The use of DNA-based therapeutics requires efficient delivery systems to transport the DNA to their place of action within the cell. To accomplish this, we investigated multiwalled carbon nanotubes (pristine MWCNT, p-MWCNT) functionalized with hydroxyl groups via 1,3-dipolar cycloaddition. In this way, we have obtained MWCNT-f-OH with improved stability in aqueous dispersions which is an advantageous property for their use in cellular environments. Afterwards, a carrier strand oligodeoxynucleotide (CS-ODN) was adsorbed to MWCNT-f-OH followed by hybridization with a therapeutic antisense oligodeoxynucleotide (AS-ODN). The amount of adsorbed CS-ODN, as well as the complementary AS-ODN and a non-complementary oligodeoxynucleotide (NS-ODN) as reference, was directly measured by radionuclide labeling of ODNs. We show that subsequent release of AS-ODNs and NS-ODNs was possible for MWCNT-f-OH above the melting temperature of AS-ODNs at 80 °C and under physiological conditions at different pH values at 37 °C. We also show a very low influence of p-MWCNT and MWCNT-f-OH on the cell viability of the bladder carcinoma (BCa) cell line EJ28 and that both MWCNT types were internalized by EJ28. Therefore, MWCNT-f-OH represents a promising carrier able to transport and release AS-ODNs inside cells.

The cellular prion protein (PrPC) is unique amongst mammalian proteins in that it not only has the capacity to aggregate (in the form of scrapie PrP; PrPSc) and cause neuronal degeneration, but can also act as an independent vector for the transmission of disease from one individual to another of the same or, in some instances, other species. Since the discovery of PrPC nearly thirty years ago, two salient questions have remained largely unanswered, namely, (i) what is the normal function of the cellular protein in the central nervous system, and (ii) what is/are the factor(s) involved in the misfolding of PrPC into PrPSc? To shed light on aspects of these questions, we undertook a discovery-based interactome investigation of PrPC in mouse neuroblastoma cells (Chapter 2), and among the candidate interactors, identified two members of the ZIP family of zinc transporters (ZIP6 and ZIP10) as possessing a PrP-like domain. Detailed analyses revealed that the LIV-1 subfamily of ZIP transporters (to which ZIPs 6 and 10 belong) are in fact the evolutionary ancestors of prions (Chapter 3). We were further able to demonstrate that PrPC likely emerged from a ZIP ancestor molecule nearly half-a-billion years ago via a retrotransposition event (Chapter 4). Moreover, biochemical investigations on ZIP10, as a model LIV-1 ZIP transporter, demonstrated that the ectodomain shedding of ZIP10 observed in prion-infected mice resembles a cellular response to transition metal starvation and suggested that prion disease in mice might phenocopy a transition metal starvation status (Chapter 5). These studies have opened a new angle to study prion biology in health and disease. Biochemical investigations on other LIV-1 ZIPs and attempts at the structural elucidation of the PrP-like domain of LIV-1 ZIP proteins are ongoing and have not been included in this thesis.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (M.Sc.). / Written for the Dept. of Experimental Medicine. Title from title page of PDF (viewed 2008/12/07). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 98-113).

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 131-147).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 132-138).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

The protein-energy malnutrition is one of the most serious nutritional problems that occur in developing countries. The energy and nutritional deficiency in early life can cause significant changes in different stages of development of central nervous system, since the cases of hyperplasia, hypertrophy and myelinization occur in more intense. The brain uses glucose as its main energy substrate. However, under certain physiological conditions, as during the weaning or during prolonged fasting, the cells may use other substrates in order to meet their metabolic needs. The transport of glucose to the nerve cells requires the presence of specific transporter proteins called GLUT s (Glucose Transporters) to perform the transport of glucose by facilitated diffusion. Besides the transporters of glucose, transporter proteins of monocarboxilatos (MCTs) are responsible for neuronal influx of lactate, MCT1 being the main responsible for providing energy to neurons during maturation of nervous tissue. The acetylcholine is a major neurotransmitter of the nervous system and is associated with the maintenance processes of attention, learning and memory. In the synaptic cleft acetylcholine is degraded by the enzyme cholinesterase, the acetylcholinesterase (AChE) and the butyrilcholinesterase (BuChE). Increased activity of fractions of AChE has been associated with states of dementia in elderly patients and the loss of cognitive function in patients with Alzheimer's Disease. This work is proposed to study the expression of neuronal glucose transporter GLUT3 and activity of the enzyme acetylcholinesterase (AChE) in brain of young adult rats (84 days of life) who were breastfed in litters formed by 6 (control group) or 12 pups (undernourished group). The results showed that the weight of the brains of malnourished rats was significantly lower (P <0001, Student s t test) compared to controls. The blood glucose levels and the expression of GLUT3 in total cortical membranes was also reduced by malnutrition (P <0001, Student s t test). The activity of AChE in different brain homogenate showed a significant interaction (P = 0019, two-way ANOVA, Tukey s test) between nutritional status and the type of fraction of the homogenate, with a significant reduction (p< 0,05) in activity this enzyme in the brain homogenate in control group. These results show for the first time that malnutrition during the period of weaning reduces the expression of GLUT3 and vi that the increased activity of acetylcholinesterase associated with decreased in the expression of the main glucose transporter in the brain could contribute to cognitive deficits and changes in brain metabolic activity. / A desnutrição energético-protéica consiste em um dos problemas nutricionais mais graves que ocorrem em países em desenvolvimento. A deficiência energética e nutricional no início da vida pode ocasionar importantes alterações nas diferentes fases de desenvolvimento do sistema nervoso central, uma vez que os processos de hiperplasia, hipertrofia e mielinização ocorrem de forma mais intensa. O cérebro utiliza a glicose como seu principal substrato energético. No entanto, em certas condições fisiológicas, como durante a fase de amamentação ou durante o jejum prolongado, as células podem utilizar outros substratos a fim de suprir suas necessidades metabólicas. O transporte de glicose para as células nervosas requer a presença de proteínas transportadoras específicas denominadas GLUTs (glucose transporters) que efetuam o transporte de glicose através de difusão facilitada. Além dos transportadores de glicose, proteínas transportadoras de monocarboxilatos (MCTs) são responsáveis pelo influxo neuronal de lactato, sendo o MCT1 o principal responsável pelo fornecimento energético aos neurônios durante o processo de maturação do tecido nervoso. A acetilcolina é um dos principais neurotransmissores do sistema nervoso, sendo associada com a manutenção de processos de atenção, aprendizagem e memória. Na fenda sináptica a acetilcolina é degradada por enzimas colinesterásicas, a acetilcolinesterase (AChE) e a Butirilcolinesterase (BuChE). O aumento da atividade de frações da AChE vem sendo associada com estados de demência em pacientes idosos e com a perda da função cognitiva em pacientes com Doença de Alzheimer. O presente trabalho se propôs a estudar a expressão do transportador neuronal de glicose GLUT3 e a atividade da AChE no cérebro de ratos adultos jovens (84 dias de vida) que foram amamentados em ninhadas formadas por 6 (grupo controle) ou 12 filhotes (grupo desnutrido). Os resultados demonstraram que o peso dos cérebros dos ratos desnutridos foi significativamente menor (P<0,001, teste t de Student) comparado ao grupo controle. Os níveis glicêmicos e a expressão do GLUT3 nas membranas corticais totais foram também diminuídos pela desnutrição (P<0,001, teste t de Student). A atividade da AChE nos diferentes homogenados do cérebro mostrou uma interação significativa (P = 0,019, ANOVA two-way, Tukey teste) entre o estado iv nutricional e o tipo de fração do homogenado, apresentando uma redução significativa ( p< 0,05) na atividade desta enzima do homogenado de células em animais controle . Esses resultados mostram pela primeira vez que a desnutrição durante o período de amamentação diminui a expressão do GLUT3 e que o aumento da atividade da acetilcolinesterase associado com a diminuição da expressão do principal trnasportador de glicose para o cérebro poderia contribuir para déficits cognitivos e alterações da atividade metabólica cerebral.

Cryptococcus gattii é um dos principais agentes de infecção oportunista em todo o mundo. Esse fungo está presente no meio ambiente e, por isso, pode infectar diversos hospedeiros, inclusive seres humanos, nematóides e células ameboides. Acanthamoeba spp. são protozoários de vida livre que fagocitam diversos organismos, especialmente bactérias e fungos. Apesar de macrófagos e amebas serem evolutivamente distantes, eles compartilham diversas etapas comuns no processo de fagocitose e eliminação do patógeno. Além disso, existem teorias de que amebas e macrófagos possuem um ancestral comum. Para averiguar se essas duas células fagocíticas apresentam estratégias antifúngicas similares, nós analisamos o mecanismo de imunidade nutricional. Essa estratégia imunológica reduz a disponibilidade de nutrientes essenciais para o patógeno, inclusive metais de transição como o zinco. Neste trabalho, nós analisamos se há modulação da homeostase de zinco em Acanthamoeba castellanii durante sua interação com C. gattii. Testes de fagocitose e taxa de replicação intracelular (IPR) realizados através da interação de amebas com a linhagem selvagem (WT) e mutante do gene ZIP1 de C. gattii. O mutante utilizado (zip1Δ) é caracterizado pela sua incapacidade de crescer sem a presença de zinco. Nós observamos que a linhagem mutante foi mais fagocitada por células de A. castellanii comparado com WT. Também, o teste de IPR mostrou que a atividade antifúngica das células hospedeiras apresentou-se mais efetiva contra as células mutantes. Entretanto, a sobrevivência de zip1Δ foi maior quando zinco extracelular (10 M) foi adicionado ao meio de interação. Esses resultados sugerem que as células criptocócicas internalizadas podem estar sofrendo uma privação da disponibilidade de zinco no interior do fagossomo. Para analisar alterações nos transportadores de zinco das células hospedeiras durante sua interação com C. gattii, análises de PCR quantitativo em tempo real (RT-qPCR) foram realizadas para os transportadores de zinco das famílias ZIP e ZnT. Uma intensa modulação de alguns genes foi observado após 3 e 24 horas pós-infecção. Além disso, análises de citometria de fluxo mostraram que os níveis de zinco livre das amebas estavam reduzidos devido a presença do fungo. Esses resultados sugerem que amebas podem modular a disponibilidade de zinco, afim de prejudicar o patógeno. / Cryptococcus gattii is one of the most important agents of opportunistic infections worldwide. They are found in the environment, where it can interact with different host types, including humans, nematodes and amoebic cells. Acanthamoeba spp. are free-living protozoa that basically feed on bacteria and yeast through phagocytosis. Macrophages and amoebae, although evolutionarily distant, share conserved mechanisms related to steps of phagocytosis and microbial killing. In addition, it has been hypothesized that amoeba and macrophage have a common ancestor. To investigate if there are similar antifungal strategies between both cellular types, we analyzed the nutritional immunity mechanism. It is a process defined as a reduction of essential nutrients availability to the pathogen, such as zinc. In this context, we investigate if amoeba cells are able to modulate zinc homeostasis during the interaction with C. gattii. Phagocytosis and intracellular replications (IPR) analysis performed through the interaction between amoebae and wild-type (WT) and mutant for the ZIP1 gene (zip1Δ) strains of C. gattii. The mutant is unable to grow in absence of zinc. We found that zip1Δ strain is more readily engulfed by A. castellanii cells compared to WT. In addition, IPR analysis showed that the antifungal activity of such host cells was more effective against the mutant cells. However, the mutant strain survival was increased when additional extracellular zinc (10 M) was added to the interaction medium. This data suggests that engulfed cryptococcal cells might have been experiencing a deprivation of zinc inside the phagosome. To further evaluate alterations of zinc transporters in host cells due to cryptococcal infection, RT-qPCR analysis was performed for the ZIP and ZnT zinc transporter families. An intense modulation of some genes was found after 3 and 24 hours’ post-infection. Furthermore, flow cytometry analysis showed that free zinc levels from amoebae are reduced by the cryptococcal presence. These results indicate that amoebae are able to modulate zinc availability to harm the pathogen.