Bacterial inter-species communication mediated by the autoinducer-2 signal

Dissertation presented to obtain the Ph.D degree in Biology by Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica, Instituto Gulbenkian de Ciência.

The extent of heterocellular communication mediated by gap junctions is predictive of bystander tumor cytotoxicity in vitro.

Herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) viral-directed enzyme prodrug gene therapy causes potent, tumor-selective cytotoxicity in animal models in which HSV-tk gene transduction is limited to a minority of tumor cells. The passage of toxic molecules from HSV-tk+ cells to neighboring HSV-tk- cells during GCV therapy is one mechanism that may account for this "bystander" cytotoxicity. To investigate whether gap junction-mediated intercellular coupling could mediate this bystander effect, we used a flow cytometry assay to quantitate the extent of heterocellular coupling between HSV-tk+ murine fibroblasts and both rodent and human tumor cell lines. Bystander tumor cytotoxicity during GCV treatment in a coculture assay was highly correlated (P < 0.001) with the extent of gap junction-mediated coupling. These findings show that gap junction-mediated intercellular coupling contributes to the in vitro bystander effect during HSV-tk/GCV therapy and that retroviral transduction of tumor cells is not required for bystander cytotoxicity.

O PERFIL DOS USUÁRIOS DA COMUNICAÇÃO MEDIADA POR COMPUTADOR UMA ABORDAGEM PSICOLÓGICA

A Comunicação Mediada por Computador (CMC) é entendida como o relacionamento interpessoal via Internet. Este modo de comunicação atinge usuários que visam interagir e conhecer pessoas sem o limite da distância. Atualmente está sendo objeto de estudos interdisciplinares que se interessam pelas influências da CMC no comportamento de seus usuários. Trata-se de um estudo caracterizado como exploratório baseado em dados coletados através de uma pesquisa bibliográfica. Seu objetivo é refletir sobre os efeitos gerados pela difusão da CMC sobre o perfil de seus usuários, além de apresentar as contribuições da Psicologia frente aos questionamentos provindos da utilização da CMC e discutir as ambivalências (ganhos e perdas) de seu uso no comportamento de seus usuários. Percebe-se que os internautas estão transformando a forma de interação social em um ambiente desprovido de limites espaço-temporais. Portanto, este trabalho estuda as novas formas de interação, e busca compreender esse novo homem, pois, se a tecnologia traz mudanças, o indivíduo muda junto com ela. Portanto, ao acompanhar todas essas transformações que têm influenciado o homem e suas novas formas de relacionamento, estão motivando o surgimento de novas formas de contribuição da Psicologia ao estudo da CMC.

O PERFIL DOS USUÁRIOS DA COMUNICAÇÃO MEDIADA POR COMPUTADOR UMA ABORDAGEM PSICOLÓGICA

A Comunicação Mediada por Computador (CMC) é entendida como o relacionamento interpessoal via Internet. Este modo de comunicação atinge usuários que visam interagir e conhecer pessoas sem o limite da distância. Atualmente está sendo objeto de estudos interdisciplinares que se interessam pelas influências da CMC no comportamento de seus usuários. Trata-se de um estudo caracterizado como exploratório baseado em dados coletados através de uma pesquisa bibliográfica. Seu objetivo é refletir sobre os efeitos gerados pela difusão da CMC sobre o perfil de seus usuários, além de apresentar as contribuições da Psicologia frente aos questionamentos provindos da utilização da CMC e discutir as ambivalências (ganhos e perdas) de seu uso no comportamento de seus usuários. Percebe-se que os internautas estão transformando a forma de interação social em um ambiente desprovido de limites espaço-temporais. Portanto, este trabalho estuda as novas formas de interação, e busca compreender esse novo homem, pois, se a tecnologia traz mudanças, o indivíduo muda junto com ela. Portanto, ao acompanhar todas essas transformações que têm influenciado o homem e suas novas formas de relacionamento, estão motivando o surgimento de novas formas de contribuição da Psicologia ao estudo da CMC.

HETEROGENEIDADE EM REDE: PRESENÇA E PRÁTICAS DE USO ENTRE ESTUDANTES DA UNIVERSIDADE METODISTA DE SÃO PAULO NAS REDES SOCIAIS

As redes sociais pela internet ganham destaque nos processos comunicacionais atuais ao permitirem uma maior interação entre os conectados. Entretanto, o conceito de rede, ao contrário do senso comum e do que se convencionou no meio acadêmico, de fato não propõe uma organização simétrica, estática e homogênea. A rede é, por natureza, dinâmica e heterogênea, assim como a sociedade é. As redes sociais e as redes digitais demonstram isso. Elas aumentam e diminuem seus nós e possuem diferentes graus de conexões, e mesmo nestes graus de conexões são diversas. O presente estudo está dividido em duas partes fundamentais. A primeira visa compreender o conceito de rede, e para isso baseia-se em Milton Santos, Pierre Musso e Albert-Lásló Barabási. Na segunda parte, a partir de uma pesquisa com estudantes universitários, há a preocupação de identificar as heterogeneidades existentes nas suas presenças nas redes e suas práticas de uso, a partir de seis categorias de análise e atividades relacionadas: relações pessoais, estudo, trabalho, ativismo, entretenimento e lazer, e informações e notícias. A questão de fundo, a respeito das possibilidades das redes, é sobre os diferentes níveis de participação, ou seja, a heterogeneidade em rede. O resultado do estudo constata a heterogeneidade, na qual os estudantes, por mais que possuam semelhanças em função da idade aproximada, da condição social e pelo fato de estudarem em uma universidade particular, são diferentes entre si, em especial, se comparados pelas áreas de formação. Não só a presença nas redes sociais se dá em níveis diferentes, quanto suas práticas também são. Portanto, o conceito de redes heterogêneas contido no pensamento de Milton Santos, Pierre Musso e Albert-Lásló Barabási, é confirmado com a pesquisa aplicada aos estudantes universitários dos cursos de graduação presencial da Universidade Metodista de São Paulo, a partir da análise de suas presenças e práticas de uso.  

O PERFIL DOS USUÁRIOS DA COMUNICAÇÃO MEDIADA POR COMPUTADOR UMA ABORDAGEM PSICOLÓGICA

A Comunicação Mediada por Computador (CMC) é entendida como o relacionamento interpessoal via Internet. Este modo de comunicação atinge usuários que visam interagir e conhecer pessoas sem o limite da distância. Atualmente está sendo objeto de estudos interdisciplinares que se interessam pelas influências da CMC no comportamento de seus usuários. Trata-se de um estudo caracterizado como exploratório baseado em dados coletados através de uma pesquisa bibliográfica. Seu objetivo é refletir sobre os efeitos gerados pela difusão da CMC sobre o perfil de seus usuários, além de apresentar as contribuições da Psicologia frente aos questionamentos provindos da utilização da CMC e discutir as ambivalências (ganhos e perdas) de seu uso no comportamento de seus usuários. Percebe-se que os internautas estão transformando a forma de interação social em um ambiente desprovido de limites espaço-temporais. Portanto, este trabalho estuda as novas formas de interação, e busca compreender esse novo homem, pois, se a tecnologia traz mudanças, o indivíduo muda junto com ela. Portanto, ao acompanhar todas essas transformações que têm influenciado o homem e suas novas formas de relacionamento, estão motivando o surgimento de novas formas de contribuição da Psicologia ao estudo da CMC.

Analysis of computer communication systems by use of digital signal processing methods

Queueing theory is an effective tool in the analysis of canputer camrunication systems. Many results in queueing analysis have teen derived in the form of Laplace and z-transform expressions. Accurate inversion of these transforms is very important in the study of computer systems, but the inversion is very often difficult. In this thesis, methods for solving some of these queueing problems, by use of digital signal processing techniques, are presented. The z-transform of the queue length distribution for the Mj GY jl system is derived. Two numerical methods for the inversion of the transfom, together with the standard numerical technique for solving transforms with multiple queue-state dependence, are presented. Bilinear and Poisson transform sequences are presented as useful ways of representing continuous-time functions in numerical computations.

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43.

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.

Selective protection of the cerebellum against intracerebroventricular LPS is mediated by local melatonin synthesis

Although melatonin is mainly produced by the pineal gland, an increasing number of extra-pineal sites of melatonin synthesis have been described. We previously demonstrated the existence of bidirectional communication between the pineal gland and the immune system that drives a switch in melatonin production from the pineal gland to peripheral organs during the mounting of an innate immune response. In the present study, we show that acute neuroinflammation induced by lipopolysaccharide (LPS) injected directly into the lateral ventricles of adult rats reduces the nocturnal peak of melatonin in the plasma and induces its synthesis in the cerebellum, though not in the cortex or hippocampus. This increase in cerebellar melatonin content requires the activation of nuclear factor kappa B (NF-κB), which positively regulates the expression of the key enzyme for melatonin synthesis, arylalkylamine N-acetyltransferase (AA-NAT). Interestingly, LPS treatment led to neuronal death in the hippocampus and cortex, but not in the cerebellum. This privileged protection of cerebellar cells was abrogated when G-protein-coupled melatonin receptors were blocked by the melatonin antagonist luzindole, suggesting that the local production of melatonin protects cerebellar neurons from LPS toxicity. This is the first demonstration of a switch between pineal and extra-pineal melatonin production in the central nervous system following a neuroinflammatory response. These results have direct implications concerning the differential susceptibility of specific brain areas to neuronal death.

Selection of Gβ Subunits with Point Mutations That Fail to Activate Specific Signaling Pathways In Vivo: Dissecting Cellular Responses Mediated by a Heterotrimeric G Protein in Dictyostelium discoideum

In Dictyostelium discoideum, a unique Gβ subunit is required for a G protein–coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Gα2, and Gβ genes completely impair all these responses. To dissect specificity in Gβγ signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gβ genes and isolated Gβ alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gβ subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gβγ by making point mutations on Gβ. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gβs and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtβγ interacts with its regulators, Gα and phosducin.

Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.

In gene therapy to treat cancer, typically only a fraction of the tumor cells can be successfully transfected with a gene. However, in the case of brain tumor therapy with the thymidine kinase gene from herpes simplex virus (HSV-tk), not only the cells transfected with the gene but also neighboring others can be killed in the presence of ganciclovir. Such a "bystander" effect is reminiscent of our previous observation that the effect of certain therapeutic agents may be enhanced by their diffusion through gap junctional intercellular communication (GJIC). Herein, we present the evidence, from in vitro studies, that gap junctions could indeed be responsible for such a gene therapy bystander effect. We used HeLa cells for this purpose, since they show very little, if any, ability to communicate through gap junctions. When HeLa cells were transfected with HSV-tk gene and cocultured with nontransfected cells, only HSV-tk-transfected HeLa cells (tk+) were killed by ganciclovir. However, when HeLa cells transfected with a gene encoding for the gap junction protein, connexin 43 (Cx43), were used, not only tk+ cells, but also tk- cells were killed, presumably due to the transfer, via Cx43-mediated GJIC, of toxic ganciclovir molecules phosphorylated by HSV-tk to the tk- cells. Such bystander effect was not observed when tk+ and tk- cells were cocultured without direct cell-cell contact between those two types of cells. Thus, our results give strong evidence that the bystander effect seen in HSV-tk gene therapy may be due to Cx-mediated GJIC.

Pupil responses associated with coloured afterimages are mediated by the magno-cellular pathway

Sustained fixation of a bright coloured stimulus will, on extinction of the stimulus and continued steady fixation, induce an afterimage whose colour is complementary to that of the initial stimulus; an effect thought to be caused by fatigue of cones and/or of cone-opponent processes to different colours. However, to date, very little is known about the specific pathway that causes the coloured afterimage. Using isoluminant coloured stimuli recent studies have shown that pupil constriction is induced by onset and offset of the stimulus, the latter being attributed specifically to the subsequent emergence of the coloured afterimage. The aim of the study was to investigate how the offset pupillary constriction is generated in terms of input signals from discrete functional elements of the magno- and/or parvo-cellular pathways, which are known principally to convey, respectively, luminance and colour signals. Changes in pupil size were monitored continuously by digital analysis of an infra-red image of the pupil while observers viewed isoluminant green pulsed, ramped or luminance masked stimuli presented on a computer monitor. It was found that the amplitude of the offset pupillary constriction decreases when a pulsed stimulus is replaced by a temporally ramped stimulus and is eliminated by a luminance mask. These findings indicate for the first time that pupillary constriction associated with a coloured afterimage is mediated by the magno-cellular pathway. © 2003 Elsevier Science Ltd. All rights reserved.

Connecting: the use of information and communication technologies by older adults in a retirement community

This is a long-term study of the use of information and communication technologies by 30 older adults (ages 70–97) living in a large retirement community. The study spanned the years of 1996 to 2008, during which time the research participants grappled with the challenges of computer use while aging 12 years. The researcher, herself a ‘mature learner,’ used a qualitative research design which included observations and open-ended interviews. Using a strategy of “intermittent immersion,” she spent an average of two weeks per visit on site and participated in the lives of the research population in numerous ways, including service as their computer tutor. With e-mail and telephone contact, she was able to continue her interactions with participants throughout the 12-year period. A long-term perspective afforded the view of the evolution, devolution or cessation of the technology use by these older adults, and this process is chronicled in detail through five individual “profiles.” Three research questions dominated the inquiry: What function do computers serve in the lives of older adults? Does computer use foster or interfere with social ties? Is social support necessary for success in the face of challenging learning tasks? In answer to the first question, it became clear that computers were valued as a symbol of competence and intelligence. Some individuals brought their computers with them when transferred to the single-room residences of assisted living or nursing care facilities. Even when use had ceased, their computers were displayed to signal that their owners were or had once been keeping up to date. In answer to the second question, computer owners socialized around computing use (with in-person family members or friends) more than, or as much as, they socialized through their computers in the digital realm of the Internet. And in answer to the third question, while the existence of social support did facilitate computer exploration, more important was the social support network generated and developed among fellow computer users.

Self-regulated learning in higher education : strategies adopted by computer programming students

Trabalho apresentado em PAEE/ALE’2016, 8th International Symposium on Project Approaches in Engineering Education (PAEE) and 14th Active Learning in Engineering Education Workshop (ALE)