Objetivo: Analisar por meio de um estudo clinico transversal a influência das forças oclusais, assim como, de outros possíveis fatores locais na morfologia de lesões cervicais não cariosas (LCNC). Material e Método: Foram avaliados 32 indivíduos que possuíam LCNC nos primeiros pré-molares superiores, totalizando uma análise de 61 dentes, além disso, todos os sujeitos da pesquisa apresentavam todos os elementos dentais. Os mesmos eram pacientes regulares do serviço odontológico da Universidade Federal de Uberlândia, de ambos os gêneros, com idades entre 21 a 64. Para confirmação da morfologia e da severidade (profundidade) das LCNC, foi realizado exame cínico e replica das mesmas. As LCNC foram classificadas em anguladas ou arredondadas e em 3 níveis de severidade. A fim de determinar a presença de forças laterais, a presença de algum tipo de hábito parafuncional, como apertamento e bruxismo, foi analisado por meio de questionário e exame clinico. Também foram analisados, a presença de interferências oclusais (IO) em Relação Cêntrica (RC) e Movimentos excursivos, por meio de exame clinico de manipulação e análise dos modelos de gesso montados em articulador, assim como informações sobre a presença de facetas de desgaste. Além disso, informações sobre o periodonto, também foram coletadas, por meio de exame clinico, da mensuração das recessões gengivais no sentido cérvico-apical e mésio-distal. Assim como, da altura e espessura da cortical óssea vestibular, realizada por meio de Tomografia Computadorizada do tipo Cone-Beam (TCCB). Resultados: Inicialmente, aplicou-se o teste de Shapiro wilk, para avaliar a normalidade dos dados. Para os dados normais, posteriormente, aplicou-se o teste de Kruskall-wallis. Enquanto, que para as variáveis que não apresentaram distribuição normal, foi aplicado o teste Mann- Whitney. Dados gerais da amostra, demonstraram que a idade apresentou associação tanto com a morfologia (p = ,016), quanto para a severidade das LCNC (p=,000). O que não foi encontrado para o gênero (morfologia, p =,139; severidade, p =,308), nem para a presença de algum distúrbio gástrico (morfologia, p =,260; severidade, p =,831). Além disso, nenhuma relação foi encontrada entre a preseça de hábitos parafuncionais ou alguma IO, com a morfologia e severidade das LCNC. Todos os sujeitos da pesquisa apresentaram facetas de desgaste, entretanto, a presença de uma ou duas facetas não influenciou a morfologia ou severidade das lesões. Já para a recessão gengival, associação significativa foi encontrada para a morfologia (p =,046). Em relação a altura e espessura da crista da cortical vestibular, a altura foi significante em relação a severidade (p= ,009), e a espessura da crista, apresentou associação com a morfologia (p=,001) e severidade (p=,044). Conclusão: Não houve associação direta entre a morfologia das LCNC e as forças oclusais, entretanto, a idade e o suporte ósseo (menor altura e cortical menos espessa), foram fatores determinantes para a prevalência de LCNC anguladas e mais severas. / Objective: Analyze through a cross clinical study the influence of occlusal forces, as well as other possible local factors in the morphology of cervical lesions noncarious (LCNC). Material and Methods: We evaluated 32 individuals who had LCNC the first premolars, totaling an analysis of 61 teeth, moreover, all the subjects had complete dental arches. They were regular patients of the dental service at the Federal University of Uberlândia, of both genders, ages 21 to 64. To confirm the morphology and severity (depth) of LCNC was held cynical examination and replicates them. The LCNC were classified into angled or rounded and three severity levels. In order to determine the presence of lateral forces, the presence of some kind of habit parafuncional as bruxism and clenching was analyzed by questionnaire and clinical examination. Were also analyzed, the presence of occlusal interferences (OI) in Relation Centrica (CR) and movements excursive through clinical examination manipulation and analysis of plaster models mounted on articulator, as well as information about the presence of wear facets. In addition, information about periodontal were also collected through clinical examination, measurement of gingival recession in cervical-apical and mesiodistally. As well as the height and thickness of the buccal bone cortical held by Computed Tomography Cone-Beam type (TCCB). Results: Initially, we applied the Shapiro Wilk test to assess the normality of the data. For normal data, later applied the Kruskal-wallis test. While that for variables without normal distribution, the Mann-Whitney test was applied. General data of the sample, showed that age was associated with both the morphology (p =. 016), as to the severity of LCNC (p =.000). What was not found for the genre (morphology, p = .139; severity, p = 308), or to the presence of some gastric disorder (morphology, p = .260; severity, p = 831). Furthermore, no relationship was found between the preseça of parafunctional habits or some IO, with the morphology and severity of LCNC. All the subjects showed wear facets, however, the presence of one or two aspects not influence the morphology or severity of the lesions. As for the gingival recession, significant association was found for morphology (p =.046). Regarding the height and thickness of the crest cortical bone, the height was significant in relation to severity (p =.009), and the thickness of the crest, was associated with the morphology (p = .001) and severity (p = .044). Conclusion: There was no direct association between the morphology of NCCL and occlusal forces, however, the age and the bony support (lower thinner height and cortical), were determining factors for the prevalence of NCCL angled and more severe.

O sistema nervoso simpático é escassamente descrito em livros na anatomia veterinária e encontra-se pouca informação a respeito de seu funcionamento em grandes mamíferos. O conceito atual da estrutura e função dos gânglios simpáticos deriva de estudos desenvolvidos no gânglio cervical superior (GCC) em animais de laboratório, devido ao seu grande tamanho, facilidade de acesso e multiplicidade de território de inervação nestas espécies. A ganglionectomia unilateral do GCC causou condição patológica associada à síndrome de Horner com sinais associados à anisocoria, enoftalmia, ptose e hipertermia de orelha. Aplicando-se métodos estereológicos, objetivamos investigar a neuroplasticidade do G CC em condições de exigência funcional em diferentes períodos de tempo. A neuroplasticidade foi investigada em vista dos aspectos morfoquantitativos, tamanho e número total de neurônios. Objetivou-se encontrar variação, ou não, no tamanho dos neurônios, na densidade neuronal, no número de neurônios secundários à ganglionectomia unilateral do GCC. Alterações macroscópicas revelaram um aumento médio de 8%, 3% e 11% em comprimento, largura e espessura para os gânglios operados no grupo I. Para o grupo II encontraram-se aumentos em 4% para comprimento e largura e 5% em espessura, enquanto que para o grupo III, 29% de aumento foram encontrados para comprimento, 4% em largura e 7% em espessura. Os gânglios controle e operados apresentaram diferença significativa (p = 0,0001) na densidade neuronal (Nv). Os gânglios operados apresentaram diminuição na densidade neuronal em média de 89% para o grupo I, 65% para o grupo II e 47% para o grupo III. Esta redução reflete a distribuição heterogênea dos neurônios no gânglio operado. Um aumento no volume neuronal global foi significativamente detectado (p = 0,0001), o gânglio operado apresentou aumentos médios de 13% para o grupo I, 24% para o grupo II e 29% para o grupo III, sugestivo de maior exigência funcional em resposta à ganglionectomia. O numero total de neurônios apresentou diferenças significativas (p = 0,0514) e dois efeitos distinto nos gânglios operados. No grupo I observou-se um aumento de 3% no numero total de neurônios enquanto que uma redução foi determinada para os grupos II (8%) e III (20%). A avaliação global dos resultados leva-nos a inferir duas hipóteses associadas e consecutivas: 1) a hipertrofia neuronal estaria associada a um mecanismo compensatório para a re-inervação contralateral sendo que a maior exigência funcional poderia levar estes neurônios à morte, seguido de hipertrofia de tecido não-neuronal cicatricial refletido na diminuição da densidade neuronal; 2) a hipertrofia neuronal estaria associada a um mecanismo compensatório à morte celular determinada pela maior exigência funcional. As alterações quantitativas principais secundárias à ganglionectomia unilateral do GCC no gânglio remanescente estão associadas à: perda significativa no número total de neurônios a partir da 8ª semana de evolução da doença, perda significativa na densidade neuronal a partir da 8ª semana e aumento significativo na área e volume neuronal. / The sympathetic nervous system is briefly described in veterinary anatomy text-books and there is little information so far concerning its function in large mammal species. The current concept of the structure and function of the sympathetic ganglia is derived from studies on the cranial cervical ganglia (CCG) carried out in laboratory animals due to very attractive characteristics of CCG in these species such as large size, accessibility and multiplicity of target organs. The CCG unilateral ganglionectomy caused a pathological condition associated with Horner´s syndrome which includes anisocoria, enophtalmos, ptosis and increase in the temperature of ear as a result of peripheral vasodilatation. Using stereology-designed methods, we aimed to study CCG neuroplasticity under experimental functional overloading along distinct periods of time. Neuroplasticity was investigated according to morphoquantitative aspects, mostly size and total number of neurons. We wanted to find out whether or not neuron size, numerical density and nerve cell numbers would vary as a result of CCG ganglionectomy. Gross anatomic differences were considered to the increase in the operated ganglia, means of 8%, 3% and 11% for length, width and thickness, respetively in the group I. For group II it was encountered means increases of 4% for length and width and 5% in thickness. Group III showed means values of 29% increase in length, 4% in width and 7% in thickness. Control and operated sheep CCG revealed significant difference (p=0.0001) in the neuronal density (Nv). The operated ganglia revealed reduction in the neuronal density of 89% for group I, 65% for group II and 47% for group III. This reduction in the operated ganglia reflects its inhomogeneous distribution of neurons. An increase in the global neuronal volume was significantly detected (p= 0.0001), the operated ganglia showed increases in the means of 13% for group I, 24% for group II and 29% for group III, suggesting a functional overload response for the unilateral ganglionectomy. The total number of neurons presented significant differences (p = 0.0514) and two distinct effects in the operated ganglia. In the group I, an increase of 3% was encountered meanwhile reduction in the total number was associated to groups II (8%) and group III (20%). An overlook of the results suggests two consecutive and associated hypotheses: 1) the hypertrophy of neurons would be associated to a compensatory mechanism for contralateral re-innervation although the functional overload would drive these neurons to cellular death, followed by hypertrophy of non-neuronal tissue as reflected in the neuronal density; 2) the neuronal hypertrophy would be associated to a compensatory mechanism to cellular death caused by the functional overload The main quantitative changes in the remaining ganglia are: Significant loss in the total number of neurons from the 8th week of evoloution of the disease; Significant decrease in the numerical density (Nv) from the 8th week and significant increase in both neuron area and neuron volume.

O objetivo deste estudo foi avaliar ex vivo a influência do alargamento cervical na determinação do instrumento apical inicial (IAI), no comprimento de trabalho (CT), dos canais radiculares de primeiros molares superiores, e determinar a forma do canal a 1 mm do ápice. Cinqüenta dentes foram divididos aleatoriamente em 5 grupos (n=10) de acordo com o preparo dos terços cervical e médio do canal: GI- sem alargamento; GII- brocas Gates- Glidden (#2, #3) (GG); GIII- AET (S1, SC, S2 e S3); GIV- GT Rotary File (20/06, 20/08, 20/10 e 20/10) (GT); GV- LA Axxess (20.06, 35.06) (LA). Os canais foram explorados com lima tipo K inserindo-se passivamente a lima 08 no CT, e limas de diâmetros sucessivamente maiores até obter a sensação de travamento. Foram feitas secções transversais no CT, analisadas em MEV e a área do IAI e a área do canal radicular foram medidas para verificar a porcentagem que o IAI ocupou no interior do canal em cada amostra. A forma do canal radicular foi classificada em circular, oval e achatada. A análise de variância indicou diferença estatisticamente significante entre a área ocupada pelo IAI entre os grupos experimentais (p<0,0001). Os resultados mostraram que, para o canal mésio-vestibular, os valores decrescentes em porcentagem que o IAI ocupou no interior do canal foram: LA Axxess (66,70 ± 7,10) > GT (44,16 ± 9,35) = AET (44,10 ± 8,88) > GG (33,17 ± 6,68) = sem alargamento (23,85 ± 6,86); para o canal disto-vestibular: LA Axxess (75,12 ± 8,56) > GT (58,68 ± 7,70) = AET (54,66 ± 7,12) > GG (39,76 ± 7,52) > sem preparo (26,90 ± 6,10); e para o canal palatino: LA Axxess (66,55 ± 11,40) > AET (51,98 ± 10,67) = GT (49,50 ± 10,05) > GG (35,70 ± 7,62) > sem preparo (21,43 ± 2,79). A forma do canal foi predominantemente achatada para o canal mésio-vestibular, circular para o canal distovestibular e oval para o canal palatino. Pode-se concluir que o preparo dos terços cervical e médio do canal radicular permite melhor determinação do IAI, e que o preparo cervical com brocas LA Axxess apresentou a melhor adaptação do IAI no CT. / The aim of this study was to investigate ex vivo the influence of cervical preflaring on determination the initial apical file (IAI), in the working length (WL), of the root canals of the maxillary first molars and to determine the shape of the canal at 1 mm from the apex. Fifty teeth were randomly divided into 5 groups (n=10) in accordance with the preflaring of the cervical and middle thirds of the canal: GI - without preflaring; GII - Gates-Glidden burs (#2, #3) (GG); GIII - AET (S1, SC, S2 and S3); GIV - GT Rotary File (20/06, 20/08, 20/10 and 20/10) (GT); GV - LA Axxess burs (20.06, 35.06) (LA). The canals were sized with the type K-file, passively inserting the file 08 in the WL and files with successively greater diameters until a binding sensation was felt. Cross sections were made in the WL, analyzed by SEM and the IAI area and the area of the root canal were measured to verify the percentage that the IAI occupied inside the canal in each sample. The shape of the root canal was classified as circular, oval and flattened. The analysis of variance indicated a statistically significant difference between the area occupied by the IAI and the experimental groups (p<0.0001). The results showed that for the mesiobuccal canal, the decreasing values by percentage that the IAI occupied inside the canal were: LA Axxess (66.70 ± 7.10) > GT (44.16 ± 9.35) = AET (44.10 ± 8.88) > GG (33.17 ± 6.68) = without preflaring (23.85 ± 6.86); for the distobuccal canal: LA Axxess (75.12 ± 8.56) > GT (58.68 ± 7.70) = AET (54.66 ± 7.12) > GG (39.76 ± 7.52) > without preflaring (26.90 ± 6.10); and for the palatal canal: LA Axxess (66.55 ± 11.40) > AET (51.98 ± 10.67) = GT (49.50 ± 10.05) > GG (35.70 ± 7.62) > without preflaring (21.43 ± 2.79). The shape of the canal was predominantly flattened for the mesiobuccal canal, circular for the distobuccal canal and oval for the palatal canal. It may be concluded that the preflaring of the cervical and middle thirds of the root canal allows better determination of the IAI and that the cervical preflaring with LA Axxess burs presented better adaptation of the IAI in the WL.

Introdução: Pacientes transplantadas renais evoluíram para uma maior sobrevida e uma melhor função renal com o advento dos imunossupressores. A rejeição do enxerto deixou de ser a principal causa de morbimortalidade, e outros problemas crônicos surgiram, como infecções e neoplasias induzidas por vírus, como as lesões precursoras de câncer de colo uterino induzidas pelo Papilomavírus Humano (HPV). Objetivo: Avaliar a prevalência de alterações no exame citopatológico do colo do útero (CP) em pacientes transplantadas renais e compará-la à prevalência de alterações no CP em pacientes imunocompetentes. Método: Estudo transversal com grupo controle de alterações do CP em pacientes transplantadas renais e em pacientes imunocompetentes, no período de maio de 2015 a agosto de 2016. Resultados: A frequência de lesão intraepitelial de baixo (LIEBG) e alto grau (LIEAG) foi três vezes maior no grupo das transplantadas em relação ao grupo controle (20,6% no grupo transplantadas vs 6,8% no grupo controle, p<0,001) considerando todos os CPs coletados após o transplante. Na avaliação ginecológica ambulatorial no período do estudo não houve diferença entre os grupos em relação ao resultado "normal" e "alterado" do CP (resultado normal em 152 pacientes transplantadas (92,1%) vs 326 pacientes (93,9%) do grupo controle). Entretanto, quando se estratificaram os resultados alterados, evidenciou-se maior frequência de LIEBG nas transplantadas (3,6% vs 0,0%, P<0,001). Conclusão: O grupo de pacientes transplantadas renais apresentou uma taxa significativamente maior de alterações no CP em relação ao grupo controle, sendo que a maior parte foi composta de LIEBG. / Introduction: Renal transplant recipients (RTR) evolved to greater survival and improved renal function with the advent of immunosuppressants. Graft rejection is no longer the leading cause of morbidity and mortality, and other chronic problems have arisen, such as virusinduced infections and neoplasms, such as HPV-induced precursor lesions of cervical cancer. Objective: To evaluate the prevalence of cervical pre-malignancies in the Pap smear (PS) in female RTR and compare to the prevalence of cervical pre-malignancies in the immunocompetent patients. Methods: Cross-sectional study with control group performed with 165 female RTR and 372 immunocompetent patients from May 2015 to August 2016. Results: This study observed a frequency 3 times higher of HSIL (CIN 2) and LSIL in the RTR group (20.6% of RTR vs. 6.8% in the control group) considering the Pap smears collected after the renal transplant. There was no difference between the groups regarding the "normal" and "altered" result from the PS in the outpatient gynecological evaluation (during the study). The exam had normal results in 152 RTR (92.1%) vs. 326 patients (93.9%) in the control group. However, when the altered results were broken down, a higher frequency of LSIL could be seen in RTR (3.6% vs 0.0%, P<0.001). Conclusion: RTR had a significantly higher rate of PS alterations in comparison with the control group and most of it was composed of LSIL.

La radiculopathie cervicale caudale a été identifiée comme cause de boiterie affectant le membre antérieur chez le cheval. Les affections dégénératives des articulations intervertébrales des processus articulaires entraînent un remodelage périarticulaire pouvant comprimer les racines du nerf spinal ou leur rameau ventral. Les objectifs de l’étude étaient de décrire la réalisation d’injections échoguidées périneurales du rameau ventral des nerfs spinaux cervicaux 7 et 8 (RV7 et RV8), d’évaluer sur des cadavres de chevaux par dissection la diffusion péri-nerveuse d’une solution colorée ainsi que de décrire chez des chevaux sains les signes cliniques associés à une anesthésie périneurale échoguidée du RV7 et RV8 individuellement. Dans l’étude post-mortem, 5 RV7 et 5 RV8 ont été visualisés échographiquement et colorés par une injection de 7 ou 14 ml de solution colorée. Une portion du tronc nerveux a été trouvée coloré pour chaque injection. La coloration était uniforme transversalement sur toute la largeur du nerf et couvrait une longueur supérieure à 2 cm pour 8 RV alors qu’une coloration de la moitié crâniale du RV sur une longueur de moins de 2 cm pour un RV7 et un RV8. L’étude sur cheval sain portait sur 4 chevaux sains sans image radiographique anormale de la colonne cervicale. Six RV7 et 8 RV8 ont été anesthésiés de la Lidocaïne 2% mélangés à du iohexol. Toutes les injections ont entraîné boiterie antérieure ipsilatérale de l’injection. Les boiteries les plus sévères correspondaient à une parésie du nerf suprascapulaire pour RV7 et à une parésie radiale pour RV8. Cette étude a montré qu’il était possible de réaliser une injection périneurale des RV 7 et RV8 chez le cheval et que l’anesthésie tronculaire des deux racines provoque une atteinte motrice essentiellement de la fonction nerveuse. Ces résultats contribuent à mieux comprendre la symptomatologie des compressions nerveuses cervicales chez le cheval. / Caudal cervical radiculopathy has been identified as a cause of frontlimb lameness in horses. Degenerative conditions of articular process joint result in periarticular remodeling responsible for compression of spinal nerve roots or their ramus ventralis (RV). The objectives of the study were to describe how to perform perineural RV injection under ultrasonographic guidance, to evaluate on cadaver perineural RV staining after a dye solution injection, as well as describe clinical signs associated with a perineural ultranosonography-guided anesthesia of RV7 and RV8 respectively. In the post-mortem study, the RV of the spinal cervical nerves was visualized in all cadavers. Eight RV had a uniform transversal staining of the nerve trunk that covered longitudinally a distance greater than 2 cm. One C7 and one C8 RV showed incomplete transversal staining with a more concentrated color on its half cranial aspect and a longitudinal coverage of less than 2 cm. The in vivo study included 6 RV7 and 8 RV8 perineural injections of a local anesthetic agent, performed on 4 horses that had no abnormal finding on cervical radiographs. All anesthetic injections (lidocaine 2% and iohexol) resulted in modifications of the locomotion with variable degree of lameness on the ipsilateral frontlimb. Severe lameness was characteristic of a suprascapular paresis for RV7 and a radial paresis for RV8. Mild to moderate lameness on the ipsilateral frontlimb included decreased anterior phase of the stride, intern circumduction of the limb and sometimes stumbling for that same frontlimb. Signs of ataxia on the hindlimbs were encountered for 3 injections. This study showed that it is possible to perform perineural injections of RV7 and RV8 in horses and that perineural anesthesia of RV7 and RV8 results in motor dysfunction. These findings constitute a contribution to understanding clinical signs associated with cervical nerve compression in horses.

子宮頸癌是女性的主要癌症殺手，而人類乳頭瘤病毒 (HPV) 則是子宮頸癌形成的必要條件之一。HPV16型及HPV18型是全球最普遍的高危型HPV；而另一方面，HPV52及HPV58兩型在東亞地區的流行程度比世界其他地區為高。 / 過往有科學研究顯示HPV病毒載量的高低是引致高度癌前病變的重要決定因素，也有研究指出病毒載量與病變的嚴重程度成正比例，但同時亦有研究指兩者並無關係。HPV基因組可以兩種物理形態存在：游離型及整合型。HPV的E2基因可對E6及E7致癌基因產生重要的調節作用，而當HPV病毒與宿主染色體整合後，可使E2基因斷裂，因而令控制E6及E7致癌基因表達的負反饋基制失效。 / 本研究假設高病毒載量及由HPV基因組整合所造成的E2基因斷裂，並非引致子宮頸癌的僅一途徑。本研究分析了在不同程度的子宮頸細胞病變下，HPV16型、18型、52型及58型的病毒載量及基因整合情況。其中，有關HPV16型的研究部份更深入地探討了E6/7 mRNA的轉錄水平、E2和LCR的序列變異及E2結合位點的甲基化情況，最終希望能找出除了病毒基因整合之外的另一種致癌機理。 / 本研究的結果顯示，在不同HPV型所引致的子宮頸細胞病變中，病毒載體及病變程度之間的關係也存有差異；而根據管家基因的數量來為細胞DNA標準化，對準確分析不同程度子宮頸細胞病變的實驗結果至關重要。本研究的一項重要發現是部份侵襲性癌細胞只含有游離型HPV基因組；而在只含游離HPV基因組的侵襲性子宮頸癌樣本中，有三種E6/E7 mRNA的抄錄本水平與只含整合型基因組的樣本相若，反映在只含游離型HPV基因組的侵襲性子宮頸癌樣本中，E6/ E7 mRNA的表達量亦有上調。最重要的是，此表達量的上調並非由基因整合或E2基因斷裂所引致。 / 在只含有游離型病毒基因組的侵襲性子宮頸癌樣本中，E6及E7致癌基因表達上調的另一種機理，很可能是HPV16啟動區內E2結合位點上的CpG位點出現甲基化。這項觀察解釋及支持了當E2蛋白因結合位點甲基化而失去對E6及E7基因轉錄的抑制功能時，E6及E7致癌蛋白仍能保持高水平，而兩種蛋白產生協同作用，令細胞轉型及出現癌變。總結之言，本實驗也肯定了HPV整合並非導致子宮頸癌形成的唯一機理。 / Cervical cancer is a major cause of cancer-related death in women worldwide. Human papillomavirus (HPV) is essential, though not sufficient, to cause cervical cancer. HPV16 and HPV18 are the most prevalent high-risk types worldwide, whereas, HPV52 and HPV58 also show a notable higher prevalence in East Asia than in other parts of the world. / Studies have suggested that HPV viral load is an important determinant for the development of high-grade lesions. While some studies observed a positive correlation between viral load and disease severity, others have reported no association. The HPV genome can exist in two physical forms, episomal or integrated. The E2 gene, encoded by HPV has an important role in the regulation of E6 and E7 viral oncogenes. When HPV integrates into the host chromosome, it may result in disruption of the E2 gene thereby its control on the expression of the E6 and E7. / The hypothesis for this study was that high viral load and disruption of E2 gene associated with integration of HPV into the host genome was not the only pathway leading to cervical cancer development. In this study, the viral load and integration profile for HPV types 16, 18, 52 and 58 among different severity of cervical lesions were analyzed. Further detailed studies were performed on HPV16 with emphases on E6/E7 mRNA transcript levels, E2 and LCR sequence variation and the methylation status of two E2 binding sites. The ultimate aim was to determine what other alternative mechanisms exist apart from viral integration to drive the oncogenicity of HPV that lead to the development of cervical cancer. / The results showed that the relationship between viral load and disease varied between different HPV types and that normalization of cellular DNA input using a housekeeping gene was crucial for accurate interpretation among different cervical lesion grades. A key finding from this study was that a substantial proportion of invasive cervical carcinomas were found to contain the purely episomal form of the HPV genome. The levels of the three E6/E7 mRNA transcripts species in invasive cervical carcinomas containing the pure episomal form of the viral genome were found to be similar to those with pure integrated forms. This observation suggested that invasive cervical carcinoma samples containing the episomal form of the HPV genome were also mediated by the up-regulated E6/E7 mRNA expression. More importantly, this up-regulation in E6/E7 mRNA expression did not depend on integration and disruption of the E2 gene. / The alternative mechanism that up-regulated of the expression of E6 and E7 oncogene found in invasive cervical carcinoma samples harbouring the episomal form of the viral genome was likely to be a consequence of methylation of CpG sites in the two E2 binding sites at the promoter region of HPV16. This observation explained and supported that the repressive role of E2 on E6 and E7 transcriptional regulation was abolished due to methylation of the E2 binding sites, and that a sustained level of the E6 and E7 oncoproteins was maintained, working in synergy in cell transformation and in carcinogenesis. These observations confirmed the hypothesis that HPV integration was not the only mechanism leading to the development of cervical cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheung, Lai Ken Jo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 233-248). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.I / Abstract of thesis --- p.IV / 論文摘要 --- p.VII / Publications --- p.IX / Contents --- p.X / Figures --- p.XV / Tables --- p.XVIII / Abbreviations --- p.XIX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cervical Cancer --- p.2 / Chapter 1.1.1 --- Cervical Cytology Screening --- p.3 / Chapter 1.1.2 --- Classification System for Cervical Squamous Cell Dysplasia --- p.4 / Chapter 1.1.3 --- Histological Grading of Cervical Lesions --- p.6 / Chapter 1.1.4 --- Development of Cervical Cancer --- p.6 / Chapter 1.2 --- Structure of HPV --- p.7 / Chapter 1.1.1 --- HPV Genome Organization --- p.8 / Chapter 1.1.2 --- The E1 Protein --- p.10 / Chapter 1.1.3 --- The E2 Protein --- p.10 / Chapter 1.1.4 --- The E4 Protein --- p.13 / Chapter 1.1.5 --- The E5 Protein --- p.13 / Chapter 1.1.6 --- The E6 Protein --- p.14 / Chapter 1.1.7 --- The E7 Protein --- p.14 / Chapter 1.1.8 --- The L1 Protein --- p.15 / Chapter 1.1.9 --- The L2 Protein --- p.16 / Chapter 1.1.10 --- The Long Control Region --- p.17 / Chapter 1.3 --- HPV and Cervical Cancer --- p.19 / Chapter 1.3.1 --- HPV is an Etiological Cause of Cervical Cancer --- p.19 / Chapter 1.3.2 --- Establishment of HPV Infection --- p.20 / Chapter 1.3.3 --- Regulation and Control of HPV Viral Gene Transcription --- p.23 / Chapter 1.3.4 --- Viral Oncogene Expression by Alternative RNA Splicing --- p.23 / Chapter 1.3.5 --- DNA Methylation in Viral Oncogene Expression --- p.24 / Chapter 1.3.6 --- The Roles of E6 and E7 Protein in Cervical Carcinogenesis --- p.26 / Chapter Chapter 2 --- Controversies and Hypothesis --- p.33 / Chapter 2.1 --- Controversies in Mechanism of Cervical Carcinogenesis --- p.34 / Chapter 2.1.1 --- Viral Integration and Risk of Cervical Cancer Development --- p.34 / Chapter 2.1.2 --- Viral Load and Risk of Cervical Cancer Development --- p.35 / Chapter 2.2 --- Hypothesis of Study --- p.37 / Chapter 2.2.1 --- Study Design --- p.38 / Chapter Chapter 3 --- Materials and Methods --- p.41 / Chapter 3.1 --- Patient Recruitment and Sample Preparation --- p.42 / Chapter 3.1.1 --- Study subject recruitment --- p.42 / Chapter 3.1.2 --- Collection of cytology samples --- p.43 / Chapter 3.1.3 --- Collection of cervical biopsy samples --- p.44 / Chapter 3.2 --- Nucleic Acid Extraction and Preparation --- p.44 / Chapter 3.2.1 --- Extraction of DNA from cervical cytology samples --- p.44 / Chapter 3.2.2 --- Extraction of DNA from cervical biopsy samples --- p.45 / Chapter 3.2.3 --- Extraction of RNA from cervical cytology samples --- p.45 / Chapter 3.2.4 --- Extraction of RNA from cervical biopsy samples --- p.46 / Chapter 3.3 --- Detection and Genotyping of Human Papillomavirus --- p.46 / Chapter 3.4 --- Determination of Viral Load using Real-Time Polymerase Chain Reaction --- p.47 / Chapter 3.4.1 --- Optimization of HPV16, 18, 52 and 58 E7 real-time PCR --- p.48 / Chapter 3.4.2 --- Optimization of housekeeping gene real-time PCR --- p.50 / Chapter 3.4.3 --- Determination of HPV16, 18, 52 and 58 viral load --- p.50 / Chapter 3.5 --- Determination of HPV Genome Physical Status --- p.53 / Chapter 3.5.1 --- HPV E2 gene primer design --- p.53 / Chapter 3.5.2 --- Optimization of HPV16, 18, 52 and 58 E2 Real-time PCR --- p.56 / Chapter 3.5.3 --- Determination of the HPV genome physical status --- p.59 / Chapter 3.6 --- Evaluation of Housekeeping Genes for Normalization of Viral Gene Expression --- p.62 / Chapter 3.6.1 --- Optimization of housekeeping gene real-time PCR --- p.62 / Chapter 3.6.2 --- Quantitation of RNA and DNase treatment --- p.66 / Chapter 3.6.3 --- cDNA synthesis from the extracted RNA --- p.67 / Chapter 3.6.4 --- Detection of five housekeeping gene levels from cervical cytology samples by real-time PCR --- p.67 / Chapter 3.6.5 --- Data analyses --- p.68 / Chapter 3.7 --- Quantitation of HPV16 mRNA Transcripts --- p.69 / Chapter 3.7.1 --- Preparation of RNA from CaSki cells --- p.69 / Chapter 3.7.2 --- Amplification of mRNA transcripts from CaSki cells --- p.69 / Chapter 3.7.3 --- Amplification of artificial mRNA transcript E6*II --- p.73 / Chapter 3.7.4 --- Gel purification of mRNA transcript amplicons --- p.73 / Chapter 3.7.5 --- Cloning of E6 mRNA transcripts --- p.74 / Chapter 3.7.6 --- Confirmation of the mRNA transcript inserts --- p.74 / Chapter 3.8 --- Quantitation HPV16 E6 mRNA Transcript Levels Using Real-Time PCR --- p.79 / Chapter 3.8.1 --- mRNA transcript primer and probe design --- p.79 / Chapter 3.8.2 --- Optimization of real-time PCR for the detection of mRNA transcripts --- p.82 / Chapter 3.8.3 --- Determination of mRNA transcript levels from invasive carcinomas --- p.83 / Chapter 3.8.4 --- Normalization of mRNA transcript expression with a housekeeping gene --- p.84 / Chapter 3.9 --- Sequence Variation of the HPV16 E2 and Long Control Region --- p.84 / Chapter 3.9.1 --- Identification of sequence variation of the E2 gene --- p.84 / Chapter 3.9.2 --- Identification of sequence variation of the long control region --- p.87 / Chapter 3.1 --- Detection of Methylation Status of E2BS1 and E2BS2 on the LCR using Pyrosequencing --- p.87 / Chapter 3.10.1 --- Bisulfite DNA conversion --- p.87 / Chapter 3.10.2 --- Amplification of E2 binding site regions on the LCR --- p.88 / Chapter 3.10.3 --- Purification of PCR product prior to pyrosequencing --- p.92 / Chapter 3.10.4 --- Quantitation of methylation using pyrosequencing --- p.92 / Chapter Chapter 4 --- Results --- p.93 / Chapter Hypothesis 1 --- p.94 / Chapter Results of Study Part: 1 --- p.95 / Chapter 4.1 --- Human Papillomavirus Type 16 Viral Load and Genome Physical Status --- p.96 / Chapter 4.1.1 --- E7 viral load --- p.96 / Chapter 4.1.2 --- Viral genome physical status --- p.100 / Chapter 4.1.3 --- E2 disruption site --- p.105 / Chapter 4.2 --- Human Papillomavirus Type 18 Viral Load and Genome Physical Status --- p.107 / Chapter 4.2.1 --- E7 viral load --- p.107 / Chapter 4.2.2 --- Viral genome physical status --- p.110 / Chapter 4.2.3 --- E2 disruption site --- p.113 / Chapter 4.2.4 --- Infection status --- p.116 / Chapter 4.2.5 --- Adeno/adenosquamous carcinoma versus squamous cell carcinoma --- p.119 / Chapter 4.3 --- Human Papillomvirus Type 52 Viral Load and Genome Physical Status --- p.120 / Chapter 4.3.1 --- E7 viral load --- p.120 / Chapter 4.3.2 --- Viral genome physical status --- p.123 / Chapter 4.3.3 --- E2 disruption site --- p.126 / Chapter 4.3.4 --- Infection status --- p.129 / Chapter 4.4 --- Human Papillomavirus Type 58 Viral Load and Genome Physical Status --- p.131 / Chapter 4.4.1 --- E7 viral load --- p.131 / Chapter 4.4.2 --- Viral genome physical status --- p.133 / Chapter 4.4.3 --- E2 disruption site --- p.134 / Chapter 4.4.4 --- Infection status --- p.137 / Chapter 4.5 --- Summary of Study Part 1: --- p.140 / Chapter Hypothesis 2 --- p.141 / Chapter Results of Study Part 2: --- p.142 / Chapter 4.6 --- Housekeeping Gene mRNA Expression Level --- p.143 / Chapter 4.6.1 --- Expression levels across different grades of cervical lesion --- p.143 / Chapter 4.6.2 --- Expression stability of housekeeping genes --- p.145 / Chapter 4.7 --- Summary of Study Part 2: --- p.149 / Chapter Results of Study Part: 3 --- p.150 / Chapter 4.8 --- HPV16 mRNA Transcript Expression Level --- p.151 / Chapter 4.8.1 --- HPV16 viral genome physical status --- p.151 / Chapter 4.8.2 --- HPV16 E2 disruption site --- p.151 / Chapter 4.8.3 --- Expression level of E6/E7 mRNA transcripts --- p.155 / Chapter 4.8.4 --- Expression level of E6/E7 mRNA transcripts and viral genome physical status --- p.157 / Chapter 4.8.5 --- Expression level of E6/E7 mRNA transcripts and E2 gene disruption status --- p.161 / Chapter 4.9 --- Summary of Study Part 3: --- p.163 / Chapter Hypothesis 3 --- p.165 / Chapter Results of Study Part 4: --- p.166 / Chapter 4.1 --- HPV 16 E2 Gene Sequence Variation --- p.167 / Chapter 4.10.1 --- Sequence variation of E2 gene --- p.167 / Chapter 4.10.2 --- Sequence variation and viral genome physical status --- p.168 / Chapter 4.10.3 --- Sequence variation in the E2 binding sites --- p.169 / Chapter 4.10.4 --- Sequence variations of E2 in HPV16 cancer derived cell lines --- p.170 / Chapter 4.11 --- HPV16 Long Control Region Sequence Variation --- p.174 / Chapter 4.11.1 --- Sequence variation of LCR --- p.174 / Chapter 4.11.2 --- Sequence variation and viral genome physical status --- p.175 / Chapter 4.11.3 --- Sequence variation in E2 binding sites --- p.176 / Chapter 4.11.4 --- Sequence variation of LCR in HPV16 cancer derived cell lines --- p.176 / Chapter 4.12 --- Summary of Study Part 4: --- p.183 / Chapter Hypothesis 4 --- p.185 / Chapter 4.13 --- Methylation Status of E2 Binding Sites --- p.187 / Chapter 4.13.1 --- Proportion methylation in E2 binding sites --- p.187 / Chapter 4.13.2 --- Methylation in invasive carcinomas according to the viral genome physical status --- p.191 / Chapter 4.14 --- Summary of Study Part 5: --- p.195 / Chapter Chapter 5 --- Discussion --- p.196 / Chapter 5.1 --- Viral Load --- p.197 / Chapter 5.2 --- Viral Integration --- p.200 / Chapter 5.2.1 --- HPV16 Viral Load and Physical Status --- p.201 / Chapter 5.2.2 --- HPV18 Viral Load and Physical Status --- p.204 / Chapter 5.2.3 --- HPV52 Viral Load and Physical Status --- p.207 / Chapter 5.2.4 --- HPV58 Viral Load and Physical Status --- p.210 / Chapter 5.2.5 --- Viral Load and Physical Status Summary --- p.214 / Chapter 5.3 --- HPV16 E6/E7 mRNA Transcript and Genome Physical Status --- p.215 / Chapter 5.4 --- HPV16 E2 Sequence Variation and Genome Physical Status --- p.218 / Chapter 5.5 --- HPV16 LCR Sequence Variation and Genome Physical Status --- p.222 / Chapter 5.6 --- Methylation of HPV16 E2 Binding Sites and Genome Physical Status --- p.225 / Chapter 5.7 --- Conclusions --- p.230 / Chapter 5.8 --- Implication of Current Findings and Future Work --- p.231 / References --- p.233

O objetivo deste estudo foi avaliar ex vivo a influência do alargamento cervical na determinação do instrumento apical inicial (IAI), no comprimento de trabalho (CT), dos canais radiculares de primeiros molares superiores, e determinar a forma do canal a 1 mm do ápice. Cinqüenta dentes foram divididos aleatoriamente em 5 grupos (n=10) de acordo com o preparo dos terços cervical e médio do canal: GI- sem alargamento; GII- brocas Gates- Glidden (#2, #3) (GG); GIII- AET (S1, SC, S2 e S3); GIV- GT Rotary File (20/06, 20/08, 20/10 e 20/10) (GT); GV- LA Axxess (20.06, 35.06) (LA). Os canais foram explorados com lima tipo K inserindo-se passivamente a lima 08 no CT, e limas de diâmetros sucessivamente maiores até obter a sensação de travamento. Foram feitas secções transversais no CT, analisadas em MEV e a área do IAI e a área do canal radicular foram medidas para verificar a porcentagem que o IAI ocupou no interior do canal em cada amostra. A forma do canal radicular foi classificada em circular, oval e achatada. A análise de variância indicou diferença estatisticamente significante entre a área ocupada pelo IAI entre os grupos experimentais (p<0,0001). Os resultados mostraram que, para o canal mésio-vestibular, os valores decrescentes em porcentagem que o IAI ocupou no interior do canal foram: LA Axxess (66,70 ± 7,10) > GT (44,16 ± 9,35) = AET (44,10 ± 8,88) > GG (33,17 ± 6,68) = sem alargamento (23,85 ± 6,86); para o canal disto-vestibular: LA Axxess (75,12 ± 8,56) > GT (58,68 ± 7,70) = AET (54,66 ± 7,12) > GG (39,76 ± 7,52) > sem preparo (26,90 ± 6,10); e para o canal palatino: LA Axxess (66,55 ± 11,40) > AET (51,98 ± 10,67) = GT (49,50 ± 10,05) > GG (35,70 ± 7,62) > sem preparo (21,43 ± 2,79). A forma do canal foi predominantemente achatada para o canal mésio-vestibular, circular para o canal distovestibular e oval para o canal palatino. Pode-se concluir que o preparo dos terços cervical e médio do canal radicular permite melhor determinação do IAI, e que o preparo cervical com brocas LA Axxess apresentou a melhor adaptação do IAI no CT. / The aim of this study was to investigate ex vivo the influence of cervical preflaring on determination the initial apical file (IAI), in the working length (WL), of the root canals of the maxillary first molars and to determine the shape of the canal at 1 mm from the apex. Fifty teeth were randomly divided into 5 groups (n=10) in accordance with the preflaring of the cervical and middle thirds of the canal: GI - without preflaring; GII - Gates-Glidden burs (#2, #3) (GG); GIII - AET (S1, SC, S2 and S3); GIV - GT Rotary File (20/06, 20/08, 20/10 and 20/10) (GT); GV - LA Axxess burs (20.06, 35.06) (LA). The canals were sized with the type K-file, passively inserting the file 08 in the WL and files with successively greater diameters until a binding sensation was felt. Cross sections were made in the WL, analyzed by SEM and the IAI area and the area of the root canal were measured to verify the percentage that the IAI occupied inside the canal in each sample. The shape of the root canal was classified as circular, oval and flattened. The analysis of variance indicated a statistically significant difference between the area occupied by the IAI and the experimental groups (p<0.0001). The results showed that for the mesiobuccal canal, the decreasing values by percentage that the IAI occupied inside the canal were: LA Axxess (66.70 ± 7.10) > GT (44.16 ± 9.35) = AET (44.10 ± 8.88) > GG (33.17 ± 6.68) = without preflaring (23.85 ± 6.86); for the distobuccal canal: LA Axxess (75.12 ± 8.56) > GT (58.68 ± 7.70) = AET (54.66 ± 7.12) > GG (39.76 ± 7.52) > without preflaring (26.90 ± 6.10); and for the palatal canal: LA Axxess (66.55 ± 11.40) > AET (51.98 ± 10.67) = GT (49.50 ± 10.05) > GG (35.70 ± 7.62) > without preflaring (21.43 ± 2.79). The shape of the canal was predominantly flattened for the mesiobuccal canal, circular for the distobuccal canal and oval for the palatal canal. It may be concluded that the preflaring of the cervical and middle thirds of the root canal allows better determination of the IAI and that the cervical preflaring with LA Axxess burs presented better adaptation of the IAI in the WL.

The Papanicolaou Smear is a screening test which detects premalignant lesions of the uterine cervix. By treating these lesions, cervical cancer can be evaded. In 1988, a cytological diagnosis which communicated a state of uncertainty in the atypicality of cervical cells was first created in the Bethesda Cytology Classification scheme. This diagnosis is now known as atypical squamous cells of undetermined significance (ASC-US) and still little is known about its natural history. / This paper analyzes the results of a longitudinal study incorporating repeated regular measurements of viral and cytological endpoints as well as lifestyle and behavioural aspects, to understand the natural history of an ASC-US Pap smear and identify determinants of ASC-US acquisition and clearance. / Overall, the median duration of ASC-US is short, and is dependent on the definition of clearance since most lesions regress to normal. The factors most predictive of ASC-US acquisition but not clearance relate to HPV infection.

Licentiatvhandling (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 2 uppsatser.

O câncer cervical é considerado um grande problema de saúde pública e um dos maiores causadores de mortes relacionadas a tumores em mulheres. O principal objetivo desta tese foi aumentar a eficácia antitumoral terapêutica da proteína gDE7 por meio da associação de adjuvantes vacinais em formulações testadas em condições experimentais com a linhagem celular tumoral TC-1. A proteína gDE7 foi produzida a partir de uma linhagem de E. coli e associada a diferentes adjuvantes. A proteína gDE7 coadministrada ao poly(I:C) conferiu proteção antitumoral completa aos camundongos previamente desafiados e induziu ativação de linfócitos T CD8+ E7-específicos polifuncionais, citotóxicos e de fenótipo de memória efetora/efetor. Foi demonstrado que a proteína gDE7 ativa de forma específica a subpopulação de células dendríticas especializada na apresentação cruzada de antígenos para linfócitos T CD8+, tanto em camundongos como em seres humanos. Esses resultados abrem perspectivas para o emprego da proteína gD como plataforma vacinal para o controle de tumores induzidos pelo HPV-16. / Cervical cancer is considered a major public health problem and one of the leading causes of cancer death in women. The main goal of this thesis was the improvement of a therapeutic antitumor vaccine based on gDE7 protein in formulations admixed with adjuvants under experimental conditions with the tumor cell line TC-1. The gDE7 protein was expressed and purified from E. coli, and then tested in combination with different vaccine adjuvants. The gDE7 protein admixed with poly(I:C) conferred complete therapeutic antitumor protection to mice previously challenged with TC-1 cells and induced polyfunctional, cytotoxic E7-specific CD8+ T cells with effector/effector memory phenotype. It was also demonstrated that the gDE7 protein activated a specialized dendritic cell subset involved in specific antigen cross-presentation to CD8+ T cells, both in mice and humans. These results open perspectives for the use of the gD protein use as a vaccine platform for the control of HPV-16-induced tumors.