La surexpression de pompes d'efflux par les cellules cancéreuses permet l'élimination d'agents cytotoxiques, induisant alors une résistance à la chimiothérapie. Trois transporteurs ABC sont principalement impliqués dans cette résistance : P-gp/ABCB1, MRP1-ABCC1 et BCRP/ABCG2. La surexpression des ces transporteurs peut également être le "talon d'Achille" des cellules cancéreuses résistantes en les sensibilisant à certains composés. Ce phénomène, appelé sensibilité collatérale, pourrait constituer un nouvel outil thérapeutique conter les les cancers intrinséquement ou rendus résistants en éliminant sélectivement les cellules cancéreuse résistances. Ainsi, le S-vérapamil provoque la mort sélective par apoptose des cellules surexprimant suite à l'extrusion rapide et massive du glutathion 5GSH) intracellulaire par MRP1. Nous avons démontré que le vérapamil est capable de dépléter s"lectivement de leur contenu en GSH les tumeurs de cancer du poumon H69AR, MRP1 positives et résistantes, dès 3 heures d'exposition aiguë. Le vérapamil étant fortemnt carditoxique, nous avons développé de nouveaux agents de sensibilité collatérale, plus sélectif que le vérapamil, comme le xanthone 9, le flavonoïde 36 et le dimère de flavonoïde 4e. Enfin, grâce à l'étude de chimères MRP1/MRP2, nous avons démontré que la région comprenant les boucles L0 et L1-TM12 pourrait constituter les sites modualteurs et substrat du GSH sur MRP1. / Resistance to chemotherapy is partly due to efflux pumps expressed in the plasma membrane, which prevent the accumulation of anticancer drugs in tumor cells. Three human ABC transporters are particulary involved in this chemoresistance : P-gp/ABCB1, MRP1-ABCC1 and BCRP/ABCG2. The overexpression of these trnasporters can also be an "Achille heel" for resistant cancer cells by sensitizing them to various drugs. This phenomenom, called collateral sensitivity, could constitute a new chemotherapy to eradicate cancers becoming resistant or cancer which ara resistant prioir to any treatment. Thus, S-verapamil triggers selective apoptosis of MRP1 overexpressing correlated to the massive and rapide extrusion of cellular glutathione by MRP1. We showed that verapamil is able to selectivity deprive H69AR MRP1 positive and resistant lung tumors, as soon as 3 hours of acute exposure. Verapamil being highly cardiotoxic, we have developed new collateral sensitivity drugs, more selective than verapamil, such as xanthone 9, flavonoïdd 36 and flavonoïd dimer 4e. Finally, thanks to the characterization of MRP1/MRP2 chimera, we showed that the MRP1 region including the intracellular loop L0 L1-TM12 might constitute the substrate and the modulator binding sites for GSH.

A deficiência de zinco é considerada uma das deficiências nutricionais de maior importância epidemiológica, com grandes repercussões no contexto da saúde pública em todo o mundo. Avanços metodológicos na avaliação de aspectos moleculares constituem uma nova ferramenta para a investigação do estado nutricional dos indivíduos relativo ao zinco, por meio da avaliação da expressão gênica das proteínas transportadoras, destacando-se o ZnT1 (SLC30A1) e ZIP4 (SLC39A4), envolvidos com a manutenção da homeostase corporal do mineral. O objetivo desse trabalho foi avaliar o estado nutricional relativo ao zinco de crianças e sua relação com a expressão gênica destes transportadores. Para isso foram avaliadas 139 crianças com idade entre 32 e 76 meses matriculadas em creches da cidade de Aracaju, Sergipe. De acordo com a avaliação antropométrica, apenas 3,6% das crianças apresentaram déficit de estatura para idade e peso para idade, enquanto 27,3% delas apresentaram sobrepeso e obesidade. A deficiência de zinco plasmático foi observada em 26,6% das crianças, enquanto no eritrócito 99,3% delas encontravam-se abaixo do ponto de corte estabelecido. A análise da expressão gênica das proteínas transportadoras ZnT1 e ZIP4 apresentou relação significativa com o estado nutricional das crianças quanto ao zinco. A expressão do mRNA ZnT1 foi maior entre as crianças com deficiência, enquanto do mRNA ZIP4 foi menor. Não foram observadas relações entre o consumo de zinco e a expressão gênica. Conclui-se que a população estudada apresenta risco de deficiência em zinco, apresentando relação com a expressão gênica das proteínas transportadoras ZnT1 e ZIP4, reforçando a existência de uma regulação da absorção do mineral de acordo com o estado nutricional do indivíduo, indicando que esta análise pode ser útil na avaliação de intervenções dietéticas. / Zinc deficiency is regarded as one of the nutritional deficiencies of greatest epidemiological importance, with major repercussions in the context of public health worldwide. Methodological advances in evaluation of molecular aspects constitute a new tool for investigating the zinc nutritional status of individuals, by assessing the gene expression of transporter proteins, highlighting the ZnT1 (SLC30A1) and ZIP4 (SLC39A4), involved in maintaining body homeostasis of this mineral. This study aimed to evaluate the zinc nutritional status of children and its relation to gene expression of these transporters. Were evaluated 139 children aged between 32 and 76 months attending daycare centers in the city of Aracaju, Sergipe. According to anthropometric measures, only 3.6% of children had deficit height for age and weight for age, while 27.3% of them were overweight and obese. The plasma zinc deficiency was observed in 26.6% of children, while in erythrocytes 99.3% of them were below the established cutoff point. The ZnT1 and ZIP4 gene expression analysis showed a significant relationship with the zinc nutritional status of children. The mRNA ZnT1 expression was higher among children with zinc deficiency, while mRNA ZIP4 was lower. There was no relationship between the consumption of zinc and gene expression. In conclusion, the evaluated population is at risk for zinc deficiency, showing significant relationship with the gene expression of transporter proteins ZnT1 and ZIP4, reinforcing the existence of a regulation of the absorption of the mineral according to the individual\'s nutritional status, indicating that this analysis can be useful in evaluating dietary interventions.

Foi realizado o pirossequenciamento de duas bibliotecas de cDNA do intestino médio de Tenebrio molitor e as sequências foram submetidos à montagem através do programa Newbler. Visando sanar alguns questionamentos a respeito de muitos tipos de transportadores que pudessem estar envolvidos com funções presumíveis em tamponamento luminal, absorção de nutrientes, envolvimento em mecanismos de secreção de enzimas como a α-manosidases e secreção e absorção de água, foram analisadas sequências de interesse que pudessem esclarecer os fenômenos fisiológicos em questão. O pirossequenciamento revelou 19 sequencias de α-manosidases. Após alinhamentos múltiplos, desconfiou-se que o contig 12 era a continuação da sequência original da α-manosidase. Utilizando-se de iniciadores apropriados, a suspeita foi confirmada e uma sequência completa foi obtida e denominada de TmMan1. Através de cladogramas gerados com as sequências de todos os contigs obtidos, assim como de sequências representativas das famílias 38 e 47 das glicosídeos hidrolases, mostrou que todas a nossas sequências, exceto o contig 6 e 7, pertencem à família 38. Todas as sequências com mais de 100 reads (exceto o contig 9) tiveram a sua expressão tecidual avaliada por RT-PCR. Os resultados mostraram que só são expressos no intestino ou intestino e túbulo de Malpighi, implicando na possibilidade de serem digestivas. Dessas sequências, as únicas com peptídeo sinal são a TmMan1 (contig 12) e o contig 14 e, portanto, devem corresponder às atividades Man1 e Man2. Levando em conta o número de reads, TmMan1 deveria corresponder a Man2 e o contig 14 à Man1. É possível, embora necessite de confirmação, que os contigs 8 e 15 sejam de expressão lisossômica. Um peptídeo sintetizado que correspondia a sequencia única da TmMan1 foi usado para gerar anticorpos, que reconheceram a Man2, mas não a Man1, confirmando a identificação de TmMan1 com a Man2. Esse anticorpo foi também utilizado para imunolocalizar a TmMan1 nas células intestinais de T. molitor. Os resultados mostraram que a TmMan1 é secretada de forma apócrina pela região anterior de intestino de T. molitor. Esse trabalho é o primeiro que mostra a ocorrência de α-manosidases com especificidade similar àquelas lissossômicas, mas que são secretadas apócrinamente para fora da célula, devendo agir no lúmen intestinal, removendo resíduos de manoses de oligossacarídeos manosilados. Foram identificados 10 tipos diferentes de transportadores e na elaboração dos modelos fisiológicos só foram levados em conta aqueles expressos exclusivamente no intestino médio ou no intestino médio e túbulos de Malpighi. A V-ATPase em T.molitor parece ser uma bomba usada para energizar muitos dos transportes ao longo do intestino médio como, por exemplo, o de oligopeptídeos. Já as bombas de Na+ e K+ são responsáveis pelo equilíbrio de cargas e, portanto estão presentes na maioria dos tipos celulares. Duas sequências de cotransportadores de oligopeptídeos/H+ foram encontradas no pirossequenciamento e sua expressão é maior na região posterior, uma vez que ali é a última possibilidade de absorção dos oligopeptídeos que ainda estiverem no lúmen, remanescentes da digestão final de proteínas. Foi demonstrado que T.Molitor absorve aminoácidos e açúcares ao longo de todo o intestino médio, pois estes tipos de transportadores possuem uma expressão uniforme ao longo do intestino médio. Já a expressão dos transportadores de NH3/NH4+ em T.molitor, encontra-se confinada ás regiões onde o pH do intestino médio do inseto é mais ácida. Também há uma expressão de transportadores de cloreto que se manifesta mais intensamente na região anterior. Podemos visualizar que a distribuição dos contigs dos transportadores de bicarbonato encontra-se mais expressiva na região posterior do intestino médio. Os resultados sugerem que a acidificação na região anterior do intestino de T.molitor pode resultar da secreção de NH4+ acompanhado do íon cloreto e a alcalinização na região posterior do lançamento no lúmen de bicarbonato. A importância dos canais de cloreto é que o mesmo balanceia as cargas e desta forma pode ser útil juntamente com o transporte de NH4+, que gera uma carga no lado onde é transportado. Há absorvição de água (junto com glicose) ao longo de todo o intestino médio, enquanto que a secreção de água ocorreria apenas nos dois terços finais do intestino médio com o auxílio de aquaporinas complementado por transportadores de íons, teria como consequência a abosorção líquida de água na região anterior e uma secreção líquida no final do intestino médio. Isso esclarece qual a base molecular para a ocorrência do contrafluxo intestinal evidenciado por experimentos fisiológicos. / Pyrosequencing was performed with two cDNA libraries in the midgut of Tenebrio molitor and the sequences were subjected to assembly with the Newbler program. In order to tackle questions concerning proteins which may be involved in midgut buffering, nutrient absorption, in the secretion of enzymes such as α-mannosidases , and water absorption and secretion, sequences of interest were analyzed in order to clarify those physiological phenomena. The pyrosequencing revealed 19 sequences of α- mannosidases . After multiple alignments, it was suspected that the contig 12 was the continuation of the original sequence of the α-mannosidase. Using appropriate primers, the hypothesis was confirmed and a complete sequence was obtained and named TmMan . Through cladograms generated from the sequences of the contigs obtained, as well as of sequences representing families 38 and 47 of glycoside hydrolases, it was showed that all sequences except the contig 6 and 7 belong to family 38. All sequences with over 100 reads (except contig 9) had their tissue expression assessed by RT-PCR. The results showed that they are expressed only in the midgut or midgut and Malpighian tubules, implying the possibility of having a digestive function. Among these sequences, the only ones with a signal peptide are TmMan1 (contig 12) and contig 14 and therefore they should correspond to the activities Man1 and Man2. Taking into account the number of reads, TmMan1 should correspond to Man2 and contig 14 to Man1. It is possible, though it requires confirmation, that the contigs 8 and 15 are lysosomal. A peptide corresponding to the unique sequence TmMan1 was synthesized and used to generate antibodies that recognized Man2 , but not Man1, confirming the identification of TmMan1 with Man2. This antibody was also used to immunolocalyze TmMan1 in the midgut cells of T. molitor. The results showed that TmMan1 is secreted in an apocrine way by the anterior region of T. molitor midgut. This is the first study that shows the occurrence of α-mannosidases with similar specificity to those lysosomal, but that are secreted in an apocrine way, acting in the midgut lumen, removing mannoses from mannosylated oligosaccharides. We identified 10 different types of carriers and in the development of physiological models it was only taken into account those expressed exclusively in the midgut or midgut and Malpighian tubules . The V- ATPase in T.molitor appears to be a pump used for powering many transports along the midgut, as of oligopeptides. The Na+ and K+ pumps are responsible for charge load balancing and therefore are present in most cell types. Two sequences of oligopeptide / H+ cotransporters were found in the transcriptome and their expression is higher in the posterior region. This agrees with the fact that there is the last possibility of oligopeptides remaining in the lumen to be absorbed. It is highly probable that T.molitor absorbs amino acids and sugars throughout the midgut, once these types of carriers have a uniform expression throughout the midgut . The expression of NH3/NH4+ transporters in T.molitor is confined to the regions where the pH of the insect midgut is more acidic. There is also chloride transporter expression there. The expression of bicarbonate transporters is more significant in the posterior midgut. The results suggest that acidification in the anterior T.molitor midgut may result from the secretion of NH4+ and chloride ions together, whereas the alkalization in the posterior midgut results from bicarbonate release. There is water absortion (along with glucose) throughout the midgut , while the water secretion occurs only in the final two-thirds of the midgut with the aid of aquaporins, complemented by ion transporters. It would result in the net absorption and net secretion of water in the anterior and postertior midgut, respectively. This clarifies the molecular basis of the midgut countercurrent fluxes evidenced by physiological experiments.

ABC-Transmembrantransporter sind an der Aufnahme, Verteilung und Ausscheidung vieler Arznei- und Fremdstoffe beteiligt. Sie spielen eine Schlüsselrolle in der Pharmakokinetik und in der Ausscheidung toxischer endogener oder exogener Substanzen. Das Ziel der hier präsentierten Untersuchungen war deshalb, den Einfluss genetischer Polymorphismen des bekanntesten Vertreters dieser Proteinfamilie, MDR1 (ABCB1) zu untersuchen. Darüberhinaus sollte der ebenfalls zur ABC-Transporterfamilie gehörende hepatozelluläre Exporter für monoanionische Gallensäuren identifiziert und charakterisiert werden. MDR1 erwies sich als ein hochpolymorphes Gen mit zahlreichen Einzelbasenaustauschen (SNPs). Die meisten SNPs waren intronisch oder stumm. Für den nichtkodierenden SNP im Exon 26 3435C>T ergab sich bei homozygoten Trägern des T-Allels eine im Vergleich zum Wildtyp geringere intestinale P-Glykoprotein Expression mit einer entsprechend höheren und schnelleren Absorption von Digoxin. Die Auswertung pharmakokinetischer Profile von Digoxin in Individuen mit MDR1-Haplotypen der miteinander verbundenen SNPs in Exon 21 2677 und Exon 26 3435 untermauerte die beobachteten pharmakogenetischen Effekte. Nach oraler Einzelgabe von 1 mg Digoxin konnten wahrscheinlich aufgrund der Überschreitung der P-Glykoprotein Transportkapazität keine genotypischen Unterschiede beobachtet werden. Der biliäre Exporter für monoanionische Gallensäuren (Bsep) konnte als ein 160 kDa Glykoprotein aus einer Rattenleber cDNA-Bibliothek identifiziert werden und gehört ebenfalls zur ABC Transporter-Familie. Die transkriptionelle Regulation und Möglichkeiten der Modulation der Expression des Bsep-Gens wurden in vitro und in Tiermodellen der Cholestase untersucht. Dabei zeigte sich, dass Gallensäuren über ein proximales FXRE-Motiv die Bsep Promotoraktivität stimulierten. Arzneistoffe hatten ebenfalls einen Einfluss auf die Transkription des Bsep-Gens. Die adaptive Regulation hepatozellulärer Transporter während der Cholestase ergab eine verminderte Expression der meisten basolateralen Aufnahmetransporter und eine unveränderte oder heraufregulierte Proteinmasse kanalikulärer (apikaler) Efflux-Transporter. Dieses Regulationsmuster dient dem Schutz der Leberzelle, indem eine intrazelluläre Anreicherung toxischer Gallensäuren vermindert und der Gallefluss für eine intakte biliäre Clearance aufrechterhalten wird. / ABC transmembrane transporters are involved in absorption, distribution and excretion of diverse drugs and xenobiotics. They are key factors in pharmacokinetics and in the elimination of toxic endogenous or exogenous compounds. Therefore, the aim of the present study was to investigate the influence of genetic polymorphisms of the best known member of this protein family, MDR1 (ABCB1). In addition, the identity of another ABC transporter assumed to be the major hepatocellular export pump for monoanionic bile acids should be revealed and characterized. MDR1 turned out as a highly polymorphic gene with many single nucleotide polymorphisms (SNPs). Most of the SNPs were intronic or silent. Homozygous carriers of the non-coding SNP in exon 26 3435C>T had lower intestinal P-glycoprotein expression rates and thus enhanced absorption of the model compound digoxin as compared to wildtype controls. The analysis of pharmacokinetic profiles in different MDR1-haplotypes of the linked SNPs in exon 21 2677 and exon 26 3435 supported the above data. An oral single dose of 1 mg digoxin did not result in genotypic differences of tested genotypes, probably because this dose was above the maximal transport capacity of P-glycoprotein. The biliary export pump for monoanionic bile acids (Bsep) was identified as an 160 kDa glycoprotein of the ABC transporter family by screening a rat liver cDNA library. The transcriptional regulation and modulatory factors of Bsep (Abcb11) gene expression were analyzed in vitro and in animal models of cholestasis. The promoter activity of Bsep was stimulated by bile acids via a proximal FXRE motif. Drugs were also able to modify Bsep gene transcription. Adaptive regulation of hepatocellular transporters during cholestasis followed a pattern of diminished expression of most basolateral uptake carrier systems and maintained or even upregulated protein mass of canalicular (apical) exporters. This pattern serves as a protective mechanism of the liver cells preventing intracellular accumulation of toxic bile acids and providing unimpaired biliary flow and clearance.

A deficiência de zinco é considerada uma das deficiências nutricionais de maior importância epidemiológica, com grandes repercussões no contexto da saúde pública em todo o mundo. Avanços metodológicos na avaliação de aspectos moleculares constituem uma nova ferramenta para a investigação do estado nutricional dos indivíduos relativo ao zinco, por meio da avaliação da expressão gênica das proteínas transportadoras, destacando-se o ZnT1 (SLC30A1) e ZIP4 (SLC39A4), envolvidos com a manutenção da homeostase corporal do mineral. O objetivo desse trabalho foi avaliar o estado nutricional relativo ao zinco de crianças e sua relação com a expressão gênica destes transportadores. Para isso foram avaliadas 139 crianças com idade entre 32 e 76 meses matriculadas em creches da cidade de Aracaju, Sergipe. De acordo com a avaliação antropométrica, apenas 3,6% das crianças apresentaram déficit de estatura para idade e peso para idade, enquanto 27,3% delas apresentaram sobrepeso e obesidade. A deficiência de zinco plasmático foi observada em 26,6% das crianças, enquanto no eritrócito 99,3% delas encontravam-se abaixo do ponto de corte estabelecido. A análise da expressão gênica das proteínas transportadoras ZnT1 e ZIP4 apresentou relação significativa com o estado nutricional das crianças quanto ao zinco. A expressão do mRNA ZnT1 foi maior entre as crianças com deficiência, enquanto do mRNA ZIP4 foi menor. Não foram observadas relações entre o consumo de zinco e a expressão gênica. Conclui-se que a população estudada apresenta risco de deficiência em zinco, apresentando relação com a expressão gênica das proteínas transportadoras ZnT1 e ZIP4, reforçando a existência de uma regulação da absorção do mineral de acordo com o estado nutricional do indivíduo, indicando que esta análise pode ser útil na avaliação de intervenções dietéticas. / Zinc deficiency is regarded as one of the nutritional deficiencies of greatest epidemiological importance, with major repercussions in the context of public health worldwide. Methodological advances in evaluation of molecular aspects constitute a new tool for investigating the zinc nutritional status of individuals, by assessing the gene expression of transporter proteins, highlighting the ZnT1 (SLC30A1) and ZIP4 (SLC39A4), involved in maintaining body homeostasis of this mineral. This study aimed to evaluate the zinc nutritional status of children and its relation to gene expression of these transporters. Were evaluated 139 children aged between 32 and 76 months attending daycare centers in the city of Aracaju, Sergipe. According to anthropometric measures, only 3.6% of children had deficit height for age and weight for age, while 27.3% of them were overweight and obese. The plasma zinc deficiency was observed in 26.6% of children, while in erythrocytes 99.3% of them were below the established cutoff point. The ZnT1 and ZIP4 gene expression analysis showed a significant relationship with the zinc nutritional status of children. The mRNA ZnT1 expression was higher among children with zinc deficiency, while mRNA ZIP4 was lower. There was no relationship between the consumption of zinc and gene expression. In conclusion, the evaluated population is at risk for zinc deficiency, showing significant relationship with the gene expression of transporter proteins ZnT1 and ZIP4, reinforcing the existence of a regulation of the absorption of the mineral according to the individual\'s nutritional status, indicating that this analysis can be useful in evaluating dietary interventions.

A levedura Saccharomyces cerevisiae, amplamente utilizada na conversão de glicose e frutose a etanol, não é capaz de fermentar a xilose presente na biomassa lignocelulósica de resíduos agroindustriais. Apesar da introdução da via metabólica dessa pentose em linhagens de S. cerevisiae, a fermentação da xilose simultaneamente com outros açúcares ainda é pouco eficiente. A proposta deste trabalho foi aumentar a eficiência do consumo da xilose por linhagens de S. cerevisiae introduzindo genes de transportadores exógenos identificados em leveduras selvagens que naturalmente fermentam pentoses. A via do metabolismo da xilose foi integrada no genoma de uma linhagem industrial brasileira de S. cerevisiae usada na produção de etanol. A partir desta, linhagens isogênicas foram criadas e mostraram ser mais eficientes no metabolismo da xilose em meio sintético e capazes de co-fermentar glicose e xilose na presença de altas concentrações de inibidores resultantes da hidrólise da biomassa lignocelulósica. Os tranportadores identificados foram testados nas linhagens industriais geneticamente modificadas criadas neste estudo e em linhagens laboratoriais. Não foi possível confirmar a eficiência dos transportadores nas linhagens, embora os resultados mostraram diferenças nas curvas de crescimento das linhagens industriais expressando os transportadores. Este trabalho foi o início de um estudo dos fatores envolvidos no metabolismo da xilose e servirá como base para que futuros trabalhos sejam realizados na obtenção de uma linhagem mais eficiente para produção de etanol de segunda geração. / The yeast Saccharomyces cerevisiae, which efficiently ferments glucose and fructose to ethanol, is unable to ferment xylose present in lignocellulosic biomass of agroindustrial residues. Although the introduction of xylose metabolic pathways in S. cerevisiae strains has been described in the literature, the simultaneous fermentation of xylose and glucose in these modified strains is still very inefficient. The aim of this study was to increase the xylose consumption efficiency of S. cerevisiae by introduction of exogenous genes identified in wild yeast that naturally ferment pentose. The xylose metabolism pathway was integrated into the genome of a Brazilian industrial strain of S. cerevisiae used for the production of ethanol, which was then used to obtain isogenic modified strains. The isogenic strains showed to be more effective in xylose metabolism in synthetic medium and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting hydrolysis of lignocellulosic biomass. The transporters identified were inserted into genetically modified industrial strains of S. cerevisiae created in this study and also in laboratory strains. It was not possible to confirm the transporters efficiency in laboratory strains but the results showed differences in the growth curves of the industrial strains expressing the transporters. This work was the beginning of a study of the factors involved in xylose metabolism and it will help to prepare future work to obtain an efficient strain for lignocellulosic ethanol production.

Mollusc shells are unique adaptations that serve to protect the organisms that make them, and are a defining feature of the phylum. However the molecular underpinnings of shell forming processes are still largely unexplored. To further understand mollusc shell formation, I studied three bivalve species in this project: the blue mussel Mytilus edulis, the Pacific oyster Crassostrea gigas, and the king scallop Pecten maximus. While previous analyses of the shell proteomes showed species specificity, transcriptomes of the mantle tissues revealed more commonalities. To reconcile these differences, I studied differential gene expression in shell damage-repair experiments and during the formation of the first larval shell, to produce a comprehensive overview of shell formation processes. Expression data showed large biological variability between individuals, requiring matched-pair experimental designs to detect differential gene expression during shell repair. Loci differentially expressed during shell repair and in the larvae encoded shell matrix proteins, transmembrane transporters, and novel transcripts. A large number of shell matrix proteins, encoded in differentially expressed loci, were common in all three species during shell formation, indicating that shell forming proteins between different species may be more common than previously thought. Differential expression of transmembrane transporters during shell repair indicated that the animals may be regulating bicarbonate ions during shell formation. Finally, the experiments revealed novel transcripts, with unknown annotations to public datasets, that may putatively be involved in shell formation.

Neisseria gonorrhoeae is an obligate human pathogen that causes the common STI gonorrhea, which presents a growing threat to global health. The WHO estimated 78 million new cases of gonorrhea worldwide in 2017, with estimates of 820,000 new cases in the United States alone according to the CDC. High-frequency phase and antigenic variation inherent in N. gonorrhoeae, coupled with its natural ability to rapidly acquire and stably integrate antimicrobial resistance factors into its genome, have culminated in an infection against which there is no effective vaccine, and for which the list of viable therapeutic options is quickly shrinking. Moreover, no protective immunity against subsequent infections is elicited upon exposure to N. gonorrhoeae, which highlights the need for research of novel antimicrobial and vaccination strategies. Within the human host, N. gonorrhoeae utilizes a unique strategy to overcome host sequestration of essential nutrients – termed nutritional immunity (NI) – such as ions of trace metals. The pathogen produces a family of outer membrane proteins called TonB-dependent transporters (TdTs) capable of binding to host NI factors and stripping them of their nutritional cargo for use by the pathogen. Importantly, these TdTs are very highly conserved and expressed among Neisseria species. TbpA is a well-characterized TdT that allows N. gonorrhoeae to acquire iron from human transferrin, and recent studies from our lab have shown that TdfH is capable of binding to a zinc-sequestering S100 protein called calprotectin and stripping it of its zinc ion. The S100 proteins are EF-hand calcium-binding proteins that naturally play an integral role in Ca2+ homeostasis, but due to their ability to bind transition metals, they have also demonstrated an innate immunity role by participating in nutrient sequestration. The S100 proteins are expressed in all human cells, and all are capable of binding transition metals including zinc, manganese, and cobalt. Calprotectin, S100A7, and S100A12 have demonstrated an ability to hinder the infection potential of pathogenic E. coli, S. aureus, C. albicans, and various other pathogens via zinc sequestration. Herein, we demonstrate that N. gonorrhoeae is able to overcome this phenomenon and actually utilize these proteins as a zinc source in vitro. Furthermore, we identify S100A7 as the specific ligand for TdfJ, which utilizes this ligand to internalize zinc during infection. S100A7 growth support in vitro is dependent upon a functional TonB, TdfJ, and the cognate ABC transport system ZnuABC, and isogenic mutants incapable of producing znuA or tdfJ recover S100A7 utilization by complementation. Whole-cell binding assays and affinity pulldowns show that S100A7 binds specifically to both gonococcal and recombinant TdfJ, and growth and binding experiments show that these described phenomena are specific to human and not mouse S100A7. Finally, we show that a His-Asn double mutant S100A7 that is incapable of binding zinc cannot be utilized for growth by gonococci. These data illustrate the unique nature of the gonococcus’ ability to co-opt host defense strategies for its own purposes, and further identify the TdTs as promising targets for strategies to combat and prevent gonococcal infection.

The blood-brain barrier is formed by tightly joined capillary cells with transporter proteins and acts as to regulate the brain concentration of nutrients as well as many drugs. When developing central nervous system drugs it is necessary to measure the unbound drug concentration in the brain, i.e. the unbound brain exposure. This is to ensure that the drug reaches the site of action. Furthermore, when designing new drugs it is extremely valuable to be able to predict brain exposure from a tentative drug structure. Established methods to measure total drug concentrations are of limited (if any) utility since the pharmacologically active, unbound, concentration is not obtained. The aim of the conducted research was to develop an efficient methodology to measure unbound drug in the brain and to generate a dataset for developing computational prediction models describing the relationship between drug structure and unbound brain exposure. First it was demonstrated that unbound brain exposure can be efficiently assessed using a combination of total drug concentrations in the brain and separate measurements of drug binding in the brain slices. The in vitro brain slice method was refined and made high-throughput. Improvements were also made to the in vivo measurements of total concentrations by introducing an appropriate correction for drug in residual blood. Modeling of a 43-drug dataset in the rat showed that unbound brain exposure is related to the drug hydrogen bonding potential and not to lipid solubility, which contrasts the common understanding. Further, the drug concentrations in cerebrospinal fluid approximated unbound concentrations in the brain (r2=0.80) and were also correlated with corresponding measurements in humans (r2=0.56). Therefore, rat-derived prediction models can be used when designing drugs for humans. This thesis work has provided drug industry and academia with efficient tools to obtain and to use relevant estimates of drug exposure in the brain for evaluating drugs candidates.

Mother's milk provides multiple benefits to the offspring. However, xenobiotics transferred into breast milk may pose a risk to the nursing infant. The breast cancer resistance protein (BCRP) actively transports xenobiotics into breast milk. BCRP also transports nutrients, like riboflavin, and together with recently identified riboflavin transporters (RFT), may provide a mechanism for riboflavin secretion into breast milk. Expression of RFT in the mammary gland remained unknown. Our objective was to characterize Bcrp and Rft mRNA expression in the mammary gland of FVB/N mice, and investigate a strategy to decrease excretion of BCRP-transported xenobiotics into the milk using riboflavin intervention. Rft and Bcrp mRNA were upregulated in the mammary gland of lactating mice. An intravenous riboflavin administration significantly reduced the levels of BCRP-transported cimetidine in milk. This study demonstrates the use of riboflavin to exploit the function of mammary BCRP in order to reduce xenobiotic secretion into breast milk.