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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / As infecÃÃes respiratÃrias agudas (IRA) sÃo importantes causas de morbidade e mortalidade em todo o mundo, acometendo principalmente crianÃas menores de cinco anos de idade. Essas infecÃÃes podem ser causadas por diferentes microrganismos, porÃm os vÃrus sÃo os mais frequentes. Esse estudo teve como objetivo descrever aspectos clÃnicos e epidemiolÃgicos de IRA de etiologia viral em crianÃas de zero a 12 anos de idade atendidas em serviÃo de emergÃncia de um hospital terciÃrio da cidade de Fortaleza-CE, no perÃodo de janeiro de 2007 a dezembro de 2008. Para tanto foram coletadas 1318 amostras de secreÃÃo de nasofaringe das crianÃas. As amostras foram submetidas à tÃcnica de imunofluorescÃncia indireta para detecÃÃo dos seguintes vÃrus respiratÃrios: vÃrus sincicial respiratÃrio (VSR), metapneumovÃrus humano (MPVh), adenovÃrus, influenza A e B e parainfluenza 1, 2 e 3. Os resultados desse estudo mostraram que pelo menos um vÃrus respiratÃrio foi detectado em 383 (29,1%) amostras. O vÃrus mais prevalente foi o VSR (44,4%), tendo o mesmo apresentado um padrÃo de sazonalidade definido, com associaÃÃo a estaÃÃo chuvosa. A co-infecÃÃo ocorreu em 12 (3,1%) amostras e o VSR foi o mais frequentemente associado. A mÃdia de idade dos pacientes foi de 23 meses e nÃo houve associaÃÃo entre o gÃnero desses pacientes e a positividade dos exames, apesar da maioria das crianÃas serem do sexo masculino. Entre os diagnÃsticos clÃnicos de etiologia viral, houve predomÃnio de infecÃÃo da via aÃrea superior (IVAS) (51,2%), e em relaÃÃo ao diagnÃstico especÃfico das infecÃÃes da via aÃrea inferior (IVAI), destacou-se a pneumonia. Portanto, os resultados desse estudo ressaltam a importÃncia dos vÃrus como causadores de IRA em crianÃas na cidade de Fortaleza, com as maiores taxas ocorrendo entre os meses de marÃo a junho, diferenciando-se da regiÃo Sul do paÃs, onde as maiores taxas sÃo encontradas nos meses de julho a outubro. / Acute respiratory infections (ARI) are important causes of morbidity and mortality worldwide, affecting mainly children under five years old. These infections can be caused by different organisms, but viruses are the most frequent. This study aimed to describe clinical and epidemiological aspects of viral ARI in children 0-12 years of age treated in the emergency department of a tertiary hospital in the city of Fortaleza, from January 2007 to December 2008. Therefore, we collected 1318 samples of nasopharyngeal secretions of children. The samples were subjected to indirect immunofluorescence for detection of the following respiratory viruses: respiratory syncytial virus (RSV), human metapneumovirus (hMPV), adenovirus, influenza A and B and parainfluenza 1, 2 and 3. The results of this study showed that at least one respiratory virus was detected in 383 (29.1%) samples. The most prevalent virus was RSV (44.4%), and presented the same seasonal pattern of a defined association with the rainy season. Co-infection occurred in 12 (3.1%) samples and RSV was the most frequently associated. The average age of patients was 23 months and there was no association between gender of these patients and positivity of the tests, although most children were male. Among the clinical diagnoses of viral etiology, there was predominance of upper respiratory infection diseases (URID) (51.2%), and in relation to the specific diagnosis of the lower respiratory infections diseases (LRID), stood out pneumonia. Therefore, the results of this study highlight the importance of viruses as causes of ARI in children in Fortaleza, with the highest rates occurring between the months March to June, differing from the southern region, where the highest rates are found in the months from July to October.

Pediatric acute respiratory tract infections (ARTIs) are a leading cause of morbidity and mortality. The objectives of this study were to describe the epidemiology of polymicrobial ARTI in a hospital-based pediatric population and to investigate the association of polymicrobial infection and severity of illness. We conducted a retrospective study of 559 archived respiratory specimens from 421 children under the age of 10 years collected from March 28, 2008 through June 30, 2009 and stored by the University of Iowa Hospital and Clinics Clinical Microbiology Laboratory. Specimens were tested by immunofluorescence assay and/or viral culture at the time of collection (influenza A and B, parainfluenza [PIV] 1-3, respiratory syncytial virus [RSV], adenovirus [Ad]) and uniformly by RT-PCR (human metapneumovirus [hMPV], rhinovirus [HRV], human bocavirus [HBoV]) and PCR (Ad) for the current study. Demographic and clinical data were abstracted from electronic medical records. Results from this study suggest that polymicrobial respiratory tract infections are common in this population. A virus was identified in 61.3% of 349 respiratory specimens from children with confirmed or suspected ARTI. HRV (27.5%), RSV (18.9%), HBoV (8.3%), hMPV (7.7%), and PIV (6.6%) were the most common viruses detected. A viral coinfection was identified in 21.5% of the 214 virus-positive specimens and was most often detected for Ad (53.3% of 15 Ad-positive specimens), HBoV (51.7% of 29 HBoV-positive specimens), PIV (43.5% of 23 PIV-positive specimens), HRV (35.4% of 96 HRV-positive specimens), and RSV (34.8% of 66 RSV-positive specimens). Among the 46 specimens with dual or triple viral coinfections detected, the most frequent virus-virus combination was HRV-RSV (n=12). We hypothesized that certain host-specific risk factors were associated with the likelihood of viral coinfection. While none of the covariates in the final model were significant, the results were suggestive. Male gender (OR 1.70, 95% CI 0.83-3.46), age between 6 months to 1 year (as compared to children less than 6 months old, OR 2.15, 95% CI 0.75-6.19), and history of any chronic condition that may result in immunosuppression (OR 2.05, 95% CI 0.99-4.23) were each associated with increased odds of viral coinfection (p > 0.05). We also hypothesized that children with coinfections would be more likely to have severe ARTI. Children with viral-bacterial coinfection, as compared to children with viral mono-infection, were more likely to be admitted to an intensive care unit (OR 6.00, 95% CI 2.51-14.33) even after controlling for age, history of prematurity, urban/rural residence, and leukocytosis. This study will inform medical and public health professionals with regard to the epidemiology of polymicrobial infections and their potential importance as a cause of severe acute respiratory tract infection in children. Furthermore, results of this study may contribute to the ongoing discussion of the importance of diagnostic ability to reliably detect multiple concurrent pathogens in a single individual.

The thesis examines a mediated crisis and how The Straits Times and The Australian approach the reporting of Severe Acute Respiratory Syndrome (SARS). It looks at how this mediated crisis exemplifies the culture of the national newspaper and in turn how the national newspaper has an historical influence on the national psyche. A total of 649 reports and headlines and 141 letters about SARS in The Straits Times (including The Straits Time Interactive) were examined from April 2003 to November 2003 as were 125 headlines from The Australian. The early sections of the thesis discuss how a crisis makes news; examine how the media report a crisis and what emphasis is given to aspects such as: actors, primary definers, vocabulary, lexical choices, subjects, themes, issues and value dimension or stance. The first chapter defines crisis, journalism and crisis journalism and discusses where the latter sits within the continuing expansion and development of major theoretical frameworks, including living in a risk society. The implication here is that crisis and risk have a symbiotic relationship. Historical perspectives of news are discussed in Chapter 2, and the newspaper is placed within the context of contemporary media. The chapter discusses how newspapers are aligned with the concept of the national mind and demonstrates the roles and formations of the two newspapers in relation to the SARS crisis. Chapter 3 codes the headlines, article titles and subtitles of The Straits Times and The Australian and using content analysis of the headlines, analyses the reporting of a serious health crisis SARS that lasted from March to November, 2003. The quantification within content analysis enables a researcher to read and interpret questions that relate to the intensity of meaning in texts, their social impact, the relationships between media texts and the realities and representations they reflect (Hansen et al, 1998). The theory and method of content analysis is used in this chapter to consider differences between The Straits Times and The Australian and to exemplify the media’s representation of the narratives of SARS as it happened in the countries of Singapore and Australia. Aspects of crisis and risk, the newspaper and the national mind, narratives, presentations, and post SARS events are discussed in the last chapter. It is concluded from these discussions there is a world narrative that tells the story of how the human condition likes to live and rely on a safe social environment always being available. The relationship between a mediated crisis and risk are also discussed. In addition, it is maintained that reporting in 2003 was not just about SARS but a way of reporting that allowed one to view journalism as an aid to good governance, particularly with regard to living in a risk and crisis-ridden society.

Acute Respiratory Distress Syndrome (ARDS) is the most extreme form of acute lung injury and continues to have a significant morbidity and mortality. Unfortunately, the mechanisms involved in the recovery and repair of the lung following ARDS remain poorly understood. An understanding of these is pivotal to improving outcome from acute lung injury. Several observational studies have suggested a potential relationship between Vascular Endothelial Growth Factor (VEGF) in the lung and the development/outcome of ARDS. In this thesis, three potential mechanisms underlying these observations have been explored: 1. What is the anatomical distribution of VEGF receptor and isoform expression in normal and ARDS lung? How does this change at early and later time points following acute lung injury? 2. Are human type 2 alveolar epithelial (ATII) cells a source of and target for VEGF? How does exposure to a pro-inflammatory milieu modify their expression of VEGF isoforms and receptors? 3. Is there a relationship between a functional VEGF polymorphism and susceptibility to developing and severity of ARDS? I have demonstrated VEGF receptor expression on both sides of the alveolarcapillary membrane with upregulation in later ARDS. All three principal isoforms (VEGF121, VEGF165 and VEGF189) are expressed in normal human lung with uniform downregulation of all three in early ARDS, which normalises with increasing time following injury. I have not found any evidence of VEGF isoform switching. I have also demonstrated human ATII cells are both a significant cellular source of and a target for VEGF (via VEGF receptor expression) confirming autocrine VEGF activity in the lung. VEGF is an ATII cell survival factor. ATII cells differentially respond to pro-inflammatory stimuli by increasing VEGF isoform but not receptor expression, which may serve as a regulatory control mechanism. Finally, I have demonstrated the VEGF 936 T allele increases susceptibility to and the severity of lung injury. The T allele is associated with an increase in plasma VEGF level in ARDS patients but intra-alveolar levels are unaffected.

Acute Respiratory Distress Syndrome (ARDS) is an inflammatory disease characterized by pulmonary edema, stiff lungs and hypoxemia. Patients with ARDS are more susceptible to VILI (ventilator induced lung injury). Under mechanical ventilation, lung stress and strain are the main determinants of VILI and in patients with muscle effort patient-ventilator asynchrony may enhance this phenomenon. Ventilation modes PCV and VCV with auto-flow can minimize patient-ventilator asynchrony, but then can liberate the offer of flow and tidal volume, compromising the protective ventilatory strategy in ARDS. This study aimed to evaluate the influence of muscle effort and patient-ventilator asynchrony on pulmonary stress and strain in a mechanic lung model of acute respiratory distress syndrome. An experimental bench study was performed, using a lung simulator, ASL 5000TM, in which was configured a lung model with restrictive respiratory mechanics with complacency of 25ml/cmH2O and resistance of 10 cmH2O/L/sec. Muscle effort was adjusted in three situations: no muscular effort (Pmus = 0), with inspiratory muscle effort (Pmus = -5 cmH2O) and inspiratory and expiratory effort (Pmus = -5/+5 cmH2O), all with breathe rate (b) of 20 bpm. Five ventilators were connected to the simulator through and endotracheal tube No 8.0 mm and adjusted on VCV, VCV with Auto-flowTM (in the ventilator in which it was available) and PCV modes, all with tidal volume (VT): 420 ml, PEEP: 10 cmH2O and breath rate set in two situations: b = 15 bpm (lower than b of the respiratory muscle effort) and b = 25 bpm (higher than b of the respiratory muscle effort). Variables analyzed were: maximum VT, alveolar pressure at the end of inspiration, effective PEEP, driving pressure, transpulmonary pressure at the end of inspiration and expiration, average transpulmonary pressure, inspiratory peak flow and analysis of mechanic curves. In the studied lung model the b of the ventilator adjusted higher of the b of the patient and not the muscle effort was the main determinant for the development of patient-ventilator asynchrony, causing large variations of the VT and pulmonary pressures, intensifying the lung stress and strain. The ventilatory modes had similar behavior, although VCV Auto-flowTM and PCV have presented slightly higher values of VT and pulmonary pressures. Thus it is concluded that the proper adjustment of the programed breath rate in the assisted/controlled modes can minimize patient-ventilator asynchrony, reducing lung stress and strain. / A SÃndrome da AngÃstia RespiratÃria Aguda (SARA) à uma doenÃa inflamatÃria caracterizada por edema pulmonar, pulmÃes rÃgidos e hipoxemia. Pacientes com SARA estÃo mais suscetÃveis à VILI (ventilator induced lung injury). Sob ventilaÃÃo mecÃnica, o stress e o strain pulmonares sÃo os principais determinantes da VILI e nos pacientes com esforÃo muscular a assincronia paciente-ventilador pode potencializar este fenÃmeno. Os modos ventilatÃrios PCV e VCV com AutoFlow podem minimizar a assincronia paciente-ventilador, mas por outro lado podem liberar a oferta de fluxo e volume corrente, comprometendo a estratÃgia ventilatÃria protetora na SARA. Objetivou-se avaliar as influÃncias do esforÃo muscular e da assincronia paciente-ventilador sobre o âstrainâ e o âstressâ pulmonares em modelo pulmonar mecÃnico de sÃndrome da angÃstia respiratÃria aguda. Foi realizado um estudo experimental de bancada, utilizando um simulador de pulmÃo, ASL 5000 no qual foi configurado um modelo pulmonar com mecÃnica respiratÃria restritiva, com complacÃncia de 25ml/cmH2O e resistÃncia de 10 cmH2O/L/sec. O esforÃo muscular foi ajustado em trÃs situaÃÃes: sem esforÃo muscular (Pmus=0), com esforÃo muscular inspiratÃrio (Pmus= -5cmH2O) e esforÃo inspiratÃrio e expiratÃrio (Pmus= -5/+5 cmH2O), todos com frequÃncia respiratÃria (f) de 20rpm. Ao simulador foram conectados cinco ventiladores atravÃs de um tubo orotraqueal n 8,0 mm e ajustados nos modos VCV, VCV com sistema AutoFlow (no ventilador que tinha o sistema disponÃvel) e PCV, todos com volume corrente (VC): 420 ml, PEEP: 10 cmH2O e frequÃncia respiratÃria programada em duas situaÃÃes: f=15rpm (< que a f de esforÃo muscular respiratÃrio) e f=25rpm (> que a f de esforÃo muscular respiratÃrio). As variÃveis analisadas foram: VC mÃximo, a pressÃo alveolar no final da inspiraÃÃo, PEEP efetiva, driving pressure, pressÃo transpulmonar no final da inspiraÃÃo e expiraÃÃo, pressÃo transpulmonar mÃdia, pico de fluxo inspiratÃrio e anÃlise das curvas de mecÃnica. No modelo pulmonar estudado a f do ventilador pulmonar ajustada acima da f do paciente e nÃo o esforÃo muscular o principal determinante para o desenvolvimento de assincronia paciente ventilador, causando grandes variaÃÃes de VC e pressÃes pulmonares, o que intensificou o stress e strain pulmonares. Os modos ventilatÃrios tiveram comportamento semelhante, embora os modos VCV AutoFlow e PCV tenham apresentado valores discretamente maiores de VC e pressÃes pulmonares. Desta forma conclui-se que o ajuste adequado da frequÃncia programada nos modos assistido/controlado podem pode minimizar a assincronia paciente ventilador reduzindo o stress e strain pulmonares. Palavras-

Masters of Science / The Coronaviridae family consists of RNA viruses within the order Nidovirales. The family is classified into two genera, namely the corona- and toroviruses. Coronaviruses are enveloped, single stranded, positive sense RNA viruses with genomes ranging between 27-32kb in size. The 5’ two-thirds of the genome encodes for the 1a/b polyprotein, while the 3’ one-third of the genome encodes for the structural proteins that mediate viral entry into the host cell. These structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The nucleocapsid protein is expressed at high levels within an infected cell. Studies have shown that this protein plays a key regulatory role in different cellular pathways, including the inhibition of interferon production and the up-regulation of the AP1 signal transduction pathway, amongst others. Also, the N protein is vital in the formation of the ribonucleocapsid core by binding to the viral RNA during virion assembly. The focus of this study is the immune response in whole blood cultures to the presence of human coronavirus (HCoV) NL63 N protein. To characterise the stimulation of the immune activity against HCoV-NL63 N in blood cultures, the HCoV-NL63 N gene was expressed in a bacterial system. In this pilot study, GSTtagged N constructs were then purified and used to treat whole blood cultures from three volunteers. ELISAs were used to measure the cytokine response in these treated whole blood cultures. Results showed that the nucleocapsid protein has an inflammatory response on whole blood cultures. These results have generated vital information in the potential function of the HCoV-NL63 N protein on the immune system. It is suffice to say that the HCoV-NL63 N protein is able to elicit an effective inflammatory response within the host cell. Future studies into the cellular pathways affected by the HCoV-NL63 N protein will clarify its exact role in stimulating the host immune system.

Background: Acute respiratory infections (ARIs) represent an important cause of morbidity and mortality in children, remaining a major public health concern, especially affecting children under 5 years old from low-income countries. Unfortunately, information regarding their epidemiology is still limited in Peru. Methods: A secondary data analysis was performed from a previous cross-sectional study conducted in children with a probable diagnosis of Pertussis from January 2010 to July 2012. All samples were analyzed via Polymerase Chain Reaction (PCR) for the following etiologies: Influenza-A, Influenza-B, RSV-A, RSV-B, Adenovirus, Parainfluenza 1 virus, Parainfluenza 2 virus, Parainfluenza 3 virus, Mycoplasma pneumoniae and Chlamydia pneumoniae. Results: A total of 288 patients were included. The most common pathogen isolated was Adenovirus (49%), followed by Bordetella pertussis (41%) from our previous investigation, the most prevelant microorganisms were Mycoplasma pneumonia (26%) and Influenza-B (19.8%). Coinfections were reported in 58% of samples and the most common association was found between B. pertussis and Adenovirus (12.2%). Conclusions: There was a high prevalence of Adenovirus, Mycoplasma pneumoniae and other etiologies in patients with a probable diagnosis of pertussis. Despite the presence of persistent cough lasting at least two weeks and other clinical characteristics highly suspicious of pertussis, secondary etiologies should be considered in children under 5 years-old in order to give a proper treatment. / Revisión por pares

Objective: This study aimed to determine the prevalence of A. baumannii in children aged less than 1 year admitted with a clinical diagnosis of whooping cough. Results: A total of 225 nasopharyngeal samples from children under 1 year old hospitalized with clinical diagnosis of whooping cough were studied from January 2010 to July 2012. The presence of A. baumannii was detected in 20.89% (47/225) of the nasopharyngeal swab samples. Among the 47 patients with A. baumannii: 5 were diagnosed with A. baumannii monoinfection, 17 co-infection with bacteria, 7 co-infection with virus and 18 co-infection with bacteria + virus. It was observed that 51.6% (116/225) were children between 29 days and 3 months old, this same group had the highest overall prevalence with 53.3%. The most common co-infecting pathogens were Bordetella pertussis in 55.3%, Adenovirus in 42.6% and Mycoplasma pneumoniae in 23.4%. / Revisión por pares

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the coronavirus disease of 2019 (COVID-19) pandemic, which has infected millions of people worldwide. To date, several vaccines and antivirals have been developed against SARS-CoV-2; however, its tendency to mutate rapidly poses a continued threat to human health. As such, the development of better pan-coronavirus therapeutics is still required. Recently, the SARS-CoV-2 non-structural protein 13 (Nsp13) helicase has been shown to be an attractive therapeutic target given its high conservation rate among coronaviruses and indispensable role in viral replication. Based on this, we sought to further study the biochemical mechanisms behind Nsp13's binding and unwinding activities, along with its interactions with host cells, to provide further insight for future therapeutic development. To study the binding and unwinding activity of Nsp13, we site-specifically incorporated the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into Nsp13 to act as a bioorthogonal handle for fluorescent labelling. We identified five potential sites for AzF incorporation in Nsp13 and assessed their reactivities towards a conjugated Cy5 fluorophore through strain-promoted alkyne-azide cycloaddition (SPAAC). Further experiments were also performed to ensure that the unwinding activity was not adversely affected before these Nsp13-AzF constructs were utilized in fluorescence resonance energy transfer (FRET)-based binding assays. Ultimately, the F81AzF construct was identified to be the most suitable for monitoring the binding of Nsp13 to a series of nucleic acid substrates in a distance-dependent manner by FRET. The next steps of this project would be to implement F81AzF in single-molecule FRET (smFRET) experiments to directly monitor the positioning and dynamics of this helicase on its substrate. In addition, interactions between Nsp13 and host cellular and biological processes were investigated to provide further insight into potential mechanisms that can be exploited for novel therapeutic development. From transcriptomic profiling analyses of A549 cells, we uncovered that Nsp13 influences microRNA (miRNA) expression and signalling pathways; in particular, miR-146a, a potent mediator of inflammation and immune responses, was found to be induced upon Nsp13-overexpression. Further experiments revealed that this may lead to the inhibition of NF-κB signalling, through the repression of the upstream targets TRAF6 and IRAK1, to suppress the production of proinflammatory cytokines and facilitate viral infection. Collectively, from this work, we propose that further exploration of these miR-146a-mediated signalling pathways may present alternative strategies for antiviral investigations.