Thesis (Ph.D. (Nutrition))--North-West University, Potchefstroom Campus, 2009.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Horse breeding industry is an activity in ascension worldwide, and is responsible for generating jobs and income. In Brazil, especially in Rio Grande do Sul state, there are several horse breeding farms with high-standard equines. Although these herds are under strict sanitary control, the occurrence of respiratory diseases is an important cause of mortality in foals and reduced athletic performance. Among the respiratory diseases, equine rhodococcosis, caused by the bacterium Rhodococcus equi, is the major cause of pneumonia in foals. Rhodococcus equi is worldwide distributed, and have emerged as an important cause of economic losses due to pneumonia in young animals. However, preventive measures and effective control of the disease are still challenges to be reached. In R. equi infection, iron (Fe) is classified as an essential element not only for the bacterium multiplication, but also as a key for the expression of virulence factors. Studies have shown the presence of specific Fe uptake mechanisms in R. equi, which have been determining its survival in both saprophytic and pathogenic life styles. However, as a type of nutritional immunity, mammals, including horses, reduce the plasmatic concentration of Fe through its binding in proteins, including the transferrin (Tf). In this context, the present thesis was developed to study aspects related to metabolism and acquisition of Fe by R. equi and Fe importance in the pathogenesis of equine rhodococcosis (manuscript 1), control and treatment of infections caused by R. equi through drugs with capability to reduce the availability of intracellular Fe (manuscript 2), and genetic susceptibility to R. equi pneumonia (manuscript 3), including the assessment of polymorphisms in the equine Tf gene as risk factors related to susceptibility and/or resistance to equine rhodococcosis (manuscript 4 ). We conclude that R. equi is evolving to specialize it in the acquisition and utilization of Fe from the host, skills that should be considered as key points for the development of chemotherapeutic agents. Once R. equi encodes redundant mechanisms of acquisition and utilization of Fe, it is likely that chemotherapeutic agents will need act on multiple cellular mechanisms or be used in combination. Furthermore, the term "nutritional immunity" may be considered an important strategy to minimize antimicrobial resistance observed in R. equi. As an example of chemotherapy associated with iron metabolism, we observed that chloroquine inhibits the intracellular multiplication of R. equi, most likely due to intracellular iron deprivation. However, further studies are necessary to evaluate the chloroquine therapeutic potential against R. equi infections. We also observed important chromosomal regions positively associated with R. equi pneumonia, which seem to possess genes associated with immune response against intracellular pathogens. This observation allows us to classify the equine rhodococcosis as a disease of polygenic basis, as postulated by previous studies. Finally, we found that polymorphisms in the Tf gene, including some not described yet in the literature, occur in Brazilian Sport Horses and Brazilian Thoroughbred Horses. There is the occurrence of two alleles between the breeds studied, including heterozygosis for these alleles. We believe that there is a relationship between equine Tf variants, and genetic susceptibility to R. equi pneumonia in the breeds evaluated. Summarizing, we have demonstrated that the modulation of Fe availability may be a useful approach to control the disease. / A equideocultura é uma atividade em ascensão mundial, responsável pela geração de empregos e renda. No Brasil, em especial no Rio Grande do Sul, encontram-se diversos locais de criação de equinos de alto padrão zootécnico. Embora estes rebanhos estejam sob rigoroso controle sanitário, a ocorrência de doenças respiratórias é causa importante de mortalidade em potros e redução de seu desempenho atlético. Dentre as doenças respiratórias, a rodococose equina, causada pela bactéria Rhodococcus equi, é a principal causa de pneumonia nesta categoria animal. R. equi está distribuído mundialmente, e cresce como causa de perdas econômicas devido à pneumonia observada em animais jovens. No entanto, medidas preventivas e efetivo controle da enfermidade são ainda desafios a serem alcançados. Na infecção por R. equi, o ferro (Fe) apresenta-se como um elemento fundamental não somente para multiplicação da bactéria, mas também, como um determinante para a expressão de fatores de virulência. Estudos têm demonstrado a presença de mecanismos específicos de captação de Fe em R. equi, os quais determinam sua sobrevivência tanto durante seu estilo de vida saprófito quanto patogênico. Em contrapartida, como uma forma de imunidade nutricional, mamíferos, entre eles os equinos, diminuem a concentração plasmática de Fe através de sua ligação em proteínas, entre elas, a transferrina (Tf). Neste contexto, esta tese foi elaborada visando contemplar aspectos relacionados ao metabolismo e aquisição de Fe por R. equi e sua importância para patogenia da rodococose equina (manuscrito 1), controle e tratamento de infecções por R. equi através de drogas com capacidade de modular a disponibilidade de Fe intracelular (manuscrito 2), e susceptibilidade genética à pneumonia por R. equi (manuscrito 3), incluindo a avaliação de polimorfismos no gene da Tf equina como fatores de risco relacionados à susceptibilidade e/ou resistência genética à rodococose equina (manuscrito 4). Concluímos que R. equi está evoluindo de forma a especializar-se na aquisição e utilização de Fe a partir do hospedeiro, habilidades que devem ser consideradas como pontos chave no desenvolvimento de agentes quimioterápicos. Uma vez que R. equi codifica redundantes mecanismos de aquisição e utilização de Fe, é provável que agentes quimioterápicos deverão inibir múltiplos mecanismos ou ser utilizados em combinação. Além disso, o conceito de imunidade nutricional pode considerado uma importante estratégia para minimizar a resistência antimicrobiana observada em R. equi. Como um exemplo de quimioterápicos associados ao metabolismo de Fe, observados que chloroquine inibir a multiplicação intracelular de R. equi, muito provavelmente devido à deprivação de Fe intracelular. No entanto, ainda são necessários estudos avaliando o potencial terapêutico de chloroquine como tratamento alternativo de infecções por R. equi. Observou-se, também, importantes regiões cromossômicas positivamente associadas à pneumonia por R. equi, as quais parecem possuir genes associados à resposta imune contra patógenos intracelulares. Esta observação nos permite classificar a rodococose equina como uma enfermidade de base poligênica, como postulado por estudos anteriores. Por fim, verificamos que polimorfismos no gene da Tf, inclusive polimorfismos ainda não descritos na literatura, ocorrem em equinos das raças Brasileiro de Hipismo e Puro Sangue de Corrida, criados no Brasil. Existe a ocorrência de dois alelos entre as raças estudas, incluindo animais heterozigotos para estes alelos. Acredita-se que exista uma relação entre variantes de Tf equina e susceptibilidade genética à pneumonia por R. equi nas raças analisadas. Em suma, demonstrou-se através de diferentes estudos que a modulação da disponibilidade de Fe pode ser uma forma de controle da rodococose equina.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-05-08T12:20:16Z No. of bitstreams: 2 Dissertação - Beatriz Assis Carvalho - 2015.pdf: 4617719 bytes, checksum: b0187daca51d7151c1658696f370401e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-05-08T13:00:51Z (GMT) No. of bitstreams: 2 Dissertação - Beatriz Assis Carvalho - 2015.pdf: 4617719 bytes, checksum: b0187daca51d7151c1658696f370401e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-05-08T13:00:51Z (GMT). No. of bitstreams: 2 Dissertação - Beatriz Assis Carvalho - 2015.pdf: 4617719 bytes, checksum: b0187daca51d7151c1658696f370401e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-02-09 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / To evaluate the nutritional status of iron and its related factors in children 12 to 15 months assisted in Health Units in Goiânia, Goiás. METHODS: This is a cross-sectional study nested in research "Effectiveness of home fortification with vitamins and minerals in the prevention of iron deficiency and anemia in children under one year of age: a multicenter study in Brazilian cities ". The study was conducted with 230 children, aged between 12 and 15 months, assisted in Health Units in Goiânia, from June 2012 to February 2013. The prevalence of iron deficiency, iron deficiency anemia and anemia were assessed by the plasma means concentration of ferritin and transferrin receptor, hemoglobin and C-reactive protein. Multiple linear regression was used to estimate the effect of independent variables on the log plasma concentrations of ferritin. These variables were socioeconomic, demographic, maternal, pregnancy, anthropometric, breastfeeding, use of supplement, and biochemical parameters. RESULTS: Regarding the iron status, iron deficiency and iron deficiency anemia prevalence was 14.1% and 1.5%, respectively. Also, anemia prevalence was 5.6% of the infants studied. The predictors of ferritin were folate, vitamin B12 and the use of iron supplement at the time of collection, which each unit raised the log plasma concentration of ferritin in 0.009 mg/L, 0.001 mg/L and 0.315 mg/L, respectively. CONCLUSION: The results of this study showed low prevalence of iron deficiency and anemia in children studied. The use of iron supplements and serum concentrations of vitamin B12 and folate correlated ferritin concentrations and consequently the iron status in this population. Keywords: Iron Deficiency; Ferritins; Receptors, transferrin; Folic Acid; Vitamin B 12; Infant. / Avaliar o estado nutricional de ferro e os seus fatores relacionados em crianças de 12 a 15 meses atendidas em Unidades Básicas de Saúde de Goiânia, Goiás. MÉTODOS: Trata-se de um estudo transversal aninhado a pesquisa “Efetividade da fortificação caseira com vitaminas e minerais na prevenção da deficiência de ferro e anemia em crianças menores de um ano: estudo multicêntrico em cidades brasileiras”. O trabalho foi realizado com 230 crianças, de 12 e 15 meses, atendidas em Unidades Básicas de Saúde de Goiânia, no período de junho de 2012 a fevereiro de 2013. As prevalências de deficiência de ferro, anemia por deficiência de ferro e anemia foram avaliadas por meio da concentração plasmática de ferritina e receptor de transferrina, hemoglobina e proteína C-reativa. Foi utilizada regressão linear múltipla para estimar o efeito de variáveis independentes sobre o log das concentrações plasmáticas de ferritina. Estas variáveis foram condições socioeconômicas, demográficas, maternas, gestacionais, antropomêtricas, amamentação, uso de suplemento, e parâmetros bioquímicos. RESULTADOS: Com relação ao estado nutricional de ferro, as prevalências de deficiência de ferro e anemia por deficiência de ferro foram de 14,1% e 1,5% respectivamente. Além disso, foi encontrada prevalência de 5,6% de anemia nos lactentes estudados. Os fatores associados a ferritina foram o folato, a vitamina B12 e o uso de suplemento de ferro no momento da coleta, os quais cada unidade elevaram o log da concentração plasmática de ferritina em 0,009 μg/L, 0,001 μg/L e 0,315 μg/L, respectivamente. CONCLUSÃO: Os dados do presente estudo evidenciaram baixas prevalências de deficiência de ferro e anemia nas crianças estudadas. O uso de suplemento de ferro e as concentrações séricas das vitaminas B12 e folato correlacionaram-se as concentrações de ferritina e consequentemente, o estado nutricional de ferro nesta população.

As síndromes talassêmicas (?- e ?-talassemia) são as desordens mais comuns e frequentes associadas com eritropoese ineficaz. O desbalanço na produção das cadeias ?- e ?-globinas resulta no comprometimento da produção de eritrócitos, em anemia e aumento de progenitores eritroides no sangue periférico. Enquanto os pacientes homozigóticos afetados por essas desordens demonstram alterações características dos parâmetros relacionados a eritropoese, a relação entre grau de anemia, eritropoese alterada e disfunção do metabolismo de ferro ainda não foram investigados nos indivíduos com ?+-talassemia heterozigótica ou ?+-talassêmia. Duzentos e vinte seis indivíduos (75 do gênero feminino e 151 do gênero masculino) foram recrutados e divididos em 5 grupos: Controle (n=28), doadores de sangue regulares (DSR, n=23), ?+-talassemia heterozigótica (TAT, n=14), ?+-thalassemia (traço ?-talassêmico, TBT, n=20) e ?0-talassemia, (?-talassemia maior, BTM, n=27). As amostras foram analisadas para parâmetros hematológicos (Micros ABX 60); ferro sérico, capacidade total de ligação ao ferro e saturação de transferrina por método colorimétrico (Pointe Scientific, Inc., Canton, MI, USA), ferritina e proteína C-reativa ultra sensível por imunoensaio (Immulite 1000); receptor solúvel de transferrina, eritropoetina, fator de diferenciação do crescimento 15 (R&D Systems) e hepcidina (Intrinsic LifeSciences, La Jolla, CA) por ELISA. As razões sTfR/log ferritina e (hepcidina/ferritina)/sTfR foram calculadas para avaliar o metabolismo do ferro. sTfR/log ferritina pode distinguir depleção dos estoques de ferro de eritropoese deficiente de ferro, enquanto (hepcidina/ferritina)/sTfR pode avaliar os estímulos contrários (disponibilidade de ferro e atividade eritropoética) que controlam a síntese de hepcidina e a absorção de ferro, na ausência de estímulos inflamatórios. Foi demonstrado que TAT teve significativa redução da hepcidina e aumento do receptor solúvel de transferrina, com parâmetros hematológicos relativamente normais. Em contraste, todos os parâmetros hematológicos de TBT foram significativamente diferentes do Controle, incluindo aumento dos níveis do receptor solúvel de transferrina, ferritina, eritropoetina e fator de diferenciação do crescimento 15. Essas alterações em ambos os grupos sugerem um balanço alterado entre eritropoese e metabolismo de ferro. Os índices sTfR/log ferritina e (hepcidina/ferritina)/sTfR estão, respectivamente, aumentado e reduzido comparados ao Controle, proporcional a severidade de cada grupo talassêmico. Em conclusão, destacamos que, pela primeira vez, foram descritas alterações no metabolismo de ferro em indivíduos com ?+-talassemia heterozigótica. Esses dados demonstram que, no contexto da saúde pública, são necessários identificação e acompanhamento dos portadores de ?+-talassemia. / The thalassemia syndromes (?- and ?-thalassemia) are the most common and frequent disorders associated with ineffective erythropoiesis. Imbalance of ?- or ?-globin chain production results in impaired red blood cell synthesis, anemia and more erythroid progenitors in the blood stream. While patients affected by these disorders show definitive altered parameters related to erythropoiesis, the relationship between the degree of anemia, altered erythropoiesis and dysfunctional iron metabolism have not been investigated in both carriers of ?-thalassemia and ?-thalassemia. 226 subjects (75 females and 151 males) were recruited to this study and divided in 5 groups: Control (n=28), repeat blood donors (DSR, n=23), ?+-thalassemia heterozygous carriers (TAT, n=14), ?+-thalassemia (?-thalassemia trait, TBT, n=20) and ?0-thalassemia, (?-thalassemia major, BTM, n=27). Samples were tested for hematological parameters (Micros ABX 60); serum iron, total iron binding capacity, and transferrin saturation by the colorimetric method (Pointe Scientific, Inc., Canton, MI, USA), ferritin and high sensitive C-reactive protein by immunoassay (Immulite 1000); soluble transferrin receptor, erythropoietin and growth differentiation factor 15 (R&D Systems) and hepcidin (Intrinsic LifeSciences, La Jolla, CA) by ELISA. Were calculated the ratios sTfR/log ferritin and (hepcidin/ferritin)/sTfR to evaluate iron metabolism. sTfR/log ferritin can distinguish storage iron depletion from iron-deficient erythropoiesis, while (hepcidin/ferritin)/sTfR can be utilized to explore and quantify the opposing forces (i.e. iron availability and erythropoietic activity) regulating hepcidin synthesis and iron absorption in absence of inflammatory stimuli. We demonstrate that TAT have a significantly reduced hepcidin and increased soluble transferrin receptor levels but relatively normal hematological findings. In contrast, TBT have all hematological parameters significantly different from controls, including increased soluble transferrin receptor, ferritin, erythropoietin and growth differentiation factor 15 levels. These changings in both groups suggest an altered balance between erythropoiesis and iron metabolism. The indexes sTfR/log ferritin and (hepcidin/ferritin)/sTfR are respectively increased and reduced relative to controls, proportional to the severity of each thalassemia group. In conclusion, we emphasize that, for the first time in the literature, subjects with heterozygous ?+-thalassemia have altered iron metabolism. Our data demonstrate that within the context of public health, identification and monitoring of patients with ?+-thalassemia are needed.

L’endocytose dépendante de la clathrine (EDC) est un processus fondamental des cellules eucaryotes. Elle se caractérise par la formation d’invaginations à la membrane plasmique aboutissant à la création de petites vésicules par l’action de la dynamine. Dans le cerveau, elle est impliquée dans la dépression synaptique à long terme, un corrélat cellulaire de la mémoire. La morphologie complexe des neurones et le contrôle précis du code neuronal suggèrent qu’elle puisse être régulée spatialement et temporellement dans ces cellules. Le but de mon travail a été de développer de nouveaux outils pour visualiser et perturber l’EDC afin d’étudier ce type de régulation. Le premier de ces outils est pHuji, un senseur de pH rouge génétiquement encodable. Je l’ai utilisé avec un senseur de pH vert existant pour montrer que dans les cellules NIH- 3T3, le récepteur β2-adrénergique est internalisé dans une sous-population de vésicules contenant le récepteur à la transferrine constitutivement endocyté. Le deuxième est une nouvelle méthode d’imagerie permettant de visualiser l’activité d’endocytose de structures recouvertes de clathrine optiquement stables dans des neurones d’hippocampe. J’ai ainsi pu suivre pour la première fois la cinétique d’internalisation de récepteurs au glutamate de type AMPA dans des conditions de plasticité. Enfin, j'ai élaboré un test combinant imagerie et patch-clamp afin de développer un bloqueur peptidique spécifique de l'EDC. En utilisant des peptides dimériques, j’ai montré que la dynamine se lie à ses partenaires via des interactions multimériques. En conclusion, ce travail propose une boite à outils permettant d’élucider les mécanismes de l’EDC avec une grande résolution spatiale et temporelle. / Clathrin mediated endocytosis (CME) is a fundamental process of all eukaryotic cells. At the level of the plasma membrane, it is characterized by the formation of deep invaginations resulting in the creation of small vesicles after membrane scission by dynamin. In the central nervous system, it is involved in the expression of synaptic long term depression, a proposed cellular correlate of learning and memory. The complex morphology of neurons and the precise timing of neuronal firing suggest that endocytosis may be spatially and temporally regulated in those cells. The aim of the work presented here was to develop new tools to visualize and perturb CME in order to study such regulation. The first tool to be characterized was pHuji, a genetically encoded red pH-sensor. I used it in combination with an existing green pHsensor to demonstrate that in NIH-3T3 cells, the β2-adrenergic receptor was internalized in a subset of vesicles containing the constitutively endocytosed transferrin receptor. The second tool is a new imaging method that allowed me to monitor the endocytic activity of optically stable clathrin coated structures in hippocampal neurons. I was thus able to visualize for the first time the kinetics of internalization of AMPA-type glutamate receptors under plasticity inducing conditions. Finally, I set up an assay combining imaging and cell dialysis in order to develop a specific peptide-based inhibitor of CME. Using dimeric peptides, I found that the interplay between dynamin and its binding partners relies on multimeric interactions. Altogether, this work provides a toolbox to decipher the mechanisms of vesicle formation with high spatial and temporal resolution.

ITCH est une ligase de l’ubiquitine impliquée dans différents processus cellulaires. Elle contient une région riche en prolines (PRR, proline rich region) qui lui permet de lier le domaine SH3 (Src homology 3) d’Endophiline et d’autres protéines à domaine SH3. Plusieurs de ces protéines sont impliquées dans l’endocytose clathrine-dépendante de récepteurs tel le récepteur du facteur de croissance épidermique (EGFR, epidermal growth factor receptor). Après activation, l’EGFR est internalisé dans des vésicules enrobées de Clathrine et un complexe protéique formé par CBL, CIN85 et Endophiline participe à cet évènement. ITCH lie l’ubiquitine à CBL et à Endophiline pour vraisemblablement modifier leurs fonctions, ce qui suggère un lien direct entre cette ligase et l’endocytose de l’EGFR. Afin de déterminer le rôle d’ITCH dans ce processus, plusieurs expériences furent réalisées. Premièrement, la modalité de liaison entre la ligase ITCH et les protéines à domaine SH3 a été étudiée en détails. Une série de mutations dans la région PRR d’ITCH nous a aidé à identifier trois résidus arginines (R252, R255, R258) nécessaires pour son interaction avec Endophiline et d’autres protéines à domaine SH3. Deuxièmement, des lignées cellulaires HeLa et Cos7 furent modifiées génétiquement par CRISPR pour empêcher l’expression d’ITCH. Ces lignées cellulaires knockout furent caractérisées et utilisées dans un essai d’endocytose de l’EGFR, puis examinées par spectrométrie de masse. L’internalisation d’EGFR fut suivie en microscopie confocale à l’aide d’un ligand EGF fluorescent -/- dans les deux types de cellules ITCH . En absence d’ITCH, nous observons une diminution significative du niveau d’EGF internalisé par rapport aux cellules parentales. La surexpression d’ITCH dans les cellules ITCH-/- rétablit l’internalisation normale de l’EGF, confirmant l’implication d’ITCH dans le processus, mais la surexpression des formes mutantes de ITCH incapable de lier Endophiline ou catalytiquement inactive ne rétablit pas l’internalisation de l’EGF. Ces résultats nous permettent de conclure que l’interaction ITCH-Endophiline et la fonction catalytique de ITCH sont nécessaires pour l’endocytose de l’EGFR. Ensemble, ces deux fonctions de ITCH régulent le trafic endocytique de l’EGFR. De plus, les cellules ITCH-/- montrent un délai de dégradation de l’EGFR phosphorylé ainsi qu’une prolongation du temps d’activation de la kinase MAPK (mitogen-activated protein kinase). Finalement, pour explorer l’influence de l’absence d’ITCH sur ses substrats et partenaires moléculaires nous avons effectué une comparaison protéomique des partenaires d’interaction et des protéines ubiquitylées à partir des lysats cellulaires ITCH-/- et WT. Les résultats ont montré que le manque d’expression de la ligase ITCH altère la présence peptidique des protéines liées à la signalisation de l’EGFR, à la voie protéolytique dépendante de l'ubiquitine et à l’adhésion cellulaire. Cette étude révèle pour une première fois que la protéine ITCH est requise pour l’endocytose dépendante de la Clathrine de l’EGFR. / ITCH is a ubiquitin ligase involved in various cellular processes including endocytosis. It contains a proline rich region (PRR) which allows it to bind the SH3 domain of endophilin and other SH3 domain-containing proteins involved in Clathrin-mediated endocytosis (CME). CME is an important regulatory mechanism for growth factor receptor activity. The epidermal growth factor receptor (EGFR) is actively internalized in Clathrin-coated vesicles after activation. This endocytosis is facilitated by a protein complex formed by CBL, CIN85 and Endophilin. ITCH is known to ubiquitinate both CBL and endophilin, providing a potential functional link between the ligase and receptor internalization. In order to determine the role of ITCH in this process, several experiments were performed. First, the mapping of molecular binding sites between the ligase ITCH and SH3 domain proteins has been studied in detail. A series of mutations in the PRR region of ITCH helped us identify three arginine residues (R252, R255, R258) as necessary for its interaction with endophilin and all the tested SH3-domain containing proteins. Secondly, HeLa and Cos7 cell lines were genetically modified by CRISPR to prevent ITCH expression. These knockout cell lines were characterized for use in an EGFR endocytosis assay and for mass spectrometry analysis. EGFR internalization was monitored by confocal microscopy using fluorescent EGF ligand in both ITCH-/- cell types. In the absence of ITCH, a significant decrease in the level of internalized EGF compared to parental cells is visible. Overexpression of WT ITCH in the knockout cells restores normal internalization of EGF, confirming the involvement of ITCH in the process. Overexpression of Endophilin-binding defective or catalytically inactive ITCH does not restore the internalization of EGF in ITCH-/- cells. These results show that the ITCH- Endophilin interaction as well as the catalytic function of ITCH are necessary for the endocytosis of EGFR. In addition, ITCH-/- cells show a delay in the degradation of phosphorylated EGFR accompanied with an extended period of mitogen-activated protein kinase (MAPK) activation. In a last set of experiments, we explored the influence of the absence of ITCH on its substrates and molecular partners. We performed a proteomic comparison of ITCH-binding partners and potential substrates using the ITCH-/- and WT cell lysates. The results showed that the lack of ITCH expression alters the peptide count of proteins mainly related to EGFR signaling, theubiquitin-dependent proteolytic pathway and cell adhesion. This study shows for the first time that the protein ITCH is required for the clathrin-dependent endocytosis of EGFR.

Endocytosis is an essential function of eukaryotic cells, providing crucial nutrients and playing key roles in interactions of the plasma membrane with the environment. The classical view of the endocytic pathway, where vesicles from the plasma membrane fuse with a homogenous population of early endosomes from which cargo is sorted, has recently been challenged by the finding of multiple subpopulations of endosomes. These subpopulations vary in their content of phosphatidylinositol 3- phosphate (PI3P) and Rab binding proteins. The role of these endosomal subpopulations is unclear, as is the role of multiple PI3P effectors, which are ubiquitously expressed and highly conserved. One possibility is that the different subpopulations represent stages in the maturation of the endocytic pathway. Alternatively, endosome subpopulations may be specialized for different functions, such as preferential trafficking of specific endocytosed cargo. To determine whether specific receptors are targeted to distinct populations of endosomes, we have built a platform for total internal reflection fluorescence (TIRF) microscopy coupled with structured illumination capabilities named TESM (TIRF Epifluorescence Structured light Microscope.) In this study, TESM, along with standard biochemical and molecular biological tools, was used to analyze the dynamic distribution of two highly conserved Rab5 and PI3P effectors, EEA1 and Rabenosyn-5, and systematically study the trafficking of transferrin. Rabenosyn-5 is necessary for proper expression of the transferrin receptor as well as internalization and recycling of transferrin-transferrin receptor complexes. Results of combining TIRF with structured light Epifluorescence (SLE) indicate that the endogenous populations of EEA1 and Rabenoysn-5 are both distinct and partially overlapping. The application of antisense oligonucleotides as potential therapeutic agents requires effective methods for their delivery to the cytoplasm of target cells. In collaboration with RXi Pharmaceuticals we show the efficient cellular uptake of the antisense oligonucleotide sd-rxRNA® in the absence of delivery vehicle or protein carrier. In this study TIRF, SLE, and biochemical approaches were utilized to determine whether sd-rxRNA traffics and functions along specific endosomal pathways. Sd-rxRNA was found to traffic along the degradative pathway and require EEA1 to functionally silence its target. These new findings will help define the cellular pathways involved in RNA silencing. Neurotransmitter reuptake and reuse by neurotransmitter transport proteins is fundamental to transmitter homeostasis and synaptic signaling. In order to understand how trafficking regulates transporters in the brain and how this system may be disregulated in monoamine-related pathologies, the transporter internalization signals and their molecular partners must be defined. We utilized a yeast two-hybrid system to identify proteins that interact with the dopamine transporter (DAT) endocytic signal. The small, membrane associated, GTPase Rin was determined to specifically and functionally interact with the DAT endocytic signal, regulating constitutive and protein kinase C (PKC) – stimulated DAT endocytosis. The results presented in this study provide new insights into functions and components of endocytosis and enhance the understanding of endocytic organization.

Machupovirus är ett virus som kan orsaka hemorragisk feber hos människor. Efter utvärdering av bindning mellan designade proteiner och virala glykoproteiner skulle proteinerna potentiellt kunna användas vid framtagning av ett proteinbasserat läkemedel mot hemorragisk feber. Syftet med studien var att efter riktad evolution och framrening av optimerade varianter av proteinet AP01 undersöka inbindningen till virala glykoproteiner mellan designade AP01 proteiner och transferrinreceptorn med hjälp av flödescytometrisk undersökning. Den fysiologiska nivån av järn i kroppen upprätthålls av transferrin (Tf) och transferrinreceptorn (TfR), ett transmembranprotein bestående av tre domäner. TfR apikala domän används av glykoprotein 1 (MGP1) och Plasmodium vivax för att ta sig in i celler genom receptormedierad endocytos. Med rekombinant genteknik kan rekombinanta plasmider skapas där en gen av intresse ligeras in i en plasmid med hjälp av DNA-ligas. I studien skapades rekombinanta plasmider pET29b+/AP01 S2.1, S2.2, S2.3, S3.3, S3.4 och S3.6 som transformerades till E. coli. Erhållna resultat från sekvensering visade att samtliga sex AP01-gener hade ligerats i vektorn men sekvensering av rekombinanta plasmider visade att endast pET29b+/AP01 S2.1, S2.2, S2.3 och S3.6 hade nukleotidsekvens utan mutationer. Proteinuttryck inducerades innan proteiner renades fram med immobilized metal ion affinity chromatography (IMAC). Den uppskattade molekylvikten hos de framrenade proteinerna var 18 kDa som bestämdes med sodium dodecyl sulfate – polyacrylamid gel electrophoresis (SDS-PAGE) vilket överrenstämde med den teoretiska molekylvikten. Flödescytometri användes för att undersöka inbindningsförmågan mellan de uttryckta proteinerna och glykoprotein 1 (MGP1). Interaktionsbindningen mellan de designade proteinerna och MGP1 är bättre än interaktionen mellan originalgen AP01 och MGP1. De designade proteinerna visar på en svag effekt i den utförda ”competition assay” som gjorts vilket kan förklaras med en ej optimal struktur hos de designade proteinerna eller närvaro av BSA. / Machupovirus is a virus that can cause hemorragic fever in humans. After evaluating the binding between designed proteins and viral glycoproteins, the proteins could potentially be used in the development of a protein-based drug for hemorrhagic fever. The aim of the study was to investigate the binding to viral glycoproteins between designed AP01 proteins and the transferrin receptor after directed evolution and purification of optimized variants of the AP01 protein by means of flow cytometric examination. The physiological level of iron in the body is maintained by transferrin (Tf) and the transferrin receptor (TfR), a transmembrane protein consisting of three domains. The apical domain of TfR is used by glycoprotein 1 (MGP1) and Plasmodium vivax to enter cells through receptor mediated endocytosis. With recombinant DNA technology, recombinant plasmids can be created where a gene of interest is ligated into a plasmid using DNA ligase. In this study, recombinant plasmids pET29b+/AP01 S2.1, S2.2, S2.3, S3.3, S3.4 and S3.6 were created and transformed into E. coli. Sequencing results showed that all six AP01 genes had been ligated into the vector but sequencing of recombinant plasmids showed that only endast pET29b+/AP01 S2.1, S2.2, S2.3 and S3.6 had nucleotid sequence without mutations. Protein expression was induced before proteins were purified by immobilized metal ion affinity chromatography (IMAC). The estimated molecular weight of the purified proteins was 18 kDa as determined by sodium dodecyl sulfate – polyacrylamid gel electrophoresis (SDS-PAGE) which was consistent with the theoretical molecular weight. Flow cytometry was used to examine the binding ability between the expressed proteins and glycoprotein 1 (MGP1). The interaction binding between the designed proteins and MGP1 is better than the interaction between the original gene AP01 and MGP1. The designed proteins show a weak effect in the “competition assay” preformed, wich can be explained by a non-optimal structure how the designed proteins or the presence of BSA.

No description available.

Motivation Alcohol consumption is one of the major risk factors of cardiovascular disease (CVD). Excessive alcohol drinking is the fifth leading cause of death worldwide and the prevalence of alcohol abuse continues to increase especially in low-income areas of sub-Saharan Africa. The alarming rate of urbanisation seems to be the driving force for excessive alcohol intake in the developing world. In addition to its influence on CVD, heavy drinking also results in a number of non-cardiovascular consequences that include injury, risky sexual behaviour, violent crime and family dysfunction among black South Africans, contributing to high mortality. Moreover, the highest number of individuals with human immunodeficiency virus (HIV) infection in South Africa is partly attributable to high intake of alcohol. HIV remains a major concern in South Africa with significant funding diverted to address the pandemic. The continued increases in mortality from preventable outcomes such as stroke, myocardial infarction and renal failure are largely due to urbanisation, poverty and dysfunctional health systems working with limited budgets. These are some of the factors requiring in-depth study of the scientific aspects of alcohol intake in South Africa. Although there is enough evidence that links excessive drinking with hypertension and CVD, the markers of alcohol intake – self reporting of alcohol, gamma-glutamyltransferase (GGT) and carbohydrate deficient transferrin – are still not specific enough to isolate other confounding factors in the association of alcohol intake with CVD. The markers of alcohol that independently predict CVD and mortality need to be explored. Finally, the severe lack of longitudinal investigations on alcohol-related hypertension development and total mortality in black South Africans has compromised the early identification of risk factors associated with these outcomes. This study will therefore attempt to address the limited availability of longitudinal studies and stimulate interest for continued investigation. Aim The aim of this study was to investigate whether alcohol intake of black South Africans is related to specific measures of cardiovascular function (change in blood pressure (BP), hypertension development) and mortality over a period of 5 years. Methodology This study was based on the international Prospective Urban and Rural Epidemiology (PURE) study which includes 26 countries, investigating the cause and development of cardiovascular risk factors in low, middle and high income countries. This South African leg of the PURE study started in 2005 in which the baseline data was collected from 2021 black South Africans from rural and urban areas in Ikageng, Ganyesa and Tlakgameng in the North West Province. Eleven participants presented with missing data, leaving 2010 participants with complete datasets at baseline. However, data from these 11 participants was useful, especially for Chapter 4. All participants gave informed consent and the Ethics committee of the North-West University (Potchefstroom Campus) approved the study. The follow-up data collection was done in 2010. General health questionnaires, anthropometric measurements, lipid profiles and cardiovascular measurements were taken both at baseline and follow-up using appropriate methods. We also collected blood samples and performed biochemical analyses for lipid markers, liver enzymes, inflammatory markers and percentage carbohydrate deficient transferrin (%CDT). Finally, we obtained data on cardiovascular and non-cardiovascular mortality through verbal autopsy and death certificates. We made use of analysis of variance (ANOVA) and Chi-square tests to compare means and proportions, respectively. We used dependent t-tests and the McNemar test to compare baseline and follow-up variables. Furthermore, we employed single and partial linear regression analyses to correlate alcohol markers with each other and with the cardiovascular measures. Multiple regression analyses were used to correlate dependent variables in the study with various independent variables as required. Finally, we employed multivariable-adjusted Cox regression analyses to assess the association of the selected alcohol markers with mortality while adjusting for several independent variables. Results and Conclusions of each manuscript - With the first research article (Chapter 4), we aimed to compare self-reported alcohol intake estimates with GGT and %CDT, considering their relationship with percentage change in brachial blood pressure (BP) and central systolic blood pressure (cSBP) over 5 years. The results indicated that only self-reported alcohol intake independently predicted % change in brachial BP and cSBP. This was not found for the biochemical markers GGT and %CDT. Self-reported alcohol intake seems to be an important measure to implement by health systems in low income areas of sub-Saharan Africa, where honest reporting is expected. - Given the likely presence of high GGT levels in both alcohol consumption and non-alcoholic fatty liver disease (NAFLD), the second manuscript (Chapter 5) aimed to compare the cardiovascular and metabolic characteristics of excessive alcohol users and individuals with suspected NAFLD (confirmed with self-report, GGT and %CDT). We found that different sex and cardiometabolic profiles characterised excessive alcohol users and individuals suspected with NAFLD. Lean body mass and male sex were the dominant characteristics in excessive alcohol use while the NAFLD group had a dysmetabolic profile with obese women making up the higher proportion of this group. In excessive alcohol users systolic blood pressure and pulse pressure were independently associated with high-density lipoprotein cholesterol. Diastolic blood pressure showed a significant correlation with waist circumference. These disparate profiles may guide healthcare practitioners in primary healthcare clinics to identify individuals with elevated GGT levels who may suffer from NAFLD or alcohol overuse. These results emphasise the importance of modifiable risk factors as the main contributors to CVD and that lifestyle change should be the main focus in developing countries such as South Africa. - The third manuscript (Chapter 6) aimed to determine the measure of alcohol intake (selfreported alcohol intake, GGT and %CDT) that related best with hypertension development, cardiovascular and all-cause mortality over 5 years in the same population of black South Africans. We found that GGT was the only independent predictor of hypertension development, cardiovascular as well as all-cause mortality. Moreover, self-reporting of alcohol intake predicted incident hypertension, confirming our findings from Chapter 4. The third marker, %CDT, a highly specific marker of alcohol intake, was not related with any outcome variable, perhaps due to its low sensitivity. Although self-reported alcohol intake is useful in low-resource primary healthcare settings, measurement of GGT is encouraged due to its predictive value for hypertension and mortality. GGT represents alcohol intake, non-alcoholic steatohepatitis and obesity - all known to have severe cardiovascular consequences. Discussion and Conclusions Excessive alcohol intake remains a major concern in the development of hypertension, CVD and premature death in sub-Saharan Africa. Despite their weaknesses such as bias and nonspecificity, self-reporting of alcohol consumption and GGT emerged as reliable alcohol markers that independently predicted 5-year change in BP, hypertension development and total mortality in this population. Serum %CDT did not show any association with the mentioned cardiovascular markers. Finally, we were also able to show that black South Africans with suspected NAFLD (i.e. with high GGT levels who do not consume alcohol) are typically obese women, whereas lean men were more likely to have high alcohol consumption. Further prospective investigations are encouraged regarding (a) these mentioned associations, as well as (b) other self-reporting estimates such as quantity and frequency of drinking and (c) the use of %CDT as a highly specific marker of alcohol intake. The simultaneous presence of HIV infection in alcohol abuse in this population also warrants further investigation. / PhD (Physiology), North-West University, Potchefstroom Campus, 2015