Ocular drug delivery remains a challenge due to the anatomical and physiological barriers of the eye. Topical drops have poor penetration and side effects, and injections are highly invasive; an alternative approach is transscleral delivery. Sustained drug release systems utilizing this delivery pathway are non-invasive yet provide extended treatment periods with single doses. Specifically, such systems would be beneficial for treating pediatric populations for diseases such as retinoblastoma and myopia. A chemo-preventative treatment for retinoblastoma, a childhood ocular cancer, has been proposed utilizing CdK inhibitors such as roscovitine and R547. Myopia, a more common, chronic condition in children, can be treated with atropine, though the dosing must be tightly controlled. Three novel delivery systems have been developed in this work to deliver these drugs, generating potential treatment platforms for these conditions. Contact lenses have been investigated previously to deliver various therapeutics, as they act as a slow, equilibrating reservoir, and the prolonged corneal residence time improves bioavailability. Silicone lenses, with their ability for continuous wear, have been explored looking at various material compositions and drug loading levels and techniques to confirm the feasibility of this system for these conditions (Chapter 2). This work has been extended to include covalent tethers for roscovitine, ensuring that it is able to survive a more commercially-relevant synthesis and providing additional ways to tailor the drug release profiles (Chapter 3). Alternatively, amphiphiles, which can self-assemble in the presence of excess water, have been explored for their drug delivery potential. Novel materials consisting of oleoylethanolamide and linoleoylethanolamide were developed to exhibit cubic phase transitions at physiologically relevant conditions, and their subsequent drug release characteristics were explored (Chapter 4). Silicone-based materials have broad usages as biomaterials, and a novel approach of incorporating PEG into the material without the use of metal catalysts were explored (Chapter 5). These exhibited a significant reduction in protein fouling, and material compositions were further iterated on, and their subsequent drug delivery capabilities were examined (Chapter 6). Overall, these three approaches have been able to successfully delivery roscovitine, R547 and atropine in non-invasive manners that have the potential to be an appropriate treatment for the childhood conditions they are intended to treat. / Dissertation / Doctor of Philosophy (PhD) / Sustained ocular drug delivery is critical to minimize side effects, maximize compliance and improve the clinical outcomes experienced by the patient. This is particularly true in pediatric applications, such as retinoblastoma and myopia. The objective of this thesis is to create drug delivery systems that could be used to treat these childhood conditions, with a focus on non-invasive delivery technologies. In this project, contact lenses are explored as drug delivery vehicles for two drugs which can be used to treat retinoblastoma and myopia. Multiple materials and drug loading are explored to create a sustained drug delivery system that meets the needs of these conditions. This is then expanded into examining systems that fit within current commercial manufacturing practices, creating a delivery system that meets the demands of the disease and industry. Alternative delivery systems that could be utilized as inserts are also explored. A lipid based system, whose materials are native to the body, are explored as a unique system capable of delivering drugs in a controlled fashion under specific hydration and temperature conditions. Alternatively, a silicone-based system is explored, whose unique chemistry eliminates many of the prohibitive biological side effects seen with these materials, allowing for a sustained drug delivery system for these ocular conditions.

Retinoblastoma (Rb) é o tumor ocular mais frequente na infância e cada grande Centro deve conhecer o perfil dos seus pacientes. Foi realizado um estudo do tipo coorte retrospectivo e incluiu pacientes com Rb atendidos entre 1983 e 2012 nos Serviços de Oncologia Pediátrica, Oftalmologia e Genética Médica do Hospital de Clínicas de Porto Alegre (HCPA). De um total de 165 registros no período foram efetivamente incluídos 140 pacientes, sendo 95,0% destes provenientes de municípios do Rio Grande do Sul. Os sinais mais frequentes ao diagnóstico foram: leucocoria (73,6%) e estrabismo (20,7%). Identificamos a seguinte distribuição: doença unilateral (65,0%), bilateral (32,9%) sendo 80,4% com doença multifocal (p=0,015), trilateral (2,1%). A idade média dos pacientes por ocasião dos primeiros sinais e sintomas foi de 18,1 meses [mediana=12,0] e a idade média ao diagnóstico foi 23,5 meses [mediana=16,5]. Cinquenta pacientes (35,7%) foram diagnosticados no 1º ano de vida. O tempo de diagnóstico médio da coorte foi 5,4 meses [mediana=3,0], (amplitude=0-77,0). A idade média aos primeiros sinais e sintomas do grupo com critérios de hereditariedade foi de 12,3 meses enquanto a do grupo não hereditário foi de 21,6 meses (p=0,001), enquanto a idade média ao diagnóstico foi de 15,9 meses vs. 28 meses, respectivamente (p<0,001). Entretanto não houve diferença na sobrevida entre esses subgrupos. O estadiamento ocular dos pacientes ao diagnóstico na sua maioria foi avançado (classificação de Reese V em 76,5%, Internacional D ou E em 78,1%), sendo que 35,2% dos unilaterais e 34,8% dos bilaterais já apresentavam doença extraocular em pelo menos um olho ao diagnóstico. Quinze pacientes (10,7%) tinham doença metastática ao diagnóstico. Em relação ao tratamento, diferentes modalidades foram utilizadas, sendo a maioria dos pacientes submetidos à cirurgia, sendo esta enucleação em 88,1% e exenteração em 11,9%. Uma parcela significativa dos pacientes foi tratada com quimioterapia sistêmica (57,1%) e/ou radioterapia (37,1%). Do total de pacientes recrutados, 131 (93,6%) permaneceram vinculados ao hospital até 2012 ou até o óbito. Destes, 32 (22,9%) recidivaram, resultando em 19 óbitos com 84,2% por progressão do Rb. Uma segunda neoplasia primária esteve presente em 4,3% (N=6) e dentre esses, um paciente teve uma terceira neoplasia primária. O tempo de seguimento médio foi 323,2 meses [300,3; 346,1]. As sobrevidas nos diferentes subgrupos foram as seguintes: sobrevida global 86,4%; no não metastático 92,0%; no metastático 40,0%; entre os intraoculares 94,0%; entre os extraoculares 68,5%; entre os unilaterais e bilaterais ambos com cerca de 88,0%; entre os trilaterais (N=3) todos foram a óbito; entre os unilaterais intraoculares 94,9% e extraoculares 75,0% e entre os bilaterais intraoculares 94,5% e extraoculares 68,4%. No nosso meio, o diagnóstico de Rb ainda é feito predominantemente em estadios avançados o que reduz a sobrevida dos pacientes e o índice de preservação do olho e da visão, além de aumentar a intensidade dos tratamentos realizados e consequentemente, toxicidade e efeitos tardios destes. Avaliações clínicas e oftalmológicas periódicas nos primeiros anos de vida da criança oferecem maior oportunidade de um diagnóstico precoce e o encaminhamento rápido à um Centro de Referência multidisciplinar que contemple cuidados terciários em Oftalmologia e Oncologia Pediátrica é fundamental. Existe grande necessidade de investimentos regionais que facilitem o acesso ao diagnóstico e tratamento do Rb, o tumor ocular mais frequente na infância. / Retinoblastoma (Rb) is the most frequent ocular tumor diagnosed in children and every pediatric hospital must be familiar with its clinical presentation and patient characteristics. A retrospective cohort study was undertaken, with patients diagnosed with retinoblastoma from 1983 until 2012, treated at the Pediatric Oncology Unit, Ophthalmology Unit, and Medical Genetics Unit of the Hospital de Clínicas de Porto Alegre (HCPA). Of a total of 165 registries during this time frame, 140 patients were included in this study, with 95% of them from the state of Rio Grande do Sul. The most frequent signs and symptoms at diagnosis were: leukocoria (73.6%) and strabismus (20.7%). The following distribution was identified: unilateral disease (65.0%), bilateral disease (32.9%), being 80.4% with multifocal disease, (P=0,015), and trilateral disease (2.1%). The average age of patients at the appearance of the first sign or symptom was 18.1 months [median=12.0] and the average age at diagnosis was 23.5 months [median=16.5]. Fifty patients (35.7%) were diagnosed during their first year of age. The average time to diagnosis was of 5.4 months [median=3.0], (amplitude=0-77.0). In the hereditary retinoblastoma group, the average age at the appearance of the first sign or symptom was 12.3 months, whereas the non-hereditary group presented the first sign or symptom on average at 21.6 months (P=0,001). The average age at diagnosis was 15.9 months vs. 28 months for the hereditary and non-hereditary patients, respectively (P<0.001). However, no significant difference in overall survival was found when both groups were compared. Ocular staging at diagnosis was, for the most part, advanced disease, (Reese V classification: 76.5%, Internacional Classification of Retinoblastoma D or E in 78.1% patients), being that 35.2% of cases were comprised of unilateral disease and 34.8% of patients with bilateral disease already presented with extraocular lesions in at least one eye at diagnosis. Fifteen patients (10.7%) presented with metastasis at diagnosis. With regards to treatment, differnet modalities were employed, being that most patients underwent surgery with enucleation in 88.1% and e exenteration in 11.9%. A significant number of patients received systemic chemotherapy (57.1%) and/or radiotherapy (37.1%). Of all patients included, 131 (93.6%) remained in follow up at the hospital until 2012 or until their demise. Of these patients, 32 (22.9%) relapsed, leading to 19 deaths, 84.2% of them due to disease progression. Secondary malignancies were present in 6 patients (4.3%) and, of these, one patient presented with two different secondary malignancies. The average time of patient follow up was 323.2 months [300.3; 346.1]. Overall survival was of 86.4%, with the following time frames among the different patient subgroups: 92.0% for non-metastatic patients, 40.0% for metastatic patients, intraoculares 94.0% for patients with intraocular disease, and 68.5% for patients with extraocular lesions. With regards to unilateral or bilateral disease, overall survival was of 88.0%; for patients with trilateral disease, (N=3) all patients expired. Survival of patients with unilateral and intraocular disease was of 94.9%; patients with unilateral and extraocular disease presented a overall survival of 75.0%. Patients with bilateral intraocular lesions overall survival was of 94.5%, whereas patients with bilateral and extraocular disease had an overall survival of 68.4%. In our setting, Rb diagnosis still occurs when the patients already manifest advanced disease, which reduces considerably their overall survival and preservation of the ocular globe and vision. Moreover, late diagnosis requires more agressive treatments, and consequently leads to more frequent toxicities and late side effects. Periodic clinical and ophthalmologic evaluations during the first years of a child's life offer a greater chance of early diagnosis and referral to a multidisciplinary pediatric oncology center, which is crucial for the patient’s well being. There is much need of further investments which facilitate patient access to diagnosis and treatment for Rb, which is the most common ocular tumor in children.

Retinoblastoma (Rb) é o tumor ocular mais frequente na infância e cada grande Centro deve conhecer o perfil dos seus pacientes. Foi realizado um estudo do tipo coorte retrospectivo e incluiu pacientes com Rb atendidos entre 1983 e 2012 nos Serviços de Oncologia Pediátrica, Oftalmologia e Genética Médica do Hospital de Clínicas de Porto Alegre (HCPA). De um total de 165 registros no período foram efetivamente incluídos 140 pacientes, sendo 95,0% destes provenientes de municípios do Rio Grande do Sul. Os sinais mais frequentes ao diagnóstico foram: leucocoria (73,6%) e estrabismo (20,7%). Identificamos a seguinte distribuição: doença unilateral (65,0%), bilateral (32,9%) sendo 80,4% com doença multifocal (p=0,015), trilateral (2,1%). A idade média dos pacientes por ocasião dos primeiros sinais e sintomas foi de 18,1 meses [mediana=12,0] e a idade média ao diagnóstico foi 23,5 meses [mediana=16,5]. Cinquenta pacientes (35,7%) foram diagnosticados no 1º ano de vida. O tempo de diagnóstico médio da coorte foi 5,4 meses [mediana=3,0], (amplitude=0-77,0). A idade média aos primeiros sinais e sintomas do grupo com critérios de hereditariedade foi de 12,3 meses enquanto a do grupo não hereditário foi de 21,6 meses (p=0,001), enquanto a idade média ao diagnóstico foi de 15,9 meses vs. 28 meses, respectivamente (p<0,001). Entretanto não houve diferença na sobrevida entre esses subgrupos. O estadiamento ocular dos pacientes ao diagnóstico na sua maioria foi avançado (classificação de Reese V em 76,5%, Internacional D ou E em 78,1%), sendo que 35,2% dos unilaterais e 34,8% dos bilaterais já apresentavam doença extraocular em pelo menos um olho ao diagnóstico. Quinze pacientes (10,7%) tinham doença metastática ao diagnóstico. Em relação ao tratamento, diferentes modalidades foram utilizadas, sendo a maioria dos pacientes submetidos à cirurgia, sendo esta enucleação em 88,1% e exenteração em 11,9%. Uma parcela significativa dos pacientes foi tratada com quimioterapia sistêmica (57,1%) e/ou radioterapia (37,1%). Do total de pacientes recrutados, 131 (93,6%) permaneceram vinculados ao hospital até 2012 ou até o óbito. Destes, 32 (22,9%) recidivaram, resultando em 19 óbitos com 84,2% por progressão do Rb. Uma segunda neoplasia primária esteve presente em 4,3% (N=6) e dentre esses, um paciente teve uma terceira neoplasia primária. O tempo de seguimento médio foi 323,2 meses [300,3; 346,1]. As sobrevidas nos diferentes subgrupos foram as seguintes: sobrevida global 86,4%; no não metastático 92,0%; no metastático 40,0%; entre os intraoculares 94,0%; entre os extraoculares 68,5%; entre os unilaterais e bilaterais ambos com cerca de 88,0%; entre os trilaterais (N=3) todos foram a óbito; entre os unilaterais intraoculares 94,9% e extraoculares 75,0% e entre os bilaterais intraoculares 94,5% e extraoculares 68,4%. No nosso meio, o diagnóstico de Rb ainda é feito predominantemente em estadios avançados o que reduz a sobrevida dos pacientes e o índice de preservação do olho e da visão, além de aumentar a intensidade dos tratamentos realizados e consequentemente, toxicidade e efeitos tardios destes. Avaliações clínicas e oftalmológicas periódicas nos primeiros anos de vida da criança oferecem maior oportunidade de um diagnóstico precoce e o encaminhamento rápido à um Centro de Referência multidisciplinar que contemple cuidados terciários em Oftalmologia e Oncologia Pediátrica é fundamental. Existe grande necessidade de investimentos regionais que facilitem o acesso ao diagnóstico e tratamento do Rb, o tumor ocular mais frequente na infância. / Retinoblastoma (Rb) is the most frequent ocular tumor diagnosed in children and every pediatric hospital must be familiar with its clinical presentation and patient characteristics. A retrospective cohort study was undertaken, with patients diagnosed with retinoblastoma from 1983 until 2012, treated at the Pediatric Oncology Unit, Ophthalmology Unit, and Medical Genetics Unit of the Hospital de Clínicas de Porto Alegre (HCPA). Of a total of 165 registries during this time frame, 140 patients were included in this study, with 95% of them from the state of Rio Grande do Sul. The most frequent signs and symptoms at diagnosis were: leukocoria (73.6%) and strabismus (20.7%). The following distribution was identified: unilateral disease (65.0%), bilateral disease (32.9%), being 80.4% with multifocal disease, (P=0,015), and trilateral disease (2.1%). The average age of patients at the appearance of the first sign or symptom was 18.1 months [median=12.0] and the average age at diagnosis was 23.5 months [median=16.5]. Fifty patients (35.7%) were diagnosed during their first year of age. The average time to diagnosis was of 5.4 months [median=3.0], (amplitude=0-77.0). In the hereditary retinoblastoma group, the average age at the appearance of the first sign or symptom was 12.3 months, whereas the non-hereditary group presented the first sign or symptom on average at 21.6 months (P=0,001). The average age at diagnosis was 15.9 months vs. 28 months for the hereditary and non-hereditary patients, respectively (P<0.001). However, no significant difference in overall survival was found when both groups were compared. Ocular staging at diagnosis was, for the most part, advanced disease, (Reese V classification: 76.5%, Internacional Classification of Retinoblastoma D or E in 78.1% patients), being that 35.2% of cases were comprised of unilateral disease and 34.8% of patients with bilateral disease already presented with extraocular lesions in at least one eye at diagnosis. Fifteen patients (10.7%) presented with metastasis at diagnosis. With regards to treatment, differnet modalities were employed, being that most patients underwent surgery with enucleation in 88.1% and e exenteration in 11.9%. A significant number of patients received systemic chemotherapy (57.1%) and/or radiotherapy (37.1%). Of all patients included, 131 (93.6%) remained in follow up at the hospital until 2012 or until their demise. Of these patients, 32 (22.9%) relapsed, leading to 19 deaths, 84.2% of them due to disease progression. Secondary malignancies were present in 6 patients (4.3%) and, of these, one patient presented with two different secondary malignancies. The average time of patient follow up was 323.2 months [300.3; 346.1]. Overall survival was of 86.4%, with the following time frames among the different patient subgroups: 92.0% for non-metastatic patients, 40.0% for metastatic patients, intraoculares 94.0% for patients with intraocular disease, and 68.5% for patients with extraocular lesions. With regards to unilateral or bilateral disease, overall survival was of 88.0%; for patients with trilateral disease, (N=3) all patients expired. Survival of patients with unilateral and intraocular disease was of 94.9%; patients with unilateral and extraocular disease presented a overall survival of 75.0%. Patients with bilateral intraocular lesions overall survival was of 94.5%, whereas patients with bilateral and extraocular disease had an overall survival of 68.4%. In our setting, Rb diagnosis still occurs when the patients already manifest advanced disease, which reduces considerably their overall survival and preservation of the ocular globe and vision. Moreover, late diagnosis requires more agressive treatments, and consequently leads to more frequent toxicities and late side effects. Periodic clinical and ophthalmologic evaluations during the first years of a child's life offer a greater chance of early diagnosis and referral to a multidisciplinary pediatric oncology center, which is crucial for the patient’s well being. There is much need of further investments which facilitate patient access to diagnosis and treatment for Rb, which is the most common ocular tumor in children.

Retinoblastoma (Rb) é o tumor ocular mais frequente na infância e cada grande Centro deve conhecer o perfil dos seus pacientes. Foi realizado um estudo do tipo coorte retrospectivo e incluiu pacientes com Rb atendidos entre 1983 e 2012 nos Serviços de Oncologia Pediátrica, Oftalmologia e Genética Médica do Hospital de Clínicas de Porto Alegre (HCPA). De um total de 165 registros no período foram efetivamente incluídos 140 pacientes, sendo 95,0% destes provenientes de municípios do Rio Grande do Sul. Os sinais mais frequentes ao diagnóstico foram: leucocoria (73,6%) e estrabismo (20,7%). Identificamos a seguinte distribuição: doença unilateral (65,0%), bilateral (32,9%) sendo 80,4% com doença multifocal (p=0,015), trilateral (2,1%). A idade média dos pacientes por ocasião dos primeiros sinais e sintomas foi de 18,1 meses [mediana=12,0] e a idade média ao diagnóstico foi 23,5 meses [mediana=16,5]. Cinquenta pacientes (35,7%) foram diagnosticados no 1º ano de vida. O tempo de diagnóstico médio da coorte foi 5,4 meses [mediana=3,0], (amplitude=0-77,0). A idade média aos primeiros sinais e sintomas do grupo com critérios de hereditariedade foi de 12,3 meses enquanto a do grupo não hereditário foi de 21,6 meses (p=0,001), enquanto a idade média ao diagnóstico foi de 15,9 meses vs. 28 meses, respectivamente (p<0,001). Entretanto não houve diferença na sobrevida entre esses subgrupos. O estadiamento ocular dos pacientes ao diagnóstico na sua maioria foi avançado (classificação de Reese V em 76,5%, Internacional D ou E em 78,1%), sendo que 35,2% dos unilaterais e 34,8% dos bilaterais já apresentavam doença extraocular em pelo menos um olho ao diagnóstico. Quinze pacientes (10,7%) tinham doença metastática ao diagnóstico. Em relação ao tratamento, diferentes modalidades foram utilizadas, sendo a maioria dos pacientes submetidos à cirurgia, sendo esta enucleação em 88,1% e exenteração em 11,9%. Uma parcela significativa dos pacientes foi tratada com quimioterapia sistêmica (57,1%) e/ou radioterapia (37,1%). Do total de pacientes recrutados, 131 (93,6%) permaneceram vinculados ao hospital até 2012 ou até o óbito. Destes, 32 (22,9%) recidivaram, resultando em 19 óbitos com 84,2% por progressão do Rb. Uma segunda neoplasia primária esteve presente em 4,3% (N=6) e dentre esses, um paciente teve uma terceira neoplasia primária. O tempo de seguimento médio foi 323,2 meses [300,3; 346,1]. As sobrevidas nos diferentes subgrupos foram as seguintes: sobrevida global 86,4%; no não metastático 92,0%; no metastático 40,0%; entre os intraoculares 94,0%; entre os extraoculares 68,5%; entre os unilaterais e bilaterais ambos com cerca de 88,0%; entre os trilaterais (N=3) todos foram a óbito; entre os unilaterais intraoculares 94,9% e extraoculares 75,0% e entre os bilaterais intraoculares 94,5% e extraoculares 68,4%. No nosso meio, o diagnóstico de Rb ainda é feito predominantemente em estadios avançados o que reduz a sobrevida dos pacientes e o índice de preservação do olho e da visão, além de aumentar a intensidade dos tratamentos realizados e consequentemente, toxicidade e efeitos tardios destes. Avaliações clínicas e oftalmológicas periódicas nos primeiros anos de vida da criança oferecem maior oportunidade de um diagnóstico precoce e o encaminhamento rápido à um Centro de Referência multidisciplinar que contemple cuidados terciários em Oftalmologia e Oncologia Pediátrica é fundamental. Existe grande necessidade de investimentos regionais que facilitem o acesso ao diagnóstico e tratamento do Rb, o tumor ocular mais frequente na infância. / Retinoblastoma (Rb) is the most frequent ocular tumor diagnosed in children and every pediatric hospital must be familiar with its clinical presentation and patient characteristics. A retrospective cohort study was undertaken, with patients diagnosed with retinoblastoma from 1983 until 2012, treated at the Pediatric Oncology Unit, Ophthalmology Unit, and Medical Genetics Unit of the Hospital de Clínicas de Porto Alegre (HCPA). Of a total of 165 registries during this time frame, 140 patients were included in this study, with 95% of them from the state of Rio Grande do Sul. The most frequent signs and symptoms at diagnosis were: leukocoria (73.6%) and strabismus (20.7%). The following distribution was identified: unilateral disease (65.0%), bilateral disease (32.9%), being 80.4% with multifocal disease, (P=0,015), and trilateral disease (2.1%). The average age of patients at the appearance of the first sign or symptom was 18.1 months [median=12.0] and the average age at diagnosis was 23.5 months [median=16.5]. Fifty patients (35.7%) were diagnosed during their first year of age. The average time to diagnosis was of 5.4 months [median=3.0], (amplitude=0-77.0). In the hereditary retinoblastoma group, the average age at the appearance of the first sign or symptom was 12.3 months, whereas the non-hereditary group presented the first sign or symptom on average at 21.6 months (P=0,001). The average age at diagnosis was 15.9 months vs. 28 months for the hereditary and non-hereditary patients, respectively (P<0.001). However, no significant difference in overall survival was found when both groups were compared. Ocular staging at diagnosis was, for the most part, advanced disease, (Reese V classification: 76.5%, Internacional Classification of Retinoblastoma D or E in 78.1% patients), being that 35.2% of cases were comprised of unilateral disease and 34.8% of patients with bilateral disease already presented with extraocular lesions in at least one eye at diagnosis. Fifteen patients (10.7%) presented with metastasis at diagnosis. With regards to treatment, differnet modalities were employed, being that most patients underwent surgery with enucleation in 88.1% and e exenteration in 11.9%. A significant number of patients received systemic chemotherapy (57.1%) and/or radiotherapy (37.1%). Of all patients included, 131 (93.6%) remained in follow up at the hospital until 2012 or until their demise. Of these patients, 32 (22.9%) relapsed, leading to 19 deaths, 84.2% of them due to disease progression. Secondary malignancies were present in 6 patients (4.3%) and, of these, one patient presented with two different secondary malignancies. The average time of patient follow up was 323.2 months [300.3; 346.1]. Overall survival was of 86.4%, with the following time frames among the different patient subgroups: 92.0% for non-metastatic patients, 40.0% for metastatic patients, intraoculares 94.0% for patients with intraocular disease, and 68.5% for patients with extraocular lesions. With regards to unilateral or bilateral disease, overall survival was of 88.0%; for patients with trilateral disease, (N=3) all patients expired. Survival of patients with unilateral and intraocular disease was of 94.9%; patients with unilateral and extraocular disease presented a overall survival of 75.0%. Patients with bilateral intraocular lesions overall survival was of 94.5%, whereas patients with bilateral and extraocular disease had an overall survival of 68.4%. In our setting, Rb diagnosis still occurs when the patients already manifest advanced disease, which reduces considerably their overall survival and preservation of the ocular globe and vision. Moreover, late diagnosis requires more agressive treatments, and consequently leads to more frequent toxicities and late side effects. Periodic clinical and ophthalmologic evaluations during the first years of a child's life offer a greater chance of early diagnosis and referral to a multidisciplinary pediatric oncology center, which is crucial for the patient’s well being. There is much need of further investments which facilitate patient access to diagnosis and treatment for Rb, which is the most common ocular tumor in children.

Ornithine Aminotransferase (OAT) is expressed at high levels in liver, kidney, and retina. Tissue-specific regulation of OAT has been described for rat kidney and liver. To characterize OAT regulation in retinal lines, we studied OAT synthesis in retinoblastomas RB355 and Y79. / OAT transcription and mRNA levels in the two lines were similar, but 3-fold greater immunoreactive OAT protein and enzyme activity were observed for Y79. Characterization of polysome-associated OAT mRNAs indicated that they were translated less efficiently, due to decreased initiation, in RB355. Initiation factor eIF-4E mRNA and protein were reduced in RB355; eIF-4E overexpression in RB355 increased OAT translation and OAT protein to the level observed in Y79. / Estrogen and thyroid hormone increased OAT expression in both strains. Estrogen increased translational initiation, with no effect on transcription, whereas thyroid hormone was primarily a transcriptional activator. / An alternatively-spliced OAT mRNA was identified in these retinoblastomas; this mRNA was poorly translated and was not affected by eIF-4E overexpression, estrogen, or thyroid hormone. / This study has demonstrated several post-transcriptional regulatory mechanisms for OAT in retinal tissue.

Retinoblastoma, a rare childhood cancer of the retina, is characterized by loss of both alleles of the RB1 gene. However, additional mutational events are required for malignancy. CGH studies described common chromosomal changes indicating potential oncogenes and tumor suppressor genes. Previous work in the lab implicated Cadherin-11 (Cdh11) as a tumor suppressor after analysis in human retinoblastomas and in the simian virus 40 large T-antigen induced murine retinoblastoma model (TAg-RB) showed loss of Cdh11 expression. TAg-RB mice crossed with Cdh11-/- mice, revealed faster growing tumors in mice null for Cdh11. This thesis focused on defining the tumor suppressor role of Cdh11 in retinoblastoma progression. The results showed in vitro and in vivo evidence that Cdh11 was promoting apoptosis and not suppressing proliferation. We also observed an increase in invasion markers upon the loss of Cdh11. We conclude that Cdh11 acts as a tumor suppressor in retinoblastoma, through promotion of apoptosis.

Retinoblastoma, a rare childhood cancer of the retina, is characterized by loss of both alleles of the RB1 gene. However, additional mutational events are required for malignancy. CGH studies described common chromosomal changes indicating potential oncogenes and tumor suppressor genes. Previous work in the lab implicated Cadherin-11 (Cdh11) as a tumor suppressor after analysis in human retinoblastomas and in the simian virus 40 large T-antigen induced murine retinoblastoma model (TAg-RB) showed loss of Cdh11 expression. TAg-RB mice crossed with Cdh11-/- mice, revealed faster growing tumors in mice null for Cdh11. This thesis focused on defining the tumor suppressor role of Cdh11 in retinoblastoma progression. The results showed in vitro and in vivo evidence that Cdh11 was promoting apoptosis and not suppressing proliferation. We also observed an increase in invasion markers upon the loss of Cdh11. We conclude that Cdh11 acts as a tumor suppressor in retinoblastoma, through promotion of apoptosis.

LIMA, Diego Silva. Avaliação dos genes MLL, RB e TP53 em pacientes com síndrome mielodisplásica. 2011. 95 f. Dissertação (Mestrado em Patologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2011. / Submitted by denise santos (denise.santos@ufc.br) on 2013-12-03T12:56:53Z No. of bitstreams: 1 2011_dis_dslima.pdf: 4024143 bytes, checksum: 0a18d4cb329d542975fac3641f5bbd03 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2013-12-03T12:59:05Z (GMT) No. of bitstreams: 1 2011_dis_dslima.pdf: 4024143 bytes, checksum: 0a18d4cb329d542975fac3641f5bbd03 (MD5) / Made available in DSpace on 2013-12-03T12:59:05Z (GMT). No. of bitstreams: 1 2011_dis_dslima.pdf: 4024143 bytes, checksum: 0a18d4cb329d542975fac3641f5bbd03 (MD5) Previous issue date: 2011 / Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal disorders affecting the hematopoietic pluripotent cell, characterized by low cell counts in peripheral blood, dysplasia in one or more cell lines, inefficient hematopoiesis and increased risk of progression to acute myeloid leukemia. Although the disease can affect patients of other age groups, they are more frequent in those with advanced age with an average 60 to 75 years at diagnosis. Chromosomal abnormalities are observed in approximately 50% of all cases of MDS and are related with clinical and morphological findings. The aim of this study was to determine, through the technique of FISH (fluorescence in situ hybridization), the frequency of changes involving the MLL, RB, and TP53 genes in patients with MDS and associate these changes with cytogenetic findings. The cases included in the study were selected in the ambulatory of SMD from University Hospital Walter Cantídio. Thirty three patients were selected, 17 had aged over 60 years. 52% of patients were classified, according to WHO criteria, as refractory cytopenia with dysplasia in multiple lineages (RCDM) and 61% stratified, according to IPSS, as intermediate risk 1 (INT-1). 78% of patients had abnormalities detected by cytogenetics, among them 31% had complex karyotypes (more than 3 changes per metaphase). 18% of patients had changes at least in one of the three genes valued in this study by FISH. Three patients showed alterations of TP53 gene, being detected in two patients (records 31 and 6) the deletion of a single allele or both alleles of the gene, respectively, and in the third (record 2), we detected amplification of TP53 gene, all this changes were not detected by classical cytogenetics, because it is a less sensitive technique. 6% of patients (records 7 and 22) had rearrangement of MLL gene. In the first case, FISH discarded the gene deletion alleged by cytogenetic, proving that it was present in the genome of the patient, but in a rearranged form, and in the second case cytogenetics failed to demonstrate rearrangement of the gene. For the RB gene, FISH identified only one patient (3%) with deletion of one allele of the gene, and this change was also not detected by classical cytogenetics. During evaluating the TP53 gene, FISH allowed identification of two patients (records 5 and 10) presenting at least six extra copies of chromosome 17, probably representing a small hyperdiploid clone partially detected in the first patient and not detected in the second . In the six patients who showed abnormalities of the genes analyzed, FISH has provided information that added, changed or confirmed the result previously given by classical cytogenetics, which are a major application of this technique due to its high sensitivity compared to the traditional method. / As síndromes mielodisplásicas (SMD) representam um grupo heterogêneo de doenças clonais que acometem a célula precursora hematopoética pluripotente, caracterizando-se por baixa contagem de células no sangue periférico, displasia em uma ou mais linhagens celulares, hematopoese ineficiente, além do risco aumentado de progressão para leucemia mielóide aguda. Embora a doença possa acometer pacientes de outras faixas etárias, é mais frequente naqueles com idade avançada, com média ao diagnóstico de 60 a 75 anos. As anormalidades cromossômicas são observadas em aproximadamente 50% de todos os casos de SMD, estando, em alguns casos, relacionadas com achados clínicos e morfológicos. O objetivo deste trabalho foi determinar, através da técnica de FISH (hibridização in situ por fluorescência), a frequência de alterações envolvendo os genes MLL, RB e TP53 em pacientes com SMD e associar estas alterações com os achados citogenéticos. Os casos inseridos no estudo foram oriundos do ambulatório de SMD do Hospital Universitário Walter Cantídio. Dos 33 pacientes selecionados, 17 pertenciam ao grupo com idade acima de 60 anos. 52% dos pacientes foram classificados, segundo a OMS, como citopenia refratária com displasia em múltiplas linhagens (CRDM) e 61% estratificados, segundo o IPSS, como de risco intermediário 1 (INT-1). Um total de 78% dos pacientes apresentaram alterações citogenéticas, dentre eles 31% possuíam cariótipos complexos (mais de 3 alterações por metáfase). A técnica de FISH permitiu identificar em 18% dos pacientes alterações envolvendo um dos três genes avaliados. Três pacientes apresentaram alteração do gene TP53, sendo detectada em dois deles (registros 31 e 6) a deleção de um único alelo ou de ambos os alelos do gene, respectivamente, e no terceiro (registro 2), detectou-se a amplificação do gene TP53, sendo estas alterações não visualizadas através da citogenética clássica, por se tratar de um técnica menos sensível. Detectou-se em 6% dos pacientes (registros 7 e 22) rearranjo do gene MLL, no primeiro a FISH descartou a suposta deleção do gene alegada pela citogenética, provando que o mesmo estava presente no genoma do paciente, porém de forma rearranjada e no segundo a citogenética não conseguiu demonstrar o rearranjo do gene. Quanto ao gene RB, a FISH permitiu identificar apenas um paciente (3%) com deleção de um dos alelos do gene, sendo esta alteração também não detectada pela citogenética clássica. A FISH possibilitou identificar, durante a avaliação do gene TP53, dois pacientes (registros 5 e 10) apresentando pelo menos 6 cópias extras do cromossomo 17, devendo essa alteração se tratar de um pequeno clone hiperdiplóide detectado parcialmente no primeiro paciente e não detectado no segundo. Nos seis pacientes que apresentaram alteração dos genes avaliados, a FISH proveu informações que adicionaram, confirmaram ou alteraram o resultado previamente emitido pela citogenética clássica, sendo estas uma das principais aplicações desta técnica devido sua alta sensibilidade quando comparada ao método clássico.

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