Conductive Atomic Force Microscope (c-AFM) was used to measure the conductivity of single horse spleen ferritin (HoSF) and azotobacter vinelandii bacterial ferritin (AvBF) molecules deposited on flat gold surfaces. A 500 micron diameter gold ball was also used as a contact probe to measure the conductivity of a thin film of ferritin molecules. The average current measured for holo HoSF was 13 and 5 times larger than that measured for apo HoSF as measured by c-AFM at 1V and gold ball at 2V and respectively, which indicates that the core of ferritin is more conductive than the protein shell and that conduction through the shell is likely the main factor limiting electron transfer. With 1 volt applied, the average electrical currents through single holo HoSF and single apo HoSF molecules were 2.6 pA and 0.19 pA respectively. Measurements on holo AvBF showed it was more than 10 times as conductive as holo HoSF, indicating that the protein shell of AvBF is more conductive than that of HoSF. The increased conductivity of AvBF is attributed to heme groups in the protein shell.

Le cancer épithélial de l’ovaire est le cancer gynécologique le plus agressif avec le plus haut taux de mortalité. La croissance des cellules cancéreuses de l’ovaire est limitée par les nutriments de l’environnement, le fer étant un des éléments indispensables à leur prolifération. L’hémochromatose héréditaire est une maladie associée à une accumulation corporelle de fer. Cette maladie est liée à deux mutations majeures du gène HFE soit H63D et C282Y. Étant donnée l’influence de la protéine HFE sur l’entrée du fer dans la cellule, des mutations du gène HFE pourraient être associées à une croissance rapide des cellules cancéreuses. Des études de génotypage du gène HFE effectuées chez 526 patientes avec cancer épithélial de l’ovaire, ont révélées une fréquence allélique de la mutation C282Y significativement plus élevées chez les patientes avec tumeur ovarienne comparativement aux patientes du groupe contrôle (5.9% versus 1.3%, p = 0.02). De plus, le taux de survie des patientes avec mutations C282Y et tumeur ovarienne de G3, après 2 ans, est faible (20%) lorsque comparé à celui des patientes sans mutations (60%, p = 0.005). Une analyse de régression multivariée de Cox a démontrée un risque relatif de 3.1, suggérant que les patientes avec mutations C282Y ont 3 fois plus de chance d’avoir une faible survie (p=0.001). Également, des études de corrélation ont démontrées que les niveaux de ferritine du sérum étaient plus élevés chez les patientes avec grade avancé du cancer épithélial de l’ovaire (r = 0.445 et p= 0.00001), suggérant que ce paramètre pourrait servir comme marqueur tumoral. Afin de comprendre ces résultats, nous avons tout d’abord étudiés l’influence des mutations HFE sur les cellules cancéreuses. Pour ce faire, la lignée du cancer de l’ovaire TOV-112D, homozygote pour la mutation C282Y, a été transfectée avec les vecteurs HFEwt et HFEC282Y. Bien qu’aucune différence significative n’ait été trouvée en termes de TfR totaux, des analyses par FACS ont démontrées un phénotype de déficience de fer pour les clones stables HFEwt. In vitro, la restauration de la protéine HFE, dans la lignée TOV-112D du cancer de l’ovaire, n’influence pas la croissance cellulaire. Ensuite, nous avons étudiés l’influence des niveaux de fer sur la progression tumorale. Une expérience in vivo préliminaire a démontré une tendance à un volume tumoral supérieur dans un modèle de souris de surcharge de fer,HfeRag1-/-. De plus, les souris HfeRag1-/-, injectées avec la lignée du cancer de l’ovaire TOV-21G, ont montrées des niveaux significativement plus faibles de fer sérique comparativement à leur contrôle (fer sérique 40±7μM versus 27±6μM, p = 0.001). En conclusion, des études supplémentaires sont nécessaires afin de comprendre davantage le rôle des mutations HFE sur la progression tumorale. Notamment, les niveaux élevés de fer pourraient rendre les cellules tumorales résistantes aux traitements ou encore, augmenter la toxicité et ainsi, contribuer à un mauvais prognostique. / Epithelial ovarian cancer is the most aggressive gynecological cancer with the highest mortality rates. Growth of the ovarian cancer cells is limited by nutrients in the environment; iron being one of the elements essential to their proliferation. Hereditary hemochromatosis is a disease associated with an accumulation of body iron, and is linked to two mutations of the HFE gene including C282Y and H63D. Given the influence of HFE protein on the entry of iron in the cell, mutations in the HFE gene may be associated with rapid growth of cancer cells. By genotyping the HFE gene of 526 patients with epithelial ovarian cancer, we have found that the allelic frequency of the C282Y mutation is significantly higher in patients with ovarian cancer compared to patients in the control group (5.9% versus 1.3% p = 0.02). Moreover, the 2-year survival rate, of patients with C282Y mutations and G3 ovarian tumor, is low (20%) when compared to patients without mutations (60%, p = 0.005). A multivariate survival analysis, using Cox’s regression model, also showed a hazard ratio value of 3.1,suggesting that patients with the C282Y mutation are 3 times more likely to have a poor survival (p =0.001). As well, correlation studies have demonstrated that serum ferritin levels were higher in patients with advanced grade of ovarian cancer (r = 0.445 and p = 0.00001), suggesting that this iron parameter could serve as a tumor marker for assessing the progression of ovarian cancer. In order to investigate these findings, we first studied the influence of HFE mutations on cancer cells. The ovarian cancer cell line TOV-112D, homozygous for the C282Y mutation, was transfected with the HFEwt and HFEC282Y vector, and although there were no differences in total TfR levels, FACS analysis demonstrated an iron deficient phenotype for the HFEwt stable clones (p<0.05). In vitro,restoration of the HFE protein, in the TOV-112D ovarian cancer cell line, does not influence cell growth. We then studied the influence of iron levels on tumor progression. A preliminary in vivo experiment has demonstrated a tendency to a higher tumor volume in a mouse model of iron overload,HfeRag1-/-. Furthermore, HfeRag1-/- mice that were injected with the ovarian cancer cell line TOV-21G showed significant lower serum iron levels compared to their controls (SI 40±7μM versus 27±6μM, p = 0.001). In conclusion, more studies are required to further understand the role of HFE mutations on tumor progression. Higher iron levels may confer tumor cells to be drug resistant or increase toxicity and thus, may contribute to a bad prognostic.

<p>Belonging to an indigenous group in Mexico is usually associated with poor health, mainly as the result of social isolation from the mainstream society. The Tarahumara are no exception. They constitute the largest indigenous group in northern Mexico and one of the most marginalized ethnic minorities in North America. Health conditions are precarious, yet very little data are available to facilitate the design and implementation of programs to prevent and manage the main public health problems affecting this people. This thesis aims at overcoming part of this information gap. It presents and discusses the results from studies focusing on the nutrition of women and children carried out between 1997 and 2002.</p><p>A survey in a representative district sample of Tarahumara women of reproductive age found the highest prevalence of anemia among pregnant women in their third trimester (38.5%) and those lactating during the first 6 months after delivery (42.9%), along with a high prevalence of iron deficiency. In this study a technique was developed to collect capillary serum samples spotted onto filter paper to measure serum ferritin in remote settings. In the same study, 52.5% of adult women were overweight, suggesting a process of ‘de-Indianization’ of their traditional diet and activity patterns. This issue was followed-up in a later study based on perceptions of food and body shape using cognitive anthropological methods. Speaking Spanish emerged as a clear indication of acculturation that could be associated with an increase in the prevalence of obesity and its consequences. A nutrition survey among Tarahumara children at boarding schools found evidence of zinc, vitamin B₁₂, iron, and iodine deficiencies but found similar anthropometric status to other rural Mexicans. Finally, a qualitative assessment was carried out to identify culturally accepted foods to redesign a food aid basket aimed at alleviating malnutrition among young Tarahumara children.</p><p>The results from this thesis provide relevant data for an improved design of interventions to combat and prevent some of the nutritional problems that affect the Tarahumara. These data could also constitute a baseline to which future changes can be compared if similar sampling strategies are used. Overall, the findings highlight the importance and challenge of achieving modernization in a way that not only improves health but at the same time supports, maintains and encourages traditional cultural values. These are not only the foundations of the Tarahumara society, but in some cases also contribute to a better diet and health.</p> / <p>Die Zugehörigkeit zu einer eingeborenen Volksgemeinschaft Mexikos wird gewöhnlich mit einem schlechten Gesundheitszustand, aufgrund sozialer Isolation von der allgemeinen Gesellschaft, verbunden. Die Tarahumara-Indianer sind dabei keine Ausnahme. Sie stellen eine der größten Eingeborenengruppen im Norden des Landes dar und sind eine der ausgeschlossensten ethnischen Minderheiten in Nordamerika. Der Gesundheitszustand ist prekär, da sehr wenige Daten existieren, um die Gestaltung und Einführung von Programmen zur Prävention und Handhabung der, diese Menschen betreffenden, hauptsächlichen Probleme im Gesundheitswesen, zu ermöglichen. Diese Dissertation beabsichtigt, Teil dieses Informationsdefizits zu beseitigen. Sie präsentiert und diskutiert die Ergebnisse von im Zeitraum 1997 bis 2002 durchgeführten Studien, welche die Ernährung der Tarahumarafrauen und -kinder fokussieren.</p><p>Eine Umfrage mit einer repräsentativen Stichprobe von Frauen im gebärfähigen Alter, im größten Tarahumara-Bezirk, ergab das höchste Vorkommen von Anämie bei schwangeren Frauen im dritten Trimester (38,5%) und bei solchen, die während der ersten 6 Monate nach der Geburt stillten (42,9%), bedingt durch Eisenmangel. Bei dieser Studie wurde eine Feldtechnik für weit entfernte Gebiete entwickelt, um die Ferritin-Konzentration in Kapillar-Serum auf Filter Papier zu messen. Dieselbe Studie zeigte eine Übergewichtsprävalenz von 52,5% bei erwachsenen Frauen, was auf einen Prozess einer „Entindianisierung“ ihrer traditionellen Diät und Aktivitätsmuster zurückzuführen ist. Dieses Thema wurde bei einer späteren Studie herangezogen, bei welcher der Eindruck von Nahrung und Körperumfang mit kognitiven anthropologischen Methoden evaluiert wurde. Spanisch zu sprechen erschien als eindeutige Indikation für Akkulturation, welche mit einer Zunahme des Vorhandenseins von Übergewicht und seiner Folgen assoziiert werden könnte. Eine Studie zu Schulkindern in Eingeboreneninternaten zeigte Beweise für Zink-, Vitamin B₁₂-, Eisen- und Jodmangel, fand aber ähnliche anthropometrische Status wie bei ländlichen Mexikanerkindern. Schließlich wurde eine qualitative Studie durchgeführt, mit dem Ziel, kulturell akzeptierte Lebensmittel für die Neuentwerfung eines Warenkorbes zu identifizieren, um den Ernährungszustand von Kleinkindern zu verbessern.</p><p>Die Ergebnisse dieser Dissertation liefern relevante Daten für eine Verbesserung der Gestaltung von Programmen zur Bekämpfung und Prävention von Ernährungsproblemen, welche die Tarahumaras betreffen. Diese Informationen können auch als „Baseline“ benutzt werden, mit der zukünftige Veränderungen verglichen werden könnten, wenn ähnliche Stichprobenstrategien angewandt würden. Vor allem betonen die Ergebnisse, die Wichtigkeit und Herausforderung, eine Modernisierung zu erreichen, die nicht nur eine Verbesserung der Gesundheit mit sich bringt, sondern gleichzeitig auch, traditionelle Werte unterstützt, aufrechterhält und anregt, da diese Werte nicht nur die Grundlagen der Tarahumara- Gesellschaft sind, sondern in vielen Fällen zu einer besseren Diät und Gesundheit beisteuern.</p> / <p>Att tillhöra en infödd folkgrupp i Mexiko associeras oftast med ett dåligt hälsotillstånd, framför allt på grund av social isolering från det konventionella samhället. Tarahumara indianerna utgör inget undantag. De utgör den största gruppen av infödda i norra Mexiko och är en av de mest utsatta etniska minoriteterna i Nord Amerika. Det finns anledning att oroa sig för de rådande hälsovillkoren då mycket lite information finns tillgänglig för att underlätta utformandet och tillämpningen av program för att förebygga och handskas med de huvudsakliga hälsoproblemen som drabbar denna folkgrupp. Denna avhandling syftar till att försöka täcka upp delar av den informations brist som råder. I den presenteras och diskuteras resultaten från de studier, som inriktar sig på näringstillståndet hos tarahumara kvinnor och barn, genomförda mellan åren 1997 och 2002. </p><p>En studie i ett representativt distrikt med ett representativt urval av Tarahumara kvinnor i fertil ålder fann man högst prevalens av anemi bland de gravida kvinnorna som befann sig i sista trimestern (38,5 %) samt i gruppen ammande kvinnor under de 6 första månaderna efter förlossning (42,9 %), detta tillsammans med en hög prevalens av järnbrist. I denna studie utvecklades en metod för insamling av kapillära serum prover som droppades på filter papper för att därefter analysera serum ferritin halten vid avsides liggande sättningar. I samma studie fann man även att 52,5 % av de vuxna kvinnorna var överviktiga, vilket skulle kunna antyda om en “avindianiserings-process” av deras traditionella diet och aktivitets mönster. Detta fynd följdes upp i en senare studie som grundade sig på föreställningar om mat och kroppsform, genom att använda kognitiva antropologiska metoder. Att vara spansktalande framträdde som ett tydligt tecken på kulturförändring som skulle kunna sammankopplas med en ökning i prevalensen av övervikt och dess konsekvenser. En skolbaserad nutritions studie bland Tarahumara barn vid internatskolor visade brist på zink, vitamin B₁₂, järn och jod, dock var dessa fynd likvärdiga med uppmätta värden bland barn på den mexikanska landsbygden. Slutligen genomfördes en kvalitativ studie med avsikt att identifiera kulturellt accepterade maträtter och därigenom kunna omforma regeringens rådande sammansättning av livsmedelsbistånd, med syfte att mildra undernäringen bland unga Tarahumara barn.</p><p>Resultaten från denna avhandling ger relevanta data för en förbättrad utformning av interventionsprogram för att bekämpa och förhindra en del av de nutritions problem som drabbar Tarahumara indianerna. Dessa data skulle också kunna utgöra en referenslinje med vilken framtida förändringar kan jämföras med såvida liknande provtagnings rutiner används. Generellt, belyser resultaten vikten och utmaningen att uppnå modernisering på ett sätt som inte enbart förbättrar hälsoläget men som samtidigt upprätthåller och uppmuntrar till att behålla traditionella värderingar. Dessa utgör inte enbart grunden för Tarahumara samhället utan bidrar även därigenom i en del fall till en bättre kosthållning och bättre hälsa.</p> / <p>La pertenencia a un grupo indígena en México se asocia frecuentemente a una salud pobre principalmente como resultado del aislamiento social de la sociedad Mexicana. Los Tarahumaras no son la excepción. Constituyen el grupo indígena más grande del norte del país y una de las minorías étnicas más marginadas de Norteamérica. A pesar de que sus condiciones de salud son precarias, existe muy poca información disponible que facilite el diseño e implementación de programas para prevenir y tratar los problemas de salud pública más importantes que les aquejan. Así pues, esta tesis tiene por objeto cubrir parte de esta falta de información. Presenta y discute resultados de estudios enfocados a la nutrición de mujeres y niños llevados a cabo entre 1997 y 2002.</p><p>Una encuesta en una muestra municipal representativa de mujeres Tarahumaras en edad reproductiva mostró la más alta prevalencia de anemia en las embarazadas en el tercer trimestre (38.5%) y las lactantes durante los primeros 6 meses después del parto (42.9%) paralelamente a una alta prevalencia de deficiencia de hierro. En este estudio, se desarrolló una técnica para la toma de muestras de suero capilar en papel filtro para medir los niveles de ferritina sérica en zonas remotas. Asimismo se encontró un 52.5% de sobrepeso en las mujeres adultas, sugiriendo un proceso de “deindigenización” de los patrones dietéticos y de actividad física tradicionales. Este tópico fue seguido en un estudio posterior sobre percepciones de la alimentación y apariencia corporal de la mujer Tarahumara utilizando métodos de antropología cognoscitiva. Hablar español emergió como un claro indicio de aculturación que podría estar asociado a un incremento en la prevalencia de obesidad y sus consecuencias. Una encuesta nutricional con niños Tarahumaras de albergues escolares mostró evidencia de deficiencia de cinc, vitamina B₁₂, hierro y yodo pero encontró un estado antropométrico similar al de otros niños mexicanos del medio rural. Finalmente, se condujo una evaluación cualitativa para identificar alimentos culturalmente aceptables para rediseñar una canasta de ayuda alimentaria con el objeto de aliviar la desnutrición infantil.</p><p>Los resultados de esta tesis ofrecen información relevante para el mejoramiento del diseño de intervenciones para combatir y prevenir algunos de los problemas nutricios que afectan a los Tarahumaras. De utilizarse estrategias muestrales similares, esta información podría además constituir el punto de comparación para evaluar cambios futuros. Pero sobre todo, los hallazgos apuntan a la importancia y el desafío para alcanzar una modernización que no solo mejore la salud de los indígenas, sino que además apoye, mantenga y promueva los valores culturales tradicionales, pues estos, además de conformar los cimientos de la sociedad Tarahumara, pueden en varios casos contribuir a una mejor nutrición y salud.</p>

Background Due to small iron stores and rapid growth during the first months of life, infants with low birth weight (LBW) are at risk of iron deficiency (ID). ID in infancy is associated with irreversible impaired neurodevelopment. Preventive iron supplementation may reduce the risk of ID and benefit neurodevelopment, but there is also a possible risk of adverse effects. More than 50% of all LBW infants are born with marginally LBW (MLBW, 2000-2500g), and it is not known if they benefit from iron supplementation. Methods We randomized 285 healthy, Swedish, MLBW infants to receive 3 different doses of oral iron supplements; 0 (Placebo), 1, and 2 mg/kg/day from six weeks to six months of age. Iron status, during and after the intervention was assessed and so was the prevalence of ID and ID anemia (IDA), growth, morbidity and the interplay with iron and the erythropoetic hormones hepcidin and erythropoietin (EPO). As a proxy for conduction speed in the developing brain, auditory brainstem response (ABR) was analyzed at six months. In a follow up at 3.5 years of age, the children were assessed with a cognitive test (WPPSI-III) and a validated parental checklist of behavioral problems (CBCL), and compared to a matched reference group of 95 children born with normal birth weight. Results At six months of age, the prevalence of ID and IDA was significantly higher in the placebo group compared to the iron supplemented infants. 36% had ID in the placebo group, compared to 8% and 4 % in the 1 and 2mg/kg/day-groups, respectively. The prevalence of IDA was 10%, 3% and 0%, respectively. ABR-latencies did not correlate with the iron intake and was not increased in infants with ID or IDA. ABR wave V latencies were similar in all three groups. Hepcidin correlated to ferritin and increased in supplemented infants while EPO, which was negatively correlated to iron status indicators, decreased. At follow up there were no differences in cognitive scores between the groups but the prevalence of behavioral problems was significantly higher in the placebo group compared to those supplemented and to controls. The relative risk increase of CBCL-scores above a validated cutoff was 4.5 (1.4 – 14.2) in the placebo-group compared to supplemented children. There was no detected difference in growth or morbidity at any age. Conclusion MLBW infants are at risk of ID in infancy and behavioral problems at 3 years of age. Iron supplementation at a dose of 1-2 mg/kg/day from six weeks to six months of age reduces the risks with no adverse effects, suggesting both short and long term benefit. MLBW infants should be included in general iron supplementation programs during their first six months of life.

Belonging to an indigenous group in Mexico is usually associated with poor health, mainly as the result of social isolation from the mainstream society. The Tarahumara are no exception. They constitute the largest indigenous group in northern Mexico and one of the most marginalized ethnic minorities in North America. Health conditions are precarious, yet very little data are available to facilitate the design and implementation of programs to prevent and manage the main public health problems affecting this people. This thesis aims at overcoming part of this information gap. It presents and discusses the results from studies focusing on the nutrition of women and children carried out between 1997 and 2002. A survey in a representative district sample of Tarahumara women of reproductive age found the highest prevalence of anemia among pregnant women in their third trimester (38.5%) and those lactating during the first 6 months after delivery (42.9%), along with a high prevalence of iron deficiency. In this study a technique was developed to collect capillary serum samples spotted onto filter paper to measure serum ferritin in remote settings. In the same study, 52.5% of adult women were overweight, suggesting a process of ‘de-Indianization’ of their traditional diet and activity patterns. This issue was followed-up in a later study based on perceptions of food and body shape using cognitive anthropological methods. Speaking Spanish emerged as a clear indication of acculturation that could be associated with an increase in the prevalence of obesity and its consequences. A nutrition survey among Tarahumara children at boarding schools found evidence of zinc, vitamin B12, iron, and iodine deficiencies but found similar anthropometric status to other rural Mexicans. Finally, a qualitative assessment was carried out to identify culturally accepted foods to redesign a food aid basket aimed at alleviating malnutrition among young Tarahumara children. The results from this thesis provide relevant data for an improved design of interventions to combat and prevent some of the nutritional problems that affect the Tarahumara. These data could also constitute a baseline to which future changes can be compared if similar sampling strategies are used. Overall, the findings highlight the importance and challenge of achieving modernization in a way that not only improves health but at the same time supports, maintains and encourages traditional cultural values. These are not only the foundations of the Tarahumara society, but in some cases also contribute to a better diet and health. / Die Zugehörigkeit zu einer eingeborenen Volksgemeinschaft Mexikos wird gewöhnlich mit einem schlechten Gesundheitszustand, aufgrund sozialer Isolation von der allgemeinen Gesellschaft, verbunden. Die Tarahumara-Indianer sind dabei keine Ausnahme. Sie stellen eine der größten Eingeborenengruppen im Norden des Landes dar und sind eine der ausgeschlossensten ethnischen Minderheiten in Nordamerika. Der Gesundheitszustand ist prekär, da sehr wenige Daten existieren, um die Gestaltung und Einführung von Programmen zur Prävention und Handhabung der, diese Menschen betreffenden, hauptsächlichen Probleme im Gesundheitswesen, zu ermöglichen. Diese Dissertation beabsichtigt, Teil dieses Informationsdefizits zu beseitigen. Sie präsentiert und diskutiert die Ergebnisse von im Zeitraum 1997 bis 2002 durchgeführten Studien, welche die Ernährung der Tarahumarafrauen und -kinder fokussieren. Eine Umfrage mit einer repräsentativen Stichprobe von Frauen im gebärfähigen Alter, im größten Tarahumara-Bezirk, ergab das höchste Vorkommen von Anämie bei schwangeren Frauen im dritten Trimester (38,5%) und bei solchen, die während der ersten 6 Monate nach der Geburt stillten (42,9%), bedingt durch Eisenmangel. Bei dieser Studie wurde eine Feldtechnik für weit entfernte Gebiete entwickelt, um die Ferritin-Konzentration in Kapillar-Serum auf Filter Papier zu messen. Dieselbe Studie zeigte eine Übergewichtsprävalenz von 52,5% bei erwachsenen Frauen, was auf einen Prozess einer „Entindianisierung“ ihrer traditionellen Diät und Aktivitätsmuster zurückzuführen ist. Dieses Thema wurde bei einer späteren Studie herangezogen, bei welcher der Eindruck von Nahrung und Körperumfang mit kognitiven anthropologischen Methoden evaluiert wurde. Spanisch zu sprechen erschien als eindeutige Indikation für Akkulturation, welche mit einer Zunahme des Vorhandenseins von Übergewicht und seiner Folgen assoziiert werden könnte. Eine Studie zu Schulkindern in Eingeboreneninternaten zeigte Beweise für Zink-, Vitamin B12-, Eisen- und Jodmangel, fand aber ähnliche anthropometrische Status wie bei ländlichen Mexikanerkindern. Schließlich wurde eine qualitative Studie durchgeführt, mit dem Ziel, kulturell akzeptierte Lebensmittel für die Neuentwerfung eines Warenkorbes zu identifizieren, um den Ernährungszustand von Kleinkindern zu verbessern. Die Ergebnisse dieser Dissertation liefern relevante Daten für eine Verbesserung der Gestaltung von Programmen zur Bekämpfung und Prävention von Ernährungsproblemen, welche die Tarahumaras betreffen. Diese Informationen können auch als „Baseline“ benutzt werden, mit der zukünftige Veränderungen verglichen werden könnten, wenn ähnliche Stichprobenstrategien angewandt würden. Vor allem betonen die Ergebnisse, die Wichtigkeit und Herausforderung, eine Modernisierung zu erreichen, die nicht nur eine Verbesserung der Gesundheit mit sich bringt, sondern gleichzeitig auch, traditionelle Werte unterstützt, aufrechterhält und anregt, da diese Werte nicht nur die Grundlagen der Tarahumara- Gesellschaft sind, sondern in vielen Fällen zu einer besseren Diät und Gesundheit beisteuern. / Att tillhöra en infödd folkgrupp i Mexiko associeras oftast med ett dåligt hälsotillstånd, framför allt på grund av social isolering från det konventionella samhället. Tarahumara indianerna utgör inget undantag. De utgör den största gruppen av infödda i norra Mexiko och är en av de mest utsatta etniska minoriteterna i Nord Amerika. Det finns anledning att oroa sig för de rådande hälsovillkoren då mycket lite information finns tillgänglig för att underlätta utformandet och tillämpningen av program för att förebygga och handskas med de huvudsakliga hälsoproblemen som drabbar denna folkgrupp. Denna avhandling syftar till att försöka täcka upp delar av den informations brist som råder. I den presenteras och diskuteras resultaten från de studier, som inriktar sig på näringstillståndet hos tarahumara kvinnor och barn, genomförda mellan åren 1997 och 2002. En studie i ett representativt distrikt med ett representativt urval av Tarahumara kvinnor i fertil ålder fann man högst prevalens av anemi bland de gravida kvinnorna som befann sig i sista trimestern (38,5 %) samt i gruppen ammande kvinnor under de 6 första månaderna efter förlossning (42,9 %), detta tillsammans med en hög prevalens av järnbrist. I denna studie utvecklades en metod för insamling av kapillära serum prover som droppades på filter papper för att därefter analysera serum ferritin halten vid avsides liggande sättningar. I samma studie fann man även att 52,5 % av de vuxna kvinnorna var överviktiga, vilket skulle kunna antyda om en “avindianiserings-process” av deras traditionella diet och aktivitets mönster. Detta fynd följdes upp i en senare studie som grundade sig på föreställningar om mat och kroppsform, genom att använda kognitiva antropologiska metoder. Att vara spansktalande framträdde som ett tydligt tecken på kulturförändring som skulle kunna sammankopplas med en ökning i prevalensen av övervikt och dess konsekvenser. En skolbaserad nutritions studie bland Tarahumara barn vid internatskolor visade brist på zink, vitamin B12, järn och jod, dock var dessa fynd likvärdiga med uppmätta värden bland barn på den mexikanska landsbygden. Slutligen genomfördes en kvalitativ studie med avsikt att identifiera kulturellt accepterade maträtter och därigenom kunna omforma regeringens rådande sammansättning av livsmedelsbistånd, med syfte att mildra undernäringen bland unga Tarahumara barn. Resultaten från denna avhandling ger relevanta data för en förbättrad utformning av interventionsprogram för att bekämpa och förhindra en del av de nutritions problem som drabbar Tarahumara indianerna. Dessa data skulle också kunna utgöra en referenslinje med vilken framtida förändringar kan jämföras med såvida liknande provtagnings rutiner används. Generellt, belyser resultaten vikten och utmaningen att uppnå modernisering på ett sätt som inte enbart förbättrar hälsoläget men som samtidigt upprätthåller och uppmuntrar till att behålla traditionella värderingar. Dessa utgör inte enbart grunden för Tarahumara samhället utan bidrar även därigenom i en del fall till en bättre kosthållning och bättre hälsa. / La pertenencia a un grupo indígena en México se asocia frecuentemente a una salud pobre principalmente como resultado del aislamiento social de la sociedad Mexicana. Los Tarahumaras no son la excepción. Constituyen el grupo indígena más grande del norte del país y una de las minorías étnicas más marginadas de Norteamérica. A pesar de que sus condiciones de salud son precarias, existe muy poca información disponible que facilite el diseño e implementación de programas para prevenir y tratar los problemas de salud pública más importantes que les aquejan. Así pues, esta tesis tiene por objeto cubrir parte de esta falta de información. Presenta y discute resultados de estudios enfocados a la nutrición de mujeres y niños llevados a cabo entre 1997 y 2002. Una encuesta en una muestra municipal representativa de mujeres Tarahumaras en edad reproductiva mostró la más alta prevalencia de anemia en las embarazadas en el tercer trimestre (38.5%) y las lactantes durante los primeros 6 meses después del parto (42.9%) paralelamente a una alta prevalencia de deficiencia de hierro. En este estudio, se desarrolló una técnica para la toma de muestras de suero capilar en papel filtro para medir los niveles de ferritina sérica en zonas remotas. Asimismo se encontró un 52.5% de sobrepeso en las mujeres adultas, sugiriendo un proceso de “deindigenización” de los patrones dietéticos y de actividad física tradicionales. Este tópico fue seguido en un estudio posterior sobre percepciones de la alimentación y apariencia corporal de la mujer Tarahumara utilizando métodos de antropología cognoscitiva. Hablar español emergió como un claro indicio de aculturación que podría estar asociado a un incremento en la prevalencia de obesidad y sus consecuencias. Una encuesta nutricional con niños Tarahumaras de albergues escolares mostró evidencia de deficiencia de cinc, vitamina B12, hierro y yodo pero encontró un estado antropométrico similar al de otros niños mexicanos del medio rural. Finalmente, se condujo una evaluación cualitativa para identificar alimentos culturalmente aceptables para rediseñar una canasta de ayuda alimentaria con el objeto de aliviar la desnutrición infantil. Los resultados de esta tesis ofrecen información relevante para el mejoramiento del diseño de intervenciones para combatir y prevenir algunos de los problemas nutricios que afectan a los Tarahumaras. De utilizarse estrategias muestrales similares, esta información podría además constituir el punto de comparación para evaluar cambios futuros. Pero sobre todo, los hallazgos apuntan a la importancia y el desafío para alcanzar una modernización que no solo mejore la salud de los indígenas, sino que además apoye, mantenga y promueva los valores culturales tradicionales, pues estos, además de conformar los cimientos de la sociedad Tarahumara, pueden en varios casos contribuir a una mejor nutrición y salud.

The Dps (DNA-Binding Protein from Starved Cells) proteins are a class of stress-specific proteins with a major role in protecting DNA during the stationary phase of bacterial growth, through direct physical binding as well as ferroxidation. These proteins are characteristically dodecameric in nature. Mycobacterium smegmatis, which is the model organism used in this study has two Dps homologues- MsDps1 and MsDps2. MsDps1, that has previously been studied, is exceptional in having trimeric as well as dodecameric states in vitro. This work focuses on the functional domains of MsDps1, with respect to its oligomerisation and DNA binding property, the identification of a new Dps homologue MsDps2, the in vitro characterization of MsDps2 and elucidation of a possible function of the protein in the physiology of Mycobacterium smegmatis. The Thesis is organized as shown below: Chapter 1: The literature on the bacterial stationary phase physiology and the role of Dps has been reviewed in this chapter. It gives a brief introduction of the background of the present study including the stationary phase response of bacteria and the significance of studying bacteria under stress as apart from ideal conditions of growth, which has been the conventional approach until recently. The advantages of using Mycobacterium smegmatis as a model system, and its starvation-induced stationary phase are also discussed. An introduction to the Dps proteins as a family of proteins branched off from ferritins and nucleoid proteins is explained. A brief summary of the ferritin and nucleoid proteins is given. Similarities connecting Dps to both these protein families is described. The review of earlier work done in our laboratory on the mycobacterial MsDps1 protein is also presented. Chapter 2: involves the study of the solution properties of the protein including its ability to oligomerize in vitro. The MsDps1 protein exists in two forms, a trimer and a dodecamer. The trimer form is a unique feature of the M.smegmatis homologue. Dps proteins from other sources are characteristically dodecameric. Earlier studies have shown that the trimeric form of the protein can perform ferroxidation while the dodecamer can bind to DNA. The dodecamer can also perform ferroxidation and accumulate the oxidized iron in its negatively charged core. In this chapter, we show that the trimeric form is extremely stable, under various conditions of pHs. The protein, when over expressed in M.smegmatis, also shows the presence of the trimer, thus ruling out the effect of heterologous expression of the protein in E.coli. We further report here, the ideal conditions for dodecamerisation of the protein from trimer to dodecamer, which binds to DNA. The dodecamer once formed is also highly stable and does not revert back to the trimeric form. The structural stability of the dodecamer is expected, as it is the fully functional form of the protein that physically protects the DNA from stress. However, the high stability of the trimeric form and its precise conversion into a stable dodecamer is intriguing. It is interesting to study the functional significance in vivo of the oligomerisation process in MsDps1. In addition, we looked at the effect of over expression of the protein on the overall phenotype of Mycobacterium smegmatis, as evidenced by the colony morphology and find no visible alteration, when compared with the wild type. Chapter 3: deals with a more detailed structural analysis of the MsDps1 protein. The role of N and C termini of the protein in maintaining a stable oligomeric structure is studied by making an N-terminal deletion mutant of the protein which is found to be unable to form a dodecamer in solution. On the other hand, MsDps1 with a 16 amino acid C-terminal deletion, MsDpsΔC16, is able to form stable oligomeric structures, when the N-terminal is intact. A previous deletion reported from our laboratory with 26 amino acids deleted from the C-terminal tail, called MsDpsΔC26 showed inability to form stable oligomeric structures in vitro. Putting together all the above results, a model for the interaction of the N and C-terminal tails of the protein in maintaining a stable dodecamer is presented. A demarcation of the C-terminal tail of MsDps1 into regions determining the oligomeric stability and DNA binding was also inferred. The MsDpsΔC16 protein, does not bind to DNA although it forms a stable dodecamer. A further deletion of 10 amino acids, as seen in a previously made construct, MsDpsΔC26 disrupts both the DNA binding as well as the oligomeric stability of the protein. Chapter 4: describes the discovery of a new homolog of the Dps protein in M.smegmatis. It was named as MsDps2. Bio-informatics analysis carried out on the complete genome data of Mycobacterium smegmatis yielded a second homologue of Dps in addition to the one already present and characterized. Interestingly, out of the 300 homogues of Dps found in bacteria, only 195 are present as single copies in a bacterium. The rest exist as more than one homologue in the same bacterial genome. The basic characterization of this new Dps homologue and its confirmation as a Dps family member is the focus of this chapter. Chapter 5: deals with the possible functions of the new protein MsDps2. Electron micrography shows that the purified protein forms stable nucleoprotein-like complexes. Over expression of the MsDps2 proteins presents no difference in the colony morphology when compared with the wild-type. Western analysis shows that the MsDps2 protein is not expressed under normal conditions tested for growth. MsDps1, on the other hand shows expression under conditions of starvation and osmotic stress, as has been established previously in the laboratory. Hence, it can be inferred that the new protein MsDps2 does not perform the same function as MsDps1. However, the in vivo function of this protein remains an important question to be addressed. The appearance of in vitro nucleoid structures involving this protein under the electron microscope, suggests a possible role for this protein in the formation and stabilization of the mycobacterial nucleoid. Indeed extensive evidence for the same exists for the E.coli protein. Chapter 6: describes the results obtained from the sequence comparison of MsDps2 with other Dps proteins listed in the TIGR database. ClustalW sequence analysis, followed by the construction of a phylogenetic tree using the MEGA software, suggests that the mycobacterial Dps proteins fall into two separate groups, represented by the MsDps1 and MsDps2 homologues from Mycobacterium smegmatis. Chapter 7 Summary and Conclusions: A summary of the work presented in the thesis is given followed by the appendix sections. Appendix 1 includes list and maps of plasmids used. Appendix 2 details the theoretical DNA and protein sequences of the recombinant clones generated in the study and theoretical physical and chemical properties of the proteins studied, as calculated with the Expasy Protparam software. Appendix 3 includes raw data obtained from the bio-informatic analysis of MsDps2, obtained using ClustalW analysis.

Le cancer épithélial de l’ovaire est le cancer gynécologique le plus agressif avec le plus haut taux de mortalité. La croissance des cellules cancéreuses de l’ovaire est limitée par les nutriments de l’environnement, le fer étant un des éléments indispensables à leur prolifération. L’hémochromatose héréditaire est une maladie associée à une accumulation corporelle de fer. Cette maladie est liée à deux mutations majeures du gène HFE soit H63D et C282Y. Étant donnée l’influence de la protéine HFE sur l’entrée du fer dans la cellule, des mutations du gène HFE pourraient être associées à une croissance rapide des cellules cancéreuses. Des études de génotypage du gène HFE effectuées chez 526 patientes avec cancer épithélial de l’ovaire, ont révélées une fréquence allélique de la mutation C282Y significativement plus élevées chez les patientes avec tumeur ovarienne comparativement aux patientes du groupe contrôle (5.9% versus 1.3%, p = 0.02). De plus, le taux de survie des patientes avec mutations C282Y et tumeur ovarienne de G3, après 2 ans, est faible (20%) lorsque comparé à celui des patientes sans mutations (60%, p = 0.005). Une analyse de régression multivariée de Cox a démontrée un risque relatif de 3.1, suggérant que les patientes avec mutations C282Y ont 3 fois plus de chance d’avoir une faible survie (p=0.001). Également, des études de corrélation ont démontrées que les niveaux de ferritine du sérum étaient plus élevés chez les patientes avec grade avancé du cancer épithélial de l’ovaire (r = 0.445 et p= 0.00001), suggérant que ce paramètre pourrait servir comme marqueur tumoral. Afin de comprendre ces résultats, nous avons tout d’abord étudiés l’influence des mutations HFE sur les cellules cancéreuses. Pour ce faire, la lignée du cancer de l’ovaire TOV-112D, homozygote pour la mutation C282Y, a été transfectée avec les vecteurs HFEwt et HFEC282Y. Bien qu’aucune différence significative n’ait été trouvée en termes de TfR totaux, des analyses par FACS ont démontrées un phénotype de déficience de fer pour les clones stables HFEwt. In vitro, la restauration de la protéine HFE, dans la lignée TOV-112D du cancer de l’ovaire, n’influence pas la croissance cellulaire. Ensuite, nous avons étudiés l’influence des niveaux de fer sur la progression tumorale. Une expérience in vivo préliminaire a démontré une tendance à un volume tumoral supérieur dans un modèle de souris de surcharge de fer,HfeRag1-/-. De plus, les souris HfeRag1-/-, injectées avec la lignée du cancer de l’ovaire TOV-21G, ont montrées des niveaux significativement plus faibles de fer sérique comparativement à leur contrôle (fer sérique 40±7μM versus 27±6μM, p = 0.001). En conclusion, des études supplémentaires sont nécessaires afin de comprendre davantage le rôle des mutations HFE sur la progression tumorale. Notamment, les niveaux élevés de fer pourraient rendre les cellules tumorales résistantes aux traitements ou encore, augmenter la toxicité et ainsi, contribuer à un mauvais prognostique. / Epithelial ovarian cancer is the most aggressive gynecological cancer with the highest mortality rates. Growth of the ovarian cancer cells is limited by nutrients in the environment; iron being one of the elements essential to their proliferation. Hereditary hemochromatosis is a disease associated with an accumulation of body iron, and is linked to two mutations of the HFE gene including C282Y and H63D. Given the influence of HFE protein on the entry of iron in the cell, mutations in the HFE gene may be associated with rapid growth of cancer cells. By genotyping the HFE gene of 526 patients with epithelial ovarian cancer, we have found that the allelic frequency of the C282Y mutation is significantly higher in patients with ovarian cancer compared to patients in the control group (5.9% versus 1.3% p = 0.02). Moreover, the 2-year survival rate, of patients with C282Y mutations and G3 ovarian tumor, is low (20%) when compared to patients without mutations (60%, p = 0.005). A multivariate survival analysis, using Cox’s regression model, also showed a hazard ratio value of 3.1,suggesting that patients with the C282Y mutation are 3 times more likely to have a poor survival (p =0.001). As well, correlation studies have demonstrated that serum ferritin levels were higher in patients with advanced grade of ovarian cancer (r = 0.445 and p = 0.00001), suggesting that this iron parameter could serve as a tumor marker for assessing the progression of ovarian cancer. In order to investigate these findings, we first studied the influence of HFE mutations on cancer cells. The ovarian cancer cell line TOV-112D, homozygous for the C282Y mutation, was transfected with the HFEwt and HFEC282Y vector, and although there were no differences in total TfR levels, FACS analysis demonstrated an iron deficient phenotype for the HFEwt stable clones (p<0.05). In vitro,restoration of the HFE protein, in the TOV-112D ovarian cancer cell line, does not influence cell growth. We then studied the influence of iron levels on tumor progression. A preliminary in vivo experiment has demonstrated a tendency to a higher tumor volume in a mouse model of iron overload,HfeRag1-/-. Furthermore, HfeRag1-/- mice that were injected with the ovarian cancer cell line TOV-21G showed significant lower serum iron levels compared to their controls (SI 40±7μM versus 27±6μM, p = 0.001). In conclusion, more studies are required to further understand the role of HFE mutations on tumor progression. Higher iron levels may confer tumor cells to be drug resistant or increase toxicity and thus, may contribute to a bad prognostic.

The etiology of anemia is characterized by abnormal hemoglobin synthesis. Iron deficiency is characterized by microcytic and hipochromic red cells and low serum ferritin, being the most prevalent nutritional deficiency worldwide, responsible for iron deficiency anemia (FA). Anemia of chronic disease (ACD) is considered a clinical syndrome associated with chronic inflammation, infectious disease, neoplastic or traumatic, being the second most frequent cause of anemia. The severity of anemia correlates with the degree of pathology. Both have functional iron deficiency. The objective of this study was to evaluate hematological and inflammatory, as well as the presence of oxidative stress in patients with anemia. The blood analyzer was done by the CBC, automated hematology analyzer processed, Sysmex® (Automated Hematology Analyzer). The quantitative determination of ferritin is serum was done in IMMULITE analyzer. Levels of CRP and AOPP were performed in serum by automated Cobas MIRA® (Roche Diagnostics). Statistical analysis was performed using GraphPad Prism 5. We analyzed 70 patients with microcytic and hypochromic anemia. Of these, 29 (41.43%) were diagnosed as iron deficiency anemia and 41 (58.57%) with anemia of chronic disease. As a control group, we used samples from 44 patients with hematological parameters, serum ferritin, CRP and AOPP normal. The values of MCV, MCH and MCHC significantly lower in iron deficiency anemia. Ferritin levels showed that it can be considered both a measure of iron store as an inflammatory marker. In ACD there is increased production of inflammatory cytokines, which, in turn, increases the concentration of C-reactive protein (CRP). The results indicate that AOPP in both groups with anemia showed increased levels of this marker, which indicates the presence of oxidative stress, probably caused by increased production of free radicals and decreases in enzyme activities of the antioxidant defense system of erythrocytes. / A etiologia das anemias caracteriza-se pela síntese anormal de hemoglobina. A deficiência de ferro é caracterizada por eritrócitos microcíticos e hipocrômicos e por ferritina sérica baixa, sendo a carência nutricional mais prevalente em todo o mundo, responsável pela Anemia Ferropriva (AF). A Anemia de Doença Crônica (ADC) é considerada uma síndrome clínica, associada à inflamação crônica, doença infecciosa, traumática ou neoplásica, sendo a segunda causa mais freqüente de anemia. Ambas apresentam deficiência funcional de ferro. O objetivo deste trabalho foi avaliar parâmetros hematológicos e inflamatórios, bem como a presença de estresse oxidativo em pacientes com anemia. A análise hematológica foi feita através do hemograma, processado em analisador hematológico automatizado, Sysmex® (Automated Hematology Analyzer). O doseamento quantitativo da ferritina no soro foi feito em analisador IMMULITE. A dosagem de Proteína C-Reativa (PCR) e de Produtos da Oxidação Avançada de Proteínas (AOPP) foram realizadas no soro através do sistema automatizado Cobas MIRA® (Roche Diagnostics). A análise estatística foi realizada através do programa GraphPad Prism 5. Foram analisados 70 pacientes portadores de anemia microcítica e hipocrômica. Destes, 29 (41,43%) foram diagnosticados como anemia ferropriva e 41 (58,57%) com anemia de doença crônica. Como grupo controle, foram utilizadas amostras de 44 indivíduos com parâmetros hematológicos, níveis de ferritina, PCR e AOPP dentro da normalidade. Os valores de VCM, HCM e CHCM foram significativamente menores na anemia ferropriva. Os níveis de ferritina revelaram que ela pode ser considerada tanto uma medida das reservas de ferro quanto um marcador inflamatório. Na ADC há aumento da produção de citocinas inflamatórias, que, por sua vez, aumenta também a concentração de PCR. Os resultados do AOPP indicam que ambos os grupos com anemia apresentaram níveis aumentados deste marcador, o que indica a presença de estresse oxidativo, provavelmente causado por aumento na produção de radicais livres e declínio das atividades das enzimas do sistema de defesa antioxidante dos eritrócitos.

The DNA binding proteins under starvation (Dps) from M. smegmatis are cage-like structures which internalize iron and bind DNA. They provide resistance to the cells from free radical damage, and physically protect the DNA from the harmful effects of reactive oxygen species by DNA compaction. The work compiled in this thesis has been an effort to study oligomerization and dynamics of iron metabolism by these nano-protein compartments. Chapter 1 gives a general introduction on stress, especially oxidative stress, and the ways bacteria fight back the host resistance systems. This has been elaborated from the point of view of the Dps proteins which is the focus of our work. Also, the competition for iron among the host and pathogens, and the modes of iron trafficking of the pathogens from host organisms has been summarized. Finally, the structural aspects of ferritin family proteins to which Dps belongs, has been discussed. Chapter 2 elaborates on the oligomerization pathways of the first M. smegmatis Dps MsDps1, which exists in vitro as two oligomeric forms. The GFP-tagging has been used to locate the Dps1 proteins by live cell imaging and the over-expression of these proteins during nutrient limiting conditions has been studied. The crystal structure of a point mutant F47E in the background of MsDps1, which shows no dodecamerization in vitro, has been solved. The possible ways of dodecamerization of MsDps1 has been concluded by analyzing the intermediates via glutaraldehyde cross-linking and native electrospray mass spectrometry. Chapter 3 documents the gating machinery of iron in MsDps2 protein, the second M. smegmatis Dps protein. Through graph theoretical approaches, a tight histidine-aspartate cluster was identified at the ferritin-like trimeric pore which harbors the channel for the entry and exit of iron. Sitespecific variants of MsDps2 were generated to disrupt this ionic knot, and the mutants were further assayed for ferroxidation, iron uptake and iron release properties. Our studies in MsDps2 show the importance of counter-acting positive and negatively charged residues for efficient assimilation and dispersion of iron. Chapter 4 describes crystallization studies of MsDps2 pore variants, done in an attempt to connect the changes in functional properties described in chapter 3, with structural alterations of the point mutants. We show here that the gating mechanism happens by alterations in side chain configuration at the pore and does not alter the over-all stability of the proteins. Chapter 5 is the final section where we have employed site specific mutations and cocrystallization studies to elucidate the behaviour of MsDps2 proteins upon the addition of iron. By studying the effect of substitutions at conserved sites near ferroxidation center, we attempt to arrive at a pathway which iron atoms take to reach the ferroxidation site. Also, by crystallization of proteins loaded with varying amounts of iron we tried to map the changes in the protein structure in the presence of its ligand. Chapter 6 concludes briefly the work that has been documented in this thesis. Appendix I relates the role of N-terminal tail for DNA binding in MsDp2. Appendix II gives the technical details of a modified protein preparation and oligomerization process for his-tagged MsDps1 protein. Appendix III gives the maps of the plasmids used in this study.

The cell is the basic unit of all living organisms. It is an amazing machine capable of self-replicating, growing, and synthesizing and shuttling thousands of compounds. To perform all of these activities the cell needs energy. The original source of energy for all living beings is the Sun. The energy of the sun is collected by the autotrophs (mostly plants) through photosynthesis and stored in the chemical bonds of carbohydrates and lipids through carboxylic acid intermediates; animals use these compounds to obtain the energy for their cells. Most of the energy extracted by the cell comes from the citric acid cycle. Therefore, two crucial energy transfer checkpoints are photosynthesis and citric acid cycle. With growing need for energy, the limited supply of fossil fuel, and the search for a cleaner environment, scientists have turned to the Sun (directly or indirectly through wind, tides, biomass, etc.) to satisfy the needs of modern society trying to reach the dual Holy Grail of energy: harvesting energy through Artificial Photosynthesis and Low Temperature Biomass Oxidation. This work represents one more step toward reaching these Holy Grails. The core reagent used in our technique is ferritin. Ferritin recapitulates some of the essential features of a plant cell: it contains a semiconductor capable of charge separation, like chlorophyll, acts as a membrane to isolate compartments, and has an enzymatic activity that prevents charge build up and oxidative damage. The energy absorbed by ferritin from the artificial “solar” radiation is used to extract reducing equivalents from stable and partially oxidized compounds, mainly carboxylic acids. The energized electrons produced are then used for a number of technical applications, from synthesis of catalytically active nanoparticles, to reductive precipitation of contaminant heavy metals (i.e.: mercury), to hydrogen evolution.