O presente estudo teve por objetivo a avaliação das consequências da expansão rápida da maxila em indivíduos com fissura labiopalatina unilateral nos seguintes desfechos: rizogênese, reabsorção radicular, espessura da tábua óssea e deiscência. Este estudo de coorte prospectivo foi composto por 30 participantes, sendo 20 homens e 10 mulheres, entre 8 e 15 anos. Os participantes foram alocados em 3 grupos, conforme o tipo de atresia maxilar, e tratados com diferentes tipos de aparelho expansor: G1, Hyrax; G2, Expansor em Leque; G3, Mini-Hyrax Invertido. Foram realizadas Tomografias Computadorizadas de Feixes Cônicos imediatamente antes do tratamento e 90 dias após contenção do aparelho. Medições lineares foram obtidas pelo mesmo examinador cegado. A estatística inferencial dos dados, após testes de normalidade e homogeneidade, foi realizada pela análise de regressão multinível. As raízes que apresentavam o ápice aberto ao início do tratamento demonstraram um aumento estatisticamente significante após o tratamento. Em contrapartida, não houve alterações significativas no comprimento radicular das raízes que apresentavam o ápice fechado no início do tratamento. Uma diminuição significativa da espessura da tábua óssea vestibular, bem como um aumento significativo da deiscência pode ser observado. Não houve diferença significante entre o lado com e sem a fissura para todas as variáveis apresentadas, assim como não houve diferença entre os grupos. Os achados neste estudo permitem concluir que as forças ortopédicas da expansão rápida da maxila não são capazes de interromper o processo de desenvolvimento radicular, tampouco causar reabsorção radicular apical externa significativa em indivíduos com fissura labiopalatina unilateral. Contudo, uma diminuição significativa de volume ósseo vestibular posterior é esperada, mas não deve ser considerada importante clinicamente. / The present study aimed to evaluate the consequences of rapid maxillary expansion in subjects with unilateral cleft lip and palate in the following outcomes: root formation, root resorption, buccal bone thickness and dehiscence. This prospective cohort study comprised 30 participants, 20 male and 10 female, between 8 and 15 years old. Participants were allocated in 3 groups, according to the type of maxillary constriction, and were treated with different types of expanders: G1, hyrax; G2, fan-type; G3, inverted mini-hyrax. Cone Beam Computed Tomography scans were performed immediately before treatment and after 90 days of retention. Linear measurements were obtained by the same blinded examiner. The inferential statistics of the data, after normality and equality tests, was performed with a multilevel regression analysis. The roots presenting open apexes at the beginning of treatment showed a statistically significant increase in length after treatment. On the other hand, there were no significant changes in the root length of roots that had a closed apexes at the beginning of the treatment. A significant decrease in buccal bone thickness as well as a significant increase in dehiscence were observed. There was no significant difference between the cleft and non-cleft side for all variables, as there was no significant difference between groups. The findings in this study allow to conclude that the orthopedic forces of rapid maxillary expansion are not able to interrupt the root development process nor to cause significant external apical root resorption in subjects with unilateral cleft lip and palate. However, a significant decrease in posterior buccal bone volume is expected, but it should not be considered important in a clinical perspective.

<p>The Antiepilptic drugs (AEDs) phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), tri- and dimethadione (TMD and DMD) are known teratogens having a common malformation pattern in human and animal studies. This thesis was designed chiefly to test a hypothesis correlating the teratogenicity of these AEDs to episodes of pharmacologically induced embryonic arrhythmia and hypoxia-reoxygenation damage.</p><p>Effects on the embryonic heart were studied both after maternal administration in mice and in</p><p>mouse embryos cultured in vitro. Only AEDs, correlated with the same type of malformation as could be induced by episodes of interrupted oxygen supply to the embryo (e.g. cleft palate) caused concentration dependent bradycardia and arrhythmia. PHT and DMD had the highest potential and affected embryonic heart at clinically relevant concentration, followed by CBZ, TMD and PB. Valproate and vigabatrin not associated with hypoxia-related malformations caused neither arrhythmia nor severe bradycardia.</p><p>The results showed that the embryonic heart is extremely susceptible to PHT and DMD only</p><p>during a restricted period of development, between gestational days 9-13 (weeks 5-9 of human pregnancy).An observed genetic susceptibility to react with arrhythmia at low concentrations when exposed to PHT or to external stress, could explain why A/J strain of mice is more susceptible to develop cleft palate compared to other strains. High activities of reactive oxygen species (ROS) capturing antioxidant enzymes observed in untreated A/J embryos supported this assumption. The potential to cause embryonic arrythmia by an AED was related to the potential to inhibit the rapid component of the delayed rectifier potassium channel (I _kr ).A marked I _kr blocking activity (70%)of DMD in voltage clamping studies was observed. The I _kr inhibition occurred at similar concentrations, which causes severe arrhythmia.</p><p>The idea of a relation between teratogenicity and arrhythmia, resulting in ischemia followed by reperfusion and generation of ROS was supported by mechanistic studies. Pre-treatment with the spin-trapping agent PBN, which has the capacity to capture ROS, markedly reduced the incidence of PHT and DMD-induced cleft palate. In utero exposure to teratogenic doses of DMD and PHT resulted in hemorrhages in the embryonic palatal region. The same type of haemorrhage in the palatal region precedes orofacial clefts induced by episodic hypoxia.</p>

The Antiepilptic drugs (AEDs) phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), tri- and dimethadione (TMD and DMD) are known teratogens having a common malformation pattern in human and animal studies. This thesis was designed chiefly to test a hypothesis correlating the teratogenicity of these AEDs to episodes of pharmacologically induced embryonic arrhythmia and hypoxia-reoxygenation damage. Effects on the embryonic heart were studied both after maternal administration in mice and in mouse embryos cultured in vitro. Only AEDs, correlated with the same type of malformation as could be induced by episodes of interrupted oxygen supply to the embryo (e.g. cleft palate) caused concentration dependent bradycardia and arrhythmia. PHT and DMD had the highest potential and affected embryonic heart at clinically relevant concentration, followed by CBZ, TMD and PB. Valproate and vigabatrin not associated with hypoxia-related malformations caused neither arrhythmia nor severe bradycardia. The results showed that the embryonic heart is extremely susceptible to PHT and DMD only during a restricted period of development, between gestational days 9-13 (weeks 5-9 of human pregnancy).An observed genetic susceptibility to react with arrhythmia at low concentrations when exposed to PHT or to external stress, could explain why A/J strain of mice is more susceptible to develop cleft palate compared to other strains. High activities of reactive oxygen species (ROS) capturing antioxidant enzymes observed in untreated A/J embryos supported this assumption. The potential to cause embryonic arrythmia by an AED was related to the potential to inhibit the rapid component of the delayed rectifier potassium channel (I kr ).A marked I kr blocking activity (70%)of DMD in voltage clamping studies was observed. The I kr inhibition occurred at similar concentrations, which causes severe arrhythmia. The idea of a relation between teratogenicity and arrhythmia, resulting in ischemia followed by reperfusion and generation of ROS was supported by mechanistic studies. Pre-treatment with the spin-trapping agent PBN, which has the capacity to capture ROS, markedly reduced the incidence of PHT and DMD-induced cleft palate. In utero exposure to teratogenic doses of DMD and PHT resulted in hemorrhages in the embryonic palatal region. The same type of haemorrhage in the palatal region precedes orofacial clefts induced by episodic hypoxia.

Development of the secondary palate (SP) is a complex event despite the small area it encompasses. Problems with SP development can lead to a cleft palate, which is one of the most common birth disorders. The matrix metalloproteinases (MMPs) are required for proper SP development, but a functional role for any one of them remains unknown. MMP-25 is a candidate MMP to have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the Mmp-25 gene. The purpose of this thesis is to investigate gene expression of Mmp-25 in the developing mouse SP, whether it has a functional role in mouse SP development and begin to identify factors potentially upstream of Mmp-25 expression.<p> Mmp-25 mRNA and protein is found at all SP developmental stages in mice with highest expression at embryonic day (E) 13.5 when analyzed by quantitative real-time PCR and western blotting. Immunohistochemistry localizes MMP-25 protein primarily to the plasma membranes of palate shelf epithelial cells with secondary expression in apical mesenchymal cells. Mmp-25 knockdown with siRNA in palatal cultures resulted in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium in vitro. Mmp-25 mRNA and protein levels are significantly decreased in vitro when cultured palate shelves are incubated in growth medium with 5 ìg/ml of a TGFâ3-neutralizing antibody. Mmp-25 gene expression is highest at E12.5 and E13.5, which corresponds to increasing palate shelf growth downward alongside the tongue. Immunohistochemistry localized MMP-25 protein expression predominantly in the epithelium of the palate shelves, but also in areas of the mesenchyme that were immediately adjacent to the epithelium and apical in location. Knockdown of Mmp-25 expression resulted in palate shelf fusion being impaired and significant medial edge epithelium remaining in contacted areas. Bioneutralization of TGFâ3 resulted in a significant decrease in Mmp-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development by removing extra-cellular matrix barriers to increased palate shelf growth and place its expression downstream of TGF-â3 signaling. This is the first research to present a role for a single MMP in mouse SP development.

Development of the secondary palate (SP) is a complex event despite the small area it encompasses. Problems with SP development can lead to a cleft palate, which is one of the most common birth disorders. The matrix metalloproteinases (MMPs) are required for proper SP development, but a functional role for any one of them remains unknown. MMP-25 is a candidate MMP to have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the Mmp-25 gene. The purpose of this thesis is to investigate gene expression of Mmp-25 in the developing mouse SP, whether it has a functional role in mouse SP development and begin to identify factors potentially upstream of Mmp-25 expression.<p> Mmp-25 mRNA and protein is found at all SP developmental stages in mice with highest expression at embryonic day (E) 13.5 when analyzed by quantitative real-time PCR and western blotting. Immunohistochemistry localizes MMP-25 protein primarily to the plasma membranes of palate shelf epithelial cells with secondary expression in apical mesenchymal cells. Mmp-25 knockdown with siRNA in palatal cultures resulted in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium in vitro. Mmp-25 mRNA and protein levels are significantly decreased in vitro when cultured palate shelves are incubated in growth medium with 5 ìg/ml of a TGFâ3-neutralizing antibody. Mmp-25 gene expression is highest at E12.5 and E13.5, which corresponds to increasing palate shelf growth downward alongside the tongue. Immunohistochemistry localized MMP-25 protein expression predominantly in the epithelium of the palate shelves, but also in areas of the mesenchyme that were immediately adjacent to the epithelium and apical in location. Knockdown of Mmp-25 expression resulted in palate shelf fusion being impaired and significant medial edge epithelium remaining in contacted areas. Bioneutralization of TGFâ3 resulted in a significant decrease in Mmp-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development by removing extra-cellular matrix barriers to increased palate shelf growth and place its expression downstream of TGF-â3 signaling. This is the first research to present a role for a single MMP in mouse SP development.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 88-99). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 88-99). Also available on the Internet.

Auditory impairment in patients with craniofacial clefts has been well studied for decades. However, most previous research has only focused on middle ear disorders and related auditory consequences in this group. Studies of higher level auditory status and central auditory processing abilities of this group—particularly in children—have been unsystematic and have significant limitations, while the potentially negative impact of central auditory impairment on children should not be ignored. One important area which needs further research is the status of the central auditory nervous system (CANS) in children with non-syndromic cleft lip and/or palate (NSCLP). In order to objectively investigate possible central auditory impairment in children with NSCLP, the present research programme was initiated. Firstly, two major studies aimed to provide anatomical structural analysis and functional evaluation of the auditory structures of CANS in a group of infants with NSCLP, and compare the results to those of normal controls (Studies 1 and 2). Secondly, a pilot study (Study 3) was conducted to provide preliminary data and suggest methodology to support a major, future research programme to comprehensively investigate central auditory processing abilities in children with NSCLP. A multi-disciplinary approach that included brain magnetic resonance image (MRI) scanning, auditory evoked potentials (AEP) recording, and a central behavioural auditory test battery assessment protocol, was applied in the present research programme. Based on the results of the studies and data analysis, it was concluded that: (1) Structural abnormalities of CANS in infants with NSCLP may be primarily located in the left cerebral hemisphere and cortical abnormalities were more marked compared with those in other subcortical locations. The development and maturation of the auditory cortex in infants with NSCLP may be abnormal, compared with that in normal children; (2) Infants with NSCLP might have normal auditory sensory function at brain stem and subcortical levels, yet this group may have significant impaired auditory discriminatory function at cortical level; (3) Children with NSCLP may show normal auditory processing abilities in a quiet listening environment. However, they may be more vulnerable to background noise and have impaired auditory processing abilities in areas such as monaural low redundancy and temporal resolution ability. In summary, combining the results of MRI, AEP and behavioural measurements in the present research programme, it is suggested that children with NSCLP are at potential risk of both structural abnormalities and functional disorders of the CANS, particularly at auditory cortical level. In addition, this group might also be at risk of auditory processing impairments to some degree, particularly in noisy environments. The present research programme has made a contribution to our understanding of the central auditory status of children with NSCLP, which was not systematically investigated in previous studies, and provided information on which to base further research. The research findings should draw the attention of researchers and clinicians to improving auditory assessment and intervention for patients with craniofacial cleft disorders. Further efforts in this field in the long-term may help to develop a more sophisticated audiological evaluation and intervention approach for this population. / published_or_final_version / Speech and Hearing Sciences / Doctoral / Doctor of Philosophy

Cleft palate is one of the most common congenital malformations in humans which occurs at a frequency of approximately 1:700 live births worldwide. Sine Oculis-related homeobox 2 (Six2) is a member of the vertebrate Six gene family that encode proteins that are transcription factors. Six2 has been reported to be a downstream target of Homeobox a2 (Hoxa2), a gene that plays a direct a role in mouse secondary palate (SP) development. In my thesis, I utilized quantitative real time Polymerase Chain Reaction (qPCR), Western blot analysis and fluorescence immunohistochemisrty (IHC) to characterize the spatial and temporal distribution patterns of Six2 in the developing SP. Additionally, I also employed in vivo cell counting analysis and in vitro cell proliferation assays to investigate the role of Six2 during palate mesenchymal cell proliferation. My study examined the temporal and spatial distribution of Six2 in the developing mouse palatal mesenchyme and epithelia in both wild-type and Hoxa2 null mice. Six2 was expressed throughout the period of embryonic palatogenesis, with the highest levels of Six2 mRNA and protein observed in palatal shelves at E13.5 in both wild-type and Hoxa2 null mice. Six2 protein expression at all stages of SP development (E12.5 to E15.5) increased in the anterior to posterior (A-P) direction with highest expression in the posterior regions of the developing SP. In addition, expression of Six2 protein was higher in the oral half of the palatal mesenchyme compared to the nasal half of the palatal mesenchyme. Interestingly, Six2 protein was expressed in the nasal palatal epithelium but was completely absent from the oral palatal epithelium. Loss of the Hoxa2 gene induced up regulation of Six2 protein and mRNA in the developing palate across all stages of palatogenesis. In the Hoxa2 null mice, there was a significant increase in cell proliferation (Ki-67 positive cells) and the percentage of actively proliferating cells that were co-expressing Six2 protein (Six2/Ki-67 double positive cells) along both the A-P and oral-nasal (O-N) axes of the developing SP. Also, the highest percentage of actively proliferating cells and Six2/Ki-67 double positive cells was observed in the nasal half of the posterior palatal mesenchyme. Furthermore, Six2 siRNA knock down in mouse embryonic palatal mesenchyme (MEPM) cell cultures restored cell proliferation and Cyclin D1 expression in the Hoxa2 null cell cultures to wild-type levels. Collectively, my data reveals a novel spatial and temporal expression profile for Six2 in the developing mouse SP and the potential role it might play during the epithelial-mesenchymal cross talk that drives palatal shelf cell proliferation and out growth.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences