Periodontal disease in dogs : an experimental approach towards prevention using antimicrobial peptides

Tese de Doutoramento em Ciências Veterinárias na especialidade de Sanidade Animal.ABSTRACT - Periodontal disease (PD) is one of the most widespread inflammatory diseases in dogs. This disease is initiated by a polymicrobial biofilm (dental plaque) in the teeth surface, along with a subsequent local inflammatory response leading to periodontium damage and systemic consequences. Enterococci are opportunistic bacteria that may be found in the plaque biofilm. Their ability to act as reservoirs of resistant determinants and their previously linkage to PD-systemic consequences, makes enterococci an interesting bacterial model for antimicrobial studies. Several strategies can be used for PD control, being the removal and inhibition of dental plaque the keystone for PD prevention. Antimicrobial peptides (AMPs) are promising compounds for the management of bacterial infections with low rate of resistance incidence. Nisin is an AMP with antimicrobial activity against Gram-positive and some Gram-negative bacteria, including periodontal pathogens. Considering PD high prevalence, its consequences, and the urgent need to control antimicrobial resistant (AMR) strains and develop new antimicrobial strategies, the main goal of this project was to evaluate the potential of the AMP nisin A for PD control in dogs. First, the antimicrobial activity of nisin and of nisin incorporated in two delivery systems (guar gum biogel and a veterinary toothpaste) were evaluated against a previously characterized collection of canine oral enterococci (n=20). Nisin and nisin-biogel were able to inhibit and eradicate all isolates tested in both planktonic and biofilm forms. Supplemented toothpaste was able to inhibit 95% of the isolates tested. As such, nisin-biogel was selected for the following assays, being observed that it also presented inhibition and eradication abilities against an in vitro multispecies biofilm. Then, nisin-biogel antimicrobial ability was evaluated in the presence of canine saliva and over a 24-month storage, as well as its safety towards eukaryotic cells. Saliva hampered nisin-biogel activity, but it maintained its antimicrobial activity over the storage period at freezing, cooling and room temperatures. In addition, nisin-biogel showed no cytotoxicity at concentrations up to 200 μg/mL. Later, nisin-biogel influence on the canine oral microbiome was evaluated by next generation sequencing, revealing that one dental application of nisin-biogel promoted a reduction in bacterial diversity. Still, a diversity recovery was detected after three applications, along with a reduction in some PD-related bacterial species. Finally, nisin influence in AMR development was evaluated by determination of the mutant section window, mutants AMR profiles and horizontal gene transfer (HGT). It was observed that nisin selective pressure may induce changes in the bacteria’s AMR profile, but not influencing vanA HGT between enterococci and staphylococci. Together, these results show that nisin-biogel seems to be an appropriate approach for PD control in dogs, being important to test this product in an in vivo controlled clinical trial.RESUMO - DOENÇA PERIODONTAL EM CÃES: UMA ABORDAGEM EXPERIMENTAL PARA PREVENÇÃO USANDO PÉPTIDOS ANTIMICROBIANOS - A doença periodontal (DP) é uma das doenças inflamatórias mais frequentes em cães. Esta doença é iniciada por um biofilme polimicrobiano (placa dentária) na superfície dentária, que induz uma resposta inflamatória local e consequências sistémicas. Enterococci são bactérias oportunistas, que podem fazer parte da placa dentária. Sendo reservatórios de fatores de resistência e tendo sido relacionados com consequências sistémicas da DP, são um bom modelo bacteriano para estudos antimicrobianos. A DP pode ser controlada através de várias estratégias, sendo a inibição e remoção da placa dentária fulcrais na sua prevenção. Os péptidos antimicrobianos (PAMs) são compostos promissores para o tratamento de infeções bacterianas, estando associados a um baixo desenvolvimento de resistências. A nisina é um PAM com atividade antimicrobiana contra bactérias Gram-positivas e algumas Gram-negativas, incluído as periodontais. Considerando a elevada prevalência de DP, as suas consequências, a necessidade de controlar infeções por bactérias resistentes a antimicrobianos (RAM) e de desenvolver novos compostos, este projeto teve como objetivo avaliar o potencial antimicrobiano da nisina A para o controlo da DP em cães. Inicialmente, a atividade antimicrobiana da nisina em suspensão ou incorporada em dois veículos de entrega (biogel de goma de guar e uma pasta dentífrica veterinária) foi avaliada utilizando uma coleção de enterococci da cavidade oral de cães (n=20), previamente caracterizada. A nisina e o biogel inibiram e erradicaram todos os isolados testados, na forma planctónica e em biofilme. A pasta suplementada inibiu 95% dos isolados. Assim, o biogel foi selecionado para avaliação posterior, revelando capacidade de inibir e erradicar biofilmes polimicrobianos. Em seguida, a atividade antimicrobiana do biogel foi avaliada na presença de saliva canina e durante um período de armazenamento de 2 anos a várias temperaturas, bem como a sua toxicidade contra células eucariotas. A saliva reduziu a atividade do produto, que não foi afetada pelo armazenamento a temperaturas de congelação, refrigeração e ambiente. Além disso, não se observou citotoxicidade até à concentração de 200 μg/mL. Depois avaliou-se a influência do biogel no microbioma oral canino por sequenciação, verificando-se que uma única aplicação dentária tópica do biogel reduziu a diversidade bacteriana, aumentando após três aplicações, apesar da redução de bactérias intervenientes na DP. Por fim, avaliou-se a influência da nisina na indução de RAM, através da determinação da mutant selection window, de perfis de RAM e transferência horizontal de genes (THG). Observou-se que a exposição a nisina pode induzir alterações nos perfis de RAM, sem promover THG entre enterococci e staphylococci. Assim, os resultados demostram que a nisina-biogel constitui um composto promissor para o controlo da DP em cães, sendo importante a sua testagem in vivo.Virbac Laboratórios de Portugal, Lda, PortugalN/

Definição de metodologia de gestão do património geológico : aplicação ao arquipélago dos Açores

Tese de Doutoramento, Geologia, 12 julho de 2019, Universidade dos Açores.O arquipélago dos Açores, com um enquadramento geodinâmico singular e importante património geológico, tem nas suas paisagens vulcânicas o principal palco de desenvolvimento socioeconómico, quer do quotidiano dos seus habitantes, quer como local idílico e cada vez mais apetecível para turistas. Dada a sua condição geográfica, com territórios exíguos, ricos em recursos naturais, mas frágeis e vulneráveis a mudanças globais e antrópicas, torna-se, então, necessária a integração harmoniosa das políticas de conservação da natureza e de planeamento ambiental, contribuindo para uma gestão racional dos seus recursos. Este é um processo complexo, pois atende a caraterísticas naturais, abordadas de uma perspetiva científica, e a aspetos legais, culturais, económicos, educacionais e recreativos. O presente trabalho de doutoramento pretende dar resposta a uma lacuna existente no domínio da geoconservação, ao nível do estabelecimento de princípios e diretrizes para a gestão do património geológico, tendo em conta a diversidade dos geossítios e a variedade de ameaças que enfrenta. Estando estabelecidas as primeiras etapas de geoconservação, nomeadamente a inventariação, caracterização e avaliação dos geossítios, estão criadas as bases para a sua gestão. Urge passar-se à ação que, para ser eficaz, deve envolver diversos intervenientes no território. Assim, estabelece-se uma estratégia de monitorização dos geossítios, como forma de averiguar a sua qualidade, uso e evolução; e a análise funcional ambiental como ferramenta de apoio à decisão para a sua gestão. Esta é a base proposta para a gestão do património geológico dos Açores, que deve ser aplicada diferenciadamente a várias escalas. Com a implementação do Geoparque Açores, Geoparque Mundial da UNESCO, e com a crescente demanda turística no arquipélago, importa agir proativamente, para manter a qualidade dos geossítios do arquipélago, afinal parte do nosso património natural científico, popular e cultural. Traçam-se algumas medidas para o futuro, mas que necessitam da sua aplicação hoje!ABSTRACT: The Azores archipelago, with a unique geodynamic environment and important geological heritage, has in its volcanic landscapes the main stage of socio-economic development, both for daily life of its inhabitants, and as an idyllic and increasingly attractive place for tourists. Given its geographical situation, with scarce territories, rich in natural resources but fragile and vulnerable to global and anthropogenic changes, the harmonious integration of nature conservation and environmental planning policies is therefore necessary, contributing to a rational management of its resources. This is a complex process; therefore, it attends to natural characteristics, approached from a scientific perspective, and to legal, cultural, economic, educational and recreational aspects. The present PhD work intends to respond to a gap in geoconservation, in the establishment of principles and guidelines for the management of geological heritage, considering the diversity of geosites and the variety of threats it faces. With the establishment of the first stages of geoconservation, namely the inventory, characterization and assessment of geosites, the bases for its management are created. It is urgent to act, and in order to be effective it must involve various actors in the territory. Thus, a geosite monitoring strategy is established to assess its quality, use and evolution; and an environmental functional analysis was performed as a decision support tool for its management. This is the proposed basis for the management of the Azores' geological heritage, which must be applied differently at several scales. With the implementation of the Azores UNESCO Global Geopark and with the growing tourist demand in the archipelago, it urges us to act proactively to maintain the quality of the archipelago's geosites, after all part of our natural, scientific, popular and cultural heritage. Some measures are planned, but they need to be applied today!Projeto de doutoramento (M3.1.2/F/033/2011) financiado pelo Fundo Regional de Ciência e Tecnologia do Governo Regional dos Açores e cofinanciado pelo Fundo Social Europeu, através do Programa PRO-EMPREGO

Vergonha e Solidão na Doença Mental Crónica: contributo de um estudo exploratório

Este estudo pretende explorar a natureza da vergonha, enquanto vivência emocional, e a sua relação com a solidão nos doentes mentais crónicos. Foi avaliada a vergonha interna e externa, a solidão, e estados emocionais negativos, nomeadamente a depressão, ansiedade e stress. Nesta investigação participaram 41 utentes com diagnóstico de perturbação mental. Para avaliar as variáveis psicológicas pretendidas foi utilizada a Escala de Vergonha Interna (ISS), a Escala de Vergonha Externa (OAS), A Escala de Solidão UCLA e a Escala de Depressão, Ansiedade e Stress (DASS-21). Os resultados indicaram que os doentes mentais da nossa amostra apresentam maiores níveis de vergonha comparativamente à população geral. São as mulheres que apresentam valores mais elevados de vergonha interna, enquanto os divorciados apresentam maiores índices de vergonha externa e de solidão. Os doentes que têm uma actividade laboral manifestam menores níveis de solidão e de depressão. Quanto mais elevadas as habilitações literárias dos participantes, menor é o nível de ansiedade. No estudo de comparação entre doentes institucionalizados e não-institucionalizados, verificou-se que os primeiros apresentam níveis significativamente mais elevados de ansiedade, não se distinguindo relativamente à percepção de solidão e de vergonha (interna e externa). A análise da relação entre as variáveis evidenciou que os valores de vergonha interna e de solidão estavam associados de forma positiva e elevada à depressão e de forma moderada à ansiedade e stress. Não obstante as limitações reconhecidas, o presente estudo contribuiu para um melhor conhecimento dos estados emocionais negativos nos doentes mentais. / This study aims to explore the nature of shame, while emotional experience and its relationship with loneliness in the chronic mentally ill. We evaluated the internal and external shame, loneliness, and negative emotional states, including depression, anxiety and stress. 41 users participated in this investigation with a diagnosis of mental disorder. To assess the psychological variables was intended to use Internal Shame Scale (ISS), the Foreign Shame Scale (OAS), the UCLA Loneliness Scale and the Scale for Depression, Anxiety and Stress (DASS-21). The results indicated that the mentally ill in our sample have higher levels of shame compared to the general population. They are women who have higher levels of internal shame, while the divorced have higher rates of external shame and loneliness. Patients who have a work activity demonstrate lower levels of loneliness and depression. The higher the educational level of participants, the lower the level of anxiety. In the comparative study of institutionalized patients and non-institutionalized, it was found that the former have significantly higher levels of anxiety, not distinguishing relation to the perception of loneliness and shame (internal and external). The analysis of the relationship between the variables showed that the values of internal shame and loneliness were positively associated with depression and high and moderately to anxiety and stress. Despite the recognized limitations, this study contributes to a better understanding of negative emotional states in the mentally ill

Analisis Kesalahan Siswa dalam Menyelesaikan Soal Cerita Teorema Pythagoras pada Siswa SMP

Penelitian ini dilakukan dengan tujuan mendeskripsikan kesalahan siswa serta mengetahui faktor penyebab siswa melakukan kesalahan dalam menyelesaikan soal cerita pada materi Teorema Pythagoras. Jenis penelitian adalah kualitatif deskriptif. Subyek penelitian yaitu 3 orang siswa kelas IX B SMP St. Yosef Maubesi yang melakukan kesalahan tingkat tinggi, sedang dan rendah. Teknik pengumpulan data yaitu tes tertulis dan wawancara. Berdasarkan hasil analisis data diperoleh jenis kesalahan yang dilakukan siswa  yaitu (1) kesalahan membaca, (2) kesalahan memahami masalah, (3) kesalahan transformasi, (4) kesalahan keterampilan proses. Faktor penyebab siswa melakukan kesalahan tersebut yaitu (1) siswa tidak mampu mentransformasikan soal ke dalam bentuk gambar, (2) siswa lupa menuliskan yang diketahui dan yang ditanyakan dalam jawaban, (3) siswa hanya terpaku dengan rumus yang sudah dihafalnya, (4) siswa tidak dapat membedakan perkalian biasa dengan perkalian berpangkat

Unveiling DNA methylation in Alzheimer’s disease: a review of array-based human brain studies

The intricacies of Alzheimer’s disease pathogenesis are being increasingly illuminated by the exploration of epigenetic mechanisms, particularly DNA methylation. This review comprehensively surveys recent human-centered studies that investigate whole genome DNA methylation in Alzheimer’s disease neuropathology. The examination of various brain regions reveals distinctive DNA methylation patterns that associate with the Braak stage and Alzheimer’s disease progression. The entorhinal cortex emerges as a focal point due to its early histological alterations and subsequent impact on downstream regions like the hippocampus. Notably, ANK1 hypermethylation, a protein implicated in neurofibrillary tangle formation, was recurrently identified in the entorhinal cortex. Further, the middle temporal gyrus and prefrontal cortex were shown to exhibit significant hypermethylation of genes like HOXA3, RHBDF2, and MCF2L, potentially influencing neuroinflammatory processes. The complex role of BIN1 in late-onset Alzheimer’s disease is underscored by its association with altered methylation patterns. Despite the disparities across studies, these findings highlight the intricate interplay between epigenetic modifications and Alzheimer’s disease pathology. Future research efforts should address methodological variations, incorporate diverse cohorts, and consider environmental factors to unravel the nuanced epigenetic landscape underlying Alzheimer’s disease progression

Unveiling DNA methylation in Alzheimer's disease: a review of array-based human brain studies

The intricacies of Alzheimer's disease pathogenesis are being increasingly illuminated by the exploration of epigenetic mechanisms, particularly DNA methylation. This review comprehensively surveys recent human-centered studies that investigate whole genome DNA methylation in Alzheimer's disease neuropathology. The examination of various brain regions reveals distinctive DNA methylation patterns that associate with the Braak stage and Alzheimer's disease progression. The entorhinal cortex emerges as a focal point due to its early histological alterations and subsequent impact on downstream regions like the hippocampus. Notably, ANK1 hypermethylation, a protein implicated in neurofibrillary tangle formation, was recurrently identified in the entorhinal cortex. Further, the middle temporal gyrus and prefrontal cortex were shown to exhibit significant hypermethylation of genes like HOXA3, RHBDF2, and MCF2L, potentially influencing neuroinflammatory processes. The complex role of BIN1 in late-onset Alzheimer's disease is underscored by its association with altered methylation patterns. Despite the disparities across studies, these findings highlight the intricate interplay between epigenetic modifications and Alzheimer's disease pathology. Future research efforts should address methodological variations, incorporate diverse cohorts, and consider environmental factors to unravel the nuanced epigenetic landscape underlying Alzheimer's disease progression.S

Fig Tree-Induced Phytophotodermatitis: A Case Report on the Perils of a Hobby

Phytophotodermatitis, a condition that results from sequential skin exposure to phototoxic chemicals contained within plants, followed by exposure to solar ultraviolet radiation, has been described with several plants and plant-based foods, namely members of the Moraceaefamily, which include Ficus carica L. This tree's branches, leaves, and fruit skin exude a milky sap or latex containing proteolytic enzymes and furocoumarins known to be photoirritants, easily absorbed upon skin contact. Oxygen-dependent and independent toxic reactions subsequent to sun exposure promote cell membrane damage and oedema, consequently leading to cell death. The diagnosis is confirmed with a detailed anamnesis, and photopatch testing is often useful to rule out a differential diagnosis. It is typically a self-limited condition, with few cases requiring treatment with topical or systemic corticosteroids. We report on a 55-year-old male patient who, following picking figs and pruning a fig tree while exposed to sunlight, developed erythematous and pruritic maculopapular lesions that progressed to blisters with residual hyperpigmentation. The diagnosis was further corroborated through photopatch testing, and the patient was recommended to avoid this recreational activity without symptoms' relapse. This case highlights the importance of considering phytophotodermatitis as a differential diagnosis when evaluating cases of dermatitis on exposed body surfaces and the importance of an exhaustive anamnesis. Identification of specific plant triggers and the performance of photopatch tests are essential to help confirm the diagnosis and guide avoidance recommendationsinfo:eu-repo/semantics/publishedVersio

Epigenetic silencing of OR and TAS2R genes expression in human orbitofrontal cortex at early stages of sporadic Alzheimer’s disease

Modulation of brain olfactory (OR) and taste receptor (TASR) expression was recently reported in neurological diseases. However, there is still limited evidence of these genes' expression in the human brain and the transcriptional regulation mechanisms involved remain elusive. We explored the possible expression and regulation of selected OR and TASR in the human orbitofrontal cortex (OFC) of sporadic Alzheimer's disease (AD) and non-demented control specimens using quantitative real-time RT-PCR and ELISA. Global H3K9me3 amounts were measured on OFC total histone extracts, and H3K9me3 binding at each chemoreceptor locus was examined through native chromatin immunoprecipitation. To investigate the potential interactome of the repressive histone mark H3K9me3 in OFC specimens, native nuclear complex co-immunoprecipitation (Co-IP) was combined with reverse phase-liquid chromatography coupled to mass spectrometry analysis. Interaction between H3K9me3 and MeCP2 was validated by reciprocal Co-IP, and global MeCP2 levels were quantitated. We found that OR and TAS2R genes are expressed and markedly downregulated in OFC at early stages of sporadic AD, preceding the progressive reduction in their protein levels and the appearance of AD-associated neuropathology. The expression pattern did not follow disease progression suggesting transcriptional regulation through epigenetic mechanisms. We discovered an increase of OFC global H3K9me3 levels and a substantial enrichment of this repressive signature at ORs and TAS2Rs proximal promoter at early stages of AD, ultimately lost at advanced stages. We revealed the interaction between H3K9me3 and MeCP2 at early stages and found that MeCP2 protein is increased in sporadic AD. Findings suggest MeCP2 might be implicated in OR and TAS2R transcriptional regulation through interaction with H3K9me3, and as an early event, it may uncover a novel etiopathogenetic mechanism of sporadic AD

The Survey of Lines in M31 (SLIM): The Drivers of the [CII]/TIR Variation

The ratio of the [CII] 158$\,\mu$m emission line over the total infrared emission (TIR) is often used as a proxy for the photoelectric (PE) heating efficiency ($\epsilon_{\rm PE}$) of the far-ultraviolet (FUV) photons absorbed by dust in the interstellar medium. In the nearby galaxy M31, we measure a strong radial variation of [CII]/TIR that we rule out as being due to an intrinsic variation in $\epsilon_{\rm PE}$. [CII]/TIR fails as a proxy for $\epsilon_{\rm PE}$, because the TIR measures all dust heating, not just the contribution from FUV photons capable of ejecting electrons from dust grains. Using extensive multiwavelength coverage from the FUV to far-infrared (FIR), we infer the attenuated FUV emission ($\rm UV_{att}$), and the total attenuated flux ($\rm TOT_{att}$). We find [CII]/TIR to be strongly correlated with $\rm UV_{att}$/$\rm TOT_{att}$, indicating that, in M31 at least, one of the dominant drivers for [CII]/TIR variation is the relative hardness of the absorbed stellar radiation field. We define $\rm{ \epsilon_{PE}^{UV}}$, [CII]/$\rm{ UV_{att}}$ which should be more closely related to the actual PE efficiency, which we find to be essentially constant ($1.85 \pm 0.8 \%$) in all explored fields in M31. This suggests that part of the observed variation of [CII]/TIR in other galaxies is likely due to a change in the relative hardness of the absorbed stellar radiation field, caused by a combination of variations in the stellar population, dust opacity and galaxy metallicity, although PE efficiency may also vary across a wider range of environments.Comment: 19 pages, 16 figures, accepted for publication in Ap

Epigenetic silencing of OR and TAS2R genes expression in human orbitofrontal cortex at early stages of sporadic Alzheimer’s disease

Modulation of brain olfactory (OR) and taste receptor (TASR) expression was recently reported in neurological diseases. However, there is still limited evidence of these genes’ expression in the human brain and the transcriptional regulation mechanisms involved remain elusive. We explored the possible expression and regulation of selected OR and TASR in the human orbitofrontal cortex (OFC) of sporadic Alzheimer’s disease (AD) and non-demented control specimens using quantitative real-time RT-PCR and ELISA. Global H3K9me3 amounts were measured on OFC total histone extracts, and H3K9me3 binding at each chemoreceptor locus was examined through native chromatin immunoprecipitation. To investigate the potential interactome of the repressive histone mark H3K9me3 in OFC specimens, native nuclear complex co-immunoprecipitation (Co-IP) was combined with reverse phase-liquid chromatography coupled to mass spectrometry analysis. Interaction between H3K9me3 and MeCP2 was validated by reciprocal Co-IP, and global MeCP2 levels were quantitated. We found that OR and TAS2R genes are expressed and markedly downregulated in OFC at early stages of sporadic AD, preceding the progressive reduction in their protein levels and the appearance of AD-associated neuropathology. The expression pattern did not follow disease progression suggesting transcriptional regulation through epigenetic mechanisms. We discovered an increase of OFC global H3K9me3 levels and a substantial enrichment of this repressive signature at ORs and TAS2Rs proximal promoter at early stages of AD, ultimately lost at advanced stages. We revealed the interaction between H3K9me3 and MeCP2 at early stages and found that MeCP2 protein is increased in sporadic AD. Findings suggest MeCP2 might be implicated in OR and TAS2R transcriptional regulation through interaction with H3K9me3, and as an early event, it may uncover a novel etiopathogenetic mechanism of sporadic AD. Graphical abstrac