Dissertations / Theses on the topic 'Genital diseases'

Metagenomics is a rapidly evolving field that has facilitated the expansion of microbiology into new areas of human and environmental health. Metagenomic studies have expanded the phylogenetic tree of life by increasing taxonomic resolution in individual phyla as well as adding entirely new branches of life. This revolution in microbiology has been made possible by the introduction of second-generation high-throughput sequencing, the associated methods for preparing DNA sequencing libraries, as well as new bioinformatic algorithms for analyzing these new types of data. Because of the novelty of these methods, very few have been systematically tested for their sensitivities and specificities outside of the initial development process. As the interpretation of metagenomic studies utilizing these tools depends greatly upon their efficiencies in both detection and classification, it is essential to best determine the performance of each tool. In this study, a variety of novel techniques were utilized and tested in their abilities to characterize the microbial populations in two regions of the female genital tract: ovarian cancer tissue and the vaginal microbiome. Although a diverse microbial population was initially observed in the transcriptome sequence data for ovarian cancer using next generation sequencing, we were unable to recover these microbial sequences through PCR and Sanger sequencing approaches. Optimized methods were applied to healthy vaginal microbiome samples and tested for their ability to differentiate them from a polymicrobial disease of the vagina, bacterial vaginosis. In addition to a high correlation between a microbial scoring system for bacterial vaginosis, this novel metagenomic pipeline also revealed microorganisms not yet associated with the vaginal microbiome such as specific Bifidobacteria spp., various bacteriophage, and Debaryomyces. Collectively, both of these studies provide unique insights into each disease as well as illustrate both the limitations and potential of the rapidly growing field of metagenomics.

Vulvar Vestibulitis Syndrome (VVS) is a chronic inflammatory condition affecting the vestibular epithelium of the vulva, which has been estimated to affect 15% of the female population (Goetsch, 1991). Many studies have attempted unsuccessfully, to elucidate the cause of this condition, and few advancesh ave beenm adet owards the understandingo f the associatedin flammatory responseT. he initial, and principal aim of this investigation was to characterise normal vestibular epithelium using electron microscopy. The ultrastructural characteristics of normal vestibular epithelium were compared with closely related epithelia, and with vestibular epithelia from VVS patients. Other aims included an investigation of the epidemiological characteristics of VVS; an assessmenot f vulvar sensitivity over several months, and an evaluation of ketoconazole as a non-invasive treatment for VVS. Transmission electron microscopy, confirmed that vestibular epithelium was non-keratinised, and closely resembled oral and vaginal mucosae. Intermediate cells were predominant, characterised by pale staining cytokeratin filaments and glycogen deposits. Leukocytes were present in small numbers. Using SEM, superficial cells were characterised by an interlacing network of rounded microridges. By comparison, vestibular epithelium from VVS patients demonstrated the presence of numerous, intensely staining, apoptotic-like cells. These cells were associated with membrane bound cytoplasmic lobules and leukocytes of varying types. A similar ultrastructural appearance was observed in post-treatment biopsies. However, apoptotic-like cells appeared heavily vacuolated, and the number of cytoplasmic bodies present was increased. Mature plasma cells, NK-like cells and macrophages were common in the dermis. Leukocyte counts, demonstrated a significantly greater number of leukocytes in the VVS biopsies compared with the controls, however, there was no statistical difference in the number of leukocytes in pre and post-treatment samples. The presence of apoptotic-like cells accompanied by a significant inflammatory cell infiltrate, may suggest a cell signalling defect, resulting in the pain associatedw ith VVS. Treatment with ketoconazolec ream was found to have very little effect on either the number of leukocytes or the frequency of apoptotic-like cells as quantified using image analysis. The epidemiological characteristics of VVS patients were investigated using a structured questionnaire interview. All of the VVS patients interviewed fulfilled the diagnostic criteria established by Friedrich (1987), and epidemiological findings were generally consistent with previous epidemiological reports. Unique to this study, HPV infections were rare, however recurrent Candida infections and cystitis were commonly reported. The 'Vulvar Algesiometer', was designed and developed in Plymouth, to assist diagnosis and assessmenot f VVS patients. Using this equipment, VVS patients demonstrate heightened vestibular sensitivity when compared with control patients. The utilisation of a pain measuring device the 'Vulvar Algesiometer', in accordance with the questionnaire and ultrastructural investigation has formed a novel and balanced approach to the study of VVS. This study has demonstrated several distinct features of VVS which have not previously been described, features which may be important in elucidating the cause of this condition. These features centre around the presence of apoptotic-like cells and associated cytoplasmic bodies which have not previously been described in association with VVS.

Orientadores: Viviane Hermann, Cássio Luiz Zanettini Riccetto
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Introdução: Os slings sintéticos marcaram a transição do tratamento invasivo para o tratamento minimamente invasivo da incontinência urinária de esforço feminina. Técnicas igualmente eficazes, porém com menores riscos de complicações têm sido pesquisadas. Os mini-slings, utilizando incisão única e de acesso exclusivamente vaginal podem representar uma alternativa à técnica de sling tradicional. Objetivo: Avaliar a eficácia do mini sling Ophira para o tratamento da incontinência urinária de esforço feminina. Material e Método: Foram avaliadas 49 mulheres que compareceram ao Ambulatório de Uroginecologia do HC da UNICAMP no período de abril de 2008 a maio de 2009 com queixa clínica de incontinência urinária de esforço. Todas as pacientes foram submetidas a Estudo Urodinâmico pré-operatório e avaliadas através de história clínica, exame físico, urina I e urocultura, teste de esforço, Pad test de uma hora e aplicação do questionário de qualidade de vida UDI-6. A colocação de mini sling Ophira foi realizada sob anestesia local em regime ambulatorial com alta após micção espontânea. As avaliações subsequentes foram realizadas após seis dias e um, três, seis e 12 meses após o procedimento, compreendendo exame físico, Pad test de uma hora e aplicação do UDI-6. A cura objetiva foi avaliada através do Pad test e do teste de esforço. A cura subjetiva foi avaliada pela queixa clínica e pelo questionário de qualidade de vida. Resultados: A análise da percepção subjetiva dos resultados demonstrou que, após 12 meses de seguimento, 37 pacientes (76%) referiram cura da IUE e sete (14%) melhora. O escore do questionário UDI-6, inicialmente com média de 41,29, caiu para 7,24 após 12 meses de seguimento. O Pad test de uma hora apresentou queda de 6,2g no préoperatório para 1g após o término do acompanhamento. Apenas seis pacientes apresentavam teste de esforço positivo no seguimento de 12 meses. Não houve complicações intra-operatórias. Apenas um caso de dor pós-operatória foi observado. Obteve-se taxa de extrusão do sling de 12,2%. Conclusão: O mini sling Ophira representa uma alternativa cirúrgica segura e eficaz para o tratamento da incontinência urinária de esforço feminina, no período de tempo avaliado
Abstract: Introduction: The synthetic slings marked the transition from invasive treatment to minimally invasive treatment of stress urinary incontinence. Techniques equally effective but with fewer risks of complications have been proposed. The minislings, with single incision and accessing exclusively the vaginal route represents an alternative to tradicional sling technique. Objective: To evaluate the efficacy of mini sling Ophira for the treatment of stress urinary incontinence in women. Methods: We evaluated 49 women attending the outpatient clinic of Urogynecology at the HC/UNICAMP from April 2008 to May 2009 with clinical complaints of stress urinary incontinence. All patients were initially submitted to urodynamic investigation, clinical and physical evaluation, urine analysis, stress test, 1-hour pad test and UDI- 6 Quality of life questionnaire. Mini sling Ophira was placed under local anesthesia and patients were dismissed after spontaneous voiding. Evaluation was undertaken 6 days after surgery and 1, 3, 6 and 12 months follow-up. Objective cure was assessed by Pad test and stress test. Subjective cure was assessed by QoL questionnaire. Results: Subjective analysis demonstrated that, after 12 months, 37 (76%) of patients referred themselves as cured and 7 (14%) as improved. UDI score significantly dropped from 41.29 to 7.24 and 1 hour Pad-test significantly decreased from 6.2 g to 1.0 g. Only 6 patients had persistent positive stress test. No intra operative complications occurred and only one patient complained of pain. Mesh erosion rate was 12.2%. Conclusion: Mini sling Ophira represents a safe and effective alternative to female stress urinary incontinence treatment, should the results proved to be long lasting
Mestrado
Fisiopatologia Cirúrgica
Mestre em Ciências

Chlamydia trachomatis is the most prevalent agent of bacterial sexually transmitted infections in the world. However, a profuse number of cases are unreported, as the infection is often asymptomatic. Sequelae such as pelvic inflammatory disease, an increased risk of cervical cancer, premature birth, and perinatal infections in pregnant women can occur. Inflammation occurs in the body in response to infection or injury. Although inflammation can lead to some unwanted secondary effects, such as pain, it serves to return the body to homeostasis by restoring injured tissues and eliminating pathogens. One recently identified connection between the central nervous system and the immune system that regulates inflammation is the cholinergic anti-inflammatory pathway (CAP). In the CAP, pathogen-associated molecular patterns stimulate the vagus nerve to activate the pathway, which ultimately results in acetylcholine (ACh) release, which down regulates inflammation. We hypothesized that genital chlamydial infection would increase the expression of choline acetyltransferase (ChAT), the enzyme that synthesizes ACh, in the female murine genital tract, therefore down regulating inflammation and promoting chlamydial infection. Transgenic female mice carrying a ChAT-promoter driven GFP reporter gene were vaginally infected with C. muridarum. Mice were sacrificed on days 3, 9, 15, and 21 post infection; cervical, uterine horn, and ovarian tissues were removed and embedded in paraffin. Small sections of each tissue were cut and mounted onto slides. The tissue sections were then stained for the expression of ChAT using immunohistochemical techniques. Finally, tissue sections were viewed under a microscope for positive staining and the data was analyzed. The results indicated that there is a significant increase in the number of cells that express ChAT in genital tract of chlamydia-infected mice versus non-infected mice.

Background. In sub-Saharan Africa, which has the highest prevalence of HIV-1 worldwide, most newHIV-1 infections occur by sexual transmission to women. Recent studies in non-human primates have demonstrated that pro-inflammatory cytokine production in the genital tract is necessary for immune cell recruitment and establishment of simian immunodeficiency virus (SIV) infection following vaginal inoculation. The aims of this study were to evaluate the relationships between inflammation in the female genital tract and (i) susceptibility to HIV-1 infection and (ii) subsequent disease progression in women who became infected. Additionally, genital inflammation was investigated as a mechanism for breakthrough HIV-1 infections in women who became infected even though they were using 1% tenofovir (TFV) microbicide. In the systemic compartment, the level of T cell activation and soluble markers of immune activation during HIV-1 infection are associated with disease outcome. Therefore, the relationships between plasma cytokine concentrations during early HIV-1 infection and disease progression were evaluated Methods. The participants of this study included 230 HIV-uninfected women from the CAPRISA 002cohort who were followed longitudinally for HIV-1 infection, 49 women who were enrolled during acuteHIV-1 infection and followed until 12 months post-infection and 166 HIV-uninfected women who were enrolled in the CAPRISA 004 1% TFV microbicide trial (62 of whom later became HIV-1-infected).Cytokine concentrations were measured in cervicovaginal lavage (CVL) and plasma samples from these women using Luminex and ELISA.

An association between persistent Human papillomavirus (HPV) infection in women and cervical cancer has been established. Young women are particularly at risk of acquiring sexually transmitted infections such as HPV because of risky sexual activity and physiological immaturity. While at risk though, young women have been shown to be amenable to health promoting initiatives. There are a small number of international studies concerning adolescent HPV infection and the risk factors associated with infection, but there is currently no evidence on the prevalence and risk factors for HPV in an Australian, sexually active female adolescent population. This study aimed to provide evidence of the prevalence of HPV, risk factors associated with infection and the patterns of sexual activity in a female sexually active, senior high school population in the Australian Capital Territory. Participants in this study were a convenience sample of 161 sexually active 16-19 year old females who had an HPV test who were attending a senior high school in the Australian Capital Territory. Nurses and doctors using a clinical record collected information about sexual and other risk behaviours. Self-obtained vaginal swabs were tested for HPV DNA using the polymerase chain reaction method and genotyping was undertaken. The HPV prevalence in this cohort of young women was 1 1.2%. High-risk genotypes were found in 55.5% and multiple genotypes were found in 38.8%. There was a significant association found between HPV infection and having had more than one male partner with whom vaginal intercourse had occurred in the previous six months. No statistically significant association was found between HPV and the age of coitarche, length of time young women had been sexually active, condom use, and smoking or alcohol intake. A young age at coitarche was common for this group. Smoking and alcohol use was seen in large proportions in this group. This is the first Australian study that has examined the prevalence and risk factors for genital HPV in this demographic group. The HPV prevalence is lower than in international studies in comparable groups, in similar age groups and much lower than in older women both in Australia and overseas. With the comparatively low prevalence comes an opportunity for important public health interventions for this group including routine Pap smears, vaccination against the high-risk types of HPV when this becomes available and strategies for young women to reduce their number of male sexual partners. A substantial amount of young women in this study were sexually active aged under 16 years. Whilst this was not identified as being a risk factor in this study, it is both a health and personal safety issue for these young women. There is a demonstrated need for health promotion strategies for this cohort about the consumption of safe levels of alcohol and for smoking cessation. Further research is recommended that includes a repetition of this study with a larger sample, the use of a prospective study design to identify trends in infection and examination of HPV prevalence and risk factors for a variety of populations.

Além da deficiência física, a diminuição ou perda da sensibilidade geniturinária é um dos maiores impactos para mulheres com Lesão Medular (LM). Devido à perda da mobilidade funcional e as barreiras arquitetônicas, estas muitas vezes não tem acesso aos cuidados adequados para a saúde ginecológica. Como aproximadamente 80% das lesões da medula espinal acometem indivíduos do sexo masculino, os estudos raramente focam as necessidades e questões referentes às mulheres. Objetivo: Avaliar a prevalência de infecções genitais não virais em mulheres com deficiência física por lesão medular, comparativamente às mulheres saudáveis Método: Estudo de corte transversal, caso controle. Foram estudadas 52 mulheres com LM (grupo estudo) e 57 mulheres saudáveis (grupo controle). Todas responderam a um questionário estruturado e foram submetidas à coleta de conteúdo vaginal para pesquisa de Trichomonas vaginalis e leveduras, bacterioscopia com coloração pelo método Gram, cultura geral (meio ágar sangue), cultura para fungos (meio Sabouraud) e coleta de conteúdo endocervical para pesquisa de Chlamydia trachomatis e Neisseria gonorrhorae (reação em cadeia da polimerase) e cultura para Mycoplasmas sp (meios U9, A7). Resultados: As mulheres com lesão medular, comparativamente ao grupo controle, apresentaram maior frequência de Candida sp no exame micológico direto (p= 0,017); entretanto não foi observada diferença estatisticamente significativa na frequência de isolamento de espécies fúngicas entre os grupos. O grupo estudo apresentou maior frequência de isolamento de Escherichia coli (p= 0,002) e de Corynebacterium sp (p= 0,023) e menor frequência de Lactobacillus sp (p < 0,001) em conteúdo vaginal. Em ambos os grupos não foram encontrados casos positivos para Trichomonas vaginalis. A avaliação do escore de Nugent para diagnóstico de vaginose bacteriana demonstrou maior freqüência de flora intermediária (Nugent 4-7) no grupo estudo (p= 0,039). As pesquisas de Chlamydia trachomatis e Neisseria gonorroheae foram negativas em todas as mulheres. Com relação ao isolamento de Mycoplasmas sp, os resultados foram semelhantes em ambos os grupos. Conclusão: A menor freqüência de isolamento de Lactobacillus sp e a maior freqüência de isolamento de Corynebacterium sp e de Escherichia coli na vagina em mulheres com LM, assim como a elevada frequência de flora intermediária pelo escore de Nugent verificada nas mesmas, fortemente sugerem um desequilíbrio da microbiota vaginal, diferente de uma flora dominada por Lactobacillus sp em tais mulheres. Desde que os Lactobacillus sp são essenciais para a manutenção da flora vaginal e a inibição do crescimento de outras bactérias, sua ausência relativa em mulheres com LM pode influenciar a ocorrência de infecções do trato urogenital. Adicionalmente, a mais elevada frequência de detecção de fungos pela microscopia em mulheres com LM sugere que estas podem albergar uma maior concentração vaginal desses microorganismos do que outras mulheres
Besides their physical disability, decreased or absent genitourinary sensitivity has a huge impact in women with spinal cord injury (SCI). Due to the absence of functional mobility and the architectonic barriers these women frequently do not have access to adequate gynecological care. Since about 80% of spinal cord injuries affect men, studies have rarely focused on the needs of women with SCI. Objective: To evaluate the prevalence of non-viral genital infections in women with SCI compared to mobile women. Methods: Fifty two women with SCI (study group) and 57 mobile women (control group) were evaluated in a case-control study. All answered a structured questionnaire and were submitted to the following microbiological tests: fresh examination of vaginal secretions for Trichomonas vaginalis and yeasts, Gram stain, general culture (agar-blood medium), yeast culture (Sabouraud medium) and endocervical sampling for Chlamydia trachomatis and Neisseria gonorrhorae (polymerase chain reaction) and Mycoplasmas sp. (U9, A7 medium). Results: A higher percentage of women with SCI had Candida sp detected by direct mycological examination than did women in the control group (p= 0.017). However there were no significant differences between the two groups in the frequency of yeast-positive cultures. The study group had a higher isolation frequency from the vagina of Escherichia coli (p= 0.002) and Corynebacterium sp (p= 0.023) and a lower frequency of Lactobacillus sp (p < 0.001). In both groups, there were no cases positive for T. vaginalis, C. trachomatis or N. gonorrhoeae. The evaluation of Nugent score for bacterial vaginosis showed a higher frequency of intermediate flora (Nugent score 4-7) in the study group (p= 0.039). Related to Mycoplasma sp isolation, the results were similar in both groups. Conclusion: The lower frequency of Lactobacillus sp isolation and the higher frequency of Corynebacterium sp and Escherichia coli isolation from the vagina in women with SCI, and the higher frequence of intermediate Nugent score, strongly suggests a disequilibrium of the vaginal microbiota away from a Lactobacillus sp dominated flora in these women. Since lactobacilli are essential for maintaining vaginal health and inhibiting growth of other bacteria, their relative absence in women with SCI may influence the occurrence of urogenital tract infections in these women. The higher frequency of yeast detection by microscopy in women with SCI suggests that these women may harbor a higher vaginal yeast concentration than do other women

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
No final da década de 1990 observou-se um aumento significativo de uma nova modalidade de afecção, o prolapso vaginal parcial ou total recorrente não associado à gestação, principalmente em vacas zebuínas. Em razão do alto valor comercial dos animais acometidos, inicialmente, indicou-se as técnicas convencionais como as de Caslick, Bühner ou Flessa, porém sem o sucesso esperado devido à recorrência da alteração. Considerando esta dificuldade, este trabalho objetivou descrever duas novas técnicas cirúrgicas na correção do prolapso vaginal, denominadas vaginectomia parcial e vaginopexia dorsal em vacas. A condução do estudo foi a campo, utilizando-se 812 vacas zebuínas em idade reprodutiva, alojadas em diversas propriedades de diferentes estados brasileiros. Foram selecionados animais que apresentavam a afecção e que mantiveram os parâmetros clínicos de frequência cardíaca, frequência respiratória e temperatura retal, dentro da normalidade. O diagnóstico do prolapso vaginal foi realizado por meio de anamnese e dos sinais clínicos como exposição da vagina pela rima vulvar, tenesmo, inquietação, lesões da porção evertida, retenção urinária e vaginite. A avaliação dos animais permitiu definir o estágio do prolapso, com a finalidade de eleger a técnica cirúrgica mais adequada. Os protocolos anestésicos foram cumpridos, considerando-se a técnica cirúrgica eleita. A vaginectomia parcial foi utilizada para o tratamento do prolapso vaginal de grau 1 e a vaginopexia dorsal para os de grau 2 e 3. Os resultados pós-cirúrgicos das duas técnicas indicaram alta porcentagem de recuperação (93.44% para vaginectomia parcial e 96,14% para vaginopexia dorsal) e baixo número de recidivas (6,25% e 3,66%, para vaginectomia parcial e vaginopexia dorsal, respectivamente, e baixa mortalidade (entre 0,20 a 0,31%), podendo...
At the end of the decade of 1990, a significant increase of a new affection was observed in the field. This occurrence, recurrent partial or total vaginal prolapse is not associated with gestation, is seen primarily in zebu cows where, in extreme cases, resulted in a total prolapse of the vagina, which included exteriorization of the cervix. Given the high commercial value of elite animals presenting this condition, the increase in cases observed led to attempts to solve the condition with conventional techniques such as those of Caslick, Bühner and Flessa. However, reoccurrence of the prolapse was commonly observed soon after the use of these techniques. Considering these difficulties, the aim of the present work was to develop two new surgical techniques employed in the correction of vaginal prolapse, named partial vaginectomy and dorsal vaginopexy in cows. The research was conducted in the field, using 812 zebu cows of reproductive age, maintained in several properties in different Brazilian states. The animals selected had the vaginal alteration while maintaining all clinical parameters of heart rate, respiratory rate and body temperature, all within normal range. The diagnosis of a vaginal prolapse was performed by anamnesis and analysis of the clinical signs, such as visualization of the vagina through the vulva, tenesmus, agitation, lesions in the everted portin and vaginitis. Evaluation of the animals allowed for the definition of stages of the disease, aiding the choice of an adequate surgical technique. Anesthetic protocols were performed taking into consideration the selected surgery. Partial vaginectomy was employed for the treatment of vaginal prolapses of grade 1, whereas dorsal vaginopexy was used for the grade 2 and 3 prolapses. Post-surgical results for both techniques indicated a high percentage of recovery... (Complete abstract click electronic access below)

Orientador: Gilson Hélio Toniollo
Banca: Fabiana Ferreira de Souza
Banca: Antonella Cristina Bliska Jacinto
Banca: César Roberto Esper
Banca: Francisco Guilherme Leite
Resumo: No final da década de 1990 observou-se um aumento significativo de uma nova modalidade de afecção, o prolapso vaginal parcial ou total recorrente não associado à gestação, principalmente em vacas zebuínas. Em razão do alto valor comercial dos animais acometidos, inicialmente, indicou-se as técnicas convencionais como as de Caslick, Bühner ou Flessa, porém sem o sucesso esperado devido à recorrência da alteração. Considerando esta dificuldade, este trabalho objetivou descrever duas novas técnicas cirúrgicas na correção do prolapso vaginal, denominadas vaginectomia parcial e vaginopexia dorsal em vacas. A condução do estudo foi a campo, utilizando-se 812 vacas zebuínas em idade reprodutiva, alojadas em diversas propriedades de diferentes estados brasileiros. Foram selecionados animais que apresentavam a afecção e que mantiveram os parâmetros clínicos de frequência cardíaca, frequência respiratória e temperatura retal, dentro da normalidade. O diagnóstico do prolapso vaginal foi realizado por meio de anamnese e dos sinais clínicos como exposição da vagina pela rima vulvar, tenesmo, inquietação, lesões da porção evertida, retenção urinária e vaginite. A avaliação dos animais permitiu definir o estágio do prolapso, com a finalidade de eleger a técnica cirúrgica mais adequada. Os protocolos anestésicos foram cumpridos, considerando-se a técnica cirúrgica eleita. A vaginectomia parcial foi utilizada para o tratamento do prolapso vaginal de grau 1 e a vaginopexia dorsal para os de grau 2 e 3. Os resultados pós-cirúrgicos das duas técnicas indicaram alta porcentagem de recuperação (93.44% para vaginectomia parcial e 96,14% para vaginopexia dorsal) e baixo número de recidivas (6,25% e 3,66%, para vaginectomia parcial e vaginopexia dorsal, respectivamente, e baixa mortalidade (entre 0,20 a 0,31%), podendo... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: At the end of the decade of 1990, a significant increase of a new affection was observed in the field. This occurrence, recurrent partial or total vaginal prolapse is not associated with gestation, is seen primarily in zebu cows where, in extreme cases, resulted in a total prolapse of the vagina, which included exteriorization of the cervix. Given the high commercial value of elite animals presenting this condition, the increase in cases observed led to attempts to solve the condition with conventional techniques such as those of Caslick, Bühner and Flessa. However, reoccurrence of the prolapse was commonly observed soon after the use of these techniques. Considering these difficulties, the aim of the present work was to develop two new surgical techniques employed in the correction of vaginal prolapse, named partial vaginectomy and dorsal vaginopexy in cows. The research was conducted in the field, using 812 zebu cows of reproductive age, maintained in several properties in different Brazilian states. The animals selected had the vaginal alteration while maintaining all clinical parameters of heart rate, respiratory rate and body temperature, all within normal range. The diagnosis of a vaginal prolapse was performed by anamnesis and analysis of the clinical signs, such as visualization of the vagina through the vulva, tenesmus, agitation, lesions in the everted portin and vaginitis. Evaluation of the animals allowed for the definition of stages of the disease, aiding the choice of an adequate surgical technique. Anesthetic protocols were performed taking into consideration the selected surgery. Partial vaginectomy was employed for the treatment of vaginal prolapses of grade 1, whereas dorsal vaginopexy was used for the grade 2 and 3 prolapses. Post-surgical results for both techniques indicated a high percentage of recovery... (Complete abstract click electronic access below)
Doutor

Orientador: Viviane Herrmann Rodrigues
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Objetivos: Avaliar pelo sistema de quantificação do prolapso dos órgãos pélvicos (POP-Q), preconizado pela Sociedade Internacional de Continência (ICS) os compartimentos vaginais anterior, posterior e apical em mulheres submetidas à colpofixação sacroespinhal para o tratamento do prolapso uterino ou de cúpula vaginal e analisar os sintomas urinários antes e depois da cirurgia. Sujeitos e métodos: Estudo retrospectivo realizado no Setor de Uroginecologia do Hospital Estadual Sumaré da Universidade Estadual de Campinas em 2006. Foram analisados os prontuários de 47 mulheres submetidas à colpopexia sacroespinhal entre março de 2003 e fevereiro de 2006. Foram avaliados os sintomas urinários (incontinência urinária de esforço, urgência, incontinência de urgência, noctúria e enurese noturna) no pré e pós-operatório, considerando-se sintomas presentes ou ausentes e analisados pelo Teste de qui-quadrado de Mc Nemar. Os pontos Aa, Ba, C, D, Ap, Bp, tvl, gh e pb do POP-Q foram avaliados na primeira consulta e na revisão pós-operatória. O Teste de Wilcoxon foi aplicado para comparar os pontos e os estágios do prolapso genital antes e depois da cirurgia. Complicações intra e pós-operatórias foram descritas. Resultados: A média dos pontos do POP-Q no pré e pós-operatório foi respectivamente: Aa (+0,7; -1,7); Ba (+3,2; -1,7); C (+3,2; -7,6); Ap (-0,2; -2,7) e Bp (+2,1; -2,7) (p<0.001). A taxa de cura foi 97,9% para o prolapso apical. Avaliação pré e pós-operatória do compartimento vaginal anterior foi respectivamente: estágio 1 (4,3%; 57,4%), estágio 2 (8,5%; 31,9%), estágio 3 (76,6%; 0%) e estágio 4 (10,6%; 0%). Cistocele ocorreu em 89,4% no pós-operatório. Onze de 12 mulheres que apresentavam urgência miccional tiveram melhora após a cirurgia (p=0,0039) e uma das 45 que não tinham a queixa passou a apresentá-la. Das 8 pacientes que se queixavam de incontinência de urgência, 7 apresentaram remissão do sintoma após a cirurgia (p=0,0082). Houve melhora da noctúria em 7 de 8 casos após a cirurgia (p=0,0399) e 1 dos 39 casos que eram assintomáticos desencadeou o sintoma no pós-operatório. Conclusão: A colpofixação sacroespinhal é um método eficiente para o tratamento do prolapso apical e de parede posterior levando, porém 89,4% das pacientes a apresentarem prolapso de parede anterior estágio 1 e 2 devido ao desvio posterior do eixo vaginal. Ocorreu melhora dos sintomas urinários irritativos (urgência, incontinência de urgência e noctúria) nas pacientes submetidas à fixação sacroespinhal da cúpula vaginal pelo restabelecimento do sistema de sustentação apical posterior
Abstract: Objectives: To evaluate the extent of prolapse of the anterior, posterior and apical vaginal compartments in women undergoing sacrospinous ligament fixation using the pelvic organ prolapse quantification system (POP-Q), recommended by the International Continence Society (ICS) for the treatment of uterine and vaginal vault prolapse and examine urinary symptoms before and after surgery. Subjects and methods: A study was conducted in the Urogynecology Sector of the Sumaré Municipal Hospital of the Universidade Estadual de Campinas in 2006. Medical charts of 47 women undergoing sacrospinous colpopexy between March 2003 and February 2006 were assessed. Urinary symptoms (stress urinary incontinence, urgency, incontinence of urgency, nocturia and nocturnal enuresis) were evaluated in the preoperative and postoperative period, categorizing symptoms as present or absent, and applying the Mc Nemar chi-square test for analysis. Aa, Ba, C, D, Ap, Bp, tvl, gh and pb points of POP-Q were evaluated in the first consultation and postoperative revision. Wilcoxon?s test was applied to compare points and stages of genital prolapse before and after surgery. Intraoperative and postoperative complications were described. Results: Mean POP-Q points in the preoperative and postoperative period were, respectively: Aa (+0.7; -1.7); Ba (+3.2; -1.7); C (+3.2; -7.6); Ap (-0.2; -2.7) and Bp (+2.1; -2.7) (p<0.001). The cure rate was 97.9% for apical prolapse. Preoperative and postoperative evaluation of the anterior vaginal compartment was, respectively: stage 1 (4.3%; 57.4%), stage 2 (8.5%; 31.9%), stage 3 (76.6%; 0%) and stage 4 (10.6%; 0%). Cystocele occurred in 89.4%. Eleven of 12 women with mictional urgency showed improvement of symptom after surgery (p=0.0039) and one of the 45 patients who had no previous complaint, started to suffer from the symptom. Of 8 patients whose complaint was incontinence of urgency, 7 had remission of symptom after surgery (p=0.0082). Nocturia improved in 7 out of 8 cases after surgery (p=0.0399) and the symptom was triggered postoperatively in 1 out of 39 asymptomatic women. Conclusion: Sacrospinous ligament fixation is an efficient method for the treatment of apical and posterior wall prolapse, despite leading to stage 1 and 2 anterior wall prolapse in 89.4% of women due to posterior deviation of the vaginal apex. Improvement in irritative urinary symtoms (urgency, incontinence of urgency and nocturia) took place in patients undergoing sacrospinous ligament fixation of the vaginal vault by reconstitution of the posterior apical support system
Mestrado
Cirurgia
Mestre em Cirurgia

Background: Persistent infection with high risk HPV is a necessary but insufficient cause of cervical cancer. Behavioural, viral and host factors modulate the risk of HPV persistence. In this thesis, I explore the role of the vaginal microbiota, a host factor and the presence of multiple HPV infections, a viral factor in HPV persistence. Considering the limited data on the epidemiology of HPV related diseases in low and middle-countries (LMIC), and the limited success of cervical cancer screening strategies in many LMIC, I provide data on the distribution of HPV related diseases in Nigeria and evaluate the acceptability of innovative strategies to increase cervical cancer screening uptake. Methods/Results: To achieve my aims, I implemented a longitudinal cohort study of 1,020 women in Nigeria. I begin my results chapters with two methodological papers. Attrition is an important consideration for every longitudinal cohort, particularly in LMIC, therefore, I present my findings on attrition, determinants of attrition and practical strategies to ensure low attrition in studies conducted in LMIC. Considering that sexual behaviour is an important potential confounder in all HPV studies, and the reliability of self-reported history is often questioned, I present findings on the test-retest reliability of self-reported sexual behaviour history collected in my study. Having found that attrition levels were low and that self-reported sexual behaviour history was generally reliable within my cohort, I present my findings on the association between the vaginal microbiota and persistent hrHPV; and the role of multiple HPV infections in viral persistence. I found that the vaginal microbiota was associated with persistent hrHPV in HIV negative women, but not in HIV positive women; and that multiple HPV infections did not increase the risk of viral persistence when compared to single HPV infections. Next, I present my findings on the prevalence and incidence of anogenital warts in Nigeria, with additional reports on the prevalence of cervical cancer and other HPV associated cancers using data from two population based cancer registries. Finally, I present my findings on the acceptability of innovative strategies to improve cervical cancer screening uptake in Nigeria. I found that Nigerian women had a favorable attitude to the use of HPV DNA based screening as part of routine antenatal care, however attitudes towards the use of self-sampling techniques for HPV based cervical cancer screening varied by religious affiliations. Conclusion: In my thesis, I was able to systematically investigate the epidemiology of HPV infections in a LMIC. I considered the distribution of HPV related diseases, host and viral determinants of HPV persistence and investigated control strategies to reduce the burden of cervical cancer in a LMIC. My results provide useful data for surveillance, monitoring and evaluation of control programs on HPV and cervical cancer in Nigeria and may be useful to cervical cancer control programs in other LMIC.

L’endometriosi és una patologia ginecològica benigna, la fisiopatologia de la qual és en part desconeguda, que afecta fins a un 10% de les dones en edat reproductiva. Aquesta malaltia representa una gran càrrega econòmica i social, ja que les pacients pateixen, al llarg de la seva vida fèrtil, múltiples cirurgies i hospitalitzacions, baixes laborals i problemes reproductius. S’ha descrit la implicació de factors proinflamatoris, neoangiogènics i procoagulants a la seva persistència i progressió, pel que avui en dia l’endometriosi és considerada una malaltia inflamatòria crònica. L’endometriosi profunda representa el fenotip més agressiu de la malaltia, amb una major severitat de l’afectació cínica i un maneig terapèutic més complex. Per tot això, s’ha postulat que l’endometriosi profunda podria presentar mecanismes fisiopatològics específics que condicionarien un ambient inflamatori superior als altres fenotips de la malaltia i en podrien fer d’ella una entitat diferenciada. En els últims anys, hi ha cada vegada més evidència sobre la implicació de les micropartícules circulants (cMP), el factor tissular i les trampes extracel·lulars de neutròfils o neutrophil extracellular traps (NETs) en funcions relacionades amb l’hemostàsia, la immunitat, la inflamació i l’angiogènesi, i se n’han observat nivells elevats en malalties inflamatòries i trastorns protrombòtics. A més, múltiples estudis descriuen la generació de cMP en procediments quirúrgics degut al dany cel·lular generat i la seva possible relació amb complicacions postquirúrgiques secundàries a mecanismes inflamatoris i protrombòtics; en el cas del tractament quirúrgic de l’endometriosi ovàrica la seva elevació podria estar relacionada amb l’afectació de la reserva ovàrica i la generació d’adherències postquirúrgiques. Tenint en compte tot això, l’objectiu d’aquesta tesi doctoral és l’estudi de les cMP, el factor tissular i els NETs com a nous mecanismes fisiopatològics de l’endometriosi i, en concret, de l’endometriosi profunda, així com també la investigació de la implicació de les cMP com a marcadors de dany tissular secundari al tractament quirúrgic de l’endometriosi. La present tesi doctorat consta de quatre articles publicats a la literatura científica, els resultats globals dels quals han demostrat que les pacients amb endometriosi presenten nivells plasmàtics de cMP i NETs superiors a les pacients sense endometriosi, i que aquests nivells més elevats semblen atribuir-se al subgrup de pacients amb endometriosi profunda. Entre les pacients afectades d’endometriosi profunda, els nivells de cMP han demostrat ser superiors en aquelles amb una major quantitat de teixit endometrial ectòpic. Aquestes troballes reafirmen el concepte de l’endometriosi com una malaltia inflamatòria sistèmica i amplien el coneixement sobre la seva fisiopatologia. Tant les cMP com els NETs podrien, a més, contribuir a l’estat d’hipercoagulabilitat ja descrit en aquestes pacients, per la seva implicació en fenòmens proinflamatoris i protrombòtics. Els nivells superiors de cMP i NETs en el subgrup de pacients amb endometriosi profunda estaria en concordança amb teories recents que descriuen l’endometriosi profunda com una entitat diferenciada amb mecanismes fisiopatològics específics que justificarien la seva major agressivitat i severitat. Per tot això, es podrien considerar les cMP i els NETs com a dos nous mecanismes fisiopatològics de l’endometriosi profunda. Finalment, s’ha detectat un increment transitori superior de cMP posteriorment al tractament quirúrgic laparoscòpic de l’endometriosi ovàrica unilateral mitjançant la tècnica excisional (quistectomia mitjançant stripping) en comparació amb la vaporització mitjançant làser CO2, fet que podria estar en relació amb una major generació d’adherències postquirúrgiques i un major dany del teixit ovàric sa restant. Aquest fet aporta un major coneixement sobre els efectes secundaris de les diferents tècniques quirúrgiques disponibles en l’actualitat per al tractament de l’endometriosi ovàrica i ens permet, per tant, optimitzar el maneig quirúrgic de les pacients amb endometriosi.
Endometriosis is a benign gyneacologic condition, the pathogenesis of which is still under debate. It is now considered a chronic systemic inflammatory condition and several immunological, hormonal and inflammatory factors have been described. In the last few years, new pathogenic mechanisms of endometriosis related to inflammation and coagulation pathways have been described and it has been hypothesized that patients with endometriosis could be in an inflammatory and hypercoagulable state. Among the different subtypes of endometriosis, deep infiltrating endometriosis (DIE) is recognized as the most aggressive form of the disease. Furthermore, DIE could be considered as a specific entity since it seems to present specific pathogenic features in comparison to other endometriosis phenotypes. Higher levels of circulating cell-derived microparticles (cMP), neutrophil extracellular traps (NETs) and tissue factor have been observed in many inflammatory conditions, thrombotic diseases, and malignancy. In fact, these factors have been described to be involved in inflammation, blood coagulation and angiogenesis. Moreover, several recent studies have described the generation of cMP after surgical procedures as markers of cellular damage, and have characterized their potential contribution to postsurgical complications, such as inflammation and thrombosis. This work is composed of four articles that have been published in the scientific literature. Global results obtained from them show increased cMP and NETs plasmatic levels in endometriosis patients, and these levels seem to be attributed to the subgroup of patients with DIE. Among these patients, those with larger cumulative size of endometriotic implants showed higher cMP levels. These findings suggest an increased inflammatory and/or hypercoagulable systemic status in patients with DIE. Furthermore, the comparison between laser ablation and stripping for the surgical treatment of unilateral ovarian endometriomas showed higher but temporary cMP generation in the latter group compared with laser ablation. These results suggest that the stripping technique might trigger a more pronounced short-term inflammatory and procoagulant response, which may contribute to postsurgical complications and may negatively impact on ovarian reserve in these patients.

Quantification of progesterone and 17-β estradiol in mouse serum by liquid chromatography-tandem mass spectrometry Authors: Benjamin Kennard, Allison Cobble, Amy Gravitte, Keleigh Galloway, Jen Kintner, Jennifer Hall, Stacy Brown Introduction: In the United States, Chlamydia trachomatis is a commonly appearing sexually transmitted infection1. It affects the U.S. healthcare system to a tune of about $500 million dollars annually2. In women, it generally appears asymptomatic and can lead to severe secondary complications such as pelvic inflammatory diseases or infertility1. Female sex hormones, estrogen and progesterone, are being identified to have a role in chlamydial infection. Specifically, this study aims to create quantification methods to detect levels of estrogen and progesterone in mice, infected with Chlamydia muridarum, plasma samples. Methods: Progesterone samples were prepared using solid-liquid extraction (SLE+) cartridges with ethyl acetate as the elution solvent. Estradiol samples were prepared using liquid-liquid extraction (LLE) with methyl tert-butyl ether and subsequent derivatization with DMIS. Following sample preparation, hormones were quantified in samples using LC-MS/MS with a gradient elution of 1 mM ammonium fluoride in water and acetonitrile. The separation was achieved using a UCT C18 column (100 x 21.mm, 1.8 μm particle size) maintained at 50oC. The mass spectrometer was set up to isolate molecular ions for progesterone (m/z 315.0910) and derivatized estradiol (m/z 431.1835). Quantification was facilitated by the use of deuterium-labeled internal standards and their corresponding molecular ions in the mass spectrometer (d9-progesterone; m/z 324.1230 and d5-estradiol; m/z 436.2922). Results: Several aspects of the assay presented have been optimized for maximum analyte recovery and analytical sensitivity, including column choice, mobile phase, derivatizing agents for estradiol, and extraction protocols for progesterone. The LC-MS/MS method was investigated for precision and accuracy over three separate days. The dynamic range of the progesterone assay was 5 – 100 ng/mL, with a limit of detection of 1 ng/mL. Likewise, the estradiol assay was linear in the range of 5 – 100 ng/mL, with a limit of detection of 0.5 ng/mL. The average precision, represented by % RSD was 0.74 – 8.5% and 6.3 – 13.4% for progesterone and estradiol, respectively. The accuracy of the method, represented by % error was 1.6 – 14.4% and 4.0 – 10.5% for progesterone and estradiol, respectively. Successful validation was defined as < 15% RSD and error (< 20% at the limit of quantification), per current FDA Guidelines. Conclusions: The developed LC-MS/MS method is specific for progesterone and estradiol, and the extraction is suitable for preparation of mouse serum samples. This assay could be successfully applied to hormone quantification in mouse samples to support the investigation of the link between chlamydia infection and hormone levels in female animals. References 1. Chlamydia - 2017 Sexually Transmitted Diseases Surveillance. https://www.cdc.gov/std/stats17/chlamydia.htm. Accessed October 23, 2018. 2. Owusu-Edusei K, Chesson HW, Gift TL, et al. The Estimated Direct Medical Cost of Selected Sexually Transmitted Infections in the United States, 2008. Sex Transm Dis. 2013;40(3):197-201. doi:10.1097/OLQ.0b013e318285c6d2

Orientador: Rogério Saad Hossne
Coorientador: Sidney Roberto Nadal
Banca: Fábio Vieira Teixeira
Banca: Maria Aparecida C. Arruda Henry
Resumo: O Papiloma Vírus Humano (HPV), é considerado um problema mundial de saúde pública, sendo a doença sexualmente transmissível mais prevalente. Guarda uma relação direta com o risco e a incidência do câncer do canal anal. Seu diagnóstico, tratamento e seguimento são de extrema importância. Neste sentido o escovado do canal anal tem um papel fundamental no rastreamento e seguimento das lesões HPV induzidas e consequente evolução para o câncer anal. Determinar a ocorrência de lesão HPV induzida em mulheres que participam dos programas de prevenção do câncer de colo uterino nas Unidades Básicas de Saúde (UBS) no município de Botucatu. Trata-se de um estudo transversal observacional que teve 228 mulheres submetidas ao escovado do canal anal a fim de estabelecer a ocorrência de lesão HPV induzida e suas correlações com dados sociais e comportamentais. Os 11 casos que apresentaram alteração de ASCUS e LSIL no escovado do canal anal traziam relação com estado civil, baixa escolaridade, não prática do sexo seguro, e a prática do sexo anal
Abstract: Human Papillomavirus (HPV) has been a world concern in Public Health, and it is the most prevalent sexually transmitted disease. It has a direct association with the risk and incidence of cancer in the anal canal. Its diagnosis, treatment and follow-up are extremely important. Using this approach, the smear of the anal canal has a crucial role in the screening and follow up of HPV-induced lesions and in the resulting development of anal cancer. To determine the occurrence of HPVinduced lesions in women who attended programs of uterine cervix cancer prevention in Basic Health Units (BHU) in Botucatu city. It is a cross sectional observational study, in which 228 women underwent brushing of the anal canal in order to establish the occurrence of HPV-induced lesion and its correlation with social and behavioral data. The 11 cases which had ASCUS and LSIL changes in the smear of the anal canal were associated with marital status, low education level, practice of unsafe intercourse and anal intercourse
Mestre

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Espécies de lactobacilos são os principais componentes da microbiota vaginal e a manutenção do predomínio lactobacilar é importante para proteção desse ambiente contra possíveis patógenos. A vaginose bacteriana é uma condição em que se observa a perda de lactobacilos e substituição desses microrganismos por espécies bacterianas, anaeróbias em sua maioria. Tal condição pode acarretar inúmeras complicações ginecológicas e obstétricas, como o aumento do risco de aquisição de infecções sexualmente transmissíveis, parto prematuro e baixo peso ao nascimento. O principal método utilizado para o diagnóstico da vaginose bacteriana é o proposto por Nugent et al. (1991) e se baseia na classificação da microbiota vaginal em flora normal, intermediária e vaginose bacteriana. Enquanto que o perfil imunológico e microbiológico da vaginose bacteriana tenha sido amplamente estudado, pouco se sabe sobre tais características na flora intermediária. Portanto, o objetivo desse estudo foi caracterizar a flora intermediária quanto aos níveis cérvico-vaginais de Interleucina (IL)1-beta, IL-6, IL-8, IL-10, fator de necrose tumoral (TNF)-alfa, antagonista de receptor de IL-1(IL-1ra), sialidases bacterianas e quanto às cargas de Gardnerella vaginalise de bactérias totais, além de verificar se o perfil geral observado na flora intermediária se assemelha ao de mulheres com flora normal ou com vaginose bacteriana. Foi realizado um estudo transversal que incluiu 526 mulheres não grávidas em idade reprodutiva. Deste total, foram constituídos os grupos de estudo de acordo com o padrão de flora vagina, segundo Nugent et al. Todos os 145 casos de vaginose bacteriana foram incluídos nas análises, bem como os 63 casos de flora intermediária e 145 das 318 mulheres que apresentaram flora normal. A determinação dos níveis cérvico-vaginais de citocinas, sialidases e a carga bacteriana foram realizados por, respectivamente, ELISA, ...

Orientador: Camila Marconi
Coorientador: Màrcia Guimarães da Silva
Banca: Andrea da Rocha Tristão
Banca: Ana Katherine da Silveira Gonçalves
Banca: Eliane Melo Brolazo
Banca: Cristina Maria Garcia de Lima Parada
Resumo: Espécies de lactobacilos são os principais componentes da microbiota vaginal e a manutenção do predomínio lactobacilar é importante para proteção desse ambiente contra possíveis patógenos. A vaginose bacteriana é uma condição em que se observa a perda de lactobacilos e substituição desses microrganismos por espécies bacterianas, anaeróbias em sua maioria. Tal condição pode acarretar inúmeras complicações ginecológicas e obstétricas, como o aumento do risco de aquisição de infecções sexualmente transmissíveis, parto prematuro e baixo peso ao nascimento. O principal método utilizado para o diagnóstico da vaginose bacteriana é o proposto por Nugent et al. (1991) e se baseia na classificação da microbiota vaginal em flora normal, intermediária e vaginose bacteriana. Enquanto que o perfil imunológico e microbiológico da vaginose bacteriana tenha sido amplamente estudado, pouco se sabe sobre tais características na flora intermediária. Portanto, o objetivo desse estudo foi caracterizar a flora intermediária quanto aos níveis cérvico-vaginais de Interleucina (IL)1-beta, IL-6, IL-8, IL-10, fator de necrose tumoral (TNF)-alfa, antagonista de receptor de IL-1(IL-1ra), sialidases bacterianas e quanto às cargas de Gardnerella vaginalise de bactérias totais, além de verificar se o perfil geral observado na flora intermediária se assemelha ao de mulheres com flora normal ou com vaginose bacteriana. Foi realizado um estudo transversal que incluiu 526 mulheres não grávidas em idade reprodutiva. Deste total, foram constituídos os grupos de estudo de acordo com o padrão de flora vagina, segundo Nugent et al. Todos os 145 casos de vaginose bacteriana foram incluídos nas análises, bem como os 63 casos de flora intermediária e 145 das 318 mulheres que apresentaram flora normal. A determinação dos níveis cérvico-vaginais de citocinas, sialidases e a carga bacteriana foram realizados por, respectivamente, ELISA, ...
Abstract: Not available
Doutor

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O Papiloma Vírus Humano (HPV), é considerado um problema mundial de saúde pública, sendo a doença sexualmente transmissível mais prevalente. Guarda uma relação direta com o risco e a incidência do câncer do canal anal. Seu diagnóstico, tratamento e seguimento são de extrema importância. Neste sentido o escovado do canal anal tem um papel fundamental no rastreamento e seguimento das lesões HPV induzidas e consequente evolução para o câncer anal. Determinar a ocorrência de lesão HPV induzida em mulheres que participam dos programas de prevenção do câncer de colo uterino nas Unidades Básicas de Saúde (UBS) no município de Botucatu. Trata-se de um estudo transversal observacional que teve 228 mulheres submetidas ao escovado do canal anal a fim de estabelecer a ocorrência de lesão HPV induzida e suas correlações com dados sociais e comportamentais. Os 11 casos que apresentaram alteração de ASCUS e LSIL no escovado do canal anal traziam relação com estado civil, baixa escolaridade, não prática do sexo seguro, e a prática do sexo anal
Human Papillomavirus (HPV) has been a world concern in Public Health, and it is the most prevalent sexually transmitted disease. It has a direct association with the risk and incidence of cancer in the anal canal. Its diagnosis, treatment and follow-up are extremely important. Using this approach, the smear of the anal canal has a crucial role in the screening and follow up of HPV-induced lesions and in the resulting development of anal cancer. To determine the occurrence of HPVinduced lesions in women who attended programs of uterine cervix cancer prevention in Basic Health Units (BHU) in Botucatu city. It is a cross sectional observational study, in which 228 women underwent brushing of the anal canal in order to establish the occurrence of HPV-induced lesion and its correlation with social and behavioral data. The 11 cases which had ASCUS and LSIL changes in the smear of the anal canal were associated with marital status, low education level, practice of unsafe intercourse and anal intercourse

The obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial STD agent in the US. This bacterium has a unique biphasic developmental cycle in which the infectious elementary body (EB) infects a host mucosal epithelial cell and differentiates into the replicative form (the reticulate body or RB) within a modified vacuole called an inclusion. The RB later divides and develops back into an EB and is released, perpetuating the infectious cycle. When developing chlamydiae are exposed to unfavorable environmental conditions, they deviate from the normal developmental cycle into a non-infectious but viable state termed persistence. Previous data from our laboratory indicate that i) during C. trachomatis/HSV co-infection, the chlamydiae become persistent and ii) HSV gD interaction with host cell surface is sufficient to induce this response. During viral entry, HSV gD interacts with one of four host co-receptors, one of which is the host adhesion molecule nectin-1. Interestingly, Western blotting demonstrated that nectin-1 is significantly decreased in C. trachomatis-infected HeLa cells. Additional studies indicated that active C. trachomatis replication is required for nectin-1 down-regulation and nectin-1 is likely down-regulated post-translationally. CPAF, a chlamydia-secreted protease, is responsible for degrading several host proteins. Both in vivo experiments using CPAF-specific chemical inhibitors and cell-free cleavage assays using recombinant CPAF indicate that nectin-1 is degraded by CPAF in C. trachomatis-infected cells. Further studies suggest that nectin-1 is the most likely candidate involved in triggering HSV-induced chlamydial persistence. Co-infection experiments using nectin-1-specific HSV-1 mutants suggest that nectin-1 is, indeed, required for persistence induction. Additional studies in single co-receptor-expressing CHO cells demonstrate that, despite the fact that HSV-1 enters both HVEM- and nectin-1-expressing cells, viral co-infection reduces chlamydial infectivity only in the CHO-nectin-1 cell line. These data confirm that HSV/nectin-1 interaction is sufficient for chlamydial persistence induction. Although nectin-1 ligation is known to activate Cdc42, pull-down assays indicate that Cdc42 is not activated in co-infected HeLa cells. Taken together, these data suggest that: i) HSV gD-nectin-1 binding activates a novel host epithelial cell pathway that restricts chlamydial development and ii) the chlamydiae may degrade nectin-1 to evade this inhibitory host response.

Background: The Division of Global HIV/AIDS at the Centers for Disease Control and Prevention (CDC) is working on a public health evaluation (PHE) in the eastern districts of Botswana. This PHE aims to evaluate the effectiveness of Project AIM, a group-level intervention designed to reduce HIV risk behaviors in youth ages 11 to 14, when combined with the regular Botswana Skills for Life Curriculum, a standard HIV prevention education curriculum in Botswana schools. In order to evaluate Project AIM, a self-report survey and a biological testing for herpes simplex virus type 2 (HSV-2) will be conducted. Methodology: Based on studies done on the psychosocial impact of HSV-2 diagnosis on asymptomatic individuals in the USA, the literature recommends providing pre and post counseling and education to individuals testing for genital herpes to help cope and diminish the psychosocial impact of the diagnosis. In order to prepare Botswana’s clinics and schools participating in the PHE to provide the support for newly diagnosed adolescents with HSV-2, guidance materials were developed for health care practitioners and school guidance teachers. Materials were created using Information Mapping technique to analyze, organize, and present the information, and the Microsoft Office Flesch Kinkade Grade Level (FK) tool to assess the readability levels of the materials. Results: Guidance materials were prepared using the 7±2 theoretical limit of human short-term memory information mapping rule, and the FK grade levels of 6.0 to 8.0 recommended readability scores. Guidance materials included information regarding HSV-2 symptoms, treatment, and prevention. They also included information on the PHE study, youth friendly health services, counseling and education, clinic referrals and contact information. Conclusions: The development of guidance materials for schools and clinic participants of the CDC PHE in Botswana will provide health practitioners and school guidance teachers with accurate HSV-2 information to counsel and educate student participants in this research study. The guidance materials should help students cope with potential psychosocial disorders associated with pre and post diagnosis of HSV-2.

The Polymerase Chain Reaction technic was applied to the epidemiological and immunopathological study of Chlamydia trachomatis genital infections. Compared to a reference method combining cell culture and PCR, the PCR technic shows a sensitivity of 93. 6% and a specificity of 83. 8%, while cell culture presents a sensitivity of 47. 6% and a specificity of 98%. One hundred and seventy nine clinical isolates of C. Trachomatis were serotyped using the microimmunofluorescence method and genotyped by Restriction Fragment Length Polymorphism. One hundred and forty eight strains (82. 7%) gave similar results by the two technics, but 31 strains (17. 3%) gave discrepant results. These discrepancies are explained by a lack of specificity of the monoclonal antibodies. The epidemiological results obtained by RFLP are in agreement with the results observed in different countries at different time points. In order to find new epidemiological markers, we demonstrated the presence of repetitive extragenic palindromic sequences (REP) in C. Trachomatis chromosome. The Rep-PCR amplification pattern of C. Trachomatis differs from the amplification patterns of other bacteria. The PCR was used to detect cytokines mRNA in the genital tracts of mice infected with a human strain of C. Trachomatis. We detected an increase in the transcription of IL-1a, IL-1b, IL-2, IL-6, IL-10, IL-12, TNFa, IFNg and Macrophage Inflammatory Protein genes at D4 post-infection in infected mice compared to normal mice. Transcription rates of IL-2, IL-12 and IFNg remain high at D14 while the transcription rates of the other cytokines return to a baseline level. Therefore, C. Trachomatis infection seems to induce a Th1 immune response in vivo in a mouse model.

Cette these presente une etude sur l'utilisation des anticorps monoclonaux radiomarques dans un but therapeutique en cancerologie. Des etudes de biodistribution de l'anticorps monoclonal oc125, qui possede une affinite specifique pour les cancers ovariens, ont ete realisees dans un modele animal greffe dans la cavite peritoneale avec un carcinome ovarien humain afin de determiner differents parametres tel que la specificite in vivo de l'anticorps, le mode d'injection et le type de radioelement. Des etudes de biodistribution chez les malades de l'oc125 marque a l'in111 sont analysees

The purpose of this study was to investigate women's knowledge and attitudes regarding genital human papillomavirus (n=100). Using a descriptive design, the Health Education Questionnaire was administered to 100 female patients (Mean Age = 33, SD = 7.17) at a physicians office in South Florida. The results indicated a lack of knowledge regarding genital human papillomavirus with 21 patients (21%) reported having knowledge and 79 (79%) having never heard of this disease. In addition, the group familiar with genital human papillomavirus also possessed a low level of knowledge with only 57% acknowledging an association of genital human papillomavirus and cervical cancer, 52% aware that a pap smear can detect the virus, 42% knowing that antibiotics can not treat the disease and 57% aware that it is not associated with a family history. An association was found between attitudes and health seeking behaviors. Subjects stating that they would take all measures to prevent genital human papillomavirus, were more likely to have a pap smear within the last year (Chi-square (1) = 4.33, p < .05). Higher levels of education and income were associated with increased knowledge regarding genital human papillomavirus when subjects were categorized according to sociodemographic characteristic (Chi-square (1) =9.45, p < .05; Chi-square (1) = 6.75, p < .05). There was no significant correlation between knowledge and ethnicity, marital status or age. Findings indicated the need for improved education and promotion of positive attitudes regarding human papillomaviurs in order to improve health seeking behaviors among women.

Introdução: O termo distúrbios do desenvolvimento gonadal (DDG) inclui condições congênitas nas quais o desenvolvimento gonadal é atípico. Estudos feitos em camundongos observaram que alguns genes como o Cbx2 e o Tcf21 interferem na fase inicial do desenvolvimento gonadal, afetando tanto gônadas XX quanto XY. O gene Dhh, por sua vez, codifica o fator de transcrição Dhh, produzido pelas células de Sertoli, que é fundamental para a diferenciação das células de Leydig em gônadas XY. Nos ovários, o gene FOXL2 atua na foliculogênese, sendo fundamental para a formação dos ovários. Objetivos: Analisar clinicamente e pesquisar anormalidades nos genes CBX2, TCF21, DHH e FOXL2 em pacientes portadores de distúrbios do desenvolvimento gonadal 46, XY e 46, XX. Material e Métodos: Foram estudados 60 pacientes (41 com DDG 46, XY e 19 com DDG 46, XX). A análise molecular foi realizada a partir da amplificação gênica por PCR e sequenciamento direto. Resultados: Várias alterações alélicas foram encontradas nos quatro genes, algumas ainda não descritas na literatura. Uma alteração intrônica no gene DHH foi encontrada em um paciente com DDG 46, XY e não foi encontrada em nenhum dos 360 alelos normais estudados (g.IVS2 +29G>A). Estudamos essa variante através da extração do RNA do testículo do paciente afetado, mas não encontramos alteração no RNA; portanto ela parece não ser uma mutação. No gene TCF21, a variante encontrada foi identificada em controles normais. No gene CBX2, das treze alterações encontradas, uma não foi identificada em 206 alelos normais, e há troca de aminoácidos (p.C132R / g.394 T>C). Trata-se de uma variante que pode ter relação com o fenótipo do paciente, portador de DDG 46, XY. No gene FOXL2, das três alterações encontradas, uma não foi identificada em 206 alelos normais; contudo, não há troca de aminoácidos (p.A181A / g.543 C>T). Conclusão: Esse estudo sugere que mutações nos genes CBX2, TCF21, FOXL2 e DHH são causas raras de distúrbios do desenvolvimento gonadal.
Introduction: Congenital disorders of gonadal development (DGD) include conditions whose gonadal development is atypical. Studies in mice found that some genes such as Cbx2 and Tcf21 interfere in the initial phase of gonadal development, affecting both XX and XY gonads. Dhh gene, in turn, encodes the transcription factor Dhh, produced by Sertoli cells, which is essential for the differentiation of Leydig cells in XY gonads. In the ovaries, genes as FOXL2 act in folliculogenesis, fundamental to the development of the ovaries. Objectives: To analyze patients with disorders of gonadal development (DGD) 46, XY and 46, XX and research mutations in CBX2, TCF21, DHH and FOXL2 genes. Methods: We analyzed 60 patients (41 DGD 46, XY patients and 19 DGD 46, XX patients). The whole coding region of CBX2, TCF21, DHH and FOXL2 genes were amplified by PCR and direct sequenced. Results: Several allelic variations have been found in the four genes, some not even described by literature. One intronic variation in DHH was described in one patient with 46, XY DGD and it wasnt found in any of the 360 normal control alleles studied (g.IVS2 +29G>A). We studied this variant through RNA extraction from the affected patients testes, but we didnt find any alteration in the RNA, so it doesnt seem to be a mutation. In TCF21 gene, the single variant that was found was identified in normal controls. In CBX2 gene, among the 13 alterations described, one wasnt identified in 206 normal control alleles, and there is aminoacid change (p.C132R / g.394 T>C). This is a variant that may be a mutation, causing the patients phenotype that had 46, XY DGD. In FOXL2, among the 3 variations described, one wasnt indentified in 206 normal control alleles, but there wasnt amino acid change (p.A181A / g.543 C>T).Conclusion: This study suggests that mutations in CBX2, TCF21, FOXL2 and DHH genes are rarely causes of disorders of gonadal development.

The ability of sperm cells to induce specific auto- and iso-immunity was reported as early as the turn of the century. However the mechanism controlling autoreactivity to sperm is not well known. As lymphocytes constitute the major cellular components of the immune system, determination of their anatomical location within the tissues of the male genital tract may be of considerable importance in understanding immunological infertility and other urogenital disorders. A series of monoclonal antibodies that react with human lymphoreticular cells was therefore used in an indirect immunoperoxidase technique to study their distribution throughout the male genital tract. The normal human tissues investigated were: testis, epididymis, vas deferens, prostate and seminal vesicles obtained from multiorgan transplant donors. The clinical specimens examined included surgical biopsies of testis, epididymis and prostate obtained during surgical procedures directed at the investigation and treatment of subfertile males and other patients. All normal tissues, apart from the testis, were found to contain appreciable numbers of T-lymphocytes (leu 4⁺). T-cells of the suppressor/cytotoxic phenotype (leu 2a⁺) were more abundant in the intraepithelial compartment while T-cells of the helper/inducer phenotye (leu 3a⁺) were more common in the interstitial areas. With the exception of the prostate, very few B-cells were observed. Macrophages (leu M3⁺) were identified within normal testicular tissues as well as the rest of the male genital tract. HLA-DR⁺ cells were also identified and the HLA-DR antigens were normally expressed on the lining epithelium of the rete testis, epididymis and vas deferens. Derangement of this pattern was observed in clinical specimens. Testicular biopsies from patients with testicular obstruction showed marked infiltration with lymphocytes mainly of the T-cell type. Biopsies from patients with benign prostatic hyperplasia showed increased infiltration with the helper/inducer T-cells and other cell types such as natural killer cells (leu llb⁺) and activated T-cells (IL₂-r⁺). These patterns of lymphoid cells distribution could provide an insight into both normal immunohomeostatic mechanisms and pathological events within the male genital tract. The presence and distribution of lymphocyte subpopulations and macrophages within human urothelium in health and disease was also examined. T-lymphocyte subsets and macrophages were identified in normal urothelium and shown to have a similar pattern of distribution to that seen in normal epithelium of the genital tract. The existence of these cell populations may contribute to the health and protection of urothelium, particularly in resistance to infection and tumour surviellance. The presence of leucocytes and their subpopulations was also studied in the ejaculate from 69 men with an infertile marriage and 12 fertile men. Leucocytes were found in large numbers in the fertile men compared with the patients. Lymphocytes were found in 20% of the patients. There was no correlation between leucocyte counts and growth of micro-organisms. These results cast doubt on the conventional criteria of subclinical genital tract infection, namely positive culture and excess leucocyte counts.

The aim of this study was to explore the impact of chlamydial infection from the perspective of the individual. One-off, unstructured interviews were conducted, either in the Genitourinary Medicine or the Family Planning Clinic, with 50 individuals (40 females and 10 males) who had contracted this sexually transmitted infection (STI). A grounded theory approach was used. STI's have long since been associated with moral reprobation and social sanctioning, particularly in relation to females. They are diseases that other people get, associated with specific types and behaviours. This legacy has shaped their institutional management and it largely determined the individual and interpersonal responses of the study participants. However this effect was modified by the specific location of chlamydia within the hierarchy of STI's where ranking occurs primarily on the basis of curability and visibility. In these terms chlamydia was classed as a 'little' infection. On an individual level, diagnosis of infection was strongly associated with a sense of discordance and a spoiled identity, commonly expressed as feelings of dirt and contamination. Some felt a need to feel clean following infection; the test of cure fulfilled this function marking the transition from liminality. Intentions to prevent reinfection centred on routine or relationship based strategies. Long term concerns were limited to possible female infertility. On an interpersonal level, information control decisions were influenced by fear of disapproval and potential threat to social reputation. Notification of sexual partners, which is necessary to prevent re-infection of self and infection of others, was fraught with anxieties. lt created opportunities for moral positioning and was associated with accusations of culpability and intent. The health interface influenced responses to infection. The female experience commonly included management in primary care which was associated with insensitive management and inadequate information. The GUM clinic produced anxieties concerning usage but attendance was associated with confidence in contained and comprehensive infection management. These findings are discussed in relation to policies and practices that focus disproportionately upon women, particularly the chlamydia screening programme.

Chlamydia trachomatis is a human pathogen of the genital tract and ocular epithelium. It is an obligate intracellular parasite with a unique biphasic development cycle. C.trachomatis infection is the most common bacterial sexually transmitted disease in industrialized nations. Its ability to cause chronic disease makes it a serious economic burden and health threat to developed and developing countries. Although treatable, approximately 70% of C.trachomatis infections are asymptomatic, potentially leading to the development of chronic sequelae. Furthermore, chlamydial genital tract infection has been associated with an increased risk of cervical cancer and human immunodeficiency virus infection. Consequently, the development of an efficacious vaccine is the most convenient, potentially reliable and cost effective option to control chlamydial infection and disease complications. Anti-chlamydial protective immunity is essentially mediated by a T helper, type 1 (Th1), response that is dependent upon the presentation of antigen via major histocompatibility (MHC) class II molecules. While antibody secreting cells are not critical components of the primary effector response, they have been shown to be important for clearance of re-infection. Thus an ideal vaccine would be one capable of inducing both a strong Th1 T cell response and a strong mucosal antibody response. Currently there are very few efficacious vaccine candidates that have been identified and characterized. More specifically, there is only a limited number of known T cell antigens processed and presented by the human leukocyte antigen (HLA) class II molecules. This type of antigen is going to be essential to the development of an efficacious chlamydial vaccine. In this study we have identified a number of unique vaccine candidates using a novel in silico approach. In an attempt to overcome HLA polymorphism the whole chlamydial genome was screened for proteins containing epitopes predicted to bind multiple HLA class II molecules (i.e. predicted ‘promiscuous’ T cell epitopes). A wide range of HLA class II molecules were used in this screen to identify vaccine antigens that could potentially offer broad and ethnically balanced population coverage. This analysis identified a number of novel targets and was validated by the identification of a known chlamydial T cell epitope. A selection of these target proteins was cloned, expressed and purified. Recombinant protein was screened against serum samples from patients with both acute and chronic chlamydial infections. Two novel targets, hypothetical protein CT425 and ribonucleotide reductase small chain protein (NrdB) were identified as being immunoreactive. The in vivo protective efficacy of NrdB was analyzed using a mouse model. CD4+ T cells were harvested from NrdB immunized mice and adoptively transferred to naïve mice, which were subsequently infected at the genital site. NrdB immunization was found to confer a CD4+ T cell driven degree of protection similar to that seen with CD4+ T cells primed from a live challenge. The adjuvants and route of immunization used ensured immunological responses were initiated at both the systemic and local sites of infection. Immunization elicited a predominant Th1 response with primed T cells producing high levels of interferon gamma, an essential requirement for the development of an efficacious chlamydial vaccine. Furthermore, high titres of antigen specific IgG and IgA were produced following immunization, with sera derived antibodies demonstrating neutralization properties. NrdB is a highly conserved chlamydial protein with an essential role in the replication of chlamydiae and could play a central role in a multi-subunit vaccine against chlamydial genital tract infections.

Chlamydia trachomatis is a human pathogen of the genital tract and ocular epithelium. It is an obligate intracellular parasite with a unique biphasic development cycle. C.trachomatis infection is the most common bacterial sexually transmitted disease in industrialized nations. Its ability to cause chronic disease makes it a serious economic burden and health threat to developed and developing countries. Although treatable, approximately 70% of C.trachomatis infections are asymptomatic, potentially leading to the development of chronic sequelae. Furthermore, chlamydial genital tract infection has been associated with an increased risk of cervical cancer and human immunodeficiency virus infection. Consequently, the development of an efficacious vaccine is the most convenient, potentially reliable and cost effective option to control chlamydial infection and disease complications. Anti-chlamydial protective immunity is essentially mediated by a T helper, type 1 (Th1), response that is dependent upon the presentation of antigen via major histocompatibility (MHC) class II molecules. While antibody secreting cells are not critical components of the primary effector response, they have been shown to be important for clearance of re-infection. Thus an ideal vaccine would be one capable of inducing both a strong Th1 T cell response and a strong mucosal antibody response. Currently there are very few efficacious vaccine candidates that have been identified and characterized. More specifically, there is only a limited number of known T cell antigens processed and presented by the human leukocyte antigen (HLA) class II molecules. This type of antigen is going to be essential to the development of an efficacious chlamydial vaccine. In this study we have identified a number of unique vaccine candidates using a novel in silico approach. In an attempt to overcome HLA polymorphism the whole chlamydial genome was screened for proteins containing epitopes predicted to bind multiple HLA class II molecules (i.e. predicted ‘promiscuous’ T cell epitopes). A wide range of HLA class II molecules were used in this screen to identify vaccine antigens that could potentially offer broad and ethnically balanced population coverage. This analysis identified a number of novel targets and was validated by the identification of a known chlamydial T cell epitope. A selection of these target proteins was cloned, expressed and purified. Recombinant protein was screened against serum samples from patients with both acute and chronic chlamydial infections. Two novel targets, hypothetical protein CT425 and ribonucleotide reductase small chain protein (NrdB) were identified as being immunoreactive. The in vivo protective efficacy of NrdB was analyzed using a mouse model. CD4+ T cells were harvested from NrdB immunized mice and adoptively transferred to naïve mice, which were subsequently infected at the genital site. NrdB immunization was found to confer a CD4+ T cell driven degree of protection similar to that seen with CD4+ T cells primed from a live challenge. The adjuvants and route of immunization used ensured immunological responses were initiated at both the systemic and local sites of infection. Immunization elicited a predominant Th1 response with primed T cells producing high levels of interferon gamma, an essential requirement for the development of an efficacious chlamydial vaccine. Furthermore, high titres of antigen specific IgG and IgA were produced following immunization, with sera derived antibodies demonstrating neutralization properties. NrdB is a highly conserved chlamydial protein with an essential role in the replication of chlamydiae and could play a central role in a multi-subunit vaccine against chlamydial genital tract infections.

The Book of Leviticus has been described as the ‘first hygiene text’ based upon the observation that Leviticus contains a great deal of matter relating to two conditions. The first is צרעת translated in the Septuagint as λέπρα and confused in English translations with modern leprosy. The second, זוב was misused as a generic term for a whole spectrum of genital discharges. Apart from these, Leviticus contains nothing of a ‘medical’ nature. The question arises, as to whether these terms implied any sort of medical context or whether their only significance was as markers of ritual impurity to the priesthood. In Chapter 1 this question is developed and an hypothesis arrived at. A hermeneutic and methodology for the study are introduced and discussed. Chapter 2 is a review of the state of developing ‘medical practice’ in the Ancient Near East. Chapter 3 is concerned with the ideology of the levitical priesthood and their worldview in particular in respect of the establishment and operation of practice of ritual. Chapter 4 treats on the Levitical notion of impurity considered from both taxonomical and sociological standpoints and these approaches are discussed in the context of the present study. Chapters 5 and 6 each contain a detailed ‘medical exegesis’ of chapters 13 and 15 of Leviticus dealing with צרעת and .זוב Chapter 7 contains a similar treatment of the biblical notion of blemish and addresses the question of whether this was a mark of impurity like צרעת and .זוב In Chapter 8 embodies idea of contagion in the context of the ‘hygienic’ theme in Leviticus and the priests’ concern with what might imperil sacred objects. Chapter 9 employs context logometrical analysis in a detailed study of the word צרעת and whether there was, in Ancient Israel, any relationship, adverse or synergic between the activities of the priests in preserving purity, and early healthcare practice. Chapter 10 is a discussion of how צרעת has been seen from a theological perspective. While the exact nature of צרעת remains unknown, its biblical context — levitical and non levitical — is considered in relation to modern theories of the relationship of the impurity laws, sin and the wholeness↔ healthcare dynamic. Chapter 11 is a presentation of the conclusions that may be drawn from this study in respect of the wholeness↔ holiness paradigm posited in the hypothesis. It is concluded that there is no clear evidence to suggest that the priesthood saw צרעת and זוב in any terms commensurate with modern pathology and clinical medicine. Consequently it would be wrong to suppose, as many authors have, that in the levitical context, countermeasures to these conditions, though diagnostic, were hygienic in the modern, medical, — they were not, nor were they ever envisaged to be. That some of these measures subsequently found a significant place in preventive medicine appears to have been both fortuitous and fortunate.

Le malattie mitocondriali sono disturbi genetici caratterizzati da difetti di fosforilazione ossidativa causati da mutazioni nel DNA mitocondriale, o in geni nucleari i cui prodotti sono legati alla fosforilazione ossidativa o alla biologia mitocondriale. La prima parte del progetto è stata focalizzata sulla generazione e la caratterizzazione di un modello murino di malattia mitocondriale, Ttc19ko. I pazienti con mutazioni in TTC19 sviluppano danni neurologici e deficit di complesso III. Ttc19 è una proteina mitocondriale che sembra essere coinvolta nell’assemblaggio e/o stabilità del complesso III. Abbiamo dimostrato che il modello murino Ttc19ko ha sintomi neurologici, debolezza muscolare e riduzione dell’attività locomotoria spontanea, in analogia con la malattia umana. L’analisi istologica ha rivelato alcune anomalie neurologiche con presenza di accumuli di ubiquitina e GFAP. I topi Ttc19ko hanno una riduzione complessiva del metabolismo energetico, una diminuzione del consumo di O2 e di produzione di CO2. L’attività enzimatica del complesso III è significativamente ridotta nei tessuti e ciò è legato ad un aumento della produzione di ROS. L’analisi BNGE ha mostrato una riduzione della incorporazione della subunità catalitica Rieske nel complesso assemblato. L’immunoprecipitazione di TTC19-Flag in colture cellulari trattate con amminoacidi marcati ha rivelato una maggiore interazione tra Ttc19 e le subunità del pre-complesso III, e una minore interazione con proteine Rieske e Uqcrh, entrambe assemblate tardivamente. Abbiamo inoltre dimostrato che Ttc19 rimane associata al complesso III assemblato. Nell’insieme, questi risultati suggeriscono che Ttc19 è un fattore intrinseco di assemblaggio del complesso III che interagisce con il pre-complesso III facilitando così l'incorporazione della proteina Rieske. La seconda parte del progetto è stata focalizzata sulla messa a punto di una terapia genica su un altro modello murino di malattia mitocondriale, MPV17ko. Mutazioni in MPV17 causano una sindrome epatocerebrale con deplezione del mtDNA, insufficienza epatica a esordio precoce, gravi crisi ipoglicemiche e morte. Il trapianto di fegato e l'alimentazione frequente a base di carboidrati a lento rilascio sono le uniche terapie disponibili, anche se in seguito si sviluppano danni neurologici. Il ruolo fisiologico di MPV17 non è chiaro. Abbiamo dimostrato che MPV17 fa parte di un complesso ad alto peso molecolare a composizione sconosciuta, che è essenziale per il mantenimento del mtDNA nel fegato. In dieta standard, il topo MPV17-/- non mostra quasi alcun sintomo di disfunzione epatica, ma una dieta chetogenica porta questi animali a sviluppare cirrosi e insufficienza epatica grave. Tuttavia, quando l'espressione di MPV17 è ripristinata dalla somministrazione di virus adeno-associato, si assiste ad un ricostituzione del complesso supramolecolare contenente Mpv17, ad un ripristino completo del numero di copie di mtDNA, ed alla prevenzione dell’insufficienza epatica indotta dal dieta chetogenica. Questi risultati aprono nuove prospettive terapeutiche per il trattamento delle sindromi da deplezione del mtDNA indotte da mutazioni nel gene MPV17.
Mitochondrial diseases are genetic disorders characterized by defects in oxidative phosphorylation caused by mutations in mitochondrial DNA, or in nuclear genes whose products are related to oxidative phosphorylation or mitochondrial biology. The first part of the project was focused on the generation and characterization of a mouse model of mitochondrial disease, Ttc19ko. Patients with mutations in TTC19 were characterized by neurological impairments and mitochondrial respiratory complex III deficiency. Ttc19 is a mitochondrial protein that seems to be associated to complex III assembly and/or stability. We showed that Ttc19ko mice have neurological symptoms, muscular weakness and reduction in spontaneous locomotors activity, clearly resembling the human disease. Brain also had neurological abnormalities with presence of ubiquitin and GFAP positive staining. Comprehensive lab animals monitoring system revealed a reduction in O2 consumption, CO2 production and energy expenditure in Ttc19ko mice, indicating an overall reduction of energy metabolism. Complex III activity was significantly reduced in tissues and this was linked to an increased ROS production. BNGE analysis of mitochondrial complex III showed a substantial reduction of the incorporation of the catalytic Rieske iron-sulfur protein into the fully assembled complex. A stable isotope labelling by amino acids in cell culture (SILAC) expressing TTC19-Flag followed by immunoprecipitation and mass spec analysis revealed a higher scored interaction between Ttc19 and the subunits of the pre-complexIII, and a lower scored interaction with Rieske protein and Uqcrh, both of them are late assembled subunits. We also demonstrated that Ttc19 is associated to the fully assembled complex III. Taken together, these results suggests that Ttc19 is an intrinsic assembly factor of complex III that interacts with the pre-complex III thus facilitating the incorporation of the late assembled Rieske protein. The second part of the project was focused on a gene therapy approach on a second mouse model of mitochondrial disease, MPv17ko. Mutations in hMPV17 cause a hepatocerebral form of mtDNA depletion syndrome hallmarked by early-onset liver failure, leading to premature death. Liver transplantation and frequent feeding using slow-release carbohydrates are the only available therapies, although surviving patients develop slowly progressive neuropathy. The physiological role of Mpv17 is still unclear. We showed that Mpv17 is part of a high molecular weight complex of unknown composition, which is essential for mtDNA maintenance in liver. On a standard diet, Mpv17ko mouse shows hardly any symptom of liver dysfunction, but a ketogenic diet leads these animals to liver cirrhosis and failure. However, when expression of human MPV17 is carried out by adeno-associated virus mediated gene replacement, the Mpv17ko mice are able to reconstitute the Mpv17-containing supramolecular complex, restore liver mtDNA copy number and oxidative phosphorylation proficiency and prevent liver failure induced by the KD. These results open new therapeutic perspectives for the treatment of MPV17-related liver-specific MDS.

La temperatura corporea costituisce un importante meccanismo di difesa ed è il risultato di una complessa interazione che coinvolge numerosi fattori. Nell'uomo sano, la temperatura corporea è finemente regolata; deviazioni di 0.5°C oltre il limite superiore possono indicare una condizione patologica. Numerosi agenti possono indurre ipertermia, tra cui, insufficienza cardiaca acuta/cardiomiopatia acuta da stress [1] e infarto miocardico acuto [2] sindrome neurolettica maligna [3], endocrinopatie [4, 5], disturbi del sistema nervoso centrale (SNC) [6] e patologie oncologiche [7]. Le temperature febbrili aumentano l'efficacia della risposta immunitaria durante le infezioni stimolando il sistema immunitario innato e adattativo. Questo studio ha come obiettivo quello di dimostrare come l'ipertermia possa indurre modifiche del profilo di espressione genica e di evidenziare nuovi marker precoci di prognosi/diagnosi in patologie autoimmuni e/o tumorali. Nelle cellule dendritiche, alcuni tra i geni up-regolati codificano per proteine secrete, come IGFBP6 [8]. In condizioni ipertermiche, questa proteina induce chemiotassi dei monociti, dei linfociti T, ma non dei linfociti B. Inoltre, IGFBP6 è un agonista selettivo nei neutrofili poiché aumenta sia il burst ossidativo che la degranulazione e agisce come fattore chemotattico.
Body temperature is an important defense mechanism and is the result of a complex interaction of many factors. In healthy human, the body temperature is regulated very carefully; deviations of 0.5°C above the upper limit of normal are considered to be significant indications of disease. Numerous elements may induce febrile conditions, including acute heart failure/stress cardiomyopathy [1] and acute myocardial infarction [2] neuroleptic malignant syndrome [3], endocrinopathies [4, 5], central nervous system (CNS) disorders [6] and oncological diseases [7]. Febrile temperatures increase the effectiveness of the immune response during infections by stimulating both the innate and adaptive arms of the immune system. The aim of this study is to demonstrate how hyperthermia can induce changes in the gene expression profile and highlight new early markers of prognosis/diagnosis in autoimmune and/or tumor pathologies. Among the up-regulated genes in dendritic cells, some encode secreted proteins, such as IGFBP6 [8]. This protein may have a functional role in the hyperthermic conditions as chemoattractant factor in monocytes and T cells, but not in B cells. Moreover, IGFBP6 is a selective neutrophil agonist, increasing oxidative burst and degranulation, as well as functioning as a chemotactic factor.

Kidney lesions can primarily involve the glomeruli, tubulointerstitium, or renal vessels. However, regardless of the initiating site of injury, all compartments often eventually become affected. Tubulointerstitial damage (TID) plays a central role in the progression of renal diseases, leading to an irreversible decline in renal function and ultimately resulting in end-stage renal disease (ESRD). TID is characterized by loss of renal tubules, increased number of interstitial myofibroblasts, and accumulation of extracellular matrix (ECM) in the interstitium usually with chronic inflammatory cell infiltrate. This project aimed at investigating different aspects of the progression of chronic TID in canine renal diseases. The first part of the project consisted in a morphological study describing the progression of renal lesions in canine Leishmaniosis possibly representing a model of TID progression in infectious immune-mediated glomerulonephritis. Renal biopsies taken at the beginning and after a 60-day period specific leishmanicidal treatment were evaluated. Progression of the TID was overall mild but present in half of the dogs especially those that had severe TID already at the first biopsy. The results further confirmed that the progression of the chronic TID is independent from the persistence of the causative agent. Moreover, elimination of the etiological agent, by means of the leishmanicidal treatment is not responsible for a significant improvement of renal lesions when these are severe and include irreversible changes like the presence of obsolescent glomeruli andd fibrosis. In contrast the positive effect of the treatment seems to occur in case of mild TID and is possibly related to the reduction of the inflammatory component. The second part of the project focused on the role of tubular epithelial cells (TECs) in the progression of chronic TID. Morphological diagnosis, severity of inflammation, interstitial fibrosis, HLA-DR expression by TECs and clinicopathological variables were compared in renal biopsies from dogs with spontaneous kidney diseases of varying severities and etiologies. Fibrosis, HLA-DR expression, serum creatinine concentration (SCr), and urine protein-to-creatinine ratio (UPC) were all increased in dogs with primary glomerular disease compared with dogs with acute tubular necrosis. HLA-DR expression by TECs was positively correlated to fibrosis, inflammation, UPC, and SCr. The study provided evidence of the capacity of TECs of acting as non-professional antigen presenting cells (APCs) in chronic TID, identifying a potential causative effect of the proteinuria. Moreover, this expression of TECs seems to precede and partially overlap with the process of epithelial-to-mesenchymal transition (EMT) and potentially represents a phase of the EMT process itself. The last part of the project investigated and described the progression of TID in a canine model of CKD and was conducted in partnership with the Texas A&M University (College Station, TX, USA). The included dogs were members of a single family affected by a X-linked hereditary nephropathy (XLHN) caused by a mutation in the gene encoding the α5 chain of type IV collagen, which is a crucial component of normal glomerular basement membranes (GBM). The salient clinical and pathological features of the nephropathy that occurs in male dogs with XLHN include juvenile onset of proteinuria and renal failure rapidly progressive to ESRD. Aims of the study were to examine the evolution of renal injury and the expression of selected molecules potentially involved in the progression of chronic TID. Affected dogs were characterized by progressive loss of glomeruli mostly undergoing cystic glomerular atrophy and less commonly global glomerulosclerosis. Primary lesions into the glomerulus were mesangial matrix expansion and hypercellularity. The tubulointerstitial lesions included changes typical of chronic TID, like tubular necrosis and atrophy, interstitial fibrosis and inflammation. The obtained results suggested that two different phases of the disease can be identified. The first was classified as an “early” phase (4 months of age), characterized by minimal or absence of histopathological lesions but evident proteinuria that is characterized by TGFβ, CTGF, and PDGFRα overexpression, likely produced by podocytes and TECs in response to the glomerular damage and intratubular proteinuria. The second “advanced” phase (after 6 months of age) was characterized by prominent glomerular and tubulointerstitial changes associated with an upregulation of clusterin and TIMP1 by TECs. The obtained results significantly improved the understanding of the progression of chronic TID in canine renal diseases pointing out the importance of proteinuria and possibly other molecular changes that precede the morphological changes. More data are needed to further understand the mechanisms responsible for the initiation ad promotion of the secondary TID and the major cellular and molecular players involved in order to identify early and specific markers of renal damage, improve the time of the diagnosis and eventually new targets for therapy.
Un danno renale primario può coinvolgere uno dei comparti del tessuto renale: glomerulare, tubulointerstiziale o vascolare. Tuttavia, indipendentemente dalla struttura anatomica primariamente colpita, tutte le componenti del tessuto renale possono venire secondariamente coinvolte. Il danno tubulointerstiziale cronico svolge un ruolo centrale nella progressione del danno renale e nell’irreversibile declino della funzionalità renale risultante nella Malattia Renale Cronica. Morfologicamente il danno tubulointerstiziale cronico è caratterizzato da perdita del parenchima funzionale, in cui si osserva marcata atrofia tubulare ed espansione dell’interstizio dovuto ad aumento del numero di fibroblasti e accumulo di matrice extracellulare. Spesso in associazione si rileva infiltrato infiammatorio cronico di entità variabile. Il progetto di dottorato è stato suddiviso in tre parti e ha avuto come obiettivo lo studio della progressione del danno tubulo-interstiziale cronico nelle patologie renali del cane. In una prima fase si è analizzata l’evoluzione delle lesioni renali in soggetti con glomerulonefrite immunomediata associata all’infezione da Leishmania spp. Lo studio si è svolto su 14 cani Leishmania-positivi sottoposti ad un trattamento leishmanicida specifico della durata di 60 giorni e in cui si è ottenuta una duplice biopsia renale, pre-trattamento e post-trattamento. Complessivamente si è osservata lieve progressione delle lesioni renali in metà dei soggetti, particolarmente in quei pazienti caratterizzati da prominente danno tubulo-interstiziale già alla valutazione della biopsia pretrattamento. I risultati ottenuti forniscono ulteriore supporto alla tesi secondo cui la progressione del danno tubulo-interstiziale cronico è indipendente dalla persistenza dell’agente causale. Inoltre l’eliminazione dell’agente eziologico, conseguente al trattamento leishmanicida, non sembra essere responsabile di un significativo miglioramento delle lesioni e funzionalità renale soprattutto in caso di lesioni in stadio avanzato e quindi croniche e di gravi come nel caso di glomerulosclerosi globale e fibrosi interstiziale. Al contrario un’efficacia del trattamento farmacologico si è evidenziato in presenza di un danno tubulo-interstiziale lieve ed è apparentemente imputabile ad una riduzione della componente infiammatoria. Nella seconda fase del progetto, lo studio è stato focalizzato ad esplorare il ruolo delle cellule epiteliali tubulari nella progressione del danno tubulointerstiziale cronico. Lo studio è stato svolto su biopsie renali di cane affetti da patologie di diversa natura e gravità ricercando una correlazione tra le lesioni istopatologiche e la funzionalità renale, nonché la capacità delle cellule tubulari epiteliali di agire come cellule presentanti l’antigene. Si è potuto evidenziare che cani affetti da glomerulopatie primarie presentavano più comunemente un danno tubulointerstiziale cronico con fibrosi interstiziale e innalzamento dei parametri di creatinemia e proteinuria, così come si osservava l’espressione ex novo di HLA-DR da parte delle cellule epiteliali tubulari. Ulteriormente si è osservata una correlazione positiva tra l’espressione di HLA-DR nelle cellule epiteliali, il grado di fibrosi, d’infiammazione, e i valori di proteinuria e creatinemia. Lo studio ha evidenziato la capacità delle cellule epiteliali tubulari di agire come cellule presentanti l’antigene nel danno tubulo-interstiziale cronico. Si è inoltre identificata nella proteinuria un possibile agente causale nell’indurre questa capacità. Infine questa parte dello studio ha messo in luce che l’espressione di HLA-DR nelle cellule epiteliali tubulari sembra precedere e parzialmente sovrapporsi con il fenomeno di transizione epitelio-mesenchimale delle cellule epiteliali e potrebbe rappresentarne una fase iniziale. La terza parte del progetto ha descritto la progressione del danno tubulointerstiziale cronico in un modello canino di Malattia Renale Cronica ed è stato svolto in collaborazione con la Texas A&M University (College Station, TX, USA). Lo studio è stato svolto su cani con nefropatia ereditaria legata al cromosoma X e mantenuti in condizioni sperimentali presso la Texas A&M University (College Station – Texas – USA). Tale nefropatia è dovuta ad una mutazione del gene codificante per la catena α5 del collagene di tipo IV, che rappresenta uno dei principali componenti della membrana basale glomerulare. Le caratteristiche cliniche e patologiche della malattia renale in cani affetti da nefropatia ereditaria consistono nell’insorgenza precoce di proteinuria ed insufficienza renale, rapidamente progressive a Malattia Renale Cronica. Obiettivi del lavoro sono stati quelli di esaminare l’evoluzione del danno renale da un punto di vista morfologico, clinico patologico e tramite lo studio dell’espressione genica e proteica di fattori potenzialmente coinvolti nella progressione del danno. I soggetti patologici presentavano una progressiva aumento del numero di glomeruli atrofici cistici o, meno frequentemente globalmente sclerotici. Le lesioni primarie osservate a livello glomerulare consistevano in espansione del mesangio ed ipercellulatià mesangiale. Il tubulointerstizio era caratterizzato da lesioni croniche ed aspecifiche come la necrosi ed atrofia tubulare, la fibrosi interstiziale e l’infiltrato infiammatorio cronico. I risultati ottenuti suggeriscono che si possano distinguere due fasi della malattia renale nella nefropatia ereditaria studiata. Un fase “precoce” (4 mesi di età) in cui si sono osservate da lesioni morfologiche minime o assenti ma con proteinuria conclamata. Da un punto di vista molecolare in questa fase si è evidenziata una sovra-espressione di TGFβ, CTGF, and PDGFRα probabilmente prodotti da podociti e cellule epiteliali tubulari in risposta al danno glomerulare e alla proteinuria. Una seconda fase “avanzata” (dopo i 6 mesi di età) sarebbe invece caratterizzata da lesioni glomerulari e tubulointerstiziali conclamate e da una up-regulation di clusterina e TIMP1 ad opera delle cellule epiteliali tubulari. I risultati ottenuti forniscono nuove informazioni e aumentano la conoscenza dei meccanismi di progressione del danno tubulointerstiziale cronico nelle malattie renali del cane. Dal lavoro effettuato emerge che l’insorgenza di proteinuria e l’alterata espressione di alcune molecole sembra precedere la presenza di lesioni morfologiche. Ulteriori studi sono necessari per approfondire la nostra conoscenza dei meccanismi di iniziazione e promozione del danno tubulointerstiziale cronico, delle componenti cellulari e molecolari coinvolte con l’obiettivo di identificare marcatori di danno renale precoci e specifici e possibili target terapeutici per la gestione del paziente con insufficienza renale.

Orientadores: Maria de Fatima Sonati, Maricilda Palandi de Mello
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A Doença da Hb H resulta da remoção ou inativação de três dos quatro genes da cadeia a da hemoglobina normalmente presentes no genoma diplóide e é caracterizada por anemia hemolítica crônica de intensidade moderada a grave. Os pacientes apresentam microcitose, hipocromia e poiquilocitose, com cerca de 25 a 30% de Hb Bart¿s (?4) ao nascimento e 5-30% de Hb H (ß4) na vida adulta. Embora a base molecular da doença tenha influência nos níveis de Hb H produzidos, uma heterogeneidade em relação a esse aspecto tem sido observada mesmo em pacientes com genótipos a idênticos, sugerindo que outros fatores contribuem para esta diversidade além dos determinantes talassêmicos. No presente trabalho, procuramos identificar transcritos diferencialmente expressos nos reticulócitos de dois pacientes com Doença da Hb H, irmãos, de origem étnica mista (chinesa e africana), um do sexo masculino, 21 anos de idade, com 18% de Hb H (MKS), e outro do sexo feminino, 19 anos, com 5% desta Hb (FKS), ambos com genótipo -a3.7/--SEA. O método envolveu a técnica de Differential Display Reverse Transcription-Polymerase Chain Reaction (DDRT-PCR) e a realização de Hibridização Subtrativa Supressiva (SSH). A validação dos achados foi feita pela Reação em Cadeia da Polimerase quantitativa em Tempo Real (qRT-PCR). Quatro perfis diferenciais de expressão foram selecionados, todos mais representados no paciente com maior nível de Hb H. Dois foram detectados por ambas as abordagens metodológicas: um transcrito altamente homólogo à parte do gene da PIP5KIIA (fosfatidilinositol 4-fosfato-5 quinase tipo II a) e outro ao gene da cadeia ß da hemoglobina humana. Os outros dois transcritos, selecionados apenas pela SSH, apresentararam similaridade ao gene FAM46C (Family with sequence similarity 46, member C), que corresponde a uma proteína hipotética de função indeterminada, e ao gene EIF4E-BP1 (eukariotic translation initiation factor 4E ¿ binding protein 1), que codifica uma proteína regulatória da tradução com capacidade de inibição do fator eIF4E (eukariotic translation initiation factor 4E). Na tentativa de identificar os mecanismos responsáveis pelo aumento dos transcritos da PIP5KIIA e da globina ß nos reticulócitos de MKSalguns outros genes, relacionados às vias de atuação ou ao processo de transcrição dos primeiros, tiveram sua expressão também avaliada pela qRT-PCR. Os resultados obtidos, embora não conclusivos, sugeriram que a diferença nos níveis de Hb H apresentada pelos pacientes aqui estudados está correlacionada com a taxa de síntese de cadeias ß, e que a enzima PIP5KIIA, provavelmente via sinalização celular pelo fosfatidilinositol, de alguma maneira participa de sua regulação. O significado destes achados e o papel dos transcritos dos genes FAM46C e EIF4E-BP1 devem ser futuramente investigados para uma melhor compreensão dos mecanismos de regulação da expressão dos genes da globina ß em pacientes com talassemia a
Abstract: Hb H disease results from the inactivation of three of the four a-globin genes normally present on diploid genome and it is characterized by a moderate to severe chronic hemolytic anemia. Patients usually present microcytosis, hypochromia and poikilocytosis, with 25 to 30% of Hb Bart¿s (?4) at birth and 5 to 30% of Hb H (ß4) in adult life. Although the molecular base of this disease influences the Hb H levels produced, some heterogeneity has been observed in relation to this aspect, even in patients with identical a genotypes, thus suggesting that other factors contribute to this diversity besides a-thalassemic determinants. The aim of the present study was to identify differentially expressed transcripts in the reticulocytes from two patients with Hb H disease, siblings, from Chinese and African origins, a 21-year- old male (MKS) with 18% of Hb H and a 19-year-old female (FKS) with 5% of Hb H, both with genotype -a3.7/--SEA. The methodology involved two techniques: the Differential Display Reverse Transcription-Polymerase Chain Reaction (DDRT-PCR) and the Suppression Subtractive Hybridization (SSH). Quantitative real time PCR (qRT-PCR) experiments were used to confirm some results. Four differentially expressed profiles were obtained, all of them better represented in the subject with the highest Hb H level. Two transcripts were detected by both methodological approaches, one being highly similar to PIP5KIIA gene (Phosphatidylinositol ¿ 4 phosphate 5-kinase, type II a) and the other one similar to human ß-globin gene. Two others transcripts were selected only by SSH and they showed to be to FAM 46C (Family with sequence similarity 46, member C) and EIF4E-BP1 (eukariotic translation initiation factor 4E-binding protein 1) gene homologues. In order to identify the mechanisms that are responsible for the transcripts PIP5KIIA and ß- globin increase in the reticulocytes from MKS patient, some other genes related to the transcriptional process also had its expression evaluated by qRT-PCR. Although not conclusive, our results suggest that the difference between the Hb H levels, showed by the subjects here studied, is correlated with ß-globin synthesis rate and that PIP5KIIA may participate of its regulation, probably by cell signalizing through Phosphatidylinositol. Studies, particularly involving a higher number of patients, and experiments aimed at elucidating the PIP5KIIA function in erythroid cells, should help to understand this process. The initiation factor -4E binding protein (EIF4E-BP1) and its capacity to bind to eIF4E, acts as negative regulator of cell growth. Its overexpression was detected in the patient with the highest HbH level. The significance of these findings and the role of the FAM46C and EIF4E-BP1 gene transcripts should be further investigated so that the regulating of the ß-globin gene expression in a-thalassemic patients can be better understood
Doutorado
Medicina Experimental
Doutor em Fisiopatologia Medica

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Genetic research of complex diseases is a challenging, but exciting, area of research. The early development of the research was limited, however, until the completion of the Human Genome and HapMap projects, along with the reduction in the cost of genotyping, which paves the way for understanding the genetic composition of complex diseases. In this thesis, we focus on the statistical methods for two aspects of genetic research: phenotype definition for diseases with complex etiology and methods for identifying potentially associated Single Nucleotide Polymorphisms (SNPs) and SNP-SNP interactions. With regard to phenotype definition for diseases with complex etiology, we firstly investigated the effects of different statistical phenotyping approaches on the subsequent analysis. In light of the findings, and the difficulties in validating the estimated phenotype, we proposed two different methods for reconciling phenotypes of different models using Bayesian model averaging as a coherent mechanism for accounting for model uncertainty. In the second part of the thesis, the focus is turned to the methods for identifying associated SNPs and SNP interactions. We review the use of Bayesian logistic regression with variable selection for SNP identification and extended the model for detecting the interaction effects for population based case-control studies. In this part of study, we also develop a machine learning algorithm to cope with the large scale data analysis, namely modified Logic Regression with Genetic Program (MLR-GEP), which is then compared with the Bayesian model, Random Forests and other variants of logic regression.