Orientadores: Vitor Baranauskas, Elaine Conceição de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-18T06:09:19Z (GMT). No. of bitstreams: 1 Silva_VaniaDanielaRamosda_M.pdf: 1200986 bytes, checksum: ea703367a77fd7a3b81588f401b24a2f (MD5) Previous issue date: 2011 / Resumo: A utilização da nanotecnologia emerge com potencial para aplicação na área biomédica, no diagnóstico e tratamento patológico, transporte de fármacos e transporte intracelular de moléculas. Dentre as nanopartículas, os nanotubos de carbono (NT) pertencem a uma nova classe de material para aplicação na área biomédica. Devido ao seu tamanho reduzido, os NT podem ser internalizados por diferentes células, como os fagócitos e células tumorais. Embora vários mecanismos de internalização de nanopartículas tenham sido descritos, o mecanismo pelo qual isso ocorre ainda não é bem entendido. O receptor MARCO, que é um receptor de membrana celular expresso em macrófagos, células dendríticas e algumas células endoteliais e está ligado à remoção de partículas inertes parece estar relacionado à internalização de NT. Assim, o objetivo desse estudo foi estudar a internalização de NT por macrófagos (MO), células dendríticas (DC) e células de carcinoma pulmonar de Lewis (3LL), e sua relação com a expressão do receptor MARCO. Assim como, estudar a produção de citocinas pelas DC e MO incubados com os NT. Os MO foram obtidos do baço e as DC obtidas da medula óssea de camundongos C57Bl/6, e células da linhagem 3LL foram cultivadas em nosso laboratório. As células foram incubadas com NT de parede múltipla, marcados com o corante PKH26 em diferentes tempos. DC MO e 3LL internalizam os NT, porém somente os dois primeiros tipos celulares expressaram MARCO. As células 3LL apesar de internalizarem grande quantidade de NT após 12 e 24 horas, não expressam MARCO. Ao bloquear o receptor com anticorpo anti-MARCO nas DC e MO observamos diminuição na internalização dos NT.Os resultados demonstram que DC são aquelas que mais internalizam os NT e tem a maior expressão de MARCO. Estudos sobre modificações estruturais na célula foram observadas através da espectroscopia Raman. A análise da expressão gênica para citocinas demonstraram aumento na expressão de RNAm para citocina pró-inflamatória TNF? em MO e DC incubadas com NT / Abstract: The aplication of nanotechnology emerges with potential for application in the biomedical area, pathological diagnosis and treatment, drug transport and intracellular transport of molecules. Among the nanoparticles, carbon nanotubes (NT) belong to a new class of material for application in the biomedical area. Due to its small size, the NT can be internalized by various cells such as phagocytes and tumor cells. Although several mechanisms of internalization of nanoparticles have been described, the mechanism by which this occurs is not well understood. The MARCO receptor, which is a cell membrane receptor expressed on macrophages, dendritic cells and some endothelial cells and is linked to the removal of inert particles appears to be related to the internalization of NT. Thus, the aim was to study the internalization of NT by macrophages (MO), dendritic cells (DC) cells and Lewis lung carcinoma (3LL) and its relationship with the expression of receptor MARCO. As well as studying the production of cytokines by DC and MO were incubated with NT. The MO were obtained from the spleen and the DC obtained from bone marrow of C57BL / 6 and 3LL cell lines were cultured in our laboratory. The cells were incubated with NT (multi wall) , labeled with PKH26 dye at different times. DC, MO and 3LL internalize the NT, but only the DC and MO express MARCO. 3LL cells internalize despite large amount of NT at 12 and 24 hours, but not express MARCO. By blocking the receptor with anti-MARCO in MO and DC, were decreased the internalization of NT. The results demonstrate that DC internalize more NT than other cells and have the highest expression of MARCO. Studies on structural changes in the cell were observed by Raman spectroscopy. The analysis of gene expression for cytokines showed increased expression of mRNA for proinflammatory cytokine TNF in MO and MO incubated with NT / Mestrado / Eletrônica, Microeletrônica e Optoeletrônica / Mestre em Engenharia Elétrica

A flagelina é reconhecida pelo receptor TLR5, e pelos receptores NLRs, NLRC4/Naip5. Estes últimos induzem ativação de caspase-1 e liberação de IL-1b e IL-18, além da morte do macrófago por piroptose. Para investigar os mecanismos moleculares envolvidos nesses processos, MOs peritoneais foram estimulados com flagelina de B. subtilis e S. typhimurium em sua forma livre (capaz de ativar TLR5), ou inserida em vesículas lipídicas (capaz de ativar os NLRs). Demonstramos que ambas as flagelinas citosólicas induzem produção de IL-1b e morte celular, embora a flagelina de S. typhimurium tenha mostrado maior potencial em induzir produção de IL-1b e IL-6 pelos MOs. Verificamos que a produção de IL-1b e lise celular de MOs se mostraram eventos subseqüentes à formação de poros na membrana celular. Embora a liberação de IL-1b após reconhecimento de ambas as flagelinas ser um fenômeno dependente do eixo NLRC4/caspase-1, a morte celular induzida pelas flagelinas citosólicas ocorre na ausência destas moléculas, ao contrário do que prevê a literatura atual. / Flagellin is recognized by TLR5, and NLRs NLRC4/Naip5. The latter induce activation of caspase-1 and release of IL-1b and IL-18, besides the death of macrophages by pyroptosis. To investigate the molecular mechanisms involved in these processes, peritoneal MOs were stimulated with flagellin from B. subtilis and S. typhimurium in its free form (activating TLR5), or inserted into lipid vesicles (able to activate NLRs). We demonstrated that both cytosolic flagellins induce the production of IL-1b and cell death, while the flagellin of S. typhimurium has shown greater potential to induce production of IL-1b and IL-6 by MOs. We found that the production of IL-1b and cell lysis by MOs are subsequent events proven by the formation of pores in cell membranes. Although the release of IL-1b after recognition of both flagellins is a phenomenon dependent on the axis NLRC4/caspase-1, cell death induced by cytosolic flagellin occurs in the absence of these molecules, unlike that provided by the current literature.

No description available.

Natural killer (NK) cells represent a crucial component of the innate immune system and are primarily regulated by the interactions of their activation and inhibitory receptors with ligands available on target cells. The genetically linked Ly49 and NKR-P1 family of receptors constitute two of the major regulatory receptor systems used by NK cells and have been shown to bind different ligands. Whereas the Ly49 receptors survey MHC-I ligands on target cells, the NKR-Pl receptor family members bind to various members of the C-type lectin-related (Clr) family. Interestingly, NKR-P1 and Clr haplotypes possess a stable genomic polymorphism across multiple mouse strains, suggesting that this inhibitory receptor:ligand relationship has an important role in the maintenance of host cellular cognate specificities. The NKR-P1 and Clr receptor-ligand pairs identified in mice include the NKR-P1B:Clr-b and the NKR-P1G:Clr-f interacting pairs. Previous RT-PCR and in situ RNA hybridization data generated by our laboratory determined that kidney tubular epithelium as well as the small and large intestinal epithelial cells specifically and highly expresses the Clr-f transcripts. Contrarily, the Clr-b transcripts were only detected on hematopoietic cells of various lymphoid organs and kidneys. Moreover, foregoing studies revealed that the loss of Clr-b following viral or chemical induced stress mediates NK cell killing of the target cell, suggesting a tissue-specific immune-surveillance mechanism in parallel with the global MHC-I-dependent missing-self model. However, the role of the NKR-P1B:Clr-b recognition-system have never been examined in the intestine. Additionally, the role of Clr-f in the kidney and intestines, where they are highly expressed, has not been investigated. For these reasons, I aimed in my thesis to provide a better understanding of the functional aspect of the NKR-P1B:Clr-b and NKR-P1G:Clr-f recognition systems in mediating gut mucosal and renal homeostasis, respectively. First, in order to determine the role of NKR-P1B and Clrb receptor:ligand pair as a “missing-self” immunosurveillance system in the gut, I started by identifying the expression pattern of both the receptor and ligand on various intestinal cells. My results demonstrate that NK cells do not represent the major NKR-P1B-expressing cells in the gut lamina propria. Instead, ILC3 subsets constituted the predominant cell population expressing the receptor, whereas γδT cells composed a small fraction of NKR-P1B+ lymphocytes. In addition, the NKR-P1B expression on myeloid cells was exclusive to colon macrophages and DC subsets. Interestingly, the highest percentage of NKR-P1B+ immune cells was found in the gut, which suggests the dominant role of NKR-P1B in regulating immune functions at the level of intestinal mucosa. As expected, the expression of the NKR-P1B ligand, Clr-b, appeared on all innate immune cell types in the gut. Next, using oral infection models of Salmonela typhimurium and Citrobacter rodentium, I showed that NKR-P1B-deficient NK cells, ILC3 and γδ T cells are hyporesponsive compared to their WT counterparts. In particular, gut NKR-P1B-deficient NK cells and γδT cells secreted low levels of IFNγ cytokine while infected with S.typhimurium. Importantly, the decreased IFNγ secretion by NK and γδT cells was associated with an increased dissemination of the bacterium into the knockout spleens at day 5 post-infection. Likewise, I detected a significant decrease in IL-22 cytokine production by NKR-P1B-deficient ILC3 compared to their WT counterparts at both steady state and following C.rodentium infection. Next, I address the potential role of Clr-f in the kidney. Renal tubular epithelial cells have been shown to express high levels of Clr-f transcripts. Epithelial cells constitute the major cellular component of kidney tubules and are well known to mediate metabolic waste excretion, reabsorption of essential molecules as well as other physiological functions, such as ions exchange and water retention. To determine the role of Clr-f in renal epithelial cells, I generated a Clr-f-deficient mouse with the help of two of my previous lab colleagues. Importantly, chemical analysis on urine and serum samples from knockout and WT littermates indicated that Clr-f-deficient kidneys display a decreased filtration capacity. In particular, higher creatinine levels were detected in the Clr-f deficient serum. In addition, Clr-f-deficient mice appeared to have a lower fractional excretion of sodium (FENa) in their urine filtrates in comparison to WT excreted urine. Blood pressure measurements on the same mice at 12 and 24 weeks of age revealed a hypotensive phenotype in the Clr-f-deficient mice. Furthermore, pathological assessment of Clr-f-deficient kidneys exhibited moderate and aggravated lesions of the tubular epithelium along with marked glomerular mesangiolysis. Lastly, flow cytometry analysis on isolated lymphocytes from Clr-f-deficient and WT mice demonstrated comparable immune infiltrates between the two mouse genotypes. Altogether, our data shows that the absence of Clr-f results in the development of glomerular and tubular lesions in an immune-independent manner leading to an abnormal kidney function. Additionally, the disruption of NKR-P1B:Clr-b recognition system results in abnormal innate immune cell number and function in the mouse intestine. These novel findings sheds light on the important role of Clr-f in maintaining healthy kidney morphology and function, as well as the crucial role for NKR-P1B:Clr-b interactions in mediating intestinal homeostasis at steady and infected states.

Solid tumors can hijack many of the same programs used in neurogenesis to enhance tumor growth and metastasis, thereby generating a plethora of neurogenesis-related molecules including semaphorins Among them, we have identified Semaphorin7A (SEMA7A) in breast cancer We first used to the DA-3 mammary tumor model to determine the effect of tumor-derived SEMA7A on immune cells We found that tumor-derived SEMA7A can modulate the production of proangiogenic chemokines CXCL2/MIP-2 and CXCL 1, and prometastatic MMP-9 in macrophages We next aimed to determine the expression and function of SEMA7A in mammary tumor cells We found that SEMA7A is highly expressed in both metastatic human and murine breast cancer cells We show that both TGF-β and hypoxia elicits the production of SEMA 7 A in mammary cells SEMA7 A shRNA silencing in 4T1 cells resulted in decreased mesenchymal markers MMP-3, MMP-13, Vimentin and TGF-β) SEMA7A silenced cells show increased stiffness with reduced migratory and proliferative potential In vivo, SEMA7A silenced 4T1 tumor bearing mice showed decreased tumor growth and metastasis Genetic ablation of host-derived SEMA7A synergized to further decrease the growth and metastasis of 4T1 cells Our findings suggest novel functional roles for SEMA7A in breast cancer and that SEMA7A could be a novel therapeutic target to limit tumor growth and metastasis / Includes bibliography / Dissertation (PhD)--Florida Atlantic University, 2016 / FAU Electronic Theses and Dissertations Collection

The study of the three-dimensional structures of biological macromolecules has given us significant insight into life and its mechanisms. Understanding these structures in their native contexts, a challenging but important goal, came closer to reality with the development of electron microscopy. After many years of technological development, we are now starting to understand previously intractable biological phenomena at an unprecedented resolution. One such phenomenon is how neighboring cells interact, both to communicate and send signals, and to adhere and form complex tissue structures. While the molecules that mediate such processes have long been studied in isolation, electron microscopy allows us to examine them in a more native biophysical environment; as hydrated, dynamic molecules tethered to opposed cellular membranes.Imaging unadulterated biological material using electron microscopy requires that the sample be embedded in a thin layer of vitreous ice to immobilize the molecules and protect them from the vacuum of the microscope, and thus is generally referred to as cryogenic electron microscopy (cryo-EM). Samples can be imaged using two common cryo-EM modalities: single particle analysis (SPA), where many two-dimensional projection images of molecules in solution are collected, and cryo-electron tomography (cryo-ET), where the sample is tilted as it is imaged at multiple angles to reconstruct a three-dimensional volume. In this work, I will describe how I have used both SPA and cryo-ET to understand cell surface interactions involving a variety of proteins. The first chapter will look at the cell surface molecules known as the Toll receptors, a family of molecules found in Drosophila melanogaster, with orthologs in mammals known as the Toll-like receptors (TLRs). I will focus on their role in the development of the Drosophila embryo during germ band extension, a kind of convergent extension that is a conserved process through all metazoans. Biophysical assays of the three implicated Toll receptors, Toll-2, -6, and -8, revealed both homophilic and heterophilic interactions. SPA was used to determine the structure of monomeric Toll-2 which closely resembles the overall fold of Toll, whose structure was previously solved by x-ray crystallography. Surface plasmon resonance (SPR) spectroscopy and analytical ultracentrifugation (AUC) showed Toll-6 is a dimer in solution, which I visualized using cryo-EM. The Toll-6 homodimer is a novel dimer interface for Tolls and TLRs, where molecules on the same cell surface have been shown to dimerize in the presence of a wide variety of ligands. In contrast, the Toll-6 dimer is formed in the absence of any ligand and exists in an antiparallel arrangement that could be formed by molecules on opposing cell surfaces. Together, these results provide a biochemical basis for germ band extension which may be further explored through the study of structure-based mutations. While cryo-EM SPA is a powerful tool, cryo-ET allows one to reconstruct three dimensional volumes of highly heterogeneous samples, such as the interior of cells, where molecules of interest may not exist in enough copies to facilitate averaging. This technique, where the sample is imaged multiple times as it is tilted to obtain three-dimensional information of a region of interest, was used to study cell adhesion of a different type: that mediated by the classical cadherins. These calcium-dependent adhesion molecules cluster into adherens junctions, spot-like protein densities found in a wide variety of tissues. In the second chapter, these junctions are recapitulated between synthetic liposome membranes by tethering the adherent cadherin molecules to chemically functionalized lipids. They are then imaged using cryo-ET to reveal higher-order structural details. First, this method is applied to the clustered protocadherins, a family of cadherins that mediate neuronal self-avoidance in mammals. Cryo-ET in combination with x-ray crystallography revealed that clustered protocadherins form extended one-dimensional zippers between membranes, which are a combination of strictly homophilic trans interactions coupled with promiscuous cis interactions. Neurons express unique subsets of the ~50-60 possible isoforms, and when two neuronal processes express identical subsets, which happens only when those processes are a part of the same cell, these linear chains grow and initiate a repulsive signal. If the subsets are different, the chains terminate and no repulsive signal is generated. The same technique has been used previously to study the type I classical cadherins, perhaps the most well-studied members of the cadherin superfamily. In the second half of this chapter, we extend our analysis to include the type II classical cadherins, which possess more complex expression patterns and binding specificities. Cryo-ET of type II cadherin ectodomains tethered to synthetic liposomes revealed that several representative members of this family form only moderately ordered arrays between liposomes, a finding in agreement with their role in cell sorting and migration. However, VE-cadherin, an outlier type II expressed in vascular endothelial cells where it withstands blood pressure, forms extraordinarily ordered junctions. Subtomogram averaging reveals the regularity of this two-dimensional array. In the final chapter, I describe my work on a membrane surface molecule of a different kind, one not involved in cell adhesion but viral infection. The global COVID-19 pandemic gave me the opportunity to contribute to our understanding of SARS-CoV-2 by studying the structure of neutralizing antibodies bound to the viral spike protein, perhaps the most infamous membrane surface protein. The first subchapter describes the initial isolation, neutralization, and structural analysis of antibodies isolated from convalescent COVID-19 patients. This work revealed that patients with severe COVID-19 produce potently neutralizing antibodies that target two spike protein domains: the receptor binding domain (RBD) and the N-terminal domain (NTD). RBD-directed antibodies occlude binding to ACE2, the human receptor that mediates viral fusion, but the neutralization mechanism of NTD-directed antibodies is unknown. The following two subchapters are more detailed structural studies of two specific types of antibodies. The first looks at a class of RBD-directed antibodies derived from the VH1-2 gene, which are some of the most potent and common antibodies against SARS-CoV-2. The heavy chains of these antibodies recognize almost identical epitopes, but the antibodies employ a modular approach to recognize the RBD in either of its possible conformations. The second class are antibodies that target the NTD, which our work revealed all bind to a single antigenic supersite. The final subchapter focuses on emerging SARS-CoV-2 variants and includes the structures of two antibodies that are still capable of neutralizing these new variants. They are also infrequent in the human antibody response to SARS-CoV-2, meaning they put little selective pressure on the virus to produce escape mutations, making them good candidates for monoclonal antibody therapies. Though Drosophila embryogenesis, adherens junction formation, and SARS-CoV-2 neutralization are seemingly unrelated systems, they are united by the incredible flexibility of cryo-EM to visualize biological molecules in more native environments. Whether it is the ability to study multiprotein complexes or assemblies formed between membranes, cryo-EM is a powerful technique that promises to help bridge the divide between structure and function.

No description available.

Glutamate is essential for proper brain function as it is our nervous systems principal excitatory neurotransmitter, a signal that stimulates nerve cells to send messages to other cells. Glutamate activates ionotropic glutamate receptors (iGluRs), which are linked to several neurological diseases in cases when they are improperly regulated. iGluRs are transmembrane channels that allow calcium, as well as other cations, into the post synaptic neuron upon binding of glutamate or other agonists.Interestingly, iGluR homologs in plants also mediate calcium signaling upon glutamate activation and were accordingly named glutamate receptor-like channels (GLRs). Cell signaling is critical for plant survival to mediate rapid response to growth, defense, and other environmental cues. GLRs are found in all plants and vital for their health, hardiness, and adaptation for growth and survival in unfavorable conditions, such as drought, nutrient poor soil, temperature extremes, pathogens, and predators. Plant research is important with vast applications. Firstly, crops are our primary source of nutrition. In addition, plants are used as sources of drugs that we employ for treating diseases. Some examples of plant-derived neuroactive compounds include caffeine in coffee beans, nicotine in tobacco, and opium from poppy plants. In short, optimizing plant growth is beneficial to maintaining our own survival and potentially achievable by understanding GLRs role in plant health and hardiness. Despite their importance for cell signaling and implication in plant defense and regeneration, the structural basis underlying the function of these channels remains ambiguous, representing a critical barrier to our understanding of GLR function. To address this problem, I dedicated my thesis work to study the structure of GLRs and gain insight into their function. There are 20 GLRs in the model plant organism, Arabidopsis thaliana, classified into 3 different clades (AtGLR1-3). To narrow down which AtGLRs to focus our structural studies on, we investigated clade 3 representatives, as many of these GLR3s have been extensively studied in different plant species, especially crops. For example, studying AtGLR3.4 could provide useful information to how the homolog in rice, OsGLR3.4, contributes to growth and production in rice. Studying AtGLR3.4’s structure may elucidate how agonistic or antagonistic targets bind and gate the channel, potentially revealing “druggable” targets to alter plant response for defense and regeneration. Without any structural information available for GLRs, I started my studies by first focusing on their mammalian homologs, iGluRs. I first designed multiple constructs for heterologous expression and purification from cell culture (for example HEK293S GnTI- cells). Then, I optimized protein extraction and purification to obtain pure protein samples. Purified proteins were then subjected to cryo-electron microscopy (cryo-EM) which eventually allowed us to solve the structure of AtGLR3.4, the first full-length GLR structure. AtGLR3.4’s structure revealed similarities to structures of its mammalian homologs, iGluRs. In comparison to iGluRs, our GLR structure also showed tetrameric subunit assembly, with a three-layer architecture that includes the ligand binding domain (LBD) in the middle, sandwiched between the extracellular amino terminal domain (ATD) at the top and the transmembrane domain (TMD) at the bottom. In contrast to the majority of iGluR structures, however, AtGLR3.4 displayed unique symmetry and domain arrangement with the non-swapped extracellular ATD and LBD domains. We also provided further evidence supporting ligand binding promiscuity that was previously revealed in isolated LBD crystal structures from other AtGLR3s. Surprisingly, we found endogenous glutathione bound to the ATDs and demonstrated its contribution to channel activity. It is important to fill the gaps in knowledge about GLR structure to understand how these channels are activated and gated. In doing so, we will learn more about iGluRs as well as better understand plant defense and growth, which has the potential to enhance crop production for food security and our overall survival.

There is compelling evidence that smokers are less responsive to vaccination. We reported that both therapeutic and prophylactic vaccines fail to protect and cure animals from disease due to negative effects of nicotine on DCs’ ability to generate effector T cells. We have been investigating whether vaccine formulated with TLR agonist(s) could potentially overcome the immunosuppressive effects of nicotine on human DC-NK cross-talk essential for effector T cell generation. Monocyte-derived DCs and nicDCs were stimulated with individual and combined TLR agonists prior to co-culture with purified T cells. The phenotypes and cytokine profiles of T cell were assessed using Flow Cytometry and ELISA, respectively. We found nicDCs cultured with TLR-8/7 alone or in combination with TLR-3 produce quantitatively and qualitatively similar IFN-γ producing effector T cells when compared to control DCs. Our data suggest that the addition of appropriate TLR agonist to vaccine formulation could potentially overcome the immunosuppression seen in smokers, thereby containing the spread of infectious disease to vulnerable population / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Malária é um dos principais problemas de saúde nos países em desenvolvimento sendo o Plasmodium o agente etiológico da doença. Neste trabalho foi investigada a função do Ca2+ e da sinalização purinérgica na modulação proteolítica de Plasmodium. Utilizando peptídeos com apagamento intracelular de fluorescência (FRET) analisamos a atividade proteolítica ativada por Ca2+ liberado do retículo endoplasmático ou de compartimentos ácidos e investigamos as diferentes classes de proteases envolvidas. Utilizando-se P. berghei e P. yoelii verificou-se a importância do Ca2+ na modulação proteolítica além de diferenças fisiológicas nesta modulação dentre as espécies estudadas. Foram também investigados os efeitos de ATP, adenosina e GTP extracelular na proteólise e conclui-se que receptores purinérgicos estão envolvidos na habilidade do parasita ativar proteólise intracelular. Na terceira parte da tese foi estabelecido um modelo murino nocaute para analisar a relação Plasmodium-hospedeiro e propõem-se a interação do receptor InsP3R2 e a proteína PbRACK do parasita. / Malaria is a major health problem in developing countries Here we investigate the role of Ca2+ and purines in Plasmodium protease modulation. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca2+from endoplasmatic reticulum or acidic compartments and investigated the classes of affected proteases. Experiments in P. berghei and P. yoelii indicated a fundamental role for calcium in modulating proteolysis and points out key differences in proteolytic responses between Plasmodium species. We also investigated the effects of extracellular ATP, adenosine and GTP on triggering proteolysis. The data lead us to conclude that purinergic receptor is involved in the ability of the parasite to activate intracellular proteolysis by sensing external molecules. The third part of the thesis established a new murine knockout model to analyze Plasmodium-host signaling and suggest a possible interaction between InsP3R2 receptor and the PbRACK parasite protein.