonas imperforata and P. butcheri were observed only when Fe quotas of bacterial prey were greater than 70 $ mu$mol Fe:mol C. At lower Fe:C ratios, but at constant prey biomass (C/ml), both species grew significantly slower. Minimum Fe quotas of the flagellates at these slow growth rates ($ sim$10 $ mu$mol Fe:mol C) were similar to those of iron-limited phytoplankton and bacteria. Growth rate reduction was the result of direct elemental limitation by Fe, judging from the protozoans' positive response to Fe additions and from their biochemical characteristics. Filtration and carbon ingestion rates increased under Fe-limitation, but carbon gross growth efficiency (CGGE) decreased when Paraphysomonas imperforata consumed iron-poor bacteria. Ammonium regeneration efficiency was also reduced. The decrease in CGGE was a consequence of reduced activity of the iron-dependent electron transport system, greater DOC excretion, and greater CO$ sb2$ evolution by Fe-limited flagellates. Paraphysomonas imperforata excreted Fe, even when limited by this element, and retained less of the ingested ration and thus had a higher Fe regeneration efficiency than when consuming Fe-rich bacteria. According to recent measurements of biogenic Fe:C in the subarctic Pacific, our results suggest that heterotrophic bacterivorous flagellates may experience iron-limitation in remote oceanic regions. Such limitation could profoundly affect C, N and Fe cycling in the sea.

Ovine (strains 9L and 3384Y) and bovine (strains 649, 2336 and 8025) isolates of Histophilus somni were investigated for their ability to acquire iron from haemoglobin (Hb). Bovine isolates were capable of utilizing bovine, but not ovine, porcine or human Hb as a source of iron. Ovine isolates could not obtain iron from Hb. Bovine isolates bound bovine, ovine, and human Hbs by means of the same iron-repressible receptor(s) and produced a ~120-kDa iron-repressible, outer membrane protein. Using PCR approaches, an iron-regulated operon containing hugX and hugZ homologues and a gene (hgbA) that encodes a TonB-dependent, Hb-binding proteins were identified in strains 649, 9L and 3384Y. In strains 9L and 3384Y, HgbA is truncated offering a possible explanation for their lack of utilization of Hb as an iron source. In strains 2336 and 8025, expression of HgbA was also subject to a form of phase variation.

[Truncated abstract] Iron is a trace mineral used by the body in many physiological processes that are essential to athletic performance. Commonly, the body's iron stores are compromised by exercise via several well established mechanisms. One such mechanism is the destruction of red blood cells (hemolysis), in response to the mechanical stress and circulatory strain of exercise. Although it appears that a force-dependent relationship between the heel-strike of the running gait and ground contact exists, the effects of the intensity trained at and the ground surface type trained upon have not been documented. Similarly, the effects of a cumulative training stress (i.e. multiple daily sessions) has not been examined. In addition to hemolysis, exercise also invokes an inflammatory response that results in an up-regulation of the cytokine interleukin-6 (IL-6). This cytokine is the primary mediator of hepcidin expression, a liver-produced hormone that regulates iron metabolism in the gut and in macrophages. The influence of exercise on hepcidin expression is relatively unknown, and as such it is possible that this hormone may be a mitigating factor implicated in athletic-induced iron deficiency. Therefore, the purpose of this thesis was to investigate the effect of different training frequencies, intensities and ground surfaces on the hemolytic response. In addition, the impact of exercise-induced inflammation on hepcidin expression in the 24 h post-exercise was investigated, with the aim of determining whether this hormone may be a potential new mechanism associated with athletic-induced iron deficiency. Finally, an interaction between hemolysis and hepcidin activity was examined to investigate their potential combined effect on iron status in the 24 h post-exercise. ... Venous blood and urine samples were collected pre- and immediately post-exercise, and at 3 and 24 h of recovery. Samples were analysed for circulating levels of IL-6, free Hb, Hp, serum iron, ferritin and urinary hepcidin activity. At the conclusion of both the T1 and T2 interval runs, the free Hb and serum Hp were significantly increased (p<0.05) from pre-exercise levels. Furthermore, a cumulative effect of two running sessions was shown in the T2 trial, via a further significant fall in serum Hp. The IL-6 and hepcidin activity were significantly increased after each running session (p<0.05) with no cumulative effect seen. Serum iron and ferritin were significantly increased post-exercise after each interval run (p<0.05), but were not influenced by the addition of a prior LSD run 12 h earlier. As a result, this investigation showed a cumulative effect of consecutive training sessions on RBC destruction in male athletes. Furthermore, post-exercise increases to serum iron and hepcidin, and their interaction was suggested to have potential implications for an athlete's iron status. Overall, the findings of this thesis show that hemolysis is evident at the conclusion of endurance running, and is influenced by training intensity and frequency. The results enabled a time-line for hepcidin expression post-exercise to be established, and the implications of increases to the activity of this hormone, in association with the hemolytic changes seen with endurance exercise are discussed.

Doctor of Philosophy(PhD) / Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Tuberculosis (TB) is an epidemic disease characterised by wet, persistent coughing, weight loss, fever, fatigue, and blood in the sputum. It has been reported that one in every three individuals is currently infected with Mycobacterium tuberculosis, the causative agent of TB, and that 10% of them will develop the active disease. The high prevalence and low penetrance of this disease has resulted in increased research performed to ascertain what factors play a role in susceptibility to M. tuberculosis infection. Some factors known to play a role in a minority of cases may include: HIV infection, diabetes, alcohol abuse and old age, but racial differences in susceptibility have also been observed. However, the influence of genetic factors is gaining popularity in current research. M. tuberculosis requires iron to proliferate, which it must appropriate from its host. For this reason the genes involved in the metabolism of iron in the human host are of particular interest when considering susceptibility to M. tuberculosis infection. In order to determine whether the expression of these genes may influence disease susceptibility, the promoter region of the CYBRD1, HAMP, HFE, LTF and SLC40A1 genes have been targeted for investigation. The aim of this study was to determine whether DNA variation in the promoter region of these genes involved in iron metabolism is associated with M. tuberculosis susceptibility. The study cohort consisted of 49 TB patients and 51 healthy, unrelated, population-matched controls, all of whom were Black, Xhosa-speaking individuals. Initially, 15 patient samples were randomly selected for exploratory screening, utilising semi-automated bi-directional sequencing analysis. In this manner, 40 variants were identified of which 30 were previously described. The novel variants included CYBRD1: -849 C/G, -492 A/G, -454 C/T, -397 A/C; HAMP: -323 C/T; HFE: -561 A/G, -331 A/C; and LTF: -1377 T/G, -457 T/C, -437 C/G. A number of loci demonstrated a statistically significant difference in allele and genotype frequencies, and in iron parameter levels when comparing the patient and control groups and for each variant separately. In silico analyses revealed the creation and/or abolishment of several transcription factor binding sites (TFBSs) due to the presence or absence of certain identified variants. The change in the composition of TFBSs in the promoter region may lead to differential expression of the gene. This study served as a pilot investigation to identify promoter region variants in the candidate genes involved in iron metabolism, in TB patients. The results presented here indicate that the identified variants (-1813 C/T, -1459 T/C, -238 A/G, -166 C/G [CYBRD1]; -561 A/G [HFE]; -1470 C/T, -1355 G/C and -1098 G/A [SLC40A1]) could possibly contribute to the increased absorption of iron in the TB patient group, which could subsequently increase the occurrence of pathogenic infection. The findings of this study could further aid in the elucidation of the exact mechanism(s) by which iron, its metabolism, and the genes involved affect disease susceptibility, more specifically, M. tuberculosis susceptibility. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is „n epidemiese siekte gekarakteriseer deur nat, aanhoudende hoes, gewigsverlies, moegheid, en bloed in die speeksel. Dit is gerapporteer dat een in elke drie individue tans geïnfekteer is met Mycobacterium tuberculosis, die veroorsakende agent van TB, en dat 10% van dié individue die aktiewe vorm van die siekte sal ontwikkel. Die hoë voorkoms en lae effek van hierdie siekte het daartoe gelei dat meer navorsing mettertyd gedoen is om die faktore wat „n rol mag speel in M. tuberculosis infeksie, te bepaal. Sommige faktore bekend vir hul rol in „n minderheid van gevalle sluit in: MIV infeksie, diabetes, alkoholmisbruik en bejaardheid, maar etniese verskille in vatbaarheid vir die siekte is ook al waargeneem. Die waarskynlikheid van genetiese invloed op die ontwikkeling van TB word ook meer deur navorsers ondersoek. M. tuberculosis benodig yster om te vermeerder, wat dit moet bekom vanaf die gasheer. Vir hierdie rede is die gene betrokke by yster metabolisme in die menslike gasheer veral van belang vir die oorweging van vatbaarheid vir M. tuberculosis. Om te bepaal of die uitdrukking van hierdie gene moontlik „n invloed het op vatbaarheid vir die siekte, was die promoter areas van die CYBRD1, HAMP, HFE, LTF en SLC40A1 gene geteiken. Die doel van hierdie studie was om te bepaal of DNS variasie in die promoter area van hierdie gene betrokke in yster metabolisme moontlik verband kan hou met vatbaarheid vir M. tuberculosis. Die studie kohort het uit 49 TB pasiënte en 51 gesonde, onverwante, populasie-gepaarde kontroles, waarvan almal Swart, Xhosa-sprekende individue was, bestaan. Aanvanklik was 15 pasiënt monsters lukraak gekies vir ondersoekende sifting, deur die gebruik van semi-outomatiese twee-rigting volgordebepalings. Op hierdie manier is 40 variante geïdentifiseer waarvan 30 voorheen beskryf is. Die nuwe variante sluit in CYBRD1: -849 C/G, -492 A/G, -454 C/T, -397 A/C; HAMP: -323 C/T; HFE: -561 A/G, -331 A/C; en LTF: -1377 T/G, -457 T/C, -437 C/G. „n Aantal loci het statisties betekenisvolle verskille getoon in alleel en genotipe frekwensies, en in yster parameter vlakke met die vergelyking van die pasiënt groep met die kontrole groep. In silico analise het verder die skepping en/of afskaffing van verskeie transkripsiefaktor bindingsetels (TFBSs), as gevolg van die teenwoordigheid of afwesigheid van sekere variante, getoon. Die verandering in die samestelling van TFBSs in die promoter area kan lei tot differensiële uitdrukking van die geen. Dié studie het gedien as „n voorlopige ondersoek om te bepaal of promoter area variante, geïdentifiseer in kandidaat gene betrokke by yster metabolisme, „n invloed het in die ontwikkeling van TB. Die resultate wat hier gewys word dui aan dat die geïdentifiseerde variante (-1813 C/T, -1459 T/C, -238 A/G, -166 C/G [CYBRD1]; -561 A/G [HFE]; -1470 C/T, -1355 G/C and -1098 G/A [SLC40A1]) moontlik die verhoogde opname van yster kan veroorsaak, wat later die toename van die patogeniese infeksie kan veroorsaak. Die bevindinge van hierdie studie kan moontlik bydra tot die toeligting van die presiese meganisme(s) waardeur yster, yster metabolisme, en die betrokke gene vatbaarheid vir siekte, meer spesifiek M. tuberculosis vatbaarheid, beïnvloed.

Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul / The oomycete Pythium insidiosum, classified in the kingdom Straminipila, is the agent of pythiosis, a chronic disease that can be hard to treat and life-threatening. The predisposing factors in mammals are unknown, but the iron overload is suspected to act directly or indirectly in the development of the infection. This aim of this study was: to evaluate the iron metabolism in rabbits with experimental pythiosis; to verify the antifungal activity and the mechanism of action in vitro of the iron chelator deferasirox against P. insidiosum isolates and to compared the treatment and the mechanism of action of the drug with immunotherapy in vivo; to verify the antifungal activity of micafungin, alone and in combination with deferasirox, in vitro and in vivo; to develop an experimental model of pythiosis using the fruit-fly Drosophila melanogaster with iron overload. The drug susceptibility tests included 17 P. insidiosum clinical isolates, and were performed according to the CLSI M38-A2 guidelines. The mechanism of action of deferasirox was evaluated by the XTT and DiBAC assays. For the in vivo tests, five P. insidiosum infected rabbits were included per group, as follows: infected treated with placebo, treated with immunotherapy, treated with deferasirox, treated with immunotherapy and deferasirox, treated with micafungin and treated with micafungin and deferasirox. Five uninfected rabbits were used as controls. Hematological and biochemical analyses were performed at days 0 25, 50 and 75 post-infection. The quantification of iron in the hepatocytes was performed after 50 days of treatment (day 75). The mechanism of action of deferasirox was evaluated by the quantification of the catabolic enzyme adenosine deaminase and by the histology of the subcutaneous lesions. The results show that P. insidiosum is poorly susceptible to deferasirox in vitro, with minimum inhibition concentrations (MICs) ranging from 12.5 to 50 μg/ml and minimum fungicidal concentrations between 50 and 100 μg/ml. No activity against P. insidiosum was observed for micafungin in vitro (MICs > 128 μg/ml) and in rabbits with pythiosis. Notwithstanding, synergism was observed in 88% of the isolates when micafungin was combined with deferasirox, and the lesions were decreased in comparison to the other groups (P = 0.06). In general, the rabbits showed microcytic hypochromic anemia with depleted iron stores, which was less prominent in the groups treated with immunotherapy or deferasirox. Despite the iron chelator has failed to thwart the infection, deferasirox showed toxicity against P. insidiosum hyphae in vitro and an immunomodulatory action was observed in the lesions, in a similar pattern to that seen in immunotherapy-treated rabbits. However, the use of deferasirox favored the dissemination of the disease to the lung by unrevealed mechanisms. D. melanogaster flies were resistant to P. insidiosum infection, independently of the iron overload. Conversely, toll-deficient D. melanogaster flies were susceptible to the infection, highlighting the importance of these receptors in pythiosis. In conclusion, rabbits with pythiosis showed iron deficiency anemia and, despite the chelating effect of deferasirox, the administration of the drug should be evaluated with care because of the dissemination of the disease observed in some of the treated animals. / O oomiceto Pythium insidiosum, classificado no Reino Straminipila, é o agente etiológico da pitiose, uma doença crônica, de difícil tratamento e que pode levar à morte. Os fatores predisponentes da doença em mamíferos não são conhecidos, mas suspeita-se que a sobrecarga de ferro exerça um papel direto ou indireto no desenvolvimento da infecção. Este estudo teve por objetivos: avaliar o metabolismo do ferro em coelhos com pitiose experimental; verificar a atividade antifúngica e o mecanismo de ação in vitro do quelante de ferro deferasirox frente a isolados de P. insidiosum e comparar o tratamento e o mecanismo de ação do fármaco com a imunoterapia in vivo; verificar a atividade antifúngica da micafungina, sozinha e em combinação com deferasirox, in vitro e in vivo; desenvolver um modelo experimental de pitiose utilizando-se a mosca-das-frutas Drosophila melanogaster com sobrecarga de ferro. Os testes de susceptibilidade aos fármacos foram realizados com 17 isolados clínicos de P. insidiosum, utilizando-se o protocolo de microdiluição M38-A2 do CLSI. O mecanismo de ação in vitro do deferasirox foi avaliado através da técnica de XTT e DiBAC. Para os testes in vivo, foram utilizados cinco coelhos infectados por P. insdiosum por grupo, conforme o delineamento a seguir: infectados tratados com placebo, tratados com imunoterápico, tratados com deferasirox, tratados com imunoterápico e deferasirox, tratados com micafungina e tratados com micafungina e deferasirox. Cinco coelhos não infectados foram utilizados como controle negativo. Realizaram-se avaliações hematológicas e bioquímicas, incluindo os níveis séricos de ferro, transferrina e ferritina nos dias 0, 25, 50 e 75 pós-infecção. A quantificação do ferro depositado nos hepatócitos foi realizada após 50 dias de tratamento (dia 75). O mecanismo de ação do deferasirox in vivo foi avaliado através da mensuração da atividade da enzima catabólica adenosina deaminase e da histologia das lesões subcutâneas. Os resultados mostram que P. insidiosum demonstrou ser pouco susceptível ao deferasirox in vitro, com concentrações inibitórias mínimas (CIMs) que variaram de 12,5 a 50 μg/ml e concentrações fungicidas mínimas entre 50 e 100 μg/ml. A micafungina não apresentou atividade antifúngica contra P. insidiosum in vitro (CIMs > 128 μg/ml) e nos coelhos com pitiose. Contudo, sinergismo foi observado em 88% dos isolados quando a micafungina foi associada ao deferasirox, e as lesões dos coelhos estavam diminuídas em relação aos demais grupos (P = 0,06). No geral, os coelhos infectados apresentaram anemia microcítica hipocrômica com depleção das reservas corporais de ferro, que foi menos acentuada nos grupos tratados com imunoterápico ou quelante de ferro. Apesar do deferasirox não ter sido capaz de conter a infecção, o mesmo teve ação tóxica sobre as hifas de P. insidiosum in vitro e teve ação imunomodulatória sobre as lesões, em um padrão similar ao observado nos coelhos tratados com imunoterapia. Contudo, o uso do deferasirox favoreceu a disseminação pulmonar da doença por mecanismos não compreendidos. Moscas D. melanogaster foram resistentes à infecção por P. insidiosum, independente da sobrecarga de ferro. Entretanto, D. melanogaster deficientes de receptores do tipo Toll foram susceptíveis à infecção, evidenciando a importância desses receptores na pitiose. Em conclusão, coelhos com pitiose apresentaram anemia por deficiência de ferro e, apesar do efeito quelante do deferasirox, a utilização do mesmo deve ser avaliada com cuidado devido à disseminação da doença em alguns animais.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-09-27T20:41:15Z No. of bitstreams: 1 DissWJSD.pdf: 2131217 bytes, checksum: 65c3deee90f5da124b4a13c80563af29 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-04T18:45:59Z (GMT) No. of bitstreams: 1 DissWJSD.pdf: 2131217 bytes, checksum: 65c3deee90f5da124b4a13c80563af29 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-04T18:46:07Z (GMT) No. of bitstreams: 1 DissWJSD.pdf: 2131217 bytes, checksum: 65c3deee90f5da124b4a13c80563af29 (MD5) / Made available in DSpace on 2016-10-04T18:46:15Z (GMT). No. of bitstreams: 1 DissWJSD.pdf: 2131217 bytes, checksum: 65c3deee90f5da124b4a13c80563af29 (MD5) Previous issue date: 2015-08-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Iron (Fe) is an essential micronutrient for cellular homeostasis. Structural component of proteins or enzyme cofactor, Fe has participation in important metabolic pathways that include oxidative metabolism, oxygen transport, cell proliferation and immune system function. Despite of its essentiality, Fe has a toxic potential to cells when in excess. So, a sophisticated system is needed to coordinate the process of absorption, recycling, use and storage. Mutations in genes related to homeostasis of this mineral may potentially alter the cellular distribution and storage. Furthermore, the Fe levels affect biological pathways such as carbohydrate and lipid metabolism. Iron content in cattle muscle has been associated with many sensory and technological parameters of meat quality. However, to date, studies that evaluate how the iron levels in the muscle can alter gene expression and the consequences for the metabolism in cattle are still absent. Therefore, this study aims to identify differentially expressed genes, metabolic pathways, gene interactions and potential regulatory biological mechanisms of physiological processes related to meat quality parameters. Longissimus dorsi (LD) muscle were collected at slaughter for total RNA extraction and determination of CFe by optical emission spectrometry (ICP OES). Eight Nelore steers, who are representatives of extreme value for Genetic Genomic Estimate (GEBV) for iron content (CFe), were selected from a reference population of 373 animals. The sequencing of the total mRNA of extreme animals was carried out from the next generation Illumina technology, which resulted in average l9.13 million of reads per sample after quality control and trimming. Data analysis carried out by Tuxedo Suite pipeline identified 49 annotated and differentially expressed genes (DE) (FDR <0.05) between groups of extremes for GEBV value for CFe. From the DE genes, 18 genes were up-regulated and 31 down-regulated for animals of low GEBV for CFe. Candidate genes for meat quality traits were identified in this study and they are related to transport and lipid metabolism. Other pathways identified through functional enrichment analysis include cell growth and development, function of the hematological system, among others. Canonical signaling pathways (interferon signaling, thyroid receptor activation (TR/RXR) and complement system) and canonical metabolic pathways (biosynthesis of stearate, fatty acid biosynthesis and palmitate biosynthesis) were also identified. Although this study did not identify genes with direct role in the regulation of Fe content, our results suggest biological pathways influenced by this mineral and contribute with information to the understanding of their participation in processes affecting quality of meat. This information will be useful in developing strategies that contribute to the production of better quality meat, healthy and nutritionally rich. In addition, this information may help in understanding of metabolic disorders in other species, including humans. / O ferro (Fe) é um micronutriente essencial à homeostase celular. Necessário como componente estrutural de proteínas ou cofator enzimático, o Fe participa de vias metabólicas importantes que incluem metabolismo oxidativo, transporte de oxigênio, proliferação celular e funcionamento do sistema imune. Apesar de essencial, apresenta um potencial tóxico às células quando em excesso. Por isso, é necessário um sofisticado sistema que coordene os processos de absorção, reciclagem, uso e armazenamento. Mutações em genes relacionados à homeostase desse mineral podem potencialmente alterar a sua distribuição e armazenamento celular. Ademais, os níveis de Fe afetam vias biológicas, tais como metabolismo de carboidratos e lipídeos. O conteúdo de Fe no músculo em bovinos tem sido associado a diversos parâmetros sensoriais e tecnológicos de qualidade de carne. Entretanto, até a presente data, são escassos estudos que avaliem como os níveis de ferro no músculo podem alterar a expressão gênica e quais as consequências para o metabolismo em bovinos. Portanto, o presente estudo tem como objetivo principal identificar genes diferencialmente expressos, vias metabólicas, interações gênicas e potenciais mecanismos biológicos que participam de processos fisiológicos relacionados à regulação do ferro e de parâmetros de qualidade da carne. Amostras do músculo Longissimus dorsi (LD) foram coletadas no momento do abate para extração de RNA total e determinação do conteúdo de ferro (CFe) por espectrometria de emissão óptica (ICP OES). Oito machos castrados da raça Nelore, representantes dos extremos para Valor Genético Genômico Estimado (GEBV) para CFe foram selecionados a partir de uma população referência de 373 animais. O equenciamento do mRNA total dos animais extremos foi realizado a partir da tecnologia de nova geração Illumina, o qual resultou em média 9,13 milhões de reads por amostra após o controle de qualidade. Por meio da análise de dados realizada pelo Tuxedo Suíte pipeline foram identificados 49 genes anotados diferencialmente expressos (DE) (FDR <0,05) entre os grupos de extremos para o valor de GEBV para CFe. Dentre os genes DE, 18 genes apresentaram-se up-regulated e 31 down-regulated para os animais do grupo de baixo GEBV para CFe. Genes candidatos para características de qualidade de carne foram identificados no presente estudo e estão relacionados ao transporte e metabolismo de lipídeos. Outras vias identificadas por meio das análises de enriquecimento funcional incluem crescimento e desenvolvimento celular, função do sistema hematológico, entre outras. Vias canônicas de sinalização (sinalização do interferon, ativação do receptor da tireóide (TR/RXR) e sistema complemento) e metabólicas (biossíntese do estearato, biossíntese de ácidos graxos e biossíntese do palmitato) foram também identificadas. Embora o presente estudo não tenha identificado genes com papel direto na regulação do conteúdo de Fe, nossos resultados apontam rotas biológicas influenciadas por esse mineral e contribui com informações para o entendimento da sua participação em vias que afetem a qualidade da carne. Essas informações serão úteis no desenvolvimento de estratégias que contribuam para a produção de carne de qualidade, saudável e nutricionalmente rica. Além disso, essas informações poderão auxiliar no entendimento de distúrbios metabólicos em outras espécies, inclusive a humana.

Made available in DSpace on 2014-12-17T14:03:39Z (GMT). No. of bitstreams: 1 PauloRPN_DISSERT.pdf: 1361148 bytes, checksum: 4572b4a963991f34258d766f8d1c322f (MD5) Previous issue date: 2012-04-23 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Iron is an essential element for many cellular functions, including the immune response against intracellular pathogens. In this study, we aimed evaluate the effect of iron on IRP2, IFN-?, TNF-?, IL-6, IL-10, MIG and IP10 expression in PBMC and assess the effect of the spleen parasite load on the expression of these genes in the spleen of L. infantum naturally infected dogs. Blood sample from 7 DTH+ donor was collected and PBMC was obtained. The cells were cultivated in absence (iron chelator desferroximane, DFO 10 ?M supplemented media) or in presence of iron (hemin 6 mM) for 1 h, followed by stimulation with Leishmania infatum antigen for 4 h. 44 dog spleen samples were obtained and parasite load in this organ was determinate by qPCR. Gene expression was analyzed by qPCR and cytokine production quantified by flow cytometry. In antigen stimulated cells, genes involved in immune response are significantly more expressed in presence of iron. T CD4+ and TCD8+ lymphocytes produces IFN-?, TNF-? and IL-10 possibly in iron dependent pathway. Monocytes antigen stimulated reduced TNF-?, IL-6 and IL-10 production in presence of iron. We found spleen of infected dogs IRP2 expression increases according to parasite load in that organ, while an inverse profile was found for IFN-?, TNF-? e IL-10 expression. These results suggest that T lymphocytes depends on iron to produce IFN-?, TNF-? and IL-10, while iron seems to inhibit cytokine production in monocytes. So, we propose an immunoregulatory mechanism carried out by iron during L. infantum infection in humans and dogs / O ferro ? um elemento essencial para diversas fun??es celulares, incluindo a resposta imune a pat?genos. O objetivo deste trabalho foi avaliar o efeito do ferro sobre a express?o dos genes IRP2, IFN-?, TNF-?, IL-6, IL-10, MIG e IP10 em c?lulas mononucleares de sangue perif?rico (CMSP) humano e examinar o efeito da carga parasit?ria espl?nica sobre a express?o destes genes no ba?o de c?es naturalmente infectados por L. infantum. Coletou-se amostras de sangue perif?rico de 7 indiv?duos DTH+ e obteve-se as CMSP. As c?lulas foram cultivadas na aus?ncia (quelante deferoxamina 10 ?M) ou presen?a de ferro (Hemina 6 mM) por 1 hora seguido de estimula??o com ant?geno de Leishmania infantum por 4 horas. Obteve-se o ba?o de 44 c?es e determinouse a carga parasit?ria neste ?rg?o por qPCR. A express?o g?nica foi avaliada por qPCR e a produ??o de citocinas foi quantificada por citometria de fluxo. Encontramos que na presen?a de ferro a express?o de genes envolvidos na resposta imune ? significativamente aumentada em CMSP. Linf?citos T CD4+ e TCD8+ produzem IFN-?, TNF-? e IL-10 possivelmente por uma via dependente de ferro. Em mon?citos a produ??o de TNF-?, IL-6 e IL-10 foram reduzidas em c?lulas estimuladas e cultivadas na presen?a do metal. Foi visto que no ba?o de c?es a express?o de IRP2 aumenta de acordo com a carga parasit?ria, sendo o inverso encontrado para IFN-?, TNF-? e IL-10. Os resultados sugerem que linf?citos T dependem de ferro para produzir IFN-?, TNF-? e IL-10, enquanto em mon?citos o ferro parece ter efeito inibit?rio sobre a produ??o de citocinas, sugerindo um efeito imunomodulat?rio para o ferro em humanos e c?es durante a leishmaniose visceral