THE UNIVERSITY OF MANCHESTER ABSTRACT OF THESIS submitted by Raja Moftah Moman for the Degree of Doctor of Philosophy entitled Interactions of Oral Bacteria with Host Tissues and Allochthonous Microorganism. 15th June 2017. The oral microbiome is a taxonomically diverse microbial community situated principally upon the hard and soft tissue surfaces of the mouth. It represents a readily accessible biofilm community for the investigation of bacteria-bacteria and bacteria-host interactions, which are responsible for some of the main features of oral biofilms in health and disease (colonisation resistance, antimicrobial tolerance, metabolic cross feeding, and other cooperative phenomena). In the oral cavity these relate specifically to cariogenesis and interactions with soft tissue that are responsible for periodontal disease. This doctoral thesis presents a series of investigations that consider processes for which growth in the biofilm phenotype or bacterial-bacteria or bacteria-host cell interaction are responsible. Four distinct methods were used to assess the effect of the biofilm phenotype on susceptibility of eight distinct oral hygiene actives with various modes of action. Bisphenol microbicide triclosan and the bis-biguanide chlorhexidine were most effective. All were markedly more effective against bacteria grown planktonically than the same organisms grown as biofilms illustrating antimicrobial tolerance, an important biofilm characteristic. In studies of interactions between oral isolates, bacteria previously isolated from the saliva and different oral sites of the oral cavity were tested using a modified cross streak method, in all possible pair-wise combinations. The frequency and strength of physical interactions (coaggregation) between these isolates was also assessed. The incidence of positive interactions was higher than the incidence of negative interactions (15.21% vs. 1.04%) and the incidence of coaggregation in bacteria isolated from saliva was significantly lower than for bacteria isolated from oral biofilms. Together, these data suggest that bacterial cooperation plays a greater role in oral biofilm development and maintenance than competition. With respect to putatively beneficial interactions between bacteria and host, the potential of the candidate dental probiotics L. rhamnosus GG, L. reuteri and S. salivarius to protect host tissues from damage by three Gram negative periodontal pathogens were investigated using human oral cells culture and the (invertebrate) G. mellonella model system. All probiotics inhibited the growth of the test pathogens when applied simultaneously, and significantly decreased toxicity (p P. gingivalis > A. actinomycetemcomitans) in two distinct cell lines. Whilst all probiotics conferred protection against the periodontal pathogens, L. rhamnosus GG, had the greatest protective effect, regardless of probiotic or pathogen used, followed by L. reuteri. S. salivarius was the least effective. Prophylactic treatment with probiotics conferred greater protection than treatment concomitant with pathogen challenge. The data presented in this doctoral thesis demonstrate the functional significance of interactions between taxonomically distinct bacteria and between bacteria and host tissues. Such interactions may determine the outcome of exposure to antimicrobials and are, particularly significant in health and through further research, may be harnessed for prevention and treatment of oral disease.

Although inflammation is a crucial mechanism in response to injury and pathogen clearance, inappropriate or excessive induction of the inflammatory response in pregnancy can cause initiation of the labour cascade and subsequent preterm delivery. Host Defence Peptides (HDPs) have important anti-microbial properties but are also implicated as multifunctional modulators of immunity and infection. They are predominantly secreted by mucosal epithelial cells and released by leukocytes. The specific HDPs that are the focus of this thesis are Human beta-defensin 3 (hBD3) and Human Cathelicidin (hCAP-18/LL-37). The immunomodulatory effect of HDPs in reproductive tissues in response to infection/inflammation has not been well studied. In a pregnant state, the hypothesis of this thesis is that HDPs have a dual role in preventing ascending infection, but also preventing an exacerbated inflammatory response that can cause preterm birth by initiation of the labour cascade. To explore this I determine whether bacterial stimuli can regulate HDPs expression in pregnancy tissues. I also explore what interactions HDPs have on the production/induction of important cytokines that are vital to the inflammatory response. With the aid of HDP knockout mice, the role of these peptides in infection/inflammation and continuation of pregnancy is investigated in a mouse-model of induced preterm-labour. To understand how ascending infection might be controlled by HDPs in pregnancy, I explore how HDPs regulate commensal and pathogenic bacteria. This is achieved by interrogating the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene and next generation sequencing. Results Placental explants respond to Lipopolysaccharide (LPS) challenge by increasing production of pro-inflammatory cytokines. LL-37 but not hBD3 peptide was able to modulate this inflammation by inhibiting the release of these pro-inflammatory cytokines. To establish whether HDPs are critical in the continuation of pregnancy I use a LPS induced mouse–model of preterm labour in animals lacking the genes for the HDPs, Defb14 (Defb14-/-), or Camp (Camp-/-). Intrauterine injection of LPS induced preterm labour in wildtype mice. However, the Defb14-/- and Camp-/- mice do not have an increased rate of preterm labour. Key inflammatory mediators are increased in response to LPS-induced PTL. Camp-/- animals have a similar inflammatory response to wildtype mice when given LPS during pregnancy. To understand how ascending infection might be controlled by HDPs, I interrogated the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene. I established a workflow for 16S rRNA gene sequencing on next-generation sequencing platforms and a bioinformatic pipeline for data analysis. Using this approach I was able to show the mucosal microbiome of Camp-/- animals were significantly different to that of wildtype controls, showing increased diversity in the microbes present. In murine pregnancy, there were very little global cumulative or progressive shifts in bacteria, with the exception of Candidatus arthromitus, which significantly increases with gestation compared to non-pregnancy This thesis has demonstrated that Host Defence Peptides are expressed in pregnancy tissues and have anti-inflammatory properties in response to bacterial stimuli. It is not clear whether the HDPs, hBD3 and LL-37 are fundamental to the immune defence in pregnancy by preventing excessive inflammation, Although, I have shown LL-37 may have a role in modulation of the maternal microbiota.

Ao processo de envelhecimento pode estar associado o surgimento das doenças crônicas não transmissíveis (DCNT) que, por sua vez, podem contribuir para a ocorrência dos agravos clínicos e levar os idosos ao atendimento de urgência/emergência, expondo tanto o paciente quanto seus familiares a uma situação de sofrimento e angústia pelo agravamento da sua condição de saúde e interrupção inesperada de seu cotidiano. Dessa forma, torna-se relevante conhecer como os profissionais e instituições de saúde estão recebendo e se preparando para acolher, tanto o idoso quanto o familiar que o acompanha. Os objetivos deste estudo são: descrever as características sociais e demográficas dos idosos internados na Unidade Funcional de Atendimento às urgências/emergências da Unidade de Emergência do HCFMRP/USP, bem como do seu familiar acompanhante; identificar os núcleos temáticos extraídos da experiência do familiar que acompanha o idoso nesta situação; analisar o acolhimento prestado a partir da experiência do familiar acompanhante do idoso. Trata-se de um estudo qualitativo, em que a história oral foi utilizada como método de investigação na modalidade história oral temática. Para a análise dos dados, seguiram-se os passos da técnica de Análise Indutiva de Conteúdo. A coleta de dados foi realizada no período de abril e maio de 2010. As entrevistas foram realizadas com 11 familiares acompanhantes do idoso que forneceram subsídios para a construção de três núcleos de sentidos: \"Entre a vida e a morte: a busca pelo atendimento de urgência/emergência\", \"Os desafios enfrentados pelo familiar acompanhante do idoso na situação de urgência/emergência\" e \"Alternativas para o atendimento humanizado e acolhedor em unidades de urgência/emergência sob a ótica do familiar acompanhante do idoso\". A partir da análise e discussão destas categorias, fica evidente a necessidade de que os profissionais da saúde precisam conhecer a realidade e as necessidades das pessoas atendidas. Verificou-se que a clientela está atenta aos aspectos da humanização, e a comunicação se revelou uma ferramenta de extrema importância no manejo das relações entre os profissionais e os usuários do serviço. Os conceitos e especificações sobre a humanização e o acolhimento são, na atualidade, bem fundamentados, formulados e amplamente discutidos, no entanto, é necessário que se tornem ações efetivas no âmbito das instituições de saúde. / The aging process may be associated with the emergence of chronic noncommunicable diseases (CNCD), which in turn can contribute to the occurrence of clinical diseases and the elderly to take care at emergency, exposing both the patient and their families to a situation of suffering and distress by the deterioration of his health condition and unexpected interruption of their daily lives. Therefore, it becomes relevant to know how the professionals and health institutions are receiving and preparing to welcome both the old as the family accompanying him. The objectives of this study are to describe the social and demographic characteristics of elderly inpatients in the Unit Functional assistance to emergency care of the Emergency Unit of HCFMRP/USP, as well as your family companion, to identify the core themes drawn from the experience of relatives in accompanies the elderly in this situation, analyze the reception provided from the experience of family companion for the elderly. This is a qualitative study; \"Oral History\" was used as a method of research in oral story mode. For the data analysis followed the steps of the technique of \"Inductive Analysis of Content\". Data collection was performed during April and May 2010. The interviews were conducted with 11 family companions for the elderly, which provided subsidies for the construction of three groups of meaning: \"Between life and death: the search for care at emergency\", \"The challenges faced by the elderly in family companion urgency/emergency\" and \"alternatives for humanized and friendly units at emergency from the viewpoint of the elderly family companion\". From the analysis and discussion of these categories is an evident necessity for health care professionals needs to know the reality and needs of people served. It was found that the customer is looking to aspects of humanization and communication has proved an extremely important tool in the management of relationships between professionals and service users. The concepts and details on humanization and the reception are nowadays well-founded, formulated and widely discussed; however it is necessary to become effective actions within the health institutions.

Em busca da compreensÃo da experiÃncia espiritual no contexto do acolhimento da pessoa em uso abusivo drogas e sua famÃlia, parti de uma atuacÃo no seio da EstratÃgia de SaÃde da FamÃlia-ESF, operando com a prevenÃÃo e promoÃÃo da saÃde. Elegi a perspectiva qualitativa, mediante a pesquisa-aÃÃo (BARBIER, 2007) e analisei a articulaÃÃo da dimensÃo espiritual junto aos recursos da clÃnica ampliada, no contexto do acolhimento à pessoa em uso abusivo de drogas e sua famÃlia. Para contemplar os objetivos desta pesquisa instituà o acolhimento ampliado, segundo conceituei, como um acolhimento que se estende por todo o bairro, focalizando as possibilidades de produÃÃo de saber dos grupos sociais do territÃrio (VALLA, 1998; FREIRE, 1967, 1992, 2003, 2005), visando ao fortalecimento das redes sociais de apoio terapÃutico formais e informais. Vinculei os acervos espirituais da nossa cultura (doutrina EspÃrita, como filosofia, ciÃncia e religiÃo) e da cultura da populaÃÃo ― Ãs formas de oraÃÃo nas diversas matrizes de compreensÃo da fà (VASCONCELOS, 1999, 2001, 2006, 2009, 2010). Analisei as falas dos sujeitos e suas famÃlias, complexificando o olhar do Acolhimento feito na ESF, encontrando a espiritualidade vista sobretudo como produÃÃo de sentido para a vida (KARDEC, 1804-1869, 1858, 2003, 2008; AMUI, 2011; PUTTINI, 2008, 2009), buscada nas instÃncias sociais do Grupo Acolhida, nas visitas domiciliares e no Grupo de PercepÃÃo Juvenil, que fundei na Escola Gabriel Cavalcante para aÃÃo de prevenÃÃo ao uso abusivo de drogas na interface com a promoÃÃo da saÃde mediada pela arte. Sempre buscando a perspectiva do sujeito em uso abusivo de drogas, na promoÃÃo de uma escuta qualificada, constatei a necessidade permanente de produÃao de sentido existencial e espiritual para as experiÃncias, a ser produzido pelos sujeitos no contexto do que nomeio de experiÃncia guiada (conduzida por educador junto a um grupo). Compreendi a necessidade de uma formaÃÃo continuada tomada como leitura da prÃtica, de carÃter interdisciplinar aos profissionais da unidade de saÃde e capaz de nutrir-se da prÃpria prÃxis social instaurada pela ESF no territÃrio, alimentando tambÃm os equipamentos sociais de saÃde do poder pÃblico e da comunidade. TambÃm à resultado desta pesquisa constatar a potÃncia da dimensÃo espiritual, atravÃs da fluidoterapia, como racionalidade em saÃde (ERBERELI, 2013), com o uso da tÃcnica de imposiÃÃo de mÃos (passe espÃrita), que se alia à oraÃÃo no concerto dos recursos da clÃnica ampliada, no contexto do acolhimento vivido na ESF. Sublinho ainda o valor de um trabalho com espiritualidade tomada como promoÃÃo de uma experiÃncia de si (DELORY-MOMBERGER, 2008; WARSCHAUER, 2001), que se alinha a elaboraÃÃes de projetos de vida, aspecto vital, sobretudo para as populacÃes juvenis em estado de pauperizaÃÃo. Ainda, aponto a necessidade de conceituar o sujeito das prÃticas de saÃde como sujeito biopsicossocial e espiritual / In search of understanding spiritual experience in the context of the host of the person in drug abusing and his family, I have started from the performance in the core of the Health Family Strategy (ESF), working with prevention and health promotion. I have chosen a qualitative perspective through action-research (Barbier, 2007) and I have analyzed the articulation of the spiritual dimension related to the expended clinic resources in the context of the person host in drug abusive use and family. To contemplate the objectives I came to realize expanded host according to the host which is extended to the neighborhood, focusing on the possibilities of knowledge production of the social groups in the territory (Valla, 1998; Freire 1967, 1992, 2003, 2005), aiming at social networks strengthen of formal and informal therapeutic support. I have linked the spiritual knowledge of our culture (Spiritist Doctrine as religion, philosophy and science) and from the population culture â to the praying ways in the several matrixes of faith understanding (VASCONCELOS, 1999, 2001, 2006, 2009, 2010). I have analyzed the speech of the individuals and their families complexifying the look of the host carried out in ESF, it was found the spirituality seen above all as meaning production for life (Kardec, 1804-1869, 1858, 2003, 2008; Amui 2011; PUTTINI, 2008, 2009),it was sought in the social instances of Grupo Acolhida, in the home visits and in the Grupo de PercepÃÃo Juvenil that I have founded in the Gabriel Cavalcante School for prevention action of drug abusive use in the interface with health promotion mediated by art. Always searching the perspective of the person in drug abusive use in the promotion of a qualified listening I have confirmed the permanent need of meaning production in the existential and spiritual field to the experiences to be produced by the individuals in the context of what we name guided experience (carried out by educator in a group). I have understood the need of a permanent formation taken as practical reading of an interdisciplinary nature to the health unit professionals which is capable of nurturing of the very social praxis established by ESF in the territory also feeding the social and health equipment belonging to community and Public Power. It is also result of this study is finding the potential of the spiritual dimension of fluid therapy with rationality in health (ERBERELI, 2013) using the technique of laying on hands (spiritist pass), which is allied with the prayer in the resource field of the extended clinic, in the context of host who is lived in the ESF. I emphasize the labor value with spirituality taken as a promotion of an experience of oneself. (Delory-Momberger, 2008; Warschauer, 2001), which aligns to the elaborations of life projects, vital aspect, especially for juvenile populations with impoverishment. Still, I point out the need to conceptualize the subject of health practices as biopsychosocial and spiritual subject.

Adaptive radiations provide exceptional opportunities to examine the relationships between natural selection, adaptation, and speciation. Neotropical Adelpha butterflies may represent such a radiation, characterized by extraordinary breadth in host plant use and wing color patterns. In this study, we use genome-wide RAD markers to reconstruct the complex evolutionary history of Adelpha and the closely related temperate genus, Limenitis. Despite the presence of significant missing data, a variety of phylogenetic methods produce similar and highly supported trees. These well-resolved phylogenies allow for the identification of an ecologically important shift to a toxic host plant family, as well as the confirmation of rampant wing pattern mimicry throughout the genus. Taken together, our results support the hypothesis that the colonization of novel host plants represents a key evolutionary innovation that is fueling ongoing adaptive diversification within this large, phenotypically diverse butterfly radiation.

Trichodysplasia spinulosa (TS) é uma doença proliferativa de pele observada em pacientes imunocomprometidos. Caracteriza-se pela formação de espinhas de queratina conhecidos como espículas, acantose epidérmica, dilatação do folículo piloso, queratose actínica, queda dos pelos, pápulas foliculares e, que normalmente, se manifestam na região facial do paciente e extremidades do corpo (constantemente confundida com danos por exposição prolongada ao sol). A TS resulta da infecção ativa com o poliomavírus TSassociado (TSPyV), onde observa-se alta carga viral, expressão de proteína do vírus e formação de partículas. Este estudo desenvolveu métodos moleculares de detecção e sequenciamento do genoma total e parcial de TSPyV e utilizou-se destes métodos para determinar padrões de excreção e viremia em indivíduos imunocompromentidos e imunocompetentes, bem como explorar possíveis vias de transmissão. Ainda, características genéticas e filogenéticas do TSPyV também foram determinadas. Apesar de observamos alta taxa de excreção urinaria em indivíduos imunocomprometidos (57,7%), o vírus não foi encontrado em amostras de água do meio ambiente. Ainda em termos de excreção urinária do TSPyV, apenas 1,4% dos indivíduos imunocompetentes apresentaram virúria (diferente do que se observa para os poliomavirus JCPyV e BKPyV), mas o vírus foi encontrado em leite materno, sugerindo assim a possibilidade de haver transmissão vertical do TSPyV. As análises filogenéticas revelaram a existência de 2 linhagens de vírus circulantes em nosso meio, com características distintas dos já descritos na literatura. As diferenças observadas foram suficientes para que os vírus sejam caracterizados como novos genótipos circulantes de TSPyV. / Trichodysplasia spinulosa (TS) is a proliferative skin disease seen in immunocompromised patients. It is characterized by the formation of keratin spines known as spicules, epidermal acanthosis, hair follicle dilatation, actinic keratosis, hair loss, follicular papules and, which usually manifest in the facial region and extremities of the body (constantly confounded with damage from prolonged exposure to the sun). TS results from active infection with TS-associated polyomavirus (TSPyV), where high viral load, virus protein expression and particle formation are observed. This study developed molecular methods for detection and sequencing the total and partial genome of TSPyV and, employing these methods, determined patterns of excretion and viremia in immunocompromised and immunocompetent individuals, as well as explored possible transmission pathways. Genetic and phylogenetic characteristics were also determined. Although we observed high rate of urinary shedding in immunocompromised individuals (57.7%), the virus was not found in environmental water samples. Also in terms of urinary excretion of TSPyV, only 1.4% of immunocompetent individuals presented viruria (different from what is observed for polyomaviruses JCPyV and BKPyV), but the virus was found in breast milk, thus suggesting the possibility of vertical transmission. Phylogenetic analyzes revealed the existence of 2 circulating virus strains in our country, with different characteristics from those already described in the literature. The differences seem to be sufficient to characterize the viruses as new genotypes of TSPyV.

Lodgepole pines Pinus contorta (Douglas) were treated by girdling to assess the response by an endemic population of mountain pine beetle Dendroctonus ponderosae (Hopkins) (Coleoptera: Scolytidae). Capture in 'sticky traps' indicated that the beetles were attracted while in flight towards the wounded trees. A significant difference in the landing rates of bark beetles between the treated and untreated trees indicated that the beetles were able to distinguish wounded from non-wounded trees while still in flight. Pressure chamber readings demonstrated that water stress developed in the girded trees when compared to the non-girdled trees.

WHO has reported that antibiotic resistance is the third major cause of human death all over the globe. Recent study, has focused on the development of new antibacterial resistance drugs. Herein, we tried to synthesis a series of polymers that can mimic the HDPs. HDPs can target the bacterial cell membrane and they have less chances to develop bacterial resistance. We synthesized the amphiphilic polycarbonates that are highly selective to Gram-positive bacteria, including multidrug resistant pathogens. The membrane disruption activity of these polymers was proved by fluorescence and TEM studies and the drug resistance study showed that the polymers don’t develop bacterial resistance. In order to further design the molecules that can target a broad spectrum of bacteria, we have designed a series of lipidated dendrimers that can target the Gram-positive and Gram-negative bacteria. These dendrimers mimic the HDPs and target the bacterial cell membrane. Dendrimers are reported to inhibit the formation of bacterial biofilm which makes them promising for their future development of antibiotic agents. Apart from the synthesis of polymers and dendrimers as antibacterial agents, we have designed a series of small molecular antibacterial agents that are based on the acylated reduced amide scaffold and small dimeric cyclic guanidine derivatives. These molecules display good potency against a panel of multidrug-resistant Gram-positive and Gram-negative bacterial strains. Meanwhile, they also effectively inhibit the biofilm formation. Mechanistic studies suggest that these compounds kill bacteria by compromising bacterial membranes, a mechanism analogous to that of host-defense peptides (HDPs). Lastly, we also demonstrate that these molecules have excellent in vivo activity against MRSA in a rat model. This class of compounds could lead to an appealing class of antibiotic agents combating drug-resistant bacterial strains.

The emergent resistance of bacteria against the conventional antibiotics has motivated the search for novel antimicrobial agents. Nature abounds with a number of antimicrobial peptides that are a part of our innate immune system and protect us against a variety of pathogenic bacteria. While they are broad-spectrum in their activity and show less drug-resistance induction, their intrinsic metabolic stability limits their potential therapeutic applications. Herein we describe the development of novel broad-spectrum bioactive antimicrobial peptidomimetics AA-peptides. AApeptides were designed based on chiral PNA backbone. Substitution of nucleobases yields AApeptides that are resistant to proteolysis and capable of mimicking peptides. Two types of AApeptides are discussed in this dissertation "[aacute]-AApeptides" and " ã-AApeptides" The therapeutic potential of these AApeptides were accessed by conducting antibacterial assays against a series of both gram-positive, gram-negative bacteria and fungi. These oligomers were characterized using MALDI-TOF and Circular Dichroism spectroscopy (CD). Their invitro toxicity was evaluated against human erythrocytes .We attempted to study their mechanism of action via membrane depolarization assay. We have successfully identified them as antimicrobial agents, pro-inflammatory immune response suppressing agents and as anti-biofilm agents.

No description available.