The “magic bullet” theory stated by Paul Ehrlich more than one century ago foresees that drugs need to reach their target to exert their pharmacological activity. When dealing with antibiotics, this implies not only a capacity to interact with bacteria, but also to rejoin them in the infected compartment. The latter property is directly linked to pharmacokinetics. In this Thesis, we have focused our interest on cellular pharmacokinetics of antibiotics in macrophages, reasoning that (i) this model would be relevant for predicting the access of antibiotics to intracellular bacteria, and (ii) deciphering mechanisms of drug entry, distribution, and efflux in single cells would provide pieces of information applicable to the understanding of general pharmaco-kinetics. Accumulation and distribution were studied for oritavancin, a semi-synthetic glycopeptide currently in phase III of clinical development. This molecule is characterized by a highly bactericidal activity against staphylococci, including multiresistant strains, which would be of interest for the eradication of the intracellular forms of these bacteria. We show that oritavancin enters macrophages by adsorptive endocytosis and accumulates to very large amounts in lysosomes, with cellular concentrations as high as 300-fold the extracellular ones. Accordingly, it exerts a concentration-dependent bactericidal activity against Staphylococcus aureus multiplying in phagolysosomes. In parallel, however, it also causes morphological and biochemical alterations, characterized by the deposition of material of heterogeneous aspect and the accumulation of phospholipids and cholesterol. These data point to the difficulty of dissociating high cellular accumulation and cellular toxicity and plead for the interest of in vitro models in the evaluation of the intracellular activity of antibiotics, the early assessment of drug safety profile and the orientation of further in vivo studies. Active efflux was evaluated for macrolides and quinolones, two antibiotic classes accumulating in macrophages and therefore usually considered as useful in the treatment intracellular infections. Macrophages indeed express multidrug transporters in their pericellular membrane, which can extrude a large variety of drugs presenting as common feature an amphiphatic character. We show that macrolides (and azithromycin in particular) are substrates for P-glycoprotein, while quinolones (and ciprofloxacin in particular) are substrates for an MRP-like transporter. Inhibiting these pumps increases the intracellular activity of these antibiotics by enhancing their cellular accumulation. We also describe different mode of transport for these antibiotics, which essentially reflect their variable fluxes through the membrane. These data underline the major role constitutive efflux plays in the modulation of the pharmacokinetics of drugs, and, hence, of their pharmacodynamics. They encourage the setting-up of large-scale screenings aimed at evaluating drug-transporter interactions in lead optimization processes. We may therefore conclude to the usefulness of cellular models in the study of the pharmacokinetics of drugs and of its consequences for pharmacodynamics and toxicity, and suggest the implementation of such models in the early development of new drugs.

Network decontamination is a well known mobile agent problem with many applications. We assume that all nodes of a network are contaminated (e.g., by a virus) and a set of agents is deployed to decontaminate them. An agent passing by a node decontaminates it, however a decontaminated node can be recontaminated if any of its neighbours is contaminated. In the vast literature a variety of models are considered and different assumptions are made on the power of the agents. In this thesis we study variation of the decontamination problem in mesh and tori topologies, under the assumption that when a node is decontaminated, it is immune to recontamination for a predefined amount of time t (called immunity time). After the immunity time is elapsed, recontamination can occur. We focus on three different models: mobile agents (MA), cellular automata (CA), and mobile cellular automata (MCA). The first two models are commonly studied and employed in several other contexts, the third model is introduced in this thesis for the first time. In each model we study the temporal decontamination problem (adapted to the particular setting) under a variety of assumptions on the capabilities of the decontaminating elements (agents for MA and MCA, decontaminating cells for CA). Some of the parameters we consider in this study are: visibility of the active elements, their ability to make copies of themselves, their ability to communicate, and the possibility to remember their past actions (memory). We describe several solutions in the various scenarios and we analyze their complexity. Efficiency is evaluated slightly differently in each model, but essentially the effort is in the minimization of the number of simultaneous decontaminating elements active in the system while performing the decontamination with a given immunity time.

Network decontamination is a well known mobile agent problem with many applications. We assume that all nodes of a network are contaminated (e.g., by a virus) and a set of agents is deployed to decontaminate them. An agent passing by a node decontaminates it, however a decontaminated node can be recontaminated if any of its neighbours is contaminated. In the vast literature a variety of models are considered and different assumptions are made on the power of the agents. In this thesis we study variation of the decontamination problem in mesh and tori topologies, under the assumption that when a node is decontaminated, it is immune to recontamination for a predefined amount of time t (called immunity time). After the immunity time is elapsed, recontamination can occur. We focus on three different models: mobile agents (MA), cellular automata (CA), and mobile cellular automata (MCA). The first two models are commonly studied and employed in several other contexts, the third model is introduced in this thesis for the first time. In each model we study the temporal decontamination problem (adapted to the particular setting) under a variety of assumptions on the capabilities of the decontaminating elements (agents for MA and MCA, decontaminating cells for CA). Some of the parameters we consider in this study are: visibility of the active elements, their ability to make copies of themselves, their ability to communicate, and the possibility to remember their past actions (memory). We describe several solutions in the various scenarios and we analyze their complexity. Efficiency is evaluated slightly differently in each model, but essentially the effort is in the minimization of the number of simultaneous decontaminating elements active in the system while performing the decontamination with a given immunity time.

Thesis (Ph. D.)--University of Chicago, Committee on Human Nutrition and Nutritional Biology, 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Committee on Immunology, August 2001. / Includes bibliographical references. Also available on the Internet.

Network decontamination is a well known mobile agent problem with many applications. We assume that all nodes of a network are contaminated (e.g., by a virus) and a set of agents is deployed to decontaminate them. An agent passing by a node decontaminates it, however a decontaminated node can be recontaminated if any of its neighbours is contaminated. In the vast literature a variety of models are considered and different assumptions are made on the power of the agents. In this thesis we study variation of the decontamination problem in mesh and tori topologies, under the assumption that when a node is decontaminated, it is immune to recontamination for a predefined amount of time t (called immunity time). After the immunity time is elapsed, recontamination can occur. We focus on three different models: mobile agents (MA), cellular automata (CA), and mobile cellular automata (MCA). The first two models are commonly studied and employed in several other contexts, the third model is introduced in this thesis for the first time. In each model we study the temporal decontamination problem (adapted to the particular setting) under a variety of assumptions on the capabilities of the decontaminating elements (agents for MA and MCA, decontaminating cells for CA). Some of the parameters we consider in this study are: visibility of the active elements, their ability to make copies of themselves, their ability to communicate, and the possibility to remember their past actions (memory). We describe several solutions in the various scenarios and we analyze their complexity. Efficiency is evaluated slightly differently in each model, but essentially the effort is in the minimization of the number of simultaneous decontaminating elements active in the system while performing the decontamination with a given immunity time.

Thesis (M.P.H.)--University of Michigan, 1947.

Thesis (M.P.H.)--University of Michigan, 1947.

Thesis (Ph. D.)--State University of New York at Buffalo, 2002. / "May 10, 2002." Includes bibliographical references (p. 140-150). Also available in print.

A ativação dos fagócitos é crítica para a defesa contra vários patógenos, por isso a importância de estudos que desenvolvam alternativas para a ativação dessas células. Nosso estudo visou investigar os efeitos do BAY 41-2272 na ativação de fagócitos. Para este propósito avaliamos a fagocitose, liberação de superóxido e atividade microbicida. Foram usados PBMC, neutrófilos e a linhagem celular THP-1, cultivadas na presença ou ausência de BAY 41-2272 (1mM ou 3mM) por 1h ou 48h a 37°C. A liberação de superóxido foi avaliada pelo ensaio da redução do citocromo c inibido especificamente pela superóxido dismutase; a fagocitose foi analisada através da co-cultura com partículas de Zymosan e contagem das células com partículas englobadas; e a atividade microbicida foi mensurada por meio da co-cultura com E. coli enteropatogênica seguida pela contagem das CFU formadas pelas bactérias recuperadas dos fagócitos. Além disso, fizemos a dosagem de IL-12, IFN-g e TNF-a e a análise da expressão gênica do gene CYBB. Todos os tipos celulares responderam aos tratamentos. Os PBMC, neutrófilos e THP-1 tratados com BAY 41-2272 produziram significativamente mais superóxido (cerca de 50% mais), e apresentaram significativamente maior atividade fagocítica (cerca de 54% mais), microbicida (cerca do dobro) comparados ao grupo controle não tratado, além de produzir TNFa. Ainda, o tratamento com o fármaco aumentou a expressão do gene da gp91phox nas THP-1 e PBMC. BAY 41-2272, especialmente na dose de 3mM, mostrou um grande potencial na ativação dos fagócitos em vários aspectos. Este potencial pode ser explorado na busca por novas terapias destinadas ao controle de infecções, principalmente no caso das imunodeficiências. / Phagocyte activation is critical role for host defense against several pathogens, so that studies developing alternatives for activating these cell are very important. We investigated the effects of BAY 41-2272 on phagocytes activation. For this purpose we evaluated several aspects indicating cellular activation, as phagocytosis, superoxide release and microbicidal activity. We used PBMC, neutrophils and THP-1 cell lineage cultured or not with BAY 41-2272 (1mM and 3mM) for 1h or 48h at 37°C. Superoxide release was tested by superoxide dismutase-inhibitable cytochrome c reduction assay; phagocytosis was evaluated through co-culture with zymosan particles and counting of ingested particles; and microbicidal activity was measured through co-incubation with enteropathogenic E. coli followed by counting of CFU from recovered bacteria from phagocytes. We analyzed the IL-12, IFN-g and TNF-a cytokine production and gp91phox gene expression. All cell types showed a response to treatments. PBMC, PMN and THP-1 treated with BAY 41-2272 produced significantly more superoxide (about 50% more), and presented significantly more phagocytic (about 54% more), microbicidal (about twice) activity than control group and production more TNF-a than control group. The expression of CYBB gene was more expressed on treated cells than control. BAY 41-2272, especially at 3mM, showed a great potential in activation of phagocytes in several aspects. This potential should be investigated at the light of new therapies seeking infection control, mainly in immunodeficiency.