Yes / Despite evidence inferring muscle and contractile mode-specific effects of high-fat diet (HFD), no study has yet considered the impact of HFD directly on eccentric muscle function. The present work uniquely examined the effect of 20-week HFD on the isometric, concentric and eccentric muscle function of isolated mouse soleus (SOL) and extensor digitorum longus (EDL) muscles. CD-1 female mice were randomly split into a control (n = 16) or HFD (n = 17) group and for 20 weeks consumed standard lab chow or HFD. Following this period, SOL and EDL muscles were isolated and assessments of maximal isometric force and concentric work loop (WL) power were performed. Each muscle was then subjected to either multiple concentric or eccentric WL activations. Post-fatigue recovery, as an indicator of incurred damage, was measured via assessment of concentric WL power. In the EDL, absolute concentric power and concentric power normalised to muscle mass were reduced in the HFD group (P < 0.038). HFD resulted in faster concentric fatigue and reduced eccentric activity-induced muscle damage (P < 0.05). For the SOL, maximal isometric force was increased, and maximal eccentric power normalised to muscle mass and concentric fatigue were reduced in the HFD group (P < 0.05). HFD effects on eccentric muscle function are muscle-specific and have little relationship with changes in isometric or concentric function. HFD has the potential to negatively affect the intrinsic concentric and eccentric power-producing capacity of skeletal muscle, but a lack of a within-muscle uniform response indicates disparate mechanisms of action which require further investigation.

Prevalence information on excessive gingival display in post-orthodontic patients is limited. By studying one aspect, namely the size relationship of the clinical crowns of teeth, in an orthodontic population, we can begin to quantify their need for periodontal plastic surgery. In this two part study, 200 plaster models were used as subjects, followed by a clinical exam of 31 of those subjects. These models represented patients before and directly after orthodontic therapy, and the Part 2 clinical exams were performed at least five years later. The lengths and widths of the six anterior teeth were measured and these values were compared to known ideals. This study revealed a significant increase in the length of the maxillary anterior teeth over the three examinations, yet these values were still approximately 1.5mm shorter than ideal. The mean tooth width-to-length ratio was 87-88% for maxillary central incisors, clearly below the accepted "ideal." As well, 61-71% of maxillary central incisors exceeded allowable tooth width-to-length ratios, and 61% of subjects displayed asymmetry in gingival architecture. Although this study only examined one aspect of excessive gingival display, it is the first study to show that in a predominantly young, post-orthodontic population, the prevalence of non-ideal width-to-length ratios is greater than 65%, and that the presence of asymmetry is greater than 60%. Therefore, close interaction between the periodontist and the orthodontist is necessary to diagnose these conditions in order to provide patients with all options for improving their smile.

The problem of excess gingival display is difficult to diagnose and treat. By studying one aspect of excess gingival display, namely the size relationships of the clinical crowns of teeth, we can begin to quantify reasonable goals of therapy. In this study, two hundred plaster models were used as subjects. These represented two hundred patients before and after orthodontic therapy. The six anterior teeth were measured for length and width and compared to known ideals. Teeth that did not meet ideal standards may require treatment. It was found that the mean tooth length after orthodontic therapy was approximately two millimeters shorter than ideal. The length of maxillary central incisors did not increase over the course of therapy. Eighty-five to ninety percent of maxillary central incisors exceeded allowable tooth width-to-length ratios. Twenty-nine to thirty percent of central incisors exceeded one hundred percent in their width-to-length ratio. Sixty-eight percent of patients displayed asymmetry in gingival architecture.

Introdução: Os pacientes com fissura labiopalatina, com deficiências maxilares muito severas, geralmente são tratados com avanço maxilar por meio da osteotomia tipo Le Fort I. Entretanto, a distração osteogênica com o distrator externo rígido (RED) pode funcionar como uma alternativa terapêutica para a correção da discrepância esquelética. Proposição: O objetivo do presente estudo é avaliar os efeitos do avanço maxilar por meio da distração osteogênica com distrator externo rígido (RED), associada à osteotomia tipo Le Fort I, em pacientes com fissura transforame unilateral ou bilateral, quanto à quantidade de avanço maxilar e à sua estabilidade a médio e longo prazo. Materiais e Métodos: Para a realização deste estudo longitudinal e retrospectivo, foram usadas telerradiografias em norma lateral de 9 pacientes (6 do gênero masculino e 3 do gênero feminino), onde 4 apresentaram fissura transforame unilateral e 5 apresentaram fissura transforame bilateral, submetidos ao avanço maxilar por meio da distração osteogênica com distrator externo rígido (RED). Foram estabelecidos três tempos de avaliação: fase pré-distração (T1), fase pós-distração imediata (T2) e fase pós-distração controle, com o mínimo de 1 ano após a finalização da distração (T3). A demarcação dos pontos cefalométricos e a obtenção das medidas das variáveis cefalométricas foram realizadas através do software Dolphin Imaging®, versão 11.5. Para a análise dos resultados, o teste estatístico ANOVA de medidas repetidas foi utilizado, adotando-se o nível de significância de 5%. Resultados: No início da distração, a idade média foi de 14 anos e 4 meses (idade mínima de 9 anos, e máxima de 21 anos). O período médio de distração foi de 18 dias, com uma média de ativação no distrator de 1,0mm/dia. O avanço médio da maxila medido em LVR-A, em T2, foi de 15,6mm (p<0,001), com recidiva não estatisticamente significante de 21,79% (p=0,102), em T3. O aumento médio de SNA, em T2, foi de 14,8º (p<0,001), com recidiva não estatisticamente significante de 18,90% (p=0,130), em T3. Os valores médios das medidas SN.GoMe, 1.PP e IMPA não apresentaram variação estatisticamente significante (p>0,05) entre T1, T2 e T3. Conclusão: A terapia de distração osteogênica para avanço maxilar com o RED mostrou ser eficiente, com aumentos significantes das medidas cefalométricas lineares e angulares relacionadas ao avanço maxilar, demonstrando efeito predominantemente esquelético, e estabilidade no período pós-distração médio (T3) de 1 ano e 8 meses. / Introduction: Patients with unilateral and bilateral cleft lip and palate, with significant maxillary hypoplasia are commonly treated with maxillary advancement by Le Fort I osteotomy. However, distraction osteogenesis with a rigid external distraction (RED) device can function as an alternative option for treatment of the skeletal discrepancy. Purpose: The aim of this study is to assess the effects of maxillary advancement by distraction osteogenesis using a rigid external distraction (RED) device, associated with the Le Fort I osteotomy in patients with unilateral or bilateral cleft lip and palate, as the amount of maxillary advancement and their stability in the medium and long term. Materials and Methods: To perform this retrospective longitudinal study, lateral cephalograms of 9 patients (6 males and 3 females) were used, where 4 had unilateral cleft lip and palate and 5 had bilateral cleft lip and palate, who underwent maxilla advancement by distraction osteogenesis with RED device. Three stages of evaluation were established: pre-distraction (T1), immediate post-distraction (T2) post-distraction control, with a minimum of 1 year after completion of distraction (T3). The anatomic landmarks and measurements of cephalometric variables were performed by using the Dolphin Imaging® version 11.5 software. To evaluate the results, the ANOVA test for repeated measures was used, adopting a significance level of 5%. Results: At the start of distraction, mean age was 14 years and 4 months (minimum age 9 years old and maximum of 21 years old). The mean distraction period was 18 days, with a mean rate of distractor activation in 1.0 mm / day. The mean maxillary advancement in LVR-A, at T2, was 15.6 mm (p<0.001), with no statistically significant relapse of 21.79% (p=0.102) at T3. The SNA angle increase, at T2, was 14.8º (p<0.001), with no statistically significant relapse of 18.90% (p=0.130), at T3. The mean values of SN.GoMe, IMPA and 1.PP measures showed no statistically significant variation (p>0.05) between T1, T2 and T3. Conclusion: The therapy of distraction osteogenesis for maxillary advancement with RED is efficient, with significant increases in the linear and angular cephalometric measurements related to the maxilla advancement, demonstrating predominantly skeletal effect and stability in mean post-distraction period (T3) of 1 year and 8 months.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced level of frataxin protein. Normal individuals have 5 to 40 GAA repeat sequences, whereas affected individuals have approximately 70 to more than 1000 GAA triplets. Frataxin is a mitochondrial protein involved in iron-sulphur cluster and heme biosynthesis. The reduction in frataxin expression leads to oxidative stress, mitochondrial iron accumulation and consequential cell death with the primary sites of neurons of the dorsal root ganglia and the dentate nucleus of the cerebellum. FRDA, which is the most common inherited ataxia, affecting 1:50,000 Caucasians, is characterised by neurodegeneration, cardiomyopathy, diabetes mellitus and skeletal deformities. To investigate FRDA molecular disease mechanisms and therapy, several human FXN YAC transgenic mouse models have been established: Y47R, containing normal-sized (GAA)9 repeats; YG8R and YG22R, which initially contained expanded GAA repeats of 90-190 units and 190 units, respectively, but which have subsequently been bred to now contain expanded GAA repeats of 120-220 units and 170-260 units, respectively, and YG8sR (YG8R with a small GAA band) that was recently generated from YG8R breeding. To determine the FXN transgene copy number in the enhanced GAA repeat expansion-based FRDA mouse lines, a TaqMan qPCR assay was developed. The results demonstrated that the YG22R and Y47R lines had a single copy of the FXN transgene while the YG8R line had two copies. The YG8s lines showed less than one copy of the target gene, suggesting potential deletion of the FXN gene. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. However, in the YG8s line, at least 25% of the YG8s cells had no signals, while the remaining cells showed one signal corresponding to the transgenic FXN gene. In addition, the analysis of FXN exons in YG8s rescue mice by PCR confirmed the presence of all FXN exons in these lines, suggesting the incidence of somatic mosaicism in these lines. Extended functional analysis was carried out on these mice from 4 to 12 months of age. Coordination ability of YG8R, YG8sR and YG22R ‘FRDA-like’ mice, together with Y47R and C57BL6/J wild-type control mice, was assessed using accelerating rotarod analysis. The results indicated a progressive decrease in the motor coordination of YG8R, YG22R and YG8sR mice compared to Y47R or C57BL6/J controls. Locomotor activity was also assessed using an open field beam-breaker apparatus followed by four additional functional analyses including beam-walk, hang wire, grip strength and foot print tests. The results indicated significant functional deficits in the FRDA mouse models. Glucose and insulin tolerance tests were also conducted in the FRDA mouse models, indicating glucose intolerance and insulin hypersensitivity in the aforementioned lines. To investigate the correlation between the FRDA-like pathological phenotype and frataxin deficiency in the FRDA mouse models, frataxin mRNA and protein levels as well as somatic GAA repeat instability were examined. The results indicated that somatic GAA repeats increased in the cerebellum and brain of YG22R, YG8R and YG8sR mice, together with significantly reduced levels of FXN mRNA and protein in the liver of YG8R and YG22R compared to Y47R. However, YG8sR lines showed a significant decrease in FXN mRNA in all of the examined tissues compared to Y47R human FXN and C57BL6/J mouse Fxn mRNA. Protein expression levels were also considerably reduced in all the tissues of YG8sR mice compared to Y47R. Subsequently, the telomere length of human and mouse FRDA and control fibroblasts was assessed using qPCR and Q-FISH. The results indicated that the FRDA cells had chromosomes with relatively longer telomeric repeats in comparison to the controls. FRDA cells were screened for expression of telomerase activity using the TRAP assay and a quantitative assay for hTERT mRNA expression using TaqMan qRT-PCR. The results indicated that telomerase activity was not present in the FRDA cells. To investigate whether FRDA cells maintained their telomeres by ALT associated PML bodies (APBs), co-localisation of PML bodies with telomeres was assessed in these cells using combined immunofluorescence to PML and Q-FISH for telomere detection. The results demonstrated that the FRDA cells had significantly higher co-localised PML foci with telomeric DNA compared to the normal cells. Moreover, telomere sister chromatid exchange (T-SCE) frequencies were analysed in the human FRDA cell lines using chromosome orientation FISH (CO-FISH). The results indicated a significant increase in T-SCE levels of the FRDA cell lines relative to the controls. Furthermore, growth curve and population doubling analysis of the human FRDA and control fibroblasts was carried out. The results showed that the FRDA fibroblast cell cultures underwent growth arrest with higher cumulative population doubling compared to the controls. Though, further analysis of telomere length at different passage numbers revealed that the FRDA cells lost telomeres faster than the controls. Finally, the telomere dysfunction-induced foci (TIF) assay was performed to detect DNA damage in the human FRDA fibroblast cells using an antibody against DNA damage marker γ-H2AX and a synthetic PNA probe for telomeres. The frequency of γ-H2AX foci was significantly higher in the FRDA cells compared to the controls. Similarly, the FRDA cells had greater frequencies of TIFs in comparison to the controls, suggesting induced telomere dysfunction in the FRDA cells.

Research on prosodic structure, the underlying structure organising the prosodic grouping of spoken utterances, has shown that it consists of hierarchically organised prosodic constituents. The present thesis explores the nature of this constituency, in particular the question of whether prosodic structure is comprised of a given set of qualitatively distinct domains, or of a set of domains of the same type varying only gradiently in "strength", or a possible mixture of both types of relations across prosodic levels. This question is addressed by testing how prosodic constituency (mirrored on boundary strength manipulations) is signalled acoustically via pre- and post-boundary durations, intonation contours, and two sandhi processes, namely vowel hiatus resolution and post-nasal stop voicing in Modern Greek. Results show that the phonetic signalling of boundary strength provides support for a mixture of both differences of type and strength across prosodic levels, with some levels only differing in terms of their strength. Pre-boundary durations and resolution of vowel hiatus are gradiently affected by boundary strength with shorter to longer durations from lower to higher domains, and less instances of vowel deletion higher in the hierarchy. Post-nasal stop voicing is qualitatively affected by boundary strength with almost all voicing instances occurring in the lowest constituent of the structure in the way a qualitative view of prosodic constituency would predict, and in line with research on prosodic phonology. Finally, both the alignment and scaling of intonation contours at the edges of domains is found to distinguish qualitatively the lowest domain from the higher ones. All higher phrasal domains align with respect to the boundary and their peak scaling varies consistently gradiently across speakers. When combining those two findings, support is provided for the existence of differences of strength and type within the same process. Taken together the results from these four phenomena support the postulation of an underlying prosodic structure with a limited number of qualitatively distinct domains, within which at the same time some type of recursivity or structured variability must be allowed for. It is shown that there are structural properties of speech, like the length of the utterance, influencing the organisation of utterances in a principled gradient manner, supporting the existence of differences of strength within domain types. These findings bear significance for theories of prosodic structure that have assumed either the view of solely qualitative differences, or sole boundary strength differences, as well as for future proposals on prosodic constituency. Finally, the use of Modern Greek in this thesis adds to the existing literature on a language that has been extensively used by researchers working in views supporting the existence of qualitative distinctions of type across prosodic domains, and provides the first in depth experimental analysis of post-nasal stop voicing.

Telomeres are nucleoprotein complexes located at the end of chromosomes. They have an essential role in protecting chromosome ends. Telomerase or ALT (alternative lengthening of telomeres) mechanisms maintain telomeres by compensating natural telomeric loss. We have set up a flow-FISH method and using mouse lymphoma cell lines we identified unexpectedly the presence of subpopulations of cells with different telomere lengths. Subpopulations of cells with different telomere lengths were also observed in a human ALT and non-ALT cell line. Differences in telomere length between subpopulations of cells were significant and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). By applying flow-FISH we could successfully measure telomere lengths during replicative senescence in human primary fibroblasts with different genetic defects that confer sensitivity to ionising radiation (IR). The results from this study, based on flow-FISH and Southern hybridisation measurements, revealed an accelerated rate of telomere shortening in radiosensitive fibroblasts. We also observed accelerated telomere shortening in murine BRCA1 deficient cells, another defect conferring radiosensitivity, in comparison with a BRCA1 proficient cell line. We transiently depleted BRCA1 by siRNAs in two human mammary epithelial cell lines but could not find changes in telomere length in comparison with control cells. Cytological evidence of telomere dysfunction was observed in all radiosensitive cell lines. These results suggest that mechanisms that confer sensitivity to IR may be linked with mechanisms that cause telomere dysfunction. Furthermore, we have been able to show that human ALT positive cell lines show dysfunctional telomeres as detected by either the presence of DSBs at their telomeres or cytogenetic analysis and usually cells with dysfunctional telomeres are sensitive to IR. Finally, we assessed hTERT mRNA splicing variants and telomerase activity in brain tumours, which exhibit considerable chromosome instability suggesting that DNA repair mechanisms may be impaired. We demonstrated that high levels of hTERT mRNAs and telomerase activity correlate with proliferation rate. The presence of hTERT splice variants did not strictly correlate with absence of telomerase activity but hTERT spliced transcripts were observed in some telomerase negative brain tumours suggesting that hTERT splicing may contribute to activation of ALT mechanisms.

Introdução: Os pacientes com fissura labiopalatina, com deficiências maxilares muito severas, geralmente são tratados com avanço maxilar por meio da osteotomia tipo Le Fort I. Entretanto, a distração osteogênica com o distrator externo rígido (RED) pode funcionar como uma alternativa terapêutica para a correção da discrepância esquelética. Proposição: O objetivo do presente estudo é avaliar os efeitos do avanço maxilar por meio da distração osteogênica com distrator externo rígido (RED), associada à osteotomia tipo Le Fort I, em pacientes com fissura transforame unilateral ou bilateral, quanto à quantidade de avanço maxilar e à sua estabilidade a médio e longo prazo. Materiais e Métodos: Para a realização deste estudo longitudinal e retrospectivo, foram usadas telerradiografias em norma lateral de 9 pacientes (6 do gênero masculino e 3 do gênero feminino), onde 4 apresentaram fissura transforame unilateral e 5 apresentaram fissura transforame bilateral, submetidos ao avanço maxilar por meio da distração osteogênica com distrator externo rígido (RED). Foram estabelecidos três tempos de avaliação: fase pré-distração (T1), fase pós-distração imediata (T2) e fase pós-distração controle, com o mínimo de 1 ano após a finalização da distração (T3). A demarcação dos pontos cefalométricos e a obtenção das medidas das variáveis cefalométricas foram realizadas através do software Dolphin Imaging®, versão 11.5. Para a análise dos resultados, o teste estatístico ANOVA de medidas repetidas foi utilizado, adotando-se o nível de significância de 5%. Resultados: No início da distração, a idade média foi de 14 anos e 4 meses (idade mínima de 9 anos, e máxima de 21 anos). O período médio de distração foi de 18 dias, com uma média de ativação no distrator de 1,0mm/dia. O avanço médio da maxila medido em LVR-A, em T2, foi de 15,6mm (p<0,001), com recidiva não estatisticamente significante de 21,79% (p=0,102), em T3. O aumento médio de SNA, em T2, foi de 14,8º (p<0,001), com recidiva não estatisticamente significante de 18,90% (p=0,130), em T3. Os valores médios das medidas SN.GoMe, 1.PP e IMPA não apresentaram variação estatisticamente significante (p>0,05) entre T1, T2 e T3. Conclusão: A terapia de distração osteogênica para avanço maxilar com o RED mostrou ser eficiente, com aumentos significantes das medidas cefalométricas lineares e angulares relacionadas ao avanço maxilar, demonstrando efeito predominantemente esquelético, e estabilidade no período pós-distração médio (T3) de 1 ano e 8 meses. / Introduction: Patients with unilateral and bilateral cleft lip and palate, with significant maxillary hypoplasia are commonly treated with maxillary advancement by Le Fort I osteotomy. However, distraction osteogenesis with a rigid external distraction (RED) device can function as an alternative option for treatment of the skeletal discrepancy. Purpose: The aim of this study is to assess the effects of maxillary advancement by distraction osteogenesis using a rigid external distraction (RED) device, associated with the Le Fort I osteotomy in patients with unilateral or bilateral cleft lip and palate, as the amount of maxillary advancement and their stability in the medium and long term. Materials and Methods: To perform this retrospective longitudinal study, lateral cephalograms of 9 patients (6 males and 3 females) were used, where 4 had unilateral cleft lip and palate and 5 had bilateral cleft lip and palate, who underwent maxilla advancement by distraction osteogenesis with RED device. Three stages of evaluation were established: pre-distraction (T1), immediate post-distraction (T2) post-distraction control, with a minimum of 1 year after completion of distraction (T3). The anatomic landmarks and measurements of cephalometric variables were performed by using the Dolphin Imaging® version 11.5 software. To evaluate the results, the ANOVA test for repeated measures was used, adopting a significance level of 5%. Results: At the start of distraction, mean age was 14 years and 4 months (minimum age 9 years old and maximum of 21 years old). The mean distraction period was 18 days, with a mean rate of distractor activation in 1.0 mm / day. The mean maxillary advancement in LVR-A, at T2, was 15.6 mm (p<0.001), with no statistically significant relapse of 21.79% (p=0.102) at T3. The SNA angle increase, at T2, was 14.8º (p<0.001), with no statistically significant relapse of 18.90% (p=0.130), at T3. The mean values of SN.GoMe, IMPA and 1.PP measures showed no statistically significant variation (p>0.05) between T1, T2 and T3. Conclusion: The therapy of distraction osteogenesis for maxillary advancement with RED is efficient, with significant increases in the linear and angular cephalometric measurements related to the maxilla advancement, demonstrating predominantly skeletal effect and stability in mean post-distraction period (T3) of 1 year and 8 months.