Effects of tobacco products on the attachment and growth of periodontal ligament fibroblasts

BACKGROUND: Cigarette smoking is one of the most significant risk factors in the development and further advancement of inflammatory periodontal disease, however, the role of either nicotine or its primary metabolite cotinine in the progression of periodontitis is unclear. This study aimed to investigate the effects of nicotine and cotinine on the attachment and growth of fibroblasts derived from human periodontal ligament (PDL).

METHODS: Primary cultures were prepared from the roots of extracted premolar teeth. Cells were used at both low (P3 to P5) and high (P11 to P13) passage. Cell numbers were determined over 14 days using either the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay or with a Coulter counter. Cultures were exposed to culture medium supplemented with 1) 15% fetal calf serum (FCS) only; 2) 1% FCS only; 3) 1% FCS and nicotine (concentration range 5 ng/ml to 10 mg/ml); or 4) 1% FCS and cotinine (concentration range 0.5 ng/ml to 10 microg/ml).

RESULTS: Nicotine significantly (P <0.05, by ANOVA) inhibits attachment and growth of low passage cells at concentrations >1 mg/ml and high passage PDL fibroblasts at concentrations >0.5 mg/ml. Cotinine, at the highest concentration used (10 microg/ml), appeared to inhibit attachment and growth of both low and high passage fibroblasts but this was not statistically significant (P >0.05, by ANOVA).

CONCLUSIONS: Tobacco products inhibit attachment and growth of human PDL fibroblasts. This may partly explain the role of these substances in the progression of periodontitis.

Reference gene selection in human periodontal ligament cells

Background: Real-time quantitative PCR (qPCR) is a highly sensitive and specific method which is used extensively for determining gene expression profiles in a variety of cell and tissue types. In order to obtain accurate and reliable gene expression quantification, qPCR data are generally normalised against so-called reference or housekeeping genes. Ideally, reference genes should have abundant and stable RNA transcriptomes under the experimental conditions employed. However, reference genes are often selected rather arbitrarily and indeed some have been shown to have variable expression in a variety of in vitro experimental conditions.
Objective: The objective of the current study was to investigate reference gene expression in human periodontal ligament (PDL) cells in response to treatment with lipopolysaccharide (LPS).
Method: Primary human PDL cells were grown in Dulbecco’s Modified Eagle Medium with L-glutamine supplemented with 10% fetal bovine serum, 100UI/ml penicillin and 100µg/ml streptomycin. RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). The expression of a total of 19 reference genes was studied in the presence and absence of LPS treatment using the Roche Reference Gene Panel. Data were analysed using NormFinder and Bestkeeper validation programs.
Results: Treatment of human PDL cells with LPS resulted in changes in expression of several commonly used reference genes, including GAPDH. On the other hand the reference genes β-actin, G6PDH and 18S were identified as stable genes following LPS treatment.
Conclusion: Many of the reference genes studied were robust to LPS treatment (up to 100 ng/ml). However several commonly employed reference genes, including GAPDH varied with LPS treatment, suggesting they would not be ideal candidates for normalisation in qPCR gene expression studies.

Osteoprotegerin Expression in the Dental Pulp

Objectives: The inflammatory response to pulpal injury or infection has major clinical significance. Osteoprotegerin (OPG) is a soluble decoy receptor for Receptor Activator of NF kappa B Ligand (RANKL), preventing ligand binding to its receptor (RANK), thus inhibiting clastic cell formation. The aim of the study is to investigate the expression of OPG in human dental pulp and the effects of inflammatory mediators. This study will specifically investigate the effects of Transforming Growth Factor Beta-1 (TGF-β1) and Interleukin 1-Beta (IL-1β) on the expression of OPG on pulp fibroblasts in vitro. Method: Five primary pulp fibroblast populations were obtained by explant culture of healthy pulp tissue. Triplicate cultures were grown to confluence in 12-well plates and stimulated for 48 hours with IL-1β (10ng/ml) or TGF-β1 (10ng/ml). The conditioned media was collected and OPG levels detected by ELISA (R+D Systems, UK). Results: All fibroblast populations produced quantifiable levels of OPG in a time-dependant fashion. IL-1β significantly increased the expression of OPG (p<0.05) in all cultures. In contrast, TGF-β1 had no significant effect on OPG expression levels. In addition, previous work in our laboratory demonstrated both TGF-β1 and IL-1β stimulated OPG expression by periodontal ligament fibroblasts. Conclusion: These data indicate that IL-1β-regulated expression of OPG by pulpal fibroblasts may mediate hard tissue turnover in the inflamed dental pulp.

Functional expression of TRPV4 in human periodontal ligament cells

Background: Periodontal ligament (PDL) cells are exposed to physical forces in vivo in response to mastication, parafunction, speech and orthodontic tooth movement. Although it has been shown that PDL cells perceive and respond directly to mechanical stimulation, the nature of the ion channels that mediate this mechanotransduction remain to be fully elucidated. The transient receptor potential (TRP) superfamily of ion channels is believed to play a critical role in sensory physiology, where they act as transducers for thermal, chemical and mechanical stimuli. Recent studies have shown that members of the vanilloid (TRPV) and ankyrin (TRPA) subfamilies encode mechanosensitive TRPs. The vanilloid family member TRPV4 is one such non selective calcium permeable cationic channel which has been shown to be activated by chemical ligands, hypotonicity, and mechanical stimuli. Objectives: The objective of the current study was to investigate functional expression of TRPV4 in cultured human PDL cells. Methods: Human PDL cells were grown in Dulbecco's Modified Eagle Medium with L-glutamine supplemented with 10% fetal bovine serum (FBS), 100UI/ml penicillin and 100μg/ml streptomycin. Cells in passage 4-6 were used in all experiments. TRPV4 functional expression was determined using ratiometric calcium imaging. Cultured cells were loaded with intracellular Ca2+ probe fura-2 and cells were then stimulated with the TRPV4 agonists, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), GSK1016790A or hypotonic solution. The TRPV4 antagonist RN 1734 was used to block the corresponding agonist responses. Results: PDL fibroblasts responded to application of TRPV4 agonists and hypotonic stimuli by an increase in intracellular calcium which was attenuated in the presence of the TRPV4 antagonist. Conclusions: We have shown for the first time the functional expression of the mechanosensitive TRPV4 channel in human PDL cells. The molecular identity and mechanisms of activation of mechanosensitive TRP channels in PDL cells merit further investigation.

Osteoprotegerin expression by periodontal ligament fibroblasts

Objectives: Receptor Activator of NF-kappaB ligand (RANKL), through binding to its receptor (RANK), plays an important role in osteoclast differentiation and activation. Conversely, osteoprotegerin (OPG), a decoy receptor for RANKL, inhibits osteoclastogenesis and subsequent bone turnover. Little is known about the role of resident periodontal ligament fibroblasts in regulating bone turnover. The aim of this study was to determine (i) if periodontal ligament fibroblasts produced OPG in vitro and (ii) the effects of IL-1b and TGF-b1 on OPG expression. Methods: Three human periodontal ligament fibroblast populations, developed by explant culture, were grown to confluence in 6-well plates in DMEM supplemented with 10% FCS. Cells were washed in HBSS and then cultured for an additional 48 hours in serum-free media supplemented with IL-1b or TGF-b1 at 10ng/ml. OPG expression levels in the conditioned medium were determined by ELISA (R&D Systems, UK) and confirmed by Western blot. Results: All three fibroblast strains produced quantifiable levels of OPG. Both IL-1b and, to a lesser extent, TGF-b1 significantly stimulated OPG expression in all fibroblast strains (p<0.05). Pre-incubation of samples with N-glycosidase F prior to Western blots indicated glycosylation of expressed OPG. Conclusions: These data indicate that periodontal ligament fibroblasts can regulate osteoclast activation via the RANK/RANKL signalling pathway. These fibroblasts may play an important role in regulating bone turnover both in periodontal disease and orthodontic tooth movement.

Molecular study of cell degeneration and evolution induced by mRNA mistranslation

As proteínas existentes nas células são produzidas pelo mecanismo de tradução do mRNA, no qual a informação genética contida nos genes é descodificada em cadeias polipeptídicas. O código genético, que define as regras de descodificação do genoma, minimiza os erros de tradução do mRNA, garantindo a síntese de proteínas com elevada fidelidade. Esta é essencial para a estabilidade do proteoma e para a manutenção e funcionamento dos processos celulares. Em condições fisiológicas normais, os erros da tradução do mRNA ocorrem com frequências que variam de 10-3 a 10-5 erros por codão descodificado. Situações que aumentam este erro basal geralmente estão associadas ao envelhecimento, stresse e a doenças; no entanto, em certos organismos o código genético é traduzido naturalmente com elevado erro, indicando que a síntese de proteínas aberrantes pode de algum modo ser vantajosa. A fim de estudar a resposta celular aos erros de tradução do mRNA, construímos leveduras que incorporam serina no proteoma em resposta a um codão de leucina, usando a expressão constitutiva de um tRNASer mutante. Este fenómeno genético artificial provocou uma forte diminuição da esporulação, da viabilidade e da eficiência de mating, afectando imensamente a reprodução sexual da levedura. Observou-se também uma grande heterogeneidade no tamanho e na forma das células e elevada instabilidade genómica, com o aparecimento de populações poliplóides e aneuplóides. No sentido de clarificar as bases celulares e moleculares daqueles fenótipos e compreender melhor a biologia do erro de tradução do mRNA, construímos também células de levedura que inserem serina em resposta a um codão de leucina de modo indutível e controlado. Utilizaram-se perfis de mRNA total e de mRNA associado a polissomas para elucidar a resposta celular ao erro de tradução do mRNA. Observou-se a indução de genes envolvidos na resposta ao stresse geral, stresse oxidativo e na unfolded protein response (UPR). Um aumento significativo de espécies reactivas de oxigénio (ROS) e um forte impacto negativo na capacidade das células pós-mitóticas re-iniciarem o crescimento foram também observados. Este fenótipo de perda de viabilidade celular foi resgatado por scavangers de ROS, indicando que o stresse oxidativo é a principal causa de morte celular causada pelos erros de tradução. Este estudo levanta a hipótese de que o stresse oxidativo e a acumulação de ROS, ao invés do colapso súbito do proteoma, são as principais causas da degeneração celular e das doenças humanas associadas aos erros de tradução do genoma. ABSTRACT: Proteins are synthesized through the mechanism of translation, which uses the genetic code to transform the nucleic acids based information of the genome into the amino acids based information of the proteome. The genetic code evolved in such a manner that translational errors are kept to a minimum and even when they occur their impact is minimized by similar chemical properties of the amino acids. Protein synthesis fidelity is essential for proteome stability and for functional maintenance of cellular processes. Indeed, under normal physiological conditions, mistranslation occurs at frequencies that range from 10-3 to 10-5 errors per codon decoded. Situations where this basal error frequency increases are usually associated to aging and disease. However, there are some organisms where genetic code errors occur naturally at high level, suggesting that mRNA mistranslation can somehow be beneficial. In order to study the cellular response to mRNA mistranslation, we have engineered single codon mistranslation in yeast cells, using constitutive expression of mutant tRNASer genes. These mistranslating strains inserted serines at leucine-CUG sites on a proteome wide scale due to competition between the wild type tRNALeu with the mutant tRNASer. Such mistranslation event decreased yeast sporulation, viability and mating efficiencies sharply and affected sexual reproduction strongly. High heterogeneity in cell size and shape and high instability in the genome were also observed, with the appearance of some polyploid or aneuploid cell populations. To further study the cellular and molecular basis of those phenotypes and the biology of mRNA mistranslation, we have also engineered inducible mRNA misreading in yeast and used total mRNA and polysome associated mRNA profiling to determine whether codon misreading affects gene expression. Induced mistranslation up-regulated genes involved in the general stress response, oxidative stress and in the unfolded protein response (UPR). A significant increase in reactive oxygen species (ROS) and a strong negative impact on the capacity of post-mitotic cells to re-initiate growth in fresh media were also observed. This cell viability phenotype was rescued by scavengers of ROS, indicating that oxidative stress is the main cause of cell death caused by mRNA mistranslation. This study provides strong support for the hypothesis that oxidative stress and ROS accumulation, rather than sudden proteome collapse or major proteome disruption, are the main cause of the cellular degeneration observed in human diseases associated mRNA mistranslation.

The Fenix II Study: A longitudinal study of psychopathology among burn patients

Psychological symptoms are common among burn survivors. However, knowledge about epidemiology and predictors of psychopathology has shown great heterogeneity in this population. The Fenix-II Project was the first epidemiological study on the psychopathological consequences of burn injuries developed in Spain, providing a detailed analysis of the progression of psychological symptoms during the first six months after injury. Three hundred and thirty-three patients were monitored and 183 were included in this study. Posttraumatic, depression and anxiety symptoms showed a general decreasing tendency across time. At 6 months, 34 patients showed clinically significant Posttraumatic Stress Disorder (PTSD) symptoms (20.5% of 166 patients reached at 6 months) as measured with the MINI Neuropsychiatric Interview. Within this group of patients, anxiety, depression and hyperarousal increased at 30 days, and avoidance 90 days after injury. The most accurate predictors of PTSD were found to be being burned in a Motor Vehicle Accident, risk of social exclusion, low body-image adjustment, anterior trunk location of the burn injury and life threat perception during the burn-shock period. Considering these factors, clinicians may identify patients at risk of PTSD development, allowing an adequate follow up and preventive interventions which may minimize the psychological consequences of burn injuries.

Joint inversion of magnetotelluric and seismic data for crustal characterization

Tese de doutoramento, Ciências Geofísicas e da Geoinformação (Geofisíca), Universidade de Lisboa, Faculdade de Ciências, 2014

Joint inversion of geophysical and hydrologic data during water infiltration

Tese de doutoramento, Ciências Geofísicas e da Geoinformação (Geofísica), Universidade de Lisboa, Faculdade de Ciências, 2014

Comparison of Group-based Outpatient Physiotherapy With Usual Care After Total Knee Replacement: a Feasibility Study For a Randomized Controlled Trial

Objective: To evaluate the feasibility of conducting a randomized controlled trial comparing group-based outpatient physiotherapy with usual care in patients following total knee replacement. Design: A feasibility study for a randomized controlled trial. Setting: One secondary-care hospital orthopaedic centre, Bristol, UK. Participants: A total of 46 participants undergoing primary total knee replacement. Interventions: The intervention group were offered six group-based exercise sessions after surgery. The usual care group received standard postoperative care. Participants were not blinded to group allocation. Outcome measures: Feasibility was assessed by recruitment, reasons for non-participation, attendance, and completion rates of study questionnaires that included the Lower Extremity Functional Scale and Knee Injury and Osteoarthritis Outcome Score. Results: Recruitment rate was 37%. Five patients withdrew or were no longer eligible to participate. Intervention attendance was high (73%) and 84% of group participants reported they were ‘very satisfied’ with the exercises. Return of study questionnaires at six months was lower in the usual care (75%) than in the intervention group (100%). Mean (standard deviation) Lower Extremity Functional Scale scores at six months were 45.0 (20.8) in the usual care and 57.8 (15.2) in the intervention groups. Conclusion: Recruitment and retention of participants in this feasibility study was good. Group-based physiotherapy was acceptable to participants. Questionnaire return rates were lower in the usual care group, but might be enhanced by telephone follow-up. The Lower Extremity Functional Scale had high responsiveness and completion rates. Using this outcome measure, 256 participants would be required in a full-scale randomized controlled trial.

Does Cost-Effectiveness Analysis Have a Role in US Managed Care Drug Formularies? An Empirical Study of Utilization, Costs, Outcomes, and Elasticity of Demand of a Value-Based Formulary

Thesis (Ph.D.)--University of Washington, 2015

Energy and ancillary services joint market simulation

In order to develop a flexible simulator, a variety of models for Ancillary Services (AS) negotiation has been implemented in MASCEM – a multi-agent system competitive electricity markets simulator. In some of these models, the energy and the AS are addressed simultaneously while in other models they are addressed separately. This paper presents an energy and ancillary services joint market simulation. This paper proposes a deterministic approach for solving the energy and ancillary services joint market. A case study based on the dispatch of Regulation Down, Regulation Up, Spinning Reserve, and Non-Spinning Reserve services is used to demonstrate that the use of the developed methodology is suitable for solving this kind of optimization problem. The presented case study is based on CAISO real AS market data considers fifteen bids.

Efeito da ligadura funcional na prevenção da recorrência da entorse do tornozelo numa equipa de basquetebol feminino

Introdução: O basquetebol é considerado um desporto de alto risco para a ocorrência de lesões, nomeadamente das entorses do tornozelo. Como a história de lesão anterior é um fator de risco para a ocorrência de entroses têm-se desenvolvido estratégias de prevenção para evitar as recidivas, um dos métodos possíveis é o recurso a ligaduras funcionais em “tape”. Objetivo: Neste trabalho vai-se verificar se as ligaduras funcionais em “tape” previnem a recorrência de entorse do tornozelo, numa equipa feminina de basquetebol sub-16. Métodos: A amostra é composta por 9 atletas de basquetebol sub-16 com história de entorse no tornozelo na época precedente, vão-se utilizar ligaduras funcionais no tornozelo. Resultados: A entorse teve 9 registos na época anterior. Não houve registo de qualquer lesão da tibiotársica nos treinos ou jogos, durante a aplicação das ligaduras. Conclusão: A utilização das ligaduras funcionais no grupo estudado, como método de prevenção foi eficaz, não se registando qualquer entorse no decorrer das atividades desportivas (treinos/jogos).

Influência do tipo de chuteira em variáveis preditoras do risco de entorse lateral do tornozelo

Introdução: No futebol, a entorse lateral do tornozelo (ELT) destaca-se como sendo a lesão mais prevalente. Potenciada pela variedade de chuteiras disponíveis no mercado e pela crescente utilização de relvados sintéticos, a interação entre o terreno e o calçado tem assumido elevada relevância como fator de risco para a ELT. A maior incidência de lesões na 2ª parte do jogo traduz a necessidade do estudo dessa interação durante tarefas que envolvam fadiga. Objetivo: Estudar a influência das chuteiras em variáveis preditoras do risco de ELT em relvado sintético sob duas condições: sem e com fadiga dos músculos eversores do tornozelo Métodos: Foi utilizada uma amostra de atletas saudáveis. Todos os indivíduos realizaram 3 séries de 5 saltos médio-laterais uni-podálicos, cada uma com 1 de 3 modelos de chuteiras (Turf, Hard e Firm ground) em duas condições: sem e com fadiga induzida pelo dinamómetro isocinético. Durante a tarefa, a atividade eletromiográfica do longo e curto peroniais, o valor das forças de reação do solo e o movimento do retro-pé (plano frontal), foram recolhidos e usados para calcular variáveis cinemáticas (eversão/inversão do tornozelo, o deslocamento e velocidade do centro de pressão), cinéticas (taxa de crescimento das forças de reação do solo) e neuromusculares (tempo de ativação muscular dos peroniais). Resultados: À exceção do tempo de ativação do curto peronial com o modelo Hard ground (sem fadiga vs com fadiga (p=0,050), não foram identificadas diferenças estatisticamente significativas nas variáveis preditoras de lesão, entre chuteiras, nem entre as duas condições avaliadas. Conclusão: Para o teste funcional escolhido e executado por atletas saudáveis em sintético de 3ª geração, nenhuma das chuteiras apresenta maior risco de lesão (com e sem fadiga), tendo em conta as variáveis em estudo.

Stiffness da tibiotársica em sujeitos com Acidente Vascular Encefálico - processo de raciocínio clínico

regula a posição do corpo no espaço, sendo um pré-requisito para o movimento. À periferia este processo de Controlo Postural pode ser identificado também através da variação do stiffness. O Acidente Vascular Encefálico apresenta-se como a patologia onde os sujeitos são referenciados como tendo alteração do stiffness, e poderão verificar-se modificações nesta variável no âmbito da reabilitação neuro-motora. Objetivo: Descrever o comportamento do stiffness da tibiotársica, nos dois membros inferiores, em indivíduos pós Acidente Vascular Encefálico, face a uma intervenção em fisioterapia baseada num processo de raciocínio clínico. Métodos: 5 sujeitos participaram no estudo, tendo sido implementado um programa de reabilitação para cada um dos sujeitos, por um período de 3 meses, com 2 momentos de avaliação (M0 e M1). O torque e a amplitude articular da tibiotársica foi monitorizada, através do dinamómetro isocinético, durante o movimento passivo de dorsiflexão, a diferentes velocidades (5º/s, 1º/s e 0,25º/s) A atividade eletromiográfica dos músculos Gastrocnémio Interno e Solear foi também recolhida. O valor de stiffness foi calculado através da relação torque/posição. Resultados: Em todos os sujeitos em estudo verificou-se que de uma forma geral o stiffness do membro contralateral à lesão apresentou uma modificação no sentido da diminuição em todas as amplitudes em M1. Nos sujeitos A e C, verificou-se que o stiffness do membro ipsilateral apresentou uma modificação no sentido da diminuição em M1 (em amplitudes intermédias). Nos sujeitos B, D e E o stiffness não apresentou modificações. O stiffness não variou com a velocidade. Conclusão: O stiffness apontou para uma diminuição, nos sujeitos em estudo no membro contralateral à lesão e no membro ipsilateral à lesão nos sujeitos A e C em amplitudes intermédias.