Growing resistance of bacteria towards antibiotics resulted in extensive research effort for development and application of new materials and techniques. Due to their unique properties, graphene quantum dots (GQDs) have attracted much attention and are a promising material with potential applications in many fields. One use of GQDs is as a photodynamic therapy agent that generates singlet oxygen. In this work, GQDs synthesized by focusing nanosecond laser pulses into a mixture of benzene and nickel(II) oxide were combined with methylene blue (MB) to eradicate Gram-negative Escherichia coli and Gram-positive Micrococcus luteus. Theoretical calculation of pressure evolution was calculated using the standard finite difference method. Detailed characterizations were performed with transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier-transform infrared (FTIR), UV-Visible (UV-Vis), and photoluminescence (PL) spectra. Furthermore, singlet oxygen generation from MB-GQD mixture was investigated by measuring the rate of 9,10-anthracenediyl-bis(methylene) dimalonic acid photobleaching at 400 nm. Combining MB with GQDs caused enhanced singlet oxygen generation, leading to improved bacterial deactivation rate. The (3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was used to determine if GQDs in dark conditions caused human cellular side-effects and affected cancer and noncancer cellular viability. We found that even high concentrations of GQDs do not alter viability under dark conditions. These results suggest that the MB-GQD combination is a promising photodynamic therapy agent that may be useful when antibiotics resistance is present.

Made available in DSpace on 2014-08-20T14:37:58Z (GMT). No. of bitstreams: 1 tese_tatiana_antunes.pdf: 3388546 bytes, checksum: 6c26eaf4ab24c11f046c7e7b2f9f2566 (MD5) Previous issue date: 2010-03-10 / Sporotrichosis is a subcutaneous mycosis of zoonotic character, cosmopolitan development subacute or chronic whose agent the dimorphic fungus Sporothrix schenckii affecting man and various species of animals, especially the domestic cat, being considered of interest to Public Healt. Considering the difficulties in the therapeutic treatment of ringworm in this animal species, including toxicity and the development of resistance to antifungal agents traditionally used to treat disease the study aimed evaluate the immunogenic capacity of yeast cells of S. schenckii inactivated both in immunoprophylaxis and immunotherapy of sporotrichosis. We used 160 Wistar albin rats (Rattus norvegicus) at 70 days (80 in immunoprophylaxis and 80 in immunotheraphy) were divided into four groups that were immunized three times every 15 days. The groups were divided as follows: G1 (control, mineral oil), G2 (Ag + incomplete freund adjuvant), G3 (Ag + Freund's complete adjuvant) and G4 (Ag + incomplete freund adjuvant + propolis). Preparation of the vaccine used was an isolate of S. schenckii from yeast in the form of a case of cutaneous sporotrichosis of domestic cat. The fungus was grown in medium liquid (Brain-Heart broth), incubated for 10 days at 370C and kept under constant agitation to obtain the yeast form. The culture was filtered through a double layer of sterile gauze, centrifuged, washed twice with buffered saline (PBS), homogenized and standardized in 108 cells of S. schenckii / ml. The cells were inactivated with 0.02% thimerosal and then emulsified with mineral oil, and vaccines packaged in sterilized sealed and kept at a temperature of 40C throughout the experimental period. Was used a dose of 0.1 ml/animal intramuscularly. Rats that received the vaccine as immunoprophylaxis after the three doses were challenged, and inoculated subcutaneously with 2X103 cells / ml of S. schenckii in the right footpad and evaluated for 10 days. Animals treated with immunotherapy were inoculated with the agent after 14 days and received three doses of immunogen. After the experimental period all were euthanized and necropsy to mycological examination, histopathology and counting colony forming units. The results were: immunoprophylaxis in the pathological changes showed statistically significant differences (P <0.05) in the vaccinated groups (G2, G3 and G4) compared to the control group (G1) to the point of inoculation, with no statistical difference the evaluation of internal organs. Immunotherapy clinical evaluation at the injection showed that there was no statistical difference among the four experimental groups, but at the end of the experiment the groups G1, G2, G3 and G4 had respectively 8.3%, 58.3%, 41.7 % and 50% of injuries in the process of regression and healing. In relation to injuries in other body areas only in the last week of the experiment, the G2 and G3 differ statistically (P <0.05) in the control group (G1). Pathological changes were found in the internal organs of the four experimental groups, but with a greater number of lesions in group CONT. Both in immunoprophylaxis and immunotherapy the retroisolation of the agent and count of colony forming units showed that there was growth of S. schenckii in the four experimental groups, but with less frequency and quantification of CFU at the point of inoculation and internal organs of the groups G2, G3 and G4. In histopathologic evaluation to determine the presence of granulomas and pyogranulomas focal and multifocal in the point of inoculation and internal organs of the four groups, but the animals that received immunoprophylactic changes were more restricted to the point of inoculation. The results indicate that the three vaccine formulations (AIF ACF and AIFP) used as immunoprophylactic and immunotherapy have not been effective for the remission of the lesions of experimental cutaneous sporotrichosis. / Esporotricose é uma micose subcutânea de caráter zoonótico, cosmopolita de evolução subaguda ou crônica que tem como agente etiológico o fungo dimórfico Sporothrix schenckii afetando o homem e várias espécies de animais, principalmente o felino doméstico, sendo considerada de interesse para a Saúde Pública. Considerando as dificuldades terapêuticas no tratamento da micose nessa espécie animal, incluindo toxicidade e o desenvolvimento de resistência aos antifúngicos tradicionalmente utilizados na terapia da enfermidade o estudo objetivou avaliar a capacidade imunogênica de células leveduriformes de S. schenckii inativadas tanto na imunoprofilaxia como na imunoterapia da esporotricose. Foram utilizados 160 ratos albinos wistar (Rattus norvergicus) com 70 dias (80 na imunoprofilaxia e 80 na imunoterapia) sendo divididos em quatro grupos que receberam três doses do imunógeno a cada 14 dias. Os grupos foram divididos em: G1 (controle- óleo mineral), G2 (Ag+ adjuvante incompleto de freund), G3 (Ag + adjuvante completo de freund) e G4 (Ag + adjuvante incompleto de freund + própolis). Para preparação da vacina foi utilizado um isolado de S. schenckii na forma leveduriforme proveniente de um caso de esporotricose cutânea de felino doméstico. O fungo foi cultivado em meio líquido (Brain-Heart broth), incubado durante 10 dias a 37oC e mantido sob agitação constante para obtenção da forma leveduriforme. A cultura foi filtrada em dupla camada de gaze estéril, centrifugada, lavada duas vezes com solução salina tamponada (PBS), homogeneizada e padronizada em 108 células de S. schenckii/ ml. As células foram inativadas com thimerosal a 0,02% e posteriormente emulsificadas com óleo mineral, sendo as vacinas acondicionadas em frascos estéreis fechados e mantidas a temperatura de 40C durante todo o período experimental. Foi utilizada uma dose de 0,1 ml/animal por via intramuscular. Os ratos que receberem a vacina como imunoprofilaxia após as três doses foram desafiados, sendo inoculados por via subcutânea com 2X103 células/ml de S. schenckii e avaliados durante 10 dias. Os animais tratados com imunoterápico foram inoculados com o agente e após 14 dias receberam três doses do imunógeno. Após o período experimental todos foram eutanasiados e feita necropsia para realização de análise micológica, histopatológica e contagem das Unidades Formadoras de Colônias. Os resultados obtidos foram: na imunoprofilaxia as alterações anatomopatológicas demonstraram diferença estatísticamente significativa (P<0,05) dos grupos vacinados (G2, G3 e G4) quando comparados ao grupo controle (G1) em relação ao ponto de inoculação, não havendo diferença estatística na avaliação dos órgãos internos. Na imunoterapia a avaliação clínica no ponto de inoculação evidenciou que não houve diferença estatística entre os quatro grupos experimentais, porém ao final do experimento os grupos G1, G2, G3 e G4 apresentavam respectivamente 8,3%, 58,3%, 41,7% e 50% das lesões em processo de regressão e cicatrização. Em relação a lesões em outras áreas corpóreas somente na última semana do experimento os grupos G2 e G3 diferiram estatisticamente (P<0,05) do grupo controle (G1). Alterações anatomopatológicas foram encontradas nos órgãos internos dos quatro grupos experimentais, porém com um maior numero de lesões nos animais do grupo CONT. Tanto na imunoprofilaxia quanto na imunoterapia o retroisolamento do agente e Contagem das Unidades Formadoras de colônias demonstraram que houve crescimento do S. schenckii nos quatro grupos experimentais, porém com menor freqüência e quantificação de UFCs no ponto de inoculação e órgãos internos dos grupos G2, G3 e G4. Na avaliação histopatológica foi verificada a presença de granulomas e piogranulomas focais e multifocais no ponto de inoculação e órgãos internos dos quatro grupos, porém os animais que receberam imunoprofilático as alterações ficaram mais restritas ao ponto de inoculação. Os resultados permitem concluir que as três formulações de vacinas (AIF, ACF e AIFP) utilizadas como imunoprofilático e imunoterápico não foram eficazes para a remissão completa das lesões de esporotricose cutânea experimental.

A major advantage of DNA vaccination is the ability to induce both humoral and cellular immune responses. DNA vaccines are currently used in veterinary medicine, but their tendency to display low immunogenicity in humans has hindered their usage, despite excellent tolerability and safety profiles. Various approaches have been used to improve the immunogenicity of DNA vaccines. Recent human study data re-established the value of DNA vaccines, especially in priming high-level antigen-specific antibody responses. Data suggests that innate immune responses to the DNA vaccine plasmid itself contribute to the immunogenicity of DNA vaccines, however the underlying mechanisms responsible remain unclear. In this dissertation, we investigate the role of innate immunity in shaping antigen-specific adaptive immune responses following DNA vaccination. The current belief is that the cytosolic DNA sensing pathways govern DNA vaccine immunogenicity. To date, only the type I interferon inducing STING/TBK1 regulatory pathway has been identified as required for DNA vaccine immunogenicity. Surprisingly, neither the upstream receptor nor the downstream signaling molecules in this pathway have been characterized. I therefore investigated a candidate cytosolic DNA receptor, as well as the downstream transcription factors required for generation of antigen-specific immune responses. Additionally, the effects of pro-inflammatory signaling on DNA vaccine immunogenicity have yet to be comprehensively studied. Previous studies have only provided indirect evidence for the role of inflammatory v signaling in DNA vaccination. As such, I also investigated the role of the DNA sensing AIM2 inflammasome in DNA vaccination. My data indicates that AIM2 is a key modulator in DNA vaccination via a previously unrecognized connection to type I interferon. Importantly, this marks the first time a DNA vaccine sensor has been identified. Of note, this dissertation represents a departure from many published works in the field. Whereas previous studies have mostly utilized model antigens and only focused on the adaptive immune responses generated, I analyzed the effects on innate immunity as well. Using various innate gene knockout murine models, I quantified antigen-specific humoral and T cell responses, as well as serum cytokine and chemokines following immunization with a clinically relevant DNA vaccine. Overall, this data provides a basis for understanding the mechanisms of DNA vaccination, allowing for the design of more effective vaccines.

Influenza virus is one of the major respiratory pathogens of humans as well as animals, including equines. There is an increasing evidence that bacterial infections are the most common cause of the death during influenza. In horses also, secondary bacterial pneumonia can lead to death, and surviving horses may take up to six months for the complete recovery resulting in heavy economic loss to the equine industry. Interleukin (IL)-23 mediated innate immune response has been shown to protect the host from various respiratory bacterial infections. However, studies to investigate the role of host and viral factors in the regulation of IL-23 are limited. Endoplasmic reticulum (ER) stress-induced transcription factor CHOP-10 and IFN-β has been shown to participate in the regulation of IL-23. Primary hypothesis for the current study was that influenza A virus (IAV) NS1 protein downregulates the IL-23 expression via inhibition of CHOP-10. In order to test our hypothesis, we infected the RAW264.7 cells - a murine macrophage cell line, and primary murine alveolar macrophage cells either with the wild type Influenza A virus (PR/8/34, PR8) or isogenic mutant virus lacking NS1 (delNS1). Quantitative analysis of mRNA expression revealed a significantly higher mRNA expression of IL23p19, IFN-β and CHOP-10 in delNS1 virus infected cells as compared the PR8 virus infected cells. Additionally, overexpression of CHOP-10 partially restored the expression of IL-23p19 in PR8 virus infected cells and knockdown of CHOP-10 resulted in downregulated expression of IL-23p19 in delNS1 infected cells. Taken together, these results suggest that IAV NS1 protein mediated inhibition of CHOP-10 expression leads to downregulation of IL-23 expression in macrophage cells in-vitro. Similar results were also observed in-vivo using IAV and Streptococcus zoooepidemicus (S. ze) co-infection model. In a co-infection mouse model delNS1 virus co-infection resulted in significantly higher expression of the IL-23 and IL-17. Considering the role of IL-23 in protection against respiratory bacterial pathogens, effect of exogenous supplementation of IL-23 was also investigated in the influenza and S. ze co-infection mouse model. We found that a single intranasal dose of recombinant murine IL-23 significantly improved the survival of mice co-infected with PR8 and S .ze. Overall, our study suggests that IAV infection subverts the IL-23 mediated respiratory innate immune response and restoration of IL-23 could protect from influenza-associated respiratory bacterial infections.

The Hepatitis C Virus (HCV) is a global health problem as it afflicts an estimated 170 million people worldwide and is the major cause of viral hepatitis, cirrhosis and liver cancer. HCV is a rapidly evolving virus, with 6 major genotypes and multiple subtypes. Over the past 20 years, HCV therapeutic efforts have focused on identifying the best-in-class direct acting antiviral (DAA) targeting crucial components of the viral lifecycle, The NS3/4A protease is responsible for processing the viral polyprotein, a crucial step in viral maturation, and for cleaving host factors involved in activating immunity. Thus targeting the NS3/4A constitutes a dual strategy of restoring the immune response and halting viral maturation. This high priority target has 4 FDA approved inhibitors as well as several others in clinical development. Unfortunately, the heterogeneity of the virus causes seriously therapeutic challenges, particularly the NS3/4A protease inhibitors (PIs), which suffer from both the rapid emergence of drug resistant mutants as well as a lack of pan-genotypic activity. My thesis research focused on filling two critical gaps in our structural understanding of inhibitor binding modes. The first gap in knowledge is the molecular basis by which macrocyclization of PIs improves antiviral activity. Macrocycles are hydrophobic chains used to link neighboring chemical moieties within an inhibitor and create a structurally pre-organized ligand. In HCV PIs, macrocycle come in two forms: a P1 - P3 and P2 - P4 strategy. I investigated the structural and thermodynamic basis of the role of macrocyclization in reducing resistance susceptibility. For a rigorous comparison, we designed and synthesized both a P1 - P3 and a linear analog of grazoprevir, a P2 - P4 inhibitor. I found that, while the P2 - P4 strategy is more favorable for achieving potency, it does not allow the inhibitor sufficient flexibility to accommodate resistance mutations. On the other hand, the P1 - P3 strategy strikes a better balance between potency and resistance barrier. The second gap my thesis addresses is elucidating the structural basis by which highly potent protease inhibitors function in genotype 1 but not in genotype 3, despite having an 87% sequence similarity. After mapping the amino acids responsible for this differential efficacy in genotypes 1 and 3, I engineered a 1a3a chimeric protease for crystallographic studies. My structural characterization of three PIs in complex with both the 1a3a and genotype 1 protease revealed that the loss of inhibitor efficacy in the 1a3a and GT-3 proteases is a consequence of disrupted electrostatic interactions between amino acids 168 and 155, which is critical for potent binding of quinoline and isoindoline based PIs. Here, I have revealed details of molecular and structural basis for the lack of PI efficacy against GT-3, which are needed for design of pan-genotypic inhibitors.

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRb deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3b sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

Influenza is a severe disease in humans and animals, causing upwards of 40,000 deaths every year in America alone. Influenza A virus (IAV) also causes periodic pandemics every 10 to 50 years, killing millions of people. Despite this, very few effective therapies are available. All strains of IAV are prone to developing resistance to antibodies due to the high mutation rate in the viral genome. Because of this mutation rate, a yearly vaccine must be generated before every flu season, and efficacy varies year to year. IAV has also mutated to escape several of the clinically-approved small molecule inhibitors. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of IAV. IAV attachment is mediated by many individually weak hemagglutinin–sialic acid interactions that all together make a strong attachment to a host cell. Polymerized sialic acid analogs can recreate these interactions and block infection. However, they are not ideal therapeutics due to solubility issues and in vivo toxicity. We used liposomes as a novel means for delivery of the sialic acid-containing glycan, sialylneolacto-N-tetraose c (LSTc). LSTcbearing decoy liposomes form multivalent, polymer-like interactions with IAV. Decoy liposomes competitively bind IAV in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. LSTc decoy liposomes co-localize with IAV, while control liposomes do not. Inhibition is specific, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind IAV or inhibit infectivity. LSTc decoy liposomes prevent the spread of IAV during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high-avidity interactions with IAV hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging strains.

While memory CD4 T cells are critical for effective immunity to pathogens, the mechanisms underlying their generation are poorly defined. Although extensive work has been done to examine the role of antigen (Ag) in shaping memory formation, most studies focus on the requirements during the first few days of the response known as the priming phase. Little is known about whether or not Ag re-encounter by effector T cells (late Ag) alters CD4 memory T cell formation. Since influenza infection produces a large, heterogeneous, protective CD4 memory T cell population, I used this model to examine the role of late Ag in promoting CD4 memory T cell formation. In the experiments presented in this thesis, I demonstrate that late Ag is required to rescue responding CD4 T cells from default apoptosis and to program the transition to long-lived memory. Responding cells that failed to re-encounter Ag had decreased memory marker expression and failed to produce multiple cytokines upon re-stimulation. Ag recognition is required at a defined stage, as short-term Ag presentation provided 6 days after infection is able to restore canonical memory formation even in the absence of viral infection. Finally, I find that memory CD4 T cell formation following cold-adapted influenza vaccination is boosted when Ag is administered at this stage. These findings imply that persistence of viral Ag presentation into the effector phase is the key factor that determines the efficiency of memory generation. They also suggest that administering Ag during the effector stage may improve vaccine efficacy.

Inflammation is often a protective reaction against harmful foreign agents. However, in many disease conditions, the mechanisms behind the inflammatory response are poorly understood. Often times, the inflammation causes adverse effects, such as joint pain, abdominal pain, fever, fatigue, and loss of appetite. Thus, many treatments aim to inhibit the inflammatory response in order to control adverse symptoms. Such treatments include TNFα inhibitors. However, a major risk associated with drugs inhibiting tumor necrosis factor alpha (TNFα) is serious infection, including tuberculosis (TB). Anti-TNFα therapy is used to treat patients with Crohn’s disease, for which the risk of tuberculosis may be even more concerning. Recent literature suggests Crohn’s might involve Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular TB-like bacterium. This study seeks to investigate the risk of developing TB in patients with Crohn’s disease treated with TNFα inhibitors. A meta-analysis synthesized existing evidence. Evidence came from published randomized, double-masked, placebo-controlled trials of TNFα inhibitors for treatment of adult Crohn’s disease. Twenty-three trials were identified, including 5,669 patients. The risk of tuberculosis was significantly increased in anti-TNFα treated patients, with a risk difference of 0.028 (95% confidence interval [CI], 0.0011-0.055). The odds ratio was 4.85 (95% CI, 1.02-22.99) when all studies were included and 5.85 (95% CI, 1.13-30.38) when studies reporting zero tuberculosis cases were excluded. The risk of tuberculosis is increased in patients with Crohn’s disease treated with TNFα inhibitors. The medical community should be alerted about this risk and the potential for TNFα inhibitor usage favoring granulomatous infections and worsening the patient condition.

In recent years, heterologous prime-boost vaccination constructs have emerged as a promising strategy to generate broad and protective immunity against a variety of pathogens. The utility of DNA vaccination in priming the immune system, in particular, has improved the immunogenicity of vaccines against difficult pathogens such as HIV-1. In addition, many vaccine formulations include an adjuvant to augment immune responses. However, the mechanisms and profiles of many adjuvants remain largely unknown, particularly in the context of such combination immunization approaches. My thesis research studied the effects of several adjuvants, QS-21, aluminum hydroxide, MPL, and ISCOMATRIX™ adjuvant in the context of a previously described pentavalent HIV-1 Env DNA prime-protein boost vaccine, DP6-001. In a murine model, we quantified HIV antigen-specific humoral and T cell responses, as well as pro-inflammatory serum cytokine and chemokines, both shortly after immunization and at the termination of studies. Our data indicates that each candidate adjuvant generates a unique pattern of biomarkers as well as improved immunogenicity in the context of the DP6-001 DNA prime-protein boost vaccine. Additionally, we examined the impact of several innate signaling pathways on the adaptive immunity raised by DP6-001 and adjuvants, as well as on the unique serum cytokine profiles. These studies provide valuable information in selection of an adjuvant for inclusion in future prime-boost strategies, with the goal of enhancing immunogenicity while minimizing reactogenicity. Furthermore, these studies provided insight about the utility of different current adjuvants in a prime-boost formulation, and the unique immune environment induced by DNA priming.