Gao, Si. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 77-85). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / Abstract (in Chinese) --- p.iv / List of Abbreviations --- p.v / List of Figures --- p.vii / Table of Contents --- p.ix / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1. --- Cancer --- p.1 / Chapter 1.2. --- Chemotherapy --- p.2 / Chapter 1.3. --- Cisplatin --- p.3 / Chapter 1.4. --- Cisplatin-induced nephrotoxicity --- p.4 / Chapter 1.5. --- Mechanisms of cisplatin-induced nephrotoxicity --- p.5 / Chapter 1.5.1. --- Apoptosis in cisplatin-induced nephrotoxicity --- p.5 / Chapter 1.5.2. --- MAPK activation in cisplatin-induced nephrotoxicity --- p.7 / Chapter 1.5.3. --- Oxidative stress in cisplatin-induced nephrotoxicity --- p.8 / Chapter 1.6. --- Polyphenols --- p.10 / Chapter 1.7. --- Anthocyanins --- p.10 / Chapter 1.8. --- Rose --- p.11 / Chapter 1.9. --- Cyanidin --- p.12 / Chapter 1.10. --- Objectives of this project --- p.13 / Chapter Chapter Two: --- Materials and Methods --- p.15 / Chapter 2.1. --- Materials --- p.15 / Chapter 2.2. --- Cell culture --- p.15 / Chapter 2.3. --- Drug treatment --- p.16 / Chapter 2.4. --- MTT assay --- p.16 / Chapter 2.5. --- Lactate dehydrogenase (LDH) assay --- p.16 / Chapter 2.6. --- TUNEL assay and DAPI staining --- p.17 / Chapter 2.7. --- Flow cytometric analysis --- p.17 / Chapter 2.8. --- Determination of caspase-3 activity --- p.18 / Chapter 2.9. --- Measurement of ROS generation --- p.18 / Chapter 2.10. --- Evaluation of mitochondrial membrane potential --- p.19 / Chapter 2.11. --- Single Cell Gel Electrophoresis (Comet Assay) --- p.19 / Chapter 2.12. --- Western blot analysis --- p.20 / Chapter 2.13. --- Statistical analysis --- p.21 / Chapter Chapter Three: --- Results --- p.22 / Chapter 3.1. --- Cyanidin attenuates cisplatin-induced cytotoxicity in HK-2 cells --- p.22 / Chapter 3.1.1. --- Cytotoxicity induces by cisplatin in HK-2 cells --- p.22 / Chapter 3.1.2. --- Rose extract attenuates cisplatin-induced cytotoxicity and LDH leakage --- p.26 / Chapter 3.1.3. --- Cyanidin attenuates cisplatin-induced cytotoxicity and LDH leakage --- p.26 / Chapter 3.1.4. --- Cyanidin did not affect cisplatin-induced cytotoxicity in Hela cell --- p.30 / Chapter 3.2. --- Cyanidin rescues HK-2 cells from cisplatin-induced apoptosis --- p.31 / Chapter 3.2.1. --- Cisplatin induces cell apoptosis in HK-2 cells --- p.31 / Chapter 3.2.2. --- Rose extract rescues HK-2 cells from cisplatin-induced apoptosis --- p.31 / Chapter 3.2.3. --- Cyanidin rescues HK-2 cells from cisplatin-induced apoptosis --- p.32 / Chapter 3.3. --- Cyanidin suppresses cisplatin-induced activation of caspase and cleavage of PARP --- p.38 / Chapter 3.3.1. --- Cisplatin induces activation of caspase-3 --- p.38 / Chapter 3.3.2. --- Rose extract suppresses cisplatin-induced activation of caspase-3 --- p.38 / Chapter 3.3.3. --- Cyanidin suppresses cisplatin-induced activation of caspase-3 --- p.38 / Chapter 3.3.4. --- Rose extract suppresses cisplatin-induced caspase activation and PARP cleavage --- p.41 / Chapter 3.3.5. --- Cyanidin suppresses cisplatin-induced caspase activation and PARP cleavage --- p.43 / Chapter 3.4. --- Cyanidin rescues HK-2 cells from cisplatin-induced mitochondrial dysfuntion by regulating the expression of Bcl-2 family proteins --- p.43 / Chapter 3.4.1. --- Cyanidin prevents cisplatin-induced loss of mitochondrial membrane potential (A^m) --- p.43 / Chapter 3.4.2. --- Cyanidin regulates the expression of Bcl-2 family proteins to prevent cisplatin-induced mitochondrial dysfunction --- p.44 / Chapter 3.5. --- Cyanidin reduces cisplatin-induced apoptosis by suppressing the activation of p53 --- p.46 / Chapter 3.6. --- Cyanidin inhibits ROS-mediated DNA damage in HK-2 cells --- p.48 / Chapter 3.6.1. --- Cyanidin prevents cisplatin-induced DNA damage --- p.48 / Chapter 3.6.2. --- Cyanidin inhibits cisplatin-induced accumulation of ROS --- p.48 / Chapter 3.7. --- "Cyanidin suppresses cisplatin-induced apoptosis by activation of AKT, JNK and ERK" --- p.52 / Chapter 3.7.1. --- Cisplatin activates ERK and AKT pathways --- p.52 / Chapter 3.7.2. --- Cyanidin suppresses cisplatin-induced activation of MAPKs and AKT pathways --- p.52 / Chapter 3.7.3. --- AKT and ERK Inhibitors attenuates cisplatin-induced apoptosis in HK-2 cells --- p.53 / Chapter Chapter Four: --- Discussion --- p.60 / Chapter 4.1. --- Cell model and cisplatin treatment --- p.60 / Chapter 4.2. --- Cisplatin nephrotoxicity and its renoprevention --- p.61 / Chapter 4.3. --- Rose extract prevents cisplatin-induced apoptosis in HK-2 cells --- p.62 / Chapter 4.3.1. --- Rose extract prevents cisplatin-induced apoptosis in HK-2 cells --- p.63 / Chapter 4.3.2. --- Rose extract inhibits cisplatin-induced caspase activation and PARP cleavage --- p.64 / Chapter 4.4. --- Cyanidin prevents cisplatin-induced apoptosis in HK-2 cells --- p.66 / Chapter 4.4.1. --- Cyanidin will not affect cisplatin-induced cell death in HeLa cells --- p.66 / Chapter 4.4.2. --- Cyanidin prevents cisplatin-induced apoptosis by inhibiting caspase activation and PARP cleavage in HK-2 cells --- p.66 / Chapter 4.4.3. --- Cyanidin prevents the cisplatin-induced loss of mitochondrial membrane potential by regulating Bcl-2 proteins in HK-2 cells --- p.67 / Chapter 4.4.4. --- Cyanidin suppresses cisplatin-induced total and phosphorylated p53 activation --- p.68 / Chapter 4.4.5. --- Cyanidin prevents the cisplatin-induced overproduction of intracellular ROS and subsequent DNA damage in HK-2 cells --- p.69 / Chapter 4.4.6. --- Cyanidin suppresses the cisplatin-induced activation of MAPKs and AKT pathways in HK-2 cells --- p.71 / Chapter Chapter Five: --- Conclusion --- p.74 / References --- p.77

Lai, Sze Tsai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 94-104). / Abstracts in English and Chinese. / Thesis Committees --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgments --- p.vi / List of Abbreviations --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xiv / Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Introduction to Cordyceps --- p.1 / Chapter 1.2 --- Fungal proteomics --- p.2 / Chapter 1.2.1 --- Extraction method --- p.2 / Chapter 1.2.2 --- Proteomic study of Cordyceps --- p.3 / Chapter 1.3 --- Ingredients of Cordyceps and their related biological activities --- p.5 / Chapter 1.3.1 --- Polysaccharides --- p.5 / Chapter 1.3.2 --- Nucleosides --- p.6 / Chapter 1.3.2.1 --- Cordycepin --- p.6 / Chapter 1.3.2.2 --- Adenosine --- p.8 / Chapter 1.4 --- Cordyceps and their related biological activities --- p.9 / Chapter 1.4.1 --- Cordyceps militaris --- p.9 / Chapter 1.4.2 --- Cordyceps sinensis --- p.10 / Chapter 1.5 --- Proteomic analysis of proteome change --- p.12 / Chapter 1.5.1 --- Proteomic tools used to study the change in protein expression --- p.12 / Chapter 1.5.2 --- Two-dimensional gel electrophoresis --- p.13 / Chapter 1.5.3 --- Mass spectrometry --- p.13 / Chapter 1.6 --- Objective --- p.16 / Chapter 2 --- Methodology --- p.17 / Chapter 2.1 --- Cultivation of Cordyceps militaris --- p.17 / Chapter 2.2 --- Proteomic study on Cordyceps militaris --- p.17 / Chapter 2.2.1 --- Extraction of proteins from Cordyceps militaris --- p.17 / Chapter 2.2.2 --- Protein quantification --- p.18 / Chapter 2.2.3 --- 2D Gel electrophoresis --- p.19 / Chapter 2.2.4 --- Image analysis --- p.20 / Chapter 2.2.5 --- In gel digestion and MALDI-ToF MS --- p.20 / Chapter 2.3 --- Preparation of Cordyceps extracts for anti-proliferation assay on cell lines --- p.21 / Chapter 2.3.1 --- Types of the extracts of Cordyceps --- p.21 / Chapter 2.3.2 --- Preparation of the extracts of Cordyceps --- p.21 / Chapter 2.4 --- Anti-proliferation assay on cell lines for extract screening --- p.22 / Chapter 2.4.1 --- Cell lines and culturing condition --- p.22 / Chapter 2.4.2 --- Viable cell count using trypan blue exclusion method --- p.22 / Chapter 2.4.3 --- Anti-proliferation assay on SV7 tert using MTT assay --- p.23 / Chapter 2.4.4 --- Determination of the IC5o values --- p.24 / Chapter 2.4.5 --- Statistical Analysis --- p.24 / Chapter 2.5 --- Anti-proliferation assay on other cell lines using the two screened extracts --- p.24 / Chapter 2.5.1 --- Cell lines and culturing condition --- p.24 / Chapter 2.5.2 --- "Anti-proliferation assay on on HepG2, H292, Neuro2a，WIL2-NS cells using MTT assay" --- p.25 / Chapter 2.6 --- Proteomic studies for SV7tert and Hs68 after the treatment of extracts --- p.25 / Chapter 2.6.1 --- Protein sample preparation of SV7tert and Hs68 --- p.25 / Chapter 2.6.2 --- Protein quantification --- p.26 / Chapter 2.6.3 --- 2D Gel electrophoresis --- p.26 / Chapter 2.6.4 --- Image analysis --- p.26 / Chapter 2.7 --- Western Immunoblotting --- p.26 / Chapter 2.7.1 --- Protein sample preparation of SV7tert and Hs68 --- p.26 / Chapter 2.7.2 --- SDS-PAGE --- p.27 / Chapter 2.7.3 --- Protein Blotting --- p.27 / Chapter 2.7.4 --- Membrane Blocking and Antibody Incubations --- p.28 / Chapter 2.7.5 --- Detection of Proteins --- p.28 / Chapter 3 --- Results --- p.29 / Chapter 3.1 --- Proteins identification in Cordyceps militaris --- p.29 / Chapter 3.1.1 --- 2D gel electrophoresis analysis and resolution --- p.29 / Chapter 3.1.2 --- Identification and categorization of proteins of mycelia and fruiting body of Cordyceps militaris --- p.30 / Chapter 3.2 --- Investigation of anti-proliferating activity of extracts using MTT assays on SV7tert and Hs68 cell lines --- p.44 / Chapter 3.2.1 --- Mycelia extract from Cordyceps militaris --- p.44 / Chapter 3.2.2 --- Fruiting body extract from Cordyceps militaris --- p.44 / Chapter 3.2.3 --- Mycelia extract from Cordyceps sinensis --- p.47 / Chapter 3.2.4 --- Fruiting body extract from Cordyceps sinensis --- p.47 / Chapter 3.2.5 --- Screening of extracts --- p.50 / Chapter 3.3 --- "Investigation of anti-proliferating activity of extracts using MTT assays on HepG2，H292, Neuro2a and WIL2-NS cell lines" --- p.51 / Chapter 3.3.1 --- Mycelia extract from Cordyceps militaris --- p.51 / Chapter 3.3.2 --- Fruiting body extract from Cordyceps militaris --- p.51 / Chapter 3.4 --- Changes in total protein expression profiles in SV7tert and Hs68 cell lines --- p.56 / Chapter 3.4.1 --- Corresponding extract treatment of cell lines --- p.56 / Chapter 3.4.2 --- 2D gel electrophoresis analysis of protein from cells (SV7tert or Hs68) --- p.56 / Chapter 3.4.2.1 --- SV7tert study --- p.57 / Chapter 3.4.2.2 --- Hs68 study --- p.57 / Chapter 3.4.3 --- Protein identification --- p.65 / Chapter 3.4.3.1 --- Changes in protein expressions in SV7tert after mycelia extract treatment --- p.65 / Chapter 3.4.3.2 --- Changes in protein expressions in Hs68 after mycelia extract treatment --- p.65 / Chapter 3.4.3.3 --- Changes in protein expressions in SV7tert after fruiting body extract treatment --- p.66 / Chapter 3.4.3.4 --- Changes in protein expressions in Hs68 after fruiting body extract treatment --- p.66 / Chapter 3.5 --- Western immunoblotting --- p.71 / Chapter 3.5.1 --- Corresponding extract treatment of cell lines --- p.71 / Chapter 3.5.2 --- Normalization of protein loading using anti-actin antibody --- p.73 / Chapter 3.5.3 --- Detection of caspase 3 by use of antibody --- p.74 / Chapter 3.5.4 --- Detection of cleaved caspase 3 by use of antibody --- p.74 / Chapter 4 --- Discussion --- p.77 / Chapter 4.1 --- Identification of proteins in Cordyceps militaris --- p.77 / Chapter 4.2 --- Difficulties in identifying the proteins in Cordyceps militaris --- p.80 / Chapter 4.3 --- Investigation of anti-proliferating activity of extracts --- p.80 / Chapter 4.4 --- Changes in cell total protein expression profiles in SV7tert and Hs68 cell lines --- p.81 / Chapter 4.4.1 --- Protein alterations in SV7tert treated with mycelia extract --- p.82 / Chapter 4.4.1.1 --- Heat shock protein 27 (Hsp27) --- p.82 / Chapter 4.4.1.2 --- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) --- p.83 / Chapter 4.4.2 --- Protein alterations in Hs68 with mycelia extract treatment --- p.84 / Chapter 4.4.2.1 --- Chain B of triosephosphate isomerase - Triosephophate isomerase 1 --- p.84 / Chapter 4.4.2.2 --- Glutathione transferase --- p.85 / Chapter 4.4.3 --- Protein alterations in SV7tert with fruiting body extract treatment --- p.86 / Chapter 4.4.3.1 --- Calreticulin precusor --- p.86 / Chapter 4.4.3.2 --- Nucleophosmin 1 isoform 2 (B23) --- p.87 / Chapter 4.4.3.3 --- Heat shock 70kDa protein 8 isoform 1 - Heat shock 70kDa protein (Hsp70) --- p.88 / Chapter 4.4.3.4 --- Voltage-dependent anion channel 2 (VDAC2) --- p.89 / Chapter 4.4.3.5 --- "Tumor protein, translationally controlled (TCTP)" --- p.90 / Chapter 4.4.3.6 --- RAN binding protein 1 (RANBP1) --- p.91 / Chapter 4.4.4 --- Protein alteration in Hs68 with mycelia extract treatment --- p.92 / Chapter 4.5 --- Conclusion --- p.93 / References --- p.94

A doença renal policística autossômica dominante (DRPAD) felina é caracterizada pela presença de múltiplos cistos localizados em parênquima renal e, ocasionalmente, hepáticoe pancreático, sendo uma importante causa de doença renal crônica terminal. Ela é considerada a enfermidade congênita hereditária mais prevalente dos gatos domésticos, porém, os dados epidemiológicos eclínicos existentes no Brasil são escassos, o que torna o seu controle mais difícil no país. Da mesma forma, a DRPAD em humanos constitui-se na doença renal monogênica mais comum, acometendo 1 em cada 400 a 1.000 indivíduos, com curso clínico muito semelhante a doença em gatos.No presente estudo, 252 felinos da raça Persa e mestiços de Persa do Estado de São Paulo foram avaliados através de teste genético para DRPAD. Os resultados indicaram uma prevalência de apenas 6,35% de gatos portadores da mutação para DRPAD em heterozigose, valor abaixo dos índices registrados em outros países. Do total de animais, uma coorte de 82 felinos da região metropolitana de São Paulo foi selecionada para realização de exames físico, laboratoriais, imagenológicos e genéticos. Estes animais foram separados em dois grupos de acordo com a presença (n=12) ou ausência (n=70) de alterações ultrassonográficas sugestivas de doença renal policística.A sensibilidade e a especificidade entre o teste molecular e o exame ultrassonográfico para DRPAD nos animais estudados foram ambas de 100%. Os animais com alterações genéticas e ultrassonográficas indicativas de doença renal policística autossômica dominante apresentaram aumento significativo no valor de cálcio total sérico, fração de excreção urinária de cálcio e de sódio(p=0,0219; p=0,0275; p=0,0032, respectivamente). Os demais parâmetros clínicos e laboratoriais não diferiram entre os dois grupos de animais. O exame ecocardiográfico revelou que casos de hipertrofia miocárdica foram mais frequente entre os animais positivos para DRPAD (p=0,0001). Dessa forma, é possível concluir que exames de triagem ultrassonográfica e/ou molecular devem ser utilizados para o diagnóstico de animais com DRPAD, visto que, alterações clínicas e laboratoriais sã o tardias. Exames eletro e ecocardiográficos devem ser rotineiramente realizados nos felinos císticos. Ainda, a caracterização clínica da DRPAD em gatos ressalta não apenas sua grande importância para medicina felina, mas também para a comunidade médica devido à correlação com a doença humana, representando um modelo ortólogo espontâneo, em animal de porte médio, para o estudo dessa enfermidade e validação de novos procedimentos terapêuticos. / Autosomal dominant polycystic kidney disease (ADPKD) in feline is characterized by the presence of multiple cists located in the renal parenchyma and occasionally, in liver and pancreas, and is an important cause of terminal chronic renal disease. It is considered worldwide as the most prevalent congenital illness in domestic cats. However, in Brazil the epidemiological and clinical data are barely existent, which difficultythe control of the disease. In the same way, the human ADPKD is the most common monogenic kidney disease. It affects 1 in every 400 to 1000 individuals, with a clinical course very similar to the disease in feline. In this research, 252 Persians and crossbreed cats from São Paulo state were assessed through the genetic test for ADPKD. The results indicated a prevalence of only 6.35% of cats carring the mutation for ADPKD in heterozygosis. This value is below the indexes reported in other countries. A cohort of 82 cats was selected in the metropolitan region of São Paulo to perform physical, laboratory, imaging and genetic tests. These animals were divided into two groups according to the presence (n = 12) or absence (n = 70) of ultrasound changes suggestive of polycystic kidney disease. The sensitivity and specificity between molecular tests and ultrasonography exam for ADPKD in the sample were both 100%. The animals with ultrasonography and genetic modifications, indicatives of ADPKD, presented a significant increase in the amount of total serum calcium, urinary fractional excretion of calcium and sodium(p=0,0219; p=0,0275; p=0,0032, respectively). Other clinical and laboratory parameters did not differ between the two groups of animals with and without the disease. Echocardiographic examination revealed that cases of myocardial hypertrophy were more frequent among animals positive for ADPKD (p = 0.0001). In conclusion, ultrasound exams and/ or molecular screening should be used to diagnostic animals with ADPKD, since the clinical and laboratory abnormalities are late. Electrocardiography and echocardiography examinations should be routinely performed in cystic cats. Still, the clinical characterization of ADPKD in cats not only highlights its importance to feline medicine, but also for the medical community due to the correlation with human disease, as an ortholog spontaneous model for the study of this disease.

A Insuficiência Renal Crônica (IRC) é uma alteração sistêmica, relativamente comum, onde os principais fatores de risco estão representados pelo diabetes e pela hipertensão. A disfunção acarreta redução ou limitação da capacidade de filtração glomerular dos rins, causando uremia, alterações sistêmicas diversas, especialmente cardiovasculares (hipertensão arterial, aterosclerose, pericardites, cardiomiopatias, arritmias cardíacas e hipertensão pulmonar), anemia, problemas hemostáticos e linfocitopenia. Também podem ser observadas alterações ósseas e bioquímicas, além de distúrbios gastrintestinais e dermatológicos. No âmbito bucal várias alterações tem sido descritas, tanto em tecidos moles quanto em tecidos duros (hálito urêmico, pH salivar mais alcalino; aumento na capacidade-tampão, elevada formação de cálculo dentário, aumento do número de cáries e incidência de doença periodontal), associadas aos distúrbios fisiológicos. Além disso, os pacientes portadores de enfermidades crônicas são frequentemente afetados por maiores níveis de estresse emocional, decorrente da necessidade de tratamento contínuo e limitação de suas atividades sociais, com consequente prejuízo de sua qualidade de vida. Este trabalho teve como objetivo caracterizar as manifestações orais destes pacientes, buscando estabelecer correlações entre o estado de ansiedade e depressão, qualidade de vida, valorização da saúde oral; características biodemográficas e status da insuficiência renal dos indivíduos incluídos no estudo. Nosso plano foi o de examinar pacientes que frequentam instituições especializadas em hemodiálise (duas) e, paralelamente, compor grupo controle, não portador de IRC, tabulando as variáveis propostas na investigação. A metodologia desenvolveu-se com a aplicação de questionários referentes a qualidade de vida (Sf36), indicativo de depressão (Inventário de Beck) e percepção de saúde bucal ( OHIP-14). Os dados foram coletados com o auxílio de ficha clínica específica. Os resultados indicaram níveis maiores de ansiedade e depressão, declínio na qualidade de vida e percepção de saúde bucal. Houve diferença estatisticamente significante no índice PSR quando comparados grupo teste (GT) e controle (GC). Houve maior número de queixas bucais nas mulheres do GC. Em ambos os grupos e gêneros houve relação inversa entre escore de xerostomia e nível de fluxo salivar. Conclui-se que a menor qualidade de vida influencia negativamente o sintoma de depressão que, por sua vez, reduz a percepção de saúde oral. O ciclo se fecha pela influencia negativa do distúrbio metabólico (e as sequelas medicamentosas) sobre a saúde bucal. / Chronic Kidney Disease (CKD) is a relatively common systemic dysfunction which main risk factors are represented by diabetes and hypertension. CKD implies in reduction or limitation of kidneys\' glomerular filtration capacity, causing uremia, various systemic disturbances, particularly cardiovascular (hypertension, atherosclerosis, pericarditis, cardiomyopathies, cardiac arrhythmias and pulmonary hypertension), anemia, lymphopenia and hemostatic problems. Biochemical changes in calcium metabolism can also be observed, as well as gastrointestinal and dermatological disturbances. Oral cavity disturbances may be represented either by soft tissue as hard tissue diseases (uremic breath, more alkaline saliva pH, increase in buffering capacity, high formation of dental calculus, increase in the incidence of tooth decay and periodontal disease). In addition, patients with chronic diseases are often affected by higher levels of emotional stress as a result of the need of continuous treatment that limits their social activities, impairing their quality of life as a consequence. This study aimed to characterize oral and systemic manifestations of these patients in order to establish correlations among anxiety and depression state, quality of life, appreciation of oral health, biodemographic data and the status of renal failure of the individuals included in the study. Our plan was to examine patients attending specialized institutions in hemodialysis and compare this population to a control group, no IRC. The data collected were discussed descriptively and analyzed according to appropriate statistical tests. The methodology was developed with the use of questionnaires regarding quality of life (SF36), indicative of depression (Beck Depression Inventory), and perception of oral health (OHIP-14). Data were collected with the assistance of medical record. The results indicated higher levels of anxiety and depression, impairment of quality of life and perception of oral health. There was a statistical significant difference when compared with the PSR index test (GT) and control group (CG). There was a higher number of oral complaints in women in CG. In both groups and genders there was an inverse relationship between scores of xerostomia and salivary flow rate. We conclude that the lower quality of life negatively influences depression level which, in turn, reduces the perception of oral health. The cycle completes by the negative influence of metabolic (and drug sequels) on oral health quality.

A insuficiência renal crônica (IRC) é uma afecção clínica frequente em gatos domésticos. É caracterizada por inflamação tubulointersticial, vascular, glomerular e fibrose severa. Estudos em modelos de IRC induzida em roedores têm revelado uma redução e estabilização do quadro clínico, evidenciados pela melhora nos parâmetros de função renal e redução da inflamação e da fibrose renal. Neste estudo foi testada a segurança e o efeito do transplante alogênico intra-renal e endovenosa das células-tronco mesenquimais derivadas da membrana amniótica felina (AMSCs) em gatos acometidos pela IRC natural. As AMSCs foram isoladas de âmnio de embriões coletadas em campanhas rotineiras de castração. Dez gatos, machos e fêmeas, foram incluídos neste estudo. Um gato hígido recebeu injeção intra-renal das AMSCs guiada por ultrassom em ambos rins (5x105 células/rim). Nove gatos com IRC natural receberam injeção endovenosa das AMSCs (2x106 células x 2 tratamentos). A avaliação da evolução clínica foi baseada na mensuração dos parâmetros do hemograma, bioquímica, hemogasometria, urinálise e ultrassonografia. Foi efetuada análise de variância (ANOVA) comparar diferenças entre as fases de tratamento seguido de teste de Tukey para comparação das médias entre os grupos. Na injeção intra-renal, não houve variação nos parâmetros clínicos, porém foi necessária a sedação e anestesia geral. Foi registrado elevado estresse de manipulação e ligeira hematúria após o procedimento. Os gatos com IRC que receberam injeção endovenosa das AMSCs, registraram uma variação significativa nos parâmetros de função renal (redução dos níveis de creatinina sérica, redução da proteinúria e aumento da densidade urinária). A arquitetura e morfologia renal não teve variação com o tratamento. Conclui-se que as AMSCs felinas têm um efeito renoprotetor e melhoram a função renal em gatos acometidos pela IRC, estabilizando o quadro clínico e a progressão da doença. A injeção endovenosa das AMSCs constitui uma ferramenta importante para proporcionar boa qualidade de vida aos gatos com IRC / Chronic kidney disease (CKD) is a common clinical condition in domestic cats. It is characterized by tubulointerstitial, vascular, glomerular inflammation and severe fibrosis. Studies in rodent model of induced CKD have been shown a decrease and stabilization of the clinical condition, evidenced by renal function improvement and by inflammation and renal fibrosis reduction. In this study was evaluated the safety and effect of intra-renal and intravenous infusion of allogeneic mesenchymal stem cells derived from feline amniotic membrane (AMSCs) in cats with naturally occurring CKD. The AMSCs were isolated from fetal membranes collected after routine castrations. Ten cats, male and female, were enrolled and included in this study. A healthy cat received intrarenal injection of AMSCs guided by ultrasound in both kidneys (5x105 cells/kidney). Nine cats with naturally CDK received intravenous injection of AMSCs (2x106 cells x 2 treatments). The evaluation of the clinical condition was based on the measurement of complete blood count, blood biochemistry, blood gases, urinalysis and ultrasound. Analysis of variance (ANOVA) was performed to compare differences between the phases of treatment followed by Tukey test to compare means between groups. The clinical parameters of the healthy cat (intrarenal injection) did not change, but sedation and general anesthesia was required. The number of interventions stressed the animal and he developed transient hematuria after AMSCs injection. Cats with CDK, registered a significant improvement of renal function (decrease in serum creatinine and urine protein concentrations and increase in urine specific gravity). The kidney architecture and morphology did not change with the treatment. We conclude that the feline AMSCs have a renoprotective effect and improve renal function in cats with naturally occurring CKD, stabilizing the clinical condition and disease progression. Intravenous injection of AMSCs is an important tool to provide general well-being for cats with CDK

Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. Johannesburg, 2014 / Background: The causes of renal dysfunction in children treated for childhood cancers are multifactorial and clinical manifestations of dysfunction include hypertension, proteinuria and varying degrees of renal insufficiency. This study aimed to determine the different residual effects of cancer therapy on the renal system and factors associated with the residual effects in children treated for childhood cancers. Patients and Methods: The study was a descriptive cross sectional study that assessed 130 children, between the age of 1 and 18 years, who had completed treatment at Charlotte Maxeke Johannesburg Academic Hospital and were being followed up at the paediatric oncology clinic of the hospital. Results: After a median follow-up post treatment of 2 years, the various manifestations of renal dysfunction identified in the survivors included; decreased GFR, hypomagnesaemia, hypophosphataemia, proteinuria, haematuria and hypertension. In total, 34 survivors (26.15%) had at least one manifestation of renal dysfunction after completing treatment. The most prevalent manifestation of renal dysfunction detected was decreased GFR (17.69%). Hypomagnesaemia and hypophosphataemia were present in 8 (6.15%) and 6 (4.62%) of the survivors respectively. Patients who had renal dysfunction pre-treatment were three times more likely to have renal dysfunction post-treatment. Ifosfamide, Carboplatinum, and nephrectomy were significantly associated with a reduction in GFR Conclusion: A significant number of the survivors had a decreased GFR while some of them had hypomagnesaemia and hypophosphataemia. There was a strong association between pre-treatment and post-treatment renal dysfunction. These findings are very important in terms of decision making for individual patients with respect to selecting treatment modalities and dosages and also with respect to instituting nephro-protective measures to avoid further damage to the kidneys during and after treatment.

INTRODUÇÃO: O objetivo deste estudo foi avaliar prospectivamente os anticorpos anti-HLA após o transplante renal e associar estes achados com episódios de rejeição aguda, marcação por C4d e sobrevida do enxerto. MÉTODOS: Foram avaliados 926 soros de 111 pacientes no primeiro ano pós-transplante ou até a perda do enxerto. Os anticorpos foram analisados por PRA-ELISA (Panel Reactive Antibodies by Enzyme Linked Immuno Sorbent Assay). Anticorpos anti-HLA doador-específicos foram detectados por provas-cruzadas e caracterizados pelo método de microesferas marcadas com antígenos HLA. Episódios de rejeição aguda foram classificados conforme os Critérios de Banff 97, atualizados em 2003. RESULTADOS: Conforme o PRA-ELISA pós-transplante os pacientes foram classificados em 5 Grupos: Grupo A (n=80): sem evidência de anticorpos pré e pós-transplante; Grupo B (n=8): pacientes com anticorpos de novo; Grupo C (n=5): pacientes sensibilizados que permaneceram com mesmo nível de PRA-ELISA; Grupo D (n=4): pacientes sensibilizados que elevaram o nível de PRA-ELISA e Grupo E (n=14): pacientes sensibilizados que diminuíram o nível de PRA-ELISA durante o primeiro ano pós-transplante. A incidência de rejeição aguda foi de 23,4%. Pacientes dos Grupos B, C e D apresentaram mais episódios de rejeição aguda (respectivamente, 57%; 60% e 100%) que os dos Grupos A (18%) e E (7%), (p<0,001). Rejeições ocorridas no Grupo A foram histologicamente menos severas do que as dos outros Grupos (p=0,03) e com menor incidência de C4d+ (p<0,001). Entre os pacientes com rejeição aguda, 44% deles apresentaram anticorpos no momento da rejeição, sendo que em 90% dos casos esses anticorpos foram doadorespecíficos. Rejeição mediada por células, ou seja, sem anticorpos e com C4d-, ocorreu em 56% dos casos. A incidência global de rejeição mediada por anticorpos (RMA) foi de 11%. A sobrevida do enxerto censurada para óbito foi menor em pacientes com rejeição aguda (p<0,001), especialmente naqueles com anticorpos anti-HLA doador-específicos (p<0,001), com C4d+ (p=0,003) e nos casos de RMA (p<0,003). CONCLUSÃO: Nossos dados sugerem que a monitorização dos anticorpos anti-HLA após o transplante renal pode ser útil no diagnóstico das respostas mediadas por anticorpos e tem implicações em termos de sobrevida do enxerto. / INTRODUCTION: The aim was to follow prospectively anti-HLA antibodies (Abs) after kidney transplantation and to evaluate their association with acute rejection episodes, C4d staining and graft survival. METHODS: We analyzed 926 sera from 111 transplanted patients until graft lost or during 1 year posttransplant. The antibodies were analyzed using Panel Reactive Antibodies by Enzyme Linked Immuno Sorbent Assay (PRA-ELISA). Donor-specific antibodies (DSA) were detected by crossmatch tests and characterized by single antigen beads. Acute rejections (AR) were classified by Banff 97 criteria, updated in 2003. RESULTS: According to post-transplant PRAELISA the patients were classified in 5 groups: Group A (n=80): no evidence of Abs pre and post-transplant; Group B (n=8): patients with Abs de novo; Group C (n=5): sensitized patients who sustained the same PRA-ELISA levels; Group D (n=4): sensitized patients who increased PRA-ELISA levels and Group E (n=14): sensitized patients who decreased PRA-ELISA levels during the first year. The overall incidence of acute rejection was 23,4%. Patients from Groups B, C and D had more AR (respectively, 57%; 60% and 100%) than patients from Groups A (18%) and E (7%), (p<0.001). Patients from Group A had lower Banff scores than other groups (p=0.03) and lower rates of C4d positivity on AR biopsies (p<0.001). Among patients with AR, 44% of them had antibodies which appeared/increased during the AR episodes, and 90% were DSA. AR were pure cell-mediated (C4d-/Abs-) in 56% of the cases. The overall incidence of antibody-mediated rejection (AMR) was 11%. One-year censored graft survival was lower in patients with AR (p<0.001), specially in those with DSA (p<0.001), C4d+ (p=0.003), and AMR (p<0.003). CONCLUSION: Our data suggest that monitoring of anti- HLA antibodies post-transplantation is an useful tool for the diagnosis of antibody-mediated responses, and has prognostic implications in terms of graft survival.

Introdução: O transplante renal é a terapia substitutiva de escolha para a insuficiência renal crônica. O número absoluto de transplantes renais vem aumentando mundialmente e consequentemente as complicações deste procedimento têm sido evidenciadas com maior recorrência, dentre elas a estenose na artéria renal transplantada. A técnica endovascular vem mostrando bons resultados iniciais, com boa taxa de perviedade e baixa taxa de complicações pósoperatórias quando comparadas a técnica aberta para tratamento da estenose. O objetivo deste estudo foi avaliar os resultados primários da angioplastia da artéria renal do rim transplantado secundário a processo de re-estenose. Métodos: Realizado estudo retrospectivo no período de setembro de 2009 a outubro de 2015, baseado em protocolos de seguimento pós-transplante com perfil dos pacientes submetidos a tratamento por estenose em artéria de rim transplantado e o seguimento em curto prazo com critérios clínicos, laboratoriais e ultrassonográficos. Resultados: Dentre um total de 391 transplantes, 19 pacientes foram diagnosticados com Estenose na Artéria renal transplantada. Evidenciado um tempo médio entre o transplante e o diagnóstico de 172,6 dias. As estenoses ou acotovelamentos com alterações hemodinâmicas foram evidenciadas na anastomose (47%), no terço proximal (35%) e terço médio (18%) da artéria transplantada. Todos os pacientes foram submetidos a angioplastia com balão e posicionamento de stent metálico com sucesso técnico de 94,7 %. A creatinina média evoluiu de 3,63 mg/dl para 2,69 mg/dl em 24h e para 1,81 mg/dl em 30 dias (p<0.05). A taxa de filtração glomerular melhorou de 32,66 ml/min para 41,61 ml/min após 24 horas e 51,05 ml/min após 30 dias (p<0.05). Os critérios ultrassonográficos avaliados de velocidade da artéria renal e índice reno-iliaco (368,9 cm/seg e 3,71 pré angioplastia) normalizaram e se estabilizaram durante o período estudado (211,45 cm/seg e 1,69 90 dias pós angioplastia; p<0.05). Conclusão: A abordagem endovascular utilizando de angioplastia primária e colocação de stent metálico foi segura com boa uma taxa de sucesso técnico. O procedimento foi efetivo na melhora da função renal do enxerto transplantado e na correção das alterações ultrassonográficas evidenciadas no pré-operatório. / Introduction: Renal transplantation is the substitutive therapy of choice for chronic renal failure. The absolute number of renal transplants has increased worldwide and consequently the complications of this procedure have been evidenced with greater recurrence, among them stenosis in the transplanted renal artery. The endovascular technique has shown good initial results, with good patency rate and low rate of postoperative complications when compared to the open technique for stenosis treatment. The aim of this study was to evaluate the primary results of ATP from the renal artery of the transplanted kidney secondary to the restenosis process. Methods: A retrospective study was carried out from September 2009 to October 2015, based on post-transplant follow-up protocols with a profile of patients submitted to stenosis in a transplanted kidney artery and a short-term follow-up with clinical, laboratory and ultrasonographics criteria. Results: Out of a total of 391 transplants, 19 patients were diagnosed with transplanted renal artery stenosis. An average time between transplantation and the diagnosis of 172.6 days was evidenced. Stenting or flexing with hemodynamic changes were evident in the anastomosis (47%), in the proximal third (35%) and the middle third (18%) of the transplanted artery. All patients underwent balloon angioplasty and metallic stent placement with technical success of 94.7%. Mean creatinine increased from 3.63 mg / dL to 2.69 mg / dL in 24 hours and to 1.81 mg / dL in 30 days (p <0.05). The glomerular filtration rate improved from 32.66 ml / min to 41.61 ml / min after 24 hours and 51.05 ml / min after 30 days (p <0.05). The ultrasound criteria evaluated for renal artery velocity and reno-iliac index (368.9 cm / sec and 3.71 pre angioplasty) normalized and stabilized during the study period (211.45 cm / sec and 1.69 Angioplasty, p <0.05). Conclusion: The endovascular approach using primary angioplasty and stent placement was safe with good technical success rate. The procedure was effective in improving the renal function of the transplanted graft and in correcting the ultrasound changes evidenced in the preoperative period.

Sympathetic nerves are important for renal physiology and sepsis pathophysiology. A recent study showed sprouting of sympathetic nerves in spleen of septic mice. This study was done to test if renal sprouting of sympathetic nerves also happens and to investigate renal morphology in septic mice. Cecal ligation and puncture (CLP) was used to induce sepsis and kidneys were removed for evaluation. Bowman’s space was diminished with cortical bubble cells present suggestive of acute renal pathology, however, renal function was unchanged. Acute sepsis did not affect either renal sympathetic innervation or non-neuronal cholinergic cells. Mouse kidneys had more epinephrine (EPI) than norepinephrine (NE) in both groups. This is most likely due to uptake of epinephrine by renal sympathetic nerves and may have no correlation with sepsis. In conclusion, septic mice showed minor renal pathology and no evidence of acute sympathetic nerve sprouting. Further studies are needed to understand the mechanism and consequences of elevated EPI in mice kidney.

Concern over kidney disease has increased dramatically in recent years within the medical community. It is estimated that approximately one in fifteen Americans, nearly 20 million people, experience chronic kidney disease with most of those diagnosed progressing to kidney failure. The ultimate treatment available for end stage renal failure is whole kidney transplantation. However, there are very few kidneys available for patients to receive and those patients who are fortunate enough to receive an organ must remain on immunosuppressive medication for the remainder of their lives. The United States Department of Health & Human Services have reported that 18 people die every day while on the waiting list for organ donations. The treatment is fairly successful as 69% of patients who receive a kidney transplant are still alive 5 years after the transplant. Tissue engineered organs could be a promising alternative for whole organ transplantation. The overall objective is to repopulate appropriate decellularized scaffolds from pigs, which are not immunogenic, with a patient's own cells to achieve a functional organ. Therefore, there would be an inexhaustible source of organs ready for transplantation without the risk of immune rejection. The naturally obtained scaffolds devoid of immunogens are a potential matrix to create artificial kidneys. Repopulation of decellularized rat kidneys with renal progenitor cells has been reported in previous studies. This dissertation reports the scale-up of the previous technology and building of partially functional human-sized kidneys. In the first step, we investigated various cell lysing agents and developed an automated decellularization procedure for whole porcine kidney decellularization. We also developed a preservation method for native and decellularized kidneys to avoid spoilage before and after decellularization. We also developed a decontamination procedure for whole porcine kidneys. Finally, we recellularized whole porcine kidney scaffolds with renal epithelial cells and achieved partial repopulation of the renal structure.