Modificações neurosensoriais, decorrentes de complicações relacionadas a tratamentos odontológicos, estão entre as principais causas de litígio envolvendo cirurgiões-dentistas. Um dos nervos mais acometidos nestes casos é o nervo lingual. Entre os inconvenientes apontados nas queixas judiciais estão as dificuldades na articulação dos sons da fala nessas situações. Neste contexto, o objetivo desse estudo foi verificar se há dano à função fonética resultante da ausência de sensibilidade unilateral da língua induzida por anestesia do nervo lingual. Participaram do estudo 10 sujeitos voluntários, brasileiros, do sexo masculino, com idade entre 21 e 27 anos. Foram realizadas as análises perceptual e acústica das falas gravadas antes e depois da anestesia do nervo lingual. Além disso, os sujeitos da pesquisa responderam a um questionário com o objetivo de avaliar o nível de desconforto e dificuldade na produção da fala. Após assinatura do TCLE e avaliação clínica e fonoaudiológica, os sujeitos foram divididos em dois grupos. As gravações consistiram em conjuntos de vogais e palavras. Para o segundo grupo, a leitura de um texto foi acrescentada para o estudo da fala encadeada. Para a análise perceptual foram observados os comportamentos das falas em relação à inteligibilidade, ao pitch, loudness e co-articulação. Para o segundo grupo, a fala encadeada foi analisada quanto ao ritmo, fluidez, co-articulação e velocidade de produção. Não foram observadas quaisquer diferenças nos parâmetros perceptivo auditivos definidos para o estudo. Para a análise acústica, realizou-se o estudo de cinco vogais isoladas e da fricativa sibilante /s/. Foram extraídos e comparados os valores dos dois primeiros formantes das vogais /a, / , /i/, / e /u/. Foi possível observar que, apesar de existirem diferenças nos valores absolutos dos formantes, eles se mantiveram em faixas de freqüências características das vogais faladas nos dois momentos e, além disso, nenhum indivíduo relatou qualquer dificuldade em relação à produção das vogais. Os parâmetros escolhidos para análise comparativa da consoante sibilante /s/ foram: a localização do pico espectral de maior amplitude, o coeficiente de assimetria, o coeficiente de curtose e o centro de gravidade. As análises demonstraram que os padrões desses parâmetros também foram mantidos após a aplicação da anestesia. Ademais, não foram encontradas diferenças estatisticamente significantes entre os dois momentos, para os parâmetros analisados. Apesar de 03 sujeitos (30%) relatarem dificuldades na produção de determinados fonemas, as análises das gravações de suas falas demonstraram não haver nenhuma diferença significativa entre os dois momentos. O presente trabalho ofereceu informações importantes a respeito de técnicas que podem ser utilizadas nas perícias envolvendo o dano fonético. Ficando claro que é preciso uma interpretação criteriosa dos resultados apresentados, especialmente nas análises acústicas. Os resultados encontrados permitem concluir que não houve dano fonético resultante da inibição funcional unilateral do nervo lingual para o grupo pesquisado. / Sensorineural changes due to complications related to dentistry treatments are between the main litigation causes involving dentists. One of the most affected nerves in these situations is the lingual nerve. Speech changes are related as a complication in these cases. The purpose of this study was to analyze the possible phonetic damage resulting from lingual nerve sensorial alterations induced by anesthesia. The study group consisted of 10 men, aged between 21 and 27 years. Perceptual and acoustic analyses of the speech samples recorded before and after the lingual nerve anesthesia were performed. Moreover, the subjects answered a questionnaire to discuss the level of discomfort and difficulty in speaking. The informers were divided into two groups. The recording consisted of a combination of vowels and words. A text reading was added for the second group. Perceptual analyses studied the behavior of the intelligibility, pitch, loudness and co-articulation. For the second group, the pace, fluidity, co-articulation and production speed were evaluated. There were no differences in perceptive parameters defined for the study. The acoustic analyses of isolated vowels /a/, / , /i/, / , /u/ were performed. The first two formant frequencies values were analyzed. It was possible to observe that, despite of the differences in absolute values of formants, they were located in bands of frequencies that are characteristics of the vowels spoken, in both moments. In addition, none of the subjects reported any difficulty in produce vowel sounds. The parameters chosen for acoustic analysis of /s/ sound were: location of the greater spectral peak, skewness, kurtosis and center of gravity. The analyses showed that the patterns of these parameters were maintained after the application of the anesthesia. Moreover, when the two moments were compared, no statistically significant differences for the parameters analyzed were found. Three subjects (30%) reported difficulties in the production of certain phonemes, but the analysis of their records did not show any significant difference between the two moments. This study provides important information about techniques that can be used in phonetic damage investigation. A careful interpretation of the results presented by acoustic analyses in these cases is necessary. It can be concluded that there was no phonetic damage resulting from the unilateral inhibition of the lingual nerve function for the enrolled group.

O sistema nervoso central (SNC) é uma das primeiras regiões a ser acometida durante a sepse e choque séptico, o que contribui para o aumento da taxa de morbidade e mortalidade. Pacientes em choque séptico apresentam disfunção neuronal aguda, tais como o delírio, desorientação e coma. Em longo prazo, o dano cognitivo pode ocorrer ocasionando o comprometimento do aprendizado e formação de memória. Estudos demonstram que durante a resposta inflamatória sistêmica exacerbada, mediadores inflamatórios presentes na circulação sistêmica, são capazes de chegar ao SNC e ocasionar a ativação de células gliais, conduzindo a um estado de neuroinflamação. Nesse processo, algumas estruturas do SNC, tais como o hipocampo são mais vulneráveis à ação de espécies reativas de oxigênio (ERO), e a mediadores inflamatórios produzidos de forma excessiva durante a sepse. Neste contexto, a investigação de novas estratégias terapêuticas que sejam capazes de atenuar a resposta inflamatória exacerbada se faz necessário. Assim, o presente projeto teve como objetivo investigar prováveis propriedades antioxidante e anti-inflamatória do Hidrogênio molecular (H2), bem como sua possível ação neuroprotetora em ratos submetidos à sepse polimicrobiana, induzida por ligadura e perfuração cecal (CLP). Para isso o projeto foi dividido em dois protocolos experimentais. No primeiro protocolo ratos Wistar submetidos à cirurgia de CLP ou Sham, foram submetidos ao tratamento com inalação do H2 a 2%, por um período de 1h durante 10 dias consecutivos, e logo após foram submetidos a testes comportamentais para avaliação da memória de habituação, discriminativa e aversiva. No segundo protocolo os animais foram tratados com inalação do H2 por um período de 3h, e 24h após ao término da cirurgia de CLP/Sham foram decapitados para coleta do sangue e cérebro. A partir dos resultados dos testes comportamentais observamos que o tratamento com inalação do H2 durante a sepse experimental preveniu a perda de memória e o dano cognitivo, bem como foi capaz de diminuir os níveis de citocinas pró-inflamatórias de fase aguda tais como IL-1?, IL-6 e TNF? no córtex pré-frontal e hipocampo. A estratégia também foi capaz de diminuir os níveis de TBARS no plasma. Observamos um aumento da concentração da enzima catalase nos animais tratados com H2. Em conjunto os resultados indicam que o H2 foi capaz de inibir a resposta inflamatória e prevenir o dano cognitivo, agindo como uma substância neuroprotetora em ratos submetidos ao choque séptico experimental / The central nervous system is one of the first regions to be affected during Sepsis and septic shock, which contributes to the increased rate of morbidity and mortality. Patients with severe sepsis may present acute neuronal dysfunction such as delirium, disorientation, and unconscious. In the long term, cognitive damage can occur causing the commitment of learning and memory formation. Studies show that during the exacerbated systemic inflammatory response, inflammatory mediators present in the systemic circulation, are able to reach the CNS and cause the activation of glial cells, leading to a state of neuroinflammation. In this process, some CNS structures such as the hippocampus are more vulnerable to the action of reactive oxygen species (ROS), and to inflammatory mediators produced excessively during sepsis. In this context, the investigation of new therapeutic strategies that are capable of attenuating the exacerbated inflammatory response is necessary. Thus, the present project aimed to investigate the probable antioxidant and anti-inflammatory properties of molecular hydrogen (H2), as well as its possible neuroprotective action in rats submitted to polymicrobial sepsis induced by ligature and cecal puncture (CLP). In order to check this hypothesis, the project was divided into two experimental protocols. In the first protocol Wistar rats submitted to CLP or Sham surgery were submitted to 2% H2 inhalation treatment for a period of 1h for 10 consecutive days, and soon after they underwent behavioral tests to evaluate habituation memory, discriminative and aversive. In the second protocol the animals were treated with H2 inhalation for a period of 3h and 24h, and at the end of the treatment, they were decapitated for blood and brain collection. Plasma was already used for nitrate dosage, lipid peroxidation, antioxidant enzymes and inflammatory cytokines. From the results of the behavioral tests, we observed that treatment with H2 inhalation during the experimental sepsis prevented memory loss and cognitive damage, and was able to decrease the levels of acute-phase inflammatory cytokines such as IL-1?, IL -6 and TNF? in the prefrontal cortex and hippocampus. The therapeutic strategy was also able to decrease plasma TBARS levels. We also observed an increase in the concentration of the enzyme catalase in H2-treated animals. Together the results indicate that molecular hydrogen was able to inhibit the inflammatory response and prevent cognitive damage, acting as a neuroprotective substance, in rats submitted to experimental septic shock

Reliable biomarkers of axonal damage are urgently needed in neurological diseases. Neurofilaments (Nf) are specific structural elements of neurons composed of at least three subunits: Nf light chain (NfL), Nf medium and Nf heavy chain (NfH). This PhD aimed to characterise NfL levels and their correlation with clinical features in patients with neurological diseases with a different rate of progression and following and under different treatment regimes. An important aim was also to develop a bioassay for NfL measurements in blood. Cerebrospinal fluid (CSF) NfL levels discriminated patients with a clinically isolated syndrome (CIS) (p=0.001) or multiple sclerosis (MS) (p=0.035) from healthy controls more efficiently, and was more sensitive to change after natalizumab therapy (p<0.0001) than CSF NfH (p=0.002). Further, CSF NfL levels decreased in fingolimodtreated MS patients (p=0.001), but not in those receiving placebo (p=0.433). Based on these findings, a sensitive method for the detection of NfL in serum was developed and validated. Patients with neurological diseases had higher serum NfL values than controls. In acute spinal cord injury (SCI), serum NfL levels correlated with injury severity and long-term motor outcome, and Minocycline treatment was associated with decreased NfL levels in complete SCI patients compared to placebo. Finally, I found that serum NfL levels were higher in CIS patients than in healthy controls but did not predict conversion to clinically definite MS (CDMS). Independent predictors of CDMS were instead oligoclonal bands, number of T2 lesions and age at CIS. Lower 25-OHvitamin D levels were associated with CDMS in univariate analysis, but this was attenuated in the multivariate model. In conclusion, NfL proved to be an analytically stable protein which is an important prerequisite for biomarkers. The role of NfL quantification as a surrogate measure of neuroaxonal damage is corroborated by my findings and further supports the usefulness of NfL as a putative biomarker of axonal damage in various neurological diseases.

Telomere dysfunction is one mechanism of cellular and tissue ageing. Dysfunctional telomeres in fibroblasts are recognised as DNA double-strand breaks (DSBs) and trigger the DNA damage pathway of senescence. However, telomere uncapping in normal human epidermal keratinocytes, via expression of the dominant negative mutant of the telomere repeat-binding factor 2 (TRF2!B!M), resulted in a senescent-like arrest without a significant DNA damage response (DDR). This suggests that either keratinocytes are unusually sensitive to telomere uncapping and the low DDR is sufficient to induce senescence or that dysfunctional telomeres may also be signalled through an alternative pathway. Subsequent analysis revealed genes HIST2H2BE, ICEBERG, S100A7 and HOPX as potential markers for telomere dysfunction-induced senescence (TDIS) since they were induced by telomere uncapping and seemed to be regulated by telomerase. The aim of this project was to assess the specificity of these candidate markers for TDIS and to select the most promising for use as a biomarker. To this end, keratinocytes were exposed to doses of ionising radiation, capable of generating transient or permanent damage to the DNA, or transduced with retroviral constructs expressing p14ARF, p16INK4a, p53 or TRF2!B!M and the gene expression levels of the candidates assessed after a recovery period or at the early stages of senescence. Whilst S100A7, HOPX or ICEBERG were not induced by a transient or persistent DDR or by p16INK4a, ICEBERG and HOPX were induced by p53 and p14ARF when these were ectopically expressed at higher levels. Thus, S100A7 seems to be the most specific early marker for telomere dysfunction in keratinocytes since it was selectively induced by telomere uncapping via expression of TRF2!B!M and not by DSBs or by over expression of p14ARF, p53 or p16INK4a. S100A7 may have the potential to identify cells with telomere dysfunction in human epithelia and body fluids.

Trauma is a leading cause of death worldwide with 5.8 million deaths occurring yearly. Almost 40% of trauma deaths are due to bleeding and occur in the first few hours after injury. Of the remaining severely injured patients up to 25% develop a dysregulated immune response leading to multiple organ failure (MOF). Despite improvements in trauma care, the morbidity and mortality of this condition remains very high. Massive traumatic injury can overwhelm endogenous homeostatic mechanisms even with prompt treatment. The underlying mechanisms driving MOF are also not fully elucidated. As a result, successful therapies for trauma-related MOF are lacking. Trauma causes tissue damage that releases a large number of endogenous damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs released in trauma, such as mitochondrial DNA (mtDNA), could help to explain part of the immune response in trauma given the structural similarities between mitochondria and bacteria. MtDNA, like bacterial DNA, contains an abundance of highly stimulatory unmethylated CpG DNA motifs that signal through Toll-like receptor (TLR)-9 to produce inflammation. MtDNA has been shown to be highly damaging when injected into healthy animals causing acute organ injury to develop. Elevated circulating levels of mtDNA have been reported in trauma patients but an association with clinically meaningful outcomes has not been established in a large cohort. The first aim of this PhD thesis was to determine whether mtDNA released after trauma haemorrhage is sufficient for the development of MOF. Secondly, I then aimed to determine the extent of mtDNA release with varying degrees of tissue injury and haemorrhagic shock in a clinically relevant rodent model. My final aim was to determine whether neutralising mtDNA at a clinically relevant time point in vivo would reduce the severity of organ injury in this model.

This thesis investigates the development of ultrasonic Structural Health Monitoring (SHM) systems, based on guided waves propagation, for the localization of low-velocity impacts and the detection of damage mechanisms in isotropic and anisotropic structures. For the identi- cation of the impact point, two main passive techniques were developed, an algorithm-based and an imaging-based method. The former approach is based on the dierences of the stress waves measured by a network of piezoelectric transducers surface bonded on plate-like structures. In particular, four piezoelectric sensors were used to measure the antisymmetrical A0 Lamb mode in isotropic materials, whilst six acoustic emission sensors were employed to record the wave packets in composite laminates. A joint time-frequency analysis based on the magnitude of the Continuous Wavelet Transform was used to determine the time of arrivals of the wave packets. Then, a combination of unconstrained optimization technique associated to a local Newton's iterative method was employed to solve a system of non linear equations, in order to assess the impact location coordinates and the wave group speeds. The main advantages of the proposed algorithms are that they do not require an a-priori estimation of the group velocity and the mechanical properties of the isotropic and anisotropic structures. Moreover, these algorithms proved to be very robust since they were able to converge from almost any guess point and required little computational time. In addition, this research provided a comparison between the theoretical and experimental results, showing that the impact source location and the wave velocity were predicted with reasonable accuracy. The passive imaging-based method was developed to detect in realtime the impact source in reverberant complex composite structures using only one passive sensor. This technique is based on the re- ciprocal time reversal approach, applied to a number of waveforms stored in a database containing the impulse responses of the structure. The proposed method allows achieving the optimal focalization of the acoustic emission source (impact event) as it overcomes the limitations of other ultrasonic impact localization techniques. Compared to a simple time reversal process, the robustness of this approach is experimentally demonstrated on a stiened composite plate. This thesis also extended active ultrasonic guided wave methods to the specic case of dissipative structures showing non-classical nonlinear behaviour. Indeed, an imaging method of the nonlinear signature due to impact damage in a reverberant complex anisotropic medium was developed. A novel technique called phase symmetry analysis, together with frequency modulated excitation signals, was used to characterize the third order nonlinearity of the structure by exploiting its invariant properties with the phase angle of the input waveforms. Then, a \virtual" reciprocal time reversal imaging process was employed to focus the elastic waves on the defect, by taking advantage of multiple linear scattering. Finally, the main characteristics of this technique were experimentally validated.

This thesis presents a development of Structural Health Monitoring (SHM) and Fault Diagnosis based on Artificial Immune System (AIS), a biology-inspired method motivated from the Biological Immune System (BIS). Using the antigen to model structural health or damage condition of specific characteristics and the antibody to represent an information system or a database that can identify the specific damage pattern, the AIS can detect structural damage and then take action to ensure the structural integrity. In this study the antibodies for SHM were first trained and then tested. The feature space in training includes the natural frequencies and the modal shapes extracted from the simulated structural response data including both free-vibration and seismic response data. The concepts were illustrated for a 2-DOF linear mass-spring-damper system and promising results were obtained. It has shown that the methodology can be effectively used to detect, locate, and assess damage if it occurred. Consistently good results were obtained for both feature spaces of the natural frequencies and the modal shapes extracted from both response data sets. As the only exception, some significant errors were observed in the result for the seismic response data when the second modal shape was used as the feature space. The study has shown great promises of the methodology for structural health monitoring, especially in the case when the measurement data are not sufficient. The work lays a solid foundation for future investigations on the AIS application for large-scale complex structures.

DNA repair is not a single mechanism found within cells. There exists numerous different DNA repair mechanisms that function within every type of cell. The majority of these mechanisms risk accumulating mutations. However, there are a few repair mechanisms that are known to be error-free and one of these is direct reversal repair. This study focused on two proteins highly involved in direct reversal DNA repair--ALKBH2 and ALKBH3. Previous studies have shown that in mice, these two proteins play a significant role in preventing and repairing DNA damage due to methylation as well as decreasing the frequency of mutagenic alkyl adducts. The goal of this study was to characterize the roles of the direct reversal repair proteins in human cells. We expected to see a similar phenotype to that of the Alkbh2 and Alkbh3-deficient mice. Telomerase immortalized human skin fibroblasts were targeted for the ALKBH2 and ALKBH3 alleles using a RNA-guided CRISPR-Cas9 construct that was designed to induce double stranded DNA breaks within the exons and disrupt the open reading frame, eliminating protein activity. Isolated clones were analyzed using fragment analysis and DNA sequencing to characterize any alterations in the open reading frame of the genes. Through sequencing analysis, results showed that one clone was successfully targeted for one of the ALKBH3 alleles with a single nucleotide insertion in its sequence, causing a disruption of the open reading frame. Though the ultimate goal of the experiment was not attained, we concluded that HTERTG fibroblasts can be expanded to serve as a model in which to construct targeted human cell lines that have near normal karyotypes.

Enamel damage and demineralisation are common complications associated with fixed orthodontic appliances. In particular, the clean-up of adhesive remnants after debonding is a recognised cause of enamel damage. Furthermore, fixed attachments offer retentive areas for accumulation of cariogenic bacteria leading to enamel demineralisation and formation of white spot lesions (WSLs). Bioactive glasses may be used to remove adhesives, preserving the integrity of the enamel surface, while also having the potential to induce enamel remineralisation, although their efficacy in both respects has received little attention. A systematic review evaluating the remineralisation potential of bioactive glasses was first undertaken. No prospective clinical studies were identified; however, a range of in vitro studies with heterogeneous designs were identified, largely providing encouraging results. A series of glasses was prepared with molar compositions similar to 45S5 (SylcTM; proprietary bioactive glass) but with constant fluoride, reduced silica and increased sodium and phosphate contents. These glasses were characterised in several tests and the most promising selected. This was designed with hardness lower than that of enamel and higher than orthodontic adhesives. Its effectiveness in terms of removal of composite- and glass ionomer- based orthodontic adhesives was evaluated against SylcTM and a tungsten carbide (TC) bur. This novel glass was subsequently used for remineralisation of artificially-induced orthodontic WSLs on extracted human teeth. The novel glass propelled via the air-abrasion system selectively removed adhesives without inducing tangible physical enamel damage compared to SylcTM and the conventional TC bur. It also remineralised WSLs with surface roughness and intensity of light backscattering similar to sound enamel. In addition, mineral deposits were detected on remineralised enamel surfaces; these acted as a protective layer on the enamel surface and improved its hardness. This layer was rich in calcium, phosphate, and fluoride; 19F MAS-NMR, confirmed the formation of fluorapatite. This is particularly beneficial since fluorapatite is more chemically stable than hydroxyapatite and has more resistance to acid attack. Hence, a promising bioactive glass has been developed.

Defects within the DNA damage response (DDR) pathways are common in human malignancies. This is especially true in high-grade serous ovarian cancer (HGSOC) where defects within the Homologous Recombination (HR) pathway may be present in up to 50% of tumours. Oncolytic adenovirus is a potential novel therapy for human malignancies. These viruses infect malignant cells and multiply selectively within them causing cell death and release of mature virions. Here, I have investigated the role of the DDR in determining the efficacy of the E1A-CR2 deleted adenovirus type 5 (Ad5) vector, dl922-947, in ovarian cancer. I show that infection with dl922-947 stimulates a robust DDR within the host cell, which the virus manipulates in order to ensure optimal viral replication. In a panel of HGSOC cell lines, the extent of overreplication of genomic DNA and the degree of genomic damage following infection with dl922-947 was shown to correlate closely with viral efficacy. Functional HR, however, promoted viral DNA replication and augmented overall anti-cancer efficacy. Mechanistically, both BRCA2 and RAD51 localised to viral replication centres within the infected cell nucleus. RAD51 co-localisation was also demonstrated in cells with defective HR and occurred independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Using functional assays of HR competence, I show that Ad5 infection does not alter cellular ability to repair DNA double-strand break damage via HR. These data suggest that oncolytic adenoviral therapy may be most clinically relevant in tumours with intact HR function. Using a high-throughput siRNA DNA repair screen, potential novel targets have been identified that can increase the efficacy of dl922-947 (for example: NONO) and also result in increased resistance (RPA). These results highlight the complex interplay between adenovirus and host cell. Further understanding of these pathways is vital to increase efficacy, develop biomarkers and improve patient selection into clinical trials for these therapies.