Resumo: Objetivos: dois estudos prospectivos comparativos de intervenção foram realizados para avaliar o efeito do tratamento periodontal não-cirúrgico sobre parâmetros clínicos periodontais e imunológicos do fluido sulcular gengival e do plasma sanguíneo de pacientes com periodontite crônica portadores ou não de Diabetes mellitus Tipo 2. Material e Método: Vinte e três indivíduos com Diabetes mellitus Tipo 2 com controle metabólico inadequado e periodontite crônica (grupo diabetes) e 26 indivíduos sistemicamente saudáveis com periodontite crônica (grupo controle) foram avaliados quanto a parâmetros periodontais, marcadores inflamatórios do fluido sulcular gengival (interleucinas, metaloproteinases de matriz e atividade de elastase), marcadores inflamatórios no plasma sanguíneo (interleucinas, fator de necrose tumoral alfa, proteína C-reativa e fibrinogênio) e perfil lipídico, antes e após 3 meses do tratamento periodontal. Adicionalmente foi avaliada a influência do tratamento periodontal no controle glicêmico do grupo com diabetes. Resultados: houve redução significativa de todos os marcadores inflamatórios avaliados no fluido gengival, exceto a interleucina 18, após tratamento periodontal, associado a uma melhora da condição clínica periodontal em ambos os grupos. Sistemicamente, o grupo diabetes apresentou maiores níveis de proteina C reativa e triglicérides em ambos os períodos ao se comparar com o grupo controle. O tratamento periodontal foi efetivo em reduzir os níveis de TNF-_ e fibrinogênio no grupo diabetes. Houve melhora no controle glicêmico embora não significativa. Conclusão: os resultados do presente estudo sugerem que os pacientes portadores de Diabetes mellitus Tipo 2 com controle metabólico inadequado apresentam boa resposta ao tratamento periodontal nãocirúrgico, com redução de marcadores inflamatórios no fluido gengival. / Abstract: Objetive: Two prospective comparative interventional studies were performed in order to evaluate the effect of non-surgical periodontal therapy on periodontal clinical and immunological parameters in gingival crevicular fluid (CGF) and plasma of patients with chronic periodontitis with or without type 2 Diabetes mellitus (T2DM). Material and Method: Twenty three individuals with inadequately controlled T2DM and chronic periodontitis (diabetes group) and 26 systemically healthy individuals with periodontitis (control group) were assessed for clinical parameters, inflammatory biomarkers in GCF (interleukins, matrix metalloproteinases and elastase activity), circulating markers of inflammation (interleukins, tumor necrosis factor alpha, C-reactive protein and fibrinogen and lipid profile before and 3 months after periodontal therapy. Additionally, the influence of periodontal treatment on glycemic control was evaluated in the diabetes group. Results: There was a significant reduction of all inflammatory biomarkers in GCF after therapy, except for interleukin 18 levels, and it was associated with improvement on periodontal status in both groups. Systemically, the diabetes group showed high levels of C-reactive protein and triglycerides compared with the control group in both periods. The periodontal therapy was effective in reducing TNF-_ circulating and fibrinogen in the diabetes group. The treatment did not change the glycemic control significantly. Conclusion: The results of the present study suggest that patients with inadequately controlled T2DM present a good response to nonsurgical periodontal treatment, with reduction of inflammatory biomarkers in GCF. However, 3 months after therapy, its influence on systemic inflammatory condition is limited. / Orientador: Silvana Regina Perez Orrico / Coorientador: Carlos Marcelo S. Figueiredo / Banca: Adriana Campus Passanezi Sant'ana / Banca: Ricardo Guimarães Fischer / Banca: Elaine Maria Sgavioli Massucato / Banca: Joni Augusto Cirelli / Doutor

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / The nectar of consistent or very high in high consistency pulp fruit, under Brazilian law, must contain at least 20% pulp. This is the case of bacuri nectar, fruit with high content of high consistency pulp. Studies show that the nectar bacuri is better accepted by consumers when it is produced with pulp content of between 10% and 12%. To adjust the composition of the nectar bacuri the current law and achieve consumer acceptance, Aquino (2012) used the enzymatic maceration. The author attributed these changes to the listing of compounds by hydrolysis of volatile compounds which were bound glycosidically making them volatile and increasing its amount in the unbound fraction. The aim of this study was to investigate the changes during the enzymatic maceration and pasteurization on the free and bound (glycosylated) pulp bacuri volatile compounds profile. The pulp was diluted bacuri homogenized and pasteurized enzymatically macerated in each of these steps, a sample was taken for analysis of free and volatile glycosylated. To characterize the profile of volatile free was used in the headspace SPME technique and the profile of volatile glycosides was used a column of Amberlite XAD-2. In the analysis of free volatile compounds detected in the 41 compounds were homogenized sample, 56 and 56 in the macerating pasteurized. It was observed that with the maceration, an increase of terpene compounds, ethers and hydrocarbons and reduction of aldehydes. The pasteurization, in turn, caused the decrease of terpene compounds, aldehydes and ketones and alcohols increase. The study found that maceration promoted the emergence of new compounds and also the increase in the peak area of the compounds present in the previous step. Pasteurization volatiles were practically the same, with just a reduction in the concentration of most compounds. Chemical classes with the highest losses were terpenes, alcohols and aldehydes. For the analysis of volatile compounds glycosylated compounds were detected in the 56 sample mixed, macerated at 51 and 38 in sanitized. The homogenization step was the one with the largest number of volatile compounds glycosylated. In most pulp macerated compounds decreased, reaching in some cases the traces or amounts not be detected, indicating that the enzymes used in the maceration promoted breaking the glycosidic bonds glycosylated volatile compounds, making them free. Among the free volatile compounds, terpenes and alcohols are predominant, while the profile of volatile compounds is mainly glycosides and terpene ester compound. The enzymatic maceration influence on volatile-free profile and glycosylated in free promoted the release of volatiles that were physically linked in the viscous matrix of pulp glycosylated promoted and breaking the glycosidic linkage of various compounds, with release of the respective volatiles but also by releasing more compounds that were trapped in the viscous matrix. Pasteurization promoted losses by volatilization and / or degradation of some compounds possible, but also increased the concentration of the other. / O nÃctar de frutos muito consistentes ou com alto teor de polpa de elevada consistÃncia, segundo a legislaÃÃo brasileira, deve conter no mÃnimo 20% de polpa. Esse à o caso do nÃctar de bacuri, fruto com alto teor de polpa de elevada consistÃncia. Estudos mostram que o nÃctar de bacuri à melhor aceito pelos consumidores quando o mesmo à produzido com teor de polpa entre 10% e 12%. Para adequar a composiÃÃo do nÃctar de bacuri à legislaÃÃo vigente e atingir a aceitaÃÃo dos consumidores, Aquino (2012) utilizou a maceraÃÃo enzimÃtica. O autor atribuiu essas alteraÃÃes no perfil de compostos volÃteis a hidrÃlise de compostos que estavam ligados glicosidicamente, tornando-os volÃteis e aumentando a sua quantidade na fraÃÃo livre. O objetivo desse estudo foi investigar as alteraÃÃes ocorridas durante a maceraÃÃo enzimÃtica e pasteurizaÃÃo no perfil de compostos volÃteis livres e ligados (glicosilados) da polpa de bacuri. A polpa de bacuri diluÃda foi homogeneizada, macerada enzimaticamente e pasteurizada, em cada uma dessas etapas, uma amostra foi retirada para anÃlise de volÃteis livres e glicosilados. Para caracterizar o perfil de volÃteis livres foi utilizado à tÃcnica de SPME em headspace e para o perfil de volÃteis glicosilados foi utilizado uma coluna de Amberlite XAD-2. Na anÃlise de compostos volÃteis livres foram detectados 41 compostos na amostra homogeneizada, 56 na macerada e 56 na pasteurizada. Observou-se que com a maceraÃÃo, houve aumento de compostos terpÃnicos, Ãteres e hidrocarbonetos e diminuiÃÃo de aldeÃdos. A pasteurizaÃÃo, por sua vez, provocou a diminuiÃÃo de compostos terpÃnicos, aldeÃdos e cetonas e o aumento de alcoÃis. O estudo apontou que a maceraÃÃo promoveu o surgimento de novos compostos e tambÃm o aumento na Ãrea dos picos dos compostos presentes na etapa anterior. Na pasteurizaÃÃo os compostos volÃteis permaneceram praticamente os mesmos, ocorrendo apenas uma diminuiÃÃo na concentraÃÃo da maioria dos compostos. As classes quÃmicas que apresentaram as maiores perdas foram os terpenos, alcoÃis e aldeÃdos. Para a anÃlise de compostos volÃteis glicosilados foram detectados 56 compostos na amostra homogeneizada, 51 na macerada e 38 na pasteurizada. A etapa de homogeneizaÃÃo foi a que apresentou o maior nÃmero de compostos volÃteis glicosilados. Na polpa macerada a maioria dos compostos diminuiu, chegando, em alguns casos, a quantidades traÃos ou ainda a nÃo serem detectados, indicando que as enzimas utilizadas na maceraÃÃo promoveram a quebra das ligaÃÃes glicosÃdicas dos compostos volÃteis glicosilados, tornando-os livres. Dentre os compostos volÃteis livres, os terpenos e os alcoÃis foram os predominantes, enquanto o perfil de compostos volÃteis glicosilados foi composto principalmente de Ãsteres e terpenos. A maceraÃÃo enzimÃtica influenciou no perfil de volÃteis livres e glicosilados, nos livres promoveu a liberaÃÃo de compostos volÃteis que estavam ligados fisicamente na matriz viscosa da polpa e nos glicosilados promoveu a quebra da ligaÃÃo glicosÃdica de vÃrios compostos, com a liberaÃÃo dos respectivos compostos volÃteis, como tambÃm pela liberaÃÃo de mais compostos que estavam presos na matriz viscosa. A pasteurizaÃÃo promoveu perdas por volatilizaÃÃo e/ou possÃvel degradaÃÃo de alguns compostos, como tambÃm fez aumentar a concentraÃÃo de outros.

Diabetes mellitus är en av de vanligaste endokrina sjukdomarna och de vanligaste formerna är typ I och typ II. Idag har ca 350 000 personer i Sverige diabetes och av dessa har 85-90% diabetes typ II. Typ II diabetes börjar med insulinresistens och så småningom blir det avtagande funktion av β- cellerna vilket leder till nedsatt insulinkänslighet och främsta orsakerna till typ II diabetes är övervikt och fetma. Det finns olika behandlingsrekommendationer för att behandla typ II diabetiker för att minska att sena komplikationer uppstår. Främst genom livsstilsförändringar som kost och fysisk aktivitet, men då dessa inte räcker till kan perorala läkemedel komma i efterhand och om inte det heller ger tillräcklig effekt kan insulinbehandling sättas in. Ca 50 % av typ II diabetiker får insulin efter 10 års sjukdom. Syftet med arbetet är att undersöka om det finns en god implikation av att sätta in insulin tidigare än det som redan är rekommenderat. Denna litteraturstudie är baserad på artiklar hämtade från databasen PubMed. Sammanlagt har fem randomiserade kontrollerade studier granskats. Resultaten visar att en HbA1c-sänkning med ca 1,5 - 2,0 % kan erhållas samt också en bibehållen β- cellfunktion vid insättning av insulin. Insulinbehandlingen bör sättas in så snart HbA1c går över 7,5 % istället för att vänta en viss tid. Den kan sättas in hos behandlingsnaiva personer med framträdande symtom eftersom insulin fortfarande sänker HbA1c och det finns inget som tyder på att insulin inte kan sättas in tidigare än det som är rekommenderat. Slutsatsen som dras är att stödja intensiv behandling som gör att HbA1c hålls på en så låg nivå det är möjligt och när målvärden för HbA1c inte kan hållas kan insulin med fördel sättas in hos typ II diabetiker som behandlats med perorala antidiabetika.

The species Didymopanax morototoni (araliaceae), known as morototó is used in folk medicine in some countries of Latin America and Brazil is used to regulate menstrual flow, however, studies of its chemical composition were not reported. This study evaluated the potential of extracts and fractions of D. morototoni for the control of tropical disease vectors, Aedes aegypti larvae, the mollusk Biomphalaria glabrata, inhibition of reverse transcriptase, antitumor activity against the NCI-H292 cells and K562, and as well as the elucidation of the six active ingredients. The plant material was collected in the municipality of Pilar, Alagoas State, and was subjected to a phytochemical study led by the molluscicidal bioassay. The crude extract of the plant was obtained by extraction with 90% ethanol and subjected to fractionation with solvents of increasing polarity. The structural elucidation of compounds was based on the analysis of IR spectra and NMR, experiments including 1D and 2D. Were isolated from the chloroform fraction of the stem bark acid 3-O-β-D-xilopiranosil (1→3) β- glucopyranosyl-olean-12-en-28-oic acid (DMCC1), the mixture of 3-O-β-D-glucopyranosylsitosterol and 3-O-β-D-glucopyranosyl-stigmasterol (DMCC2) and acid 3-O-β-Dglucopyranosyl (1→2) β - arabinopiranosil (4→1) β-glucopyranosyl-olean-12-en-28-oicacid (DMCC3), and the hexane fraction of the wood the mixture of steroids stigmasterol and β-sitosterol (DMM1), oleanolic acid (DMM2), ursolic acid (DMM3). The ethanolic leaf extract of D. morototoni was active against the larvae of Aedes aegypti with LC50 55.0264mg. mL-1. The ethanolic extract of the wood, stem bark and root have shown molluscicidal activity against the snail Biomphalaria glabrata. The ethanol extract of the root exhibited higher molluscicidal activity with LC50 5.336 mg. mL-1. among the compounds isolated the triterpene glycosides and DMCC1 DMCC3 showed molluscicidal activity with LC50 1.012 and 0.906 mg. mL-1 respectively. The ethanol extracts of leaf and root bark inhibited the action of the enzyme reverse transcriptase (RT) 100% and 51% respectively at a concentration of 50 mg. mL-1. Although the stem bark extract did not show significant inhibition of TR to the hexane fraction showed 79.62% inhibitory activity of 50 mg. mL-1. All substances isolated from D. morototoni inhibited RT, the mixture of 3-O-β-D-glucopyranosyl-sitosterol and 3-O-β-D-glucopyranosyl-stigmasterol showed the greatest potential inhibitor of TR with 70.41% at 10 mg. mL-1. For testing the antitumor activity was the best result for the ethanolic extract of stem bark with IC50 3.44 mg. mL-1 and K-562 IC50 8.01 mg. mL-1 in NCI-H292. This extract only the chloroform fraction was active with IC50 7.97 and 13.32 mg. mL-1 for NCIH292 cells and K562 respectively. The extract and fractions derived from the stem bark showed activity imunomudulatória with a percentage ranging from 64% to 99.25% 100 mg.mL-1. The results of phytochemical and biological activity contribute to propose the use of this plant molluscicide agent, antiviral and antitumor. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A espécie Didymopanax morototoni (Araliaceae), conhecida como morototó, é utilizada na medicina popular de alguns países da América Latina e no Brasil é usado para regular o fluxo menstrual, no entanto, estudos de sua composição química não foram relatados. O presente trabalho avaliou o potencial dos extratos e frações de D. morototoni para o controle dos vetores de doenças tropicais, larvas de Aedes aegypti, do molusco Biomphalaria glabrata, inibição da transcriptase reversa, atividade antitumoral contra as células NCI-H292 e K562 e bem como a elucidação de seis dos princípios ativos. O material vegetal foi coletado no município de Pilar, Estado de Alagoas, e foi submetido a um estudo de fitoquímica guiado pelo bioensaio moluscicida. O extrato bruto da planta foi obtido pela extração com etanol 90% e submetido a fracionamento com solventes de polaridade crescente. A elucidação estrutural dos compostos foi feita com base na análise dos espectros no IV e de RMN, incluindo experimentos 1D e 2D. Foram isolados da fração clorofórmica da casca do caule os o ácido 3-O-β-D- xilopiranosil (1→3) β- glicopiranosil-oleano-12-en-28-oico (DMCC1), a mistura de 3-O-β-D-glicopiranosil-sitosterol e 3-O-β-D-glicopiranosil-estigmasterol (DMCC2) e o ácido 3-O-β-D-glicopiranosil (1→2) β - arabinopiranosil (4→1) β-glicopiranosil- oleano-12-en-28-oico (DMCC3), e da fração hexânica da madeira a mistura dos esteróides estigmasterol e β-sitosterol (DMM1), ácido oleanólico (DMM2), ácido ursólico (DMM3). O extrato etanólico da folha de D. morototoni foi ativo frente às larvas de Aedes aegypti com CL50 55,0264 μg. mL-1. Os extratos etanólico da madeira, casca do caule e raiz apresentaram atividade moluscicida frente ao caramujo Biomphalaria glabrata. O extrato etanólico da raiz exibiu maior atividade moluscicida com CL50 5,336 μg. mL-1. Dentre os compostos isolados os triterpenos glicosilados DMCC1 e DMCC3 apresentaram atividade moluscicida com CL50 1,012 e 0,906 μg. mL-1 respectivamente. Os extratos etanólico da folha e da casca da raiz inibiram a ação da enzima transcriptase reversa (TR) 100% e 51% respectivamente na concentração de 50 μg. mL-1. Apesar do extrato da casca do caule não apresentar inibição significativa da TR a fração hexânica mostrou 79,62 % de atividade inibitória 50 μg. mL-1. Todas as substâncias isoladas de D. morototoni inibiram a TR, a mistura de 3-O-β-D-glicopiranosil-sitosterol e 3-O-β-D-glicopiranosil-estigmasterol apresentou maior potencial inibidor da TR com 70,41% a 10 μg. mL-1. Para o ensaio da atividade antitumoral o melhor resultado foi para extrato etanólico da casca do caule com CI50 3,44 μg. mL-1 em K-562 e CI50 8,01 μg. mL-1 em NCI-H292. Desse extrato apenas a fração clorofórmica foi ativa com CI50 13,32 e 7,97 μg. mL-1 para as células NCI-H292 e K562 respectivamente. O extrato e frações oriundas da casca do caule exibiram atividade imunomudulatória com percentual variando de 64% a 99,25% 100 mg. mL-1. Os resultados da atividade biológica e fitoquímica contribuem para propor a utilização dessa planta como agente moluscicida, antiviral e antitumoral.

Introdução- A hiperglicemia (HG), durante o infarto agudo do miocárdio (IAM), está associada com aumento de mortalidade hospitalar em pacientes diabéticos e não diabéticos. Entretanto, não é conhecido o mecanismo responsável por esta associação. Assim estudou-se, simultaneamente, a correlação entre a glicemia e marcadores bioquímicos relacionados ao sistema neuro-humoral de estresse, metabolismo glicídico e lipídico, sistema de coagulação e inflamatório. Métodos- 80 pacientes foram incluídos consecutiva e prospectivamente. Foram realizadas duas coletas de sangue, a primeira com 24h a 48h do início dos sintomas do IAM (fase aguda) e a segunda após 3 meses do IAM (fase crônica), sempre com 12h de jejum. Foram analisados os seguintes parâmetros: glicose, cortisol, noradrenalina, hemoglobina glicada (HbA1c), insulina, LDL minimamente modificada eletronegativa, ácidos graxos livres (AGL), adiponectina, factor VII da coagulação, fibrinogênio, inibidor do ativação do plasminogênio tipo 1, proteína C reativa ultra-sensível (PCRus), colesterol total (c) e frações e triglicérides. Nas correlações univariadas entre glicemia e as variáveis contínuas empregou-se o teste de correlação de Pearson. As análises multivariadas foram feitas através de regressão logística (variáveis qualitativas) e modelo linear generalizado (quando as variáveis independentes incluídas foram quantitativas e nominais). Resultados- Na fase aguda, a glicemia correlacionou-se significativamente com HbA1c (r=0,75, p<0,001), insulina (r=0,25, p<0,001), AGL (r=0,3, p=0,01), adiponectina (r=-0,22, p=0,05), LDL-c (r=-0,25, p=0,03), VLDL-c (r=0,24, p=0,03) e triglicérides (r=0,27, p=0,01). No modelo multivariado, as variáveis correlacionadas de forma independente com a glicemia, na fase aguda, foram: HbA1c (p<0,001), insulina (p<0,001), e AGL (p=0,013). Para analisar uma variável de confusão, a história de diabetes mellitus (DM), incluiu-se esta variável num modelo, juntamente com as variáveis acima e todas mostraram associação significativas com glicose: HbA1c (p<0,001), insulina (p=0,001), AGL (p=0,013) e história de DM (p=0,027). Na fase crônica, glicose correlacionou-se com: cortisol (r=0,31, p=0,01), noradrenalina (r=0,54, p<0,001), HbA1c (r=0,78, p<0,001) e PCRus (r=0,46, p<0,001). Na análise multivariada, somente HbA1c (p<0,001) e noradrenalina (p<0,001) mantiveram correlação independente. Conclusão- A HbA1c foi a única variável que correlacionou-se de forma significativa e independente com a glicemia, tanto na fase aguda, quanto na crônica, mostrando que a hiperglicemia, durante o IAM, pode representar uma alteração crônica, sub-diagnosticada, do metabolismo glicídico. / Introduction- Hyperglycemia (HG) is an important prognostic factor in acute myocardial infarction (AMI). However, the pathophysiology is poorly understood. So we proposed a simultaneous correlation between glycemia and biochemical markers of stress, glucose and lipid metabolism, coagulation and inflammation system. Methods- Eighty AMI patients were included prospectively. Blood were collected between 24h and 48h from the pain (acute phase), and 3 months post AMI (chronic phase), with 12-h fasting. These parameters were analyzed: glucose, cortisol, norepinephrine, hemoglobin glycated (HbA1c), insulin, minimally modified electronegative LDL, free fatty acids (FFA), adiponectin, factor VII coagulant, fibrinogen, plasminogen activator inhibitor-1, high sensitive C reaction protein (hsCRP), total cholesterol (c) and fractions and triglyceride. The relationships between glucose and continuous variables were assessed by Pearsons correlation coefficient (r) and multivariate analysis with linear regression. Results- At acute phase, glucose correlated significantly with HbA1c (r=0.75, p<0.001), insulin (r=0.25, p<0.001), FFA (r=0.3, p=0.01), adiponectin (r=-0.22, p=0.05), LDL-c (r=-0.25, p=0.03), VLDL-c (r=0.24, p=0.03) and triglyceride (r=0.27, p=0.01). In a multivariate model, variables correlated were: HbA1c (p<0.001), insulin (p<0.001), and FFA (p=0.013). At the chronic phase, glucose correlated significantly with cortisol (r=0.31, p=0.01), norepinephrine (r=0.54, p<0.001), HbA1c (r=0.78, p<0.001) and hsCRP (r=0.46, p<0,001). By multivariable analysis, only HbA1c (p<0.001) and norepinephrine (p<0.001) remained correlated. Conclusion- HbA1c was the main variable that correlated significantly and independently with glycemia at acute and chronic phases, suggesting that HG during AMI can represent an exacerbation of abnormal glucose metabolism previously not diagnosed.

Murine leukemia virus (MLV), classified as a gammaretrovirus, has been studied extensively to enhance our understanding of the biology and replication of retroviral infection. Typically referred to as a simple retrovirus, its usefulness as a model is highlighted owing to its minimal genome. The genetic material for MLV was thought to only code the basic and essential defining features of a retrovirus. Through the understanding developed from the use of simple retroviruses, the clinical and research communities were immeasurably more prepared to combat the more complex and decidedly infamous human immunodeficiency virus (HIV). Interestingly, a scenario of convergent evolution has directed MLV to encode an accessory protein, termed Glycosylated Gag (gGag), that shares functionality reminiscent of several HIV proteins. Herein, I present a dissection of a novel function of this enigmatic protein, paired with an improved understanding of the biology of MLV that was revealed by the development of small particle flow cytometry performed on viruses, also known as flow virometry. Initially, we elucidated that gGag is responsible for the resistance of MLV towards the restriction factor murine APOBEC3 (mA3). I showed that even endogenous mA3 from primary cells exhibited an enhanced enzymatic activity towards MLV with mutant gGag proteins which have lost glycosylation sites. In our following study, I illustrated that these mutants displayed a reduced viral core stability, the severity of which was correlated directly with susceptibility to mA3. These results are in line with the hypothesis that viral core stability and APOBEC3-susceptibility are directly linked. Furthermore, I showed for the first time that unprocessed gGag was associated with viral particles released from producer cells in the orientation of a type I membrane protein, with the structural regions directed within the viral core. This may be the direct evidence of how gGag improves capsid stability, a mechanism which is still unresolved. On the flip side, gGag as a type II membrane protein was observed exclusively on virus-like particles devoid of detectable envelope glycoprotein (Env). This marks a potential new function for gGag in the context of infection. Given the ubiquitous necessity of an optimized core stability for any virus, combined with the overlapping function of gGag with HIV accessory proteins, continuation of this work represents an as of yet clinically unexplored avenue for the development of HIV therapeutics. At the same time, in order to characterize individual viral particles, I played an instrumental role in developing the technique of flow virometry within our core facility. I illustrated that the Env of MLV does not significantly accumulate on extracellular vesicles (EVs) and acts as an effective marker for viral particles. With this evidence in hand, the enumeration of MLV virions was made possible. By correlating this information with an absolute viral genome determination, I was able to estimate the packaging efficiency for MLV in a quantitative manner. This information suggests that roughly 80-85% of MLV particles are missing their essential genetic information. These findings may implicate the disease progression of MLV infection may be enhanced by the use of defective-interfering particles, a theory that has been suggested for HIV. This work highlighted the fact that flow virometry is uniquely capable to discriminate viral particles from other cell-derived membraned vesicles in a highly sensitive manner. Overall, my work has unveiled new complexities of a simple retrovirus, while laying the groundwork towards both diagnostics and therapeutics for the ongoing battle with HIV.

Objetivo: Determinar la relación entre el nivel de hemoglobina glicosilada y el riesgo de desarrollar diabetes mellitus tipo 2 en el plazo de diez años, en pacientes no diabéticos de un Hospital de Lima Norte. Metodología: El presente estudio fue descriptivo, observacional, transversal y prospectivo, se realizó en el consultorio externo de un Hospital de Lima Norte, participaron 101 personas no diabéticas entre 18 y 65 años de uno u otro sexo, y se relacionó los niveles de hemoglobina glicosilada con el riesgo de desarrollar diabetes mellitus tipo 2 en el plazo de diez años mediante el test de FINDRISK. Resultados: Se estudiaron 101 pacientes no diabèticos, el 66.7% fueron del sexo femenino, el grupo etario más frecuente fue los menores de 35 años, (44.1%). El 99% de los encuetados presentaron valores normales de hemoglobina glicosilada. Los resultados del test de FINDRISK mostraron que el 29.7% de los evaluados tuvieron riesgo bajo de desarrollar diabetes tipo 2 en el plazo de 10 años, y el 21.8% tuvieron riesgo de moderado y alto. Cuando se asoció hemoglobina glicosilada con el riesgo de desarrollar diabetes s mellitus tipo 2 no se encontró asociación p=0.218. Conclusión: No se halló relación estadísticamente significativa cuando se relacionó la hemoglobina glicosilada con los riesgos de desarrollar diabetes mellitus tipo 2, en la población estudiada. / Objective: To determine the relationship between the glycosylated hemoglobin level and the risk of developing type 2 diabetes mellitus within ten years in non-diabetic patients in a Hospital in North Lima. Methodology: This study was descriptive, observational, transversal, and prospective, and was carried out in the outpatient clinic of a Hospital in Lima Norte, 101 non-diabetic persons between 18 and 65 years of age from one sex or another, And glycosylated hemoglobin levels were associated with the risk of developing type 2 diabetes mellitus within ten years using the FINDRISK test. Results: 101 non-diabetic patients were studied, 66.7% were female, the age group was the most frequent ones under 35 years of age (44.1%). 99% of respondents had normal glycosylated hemoglobin values. Results of the FINDRISK test showed that 29.7% of those evaluated had a low risk of developing type 2 diabetes within 10 years, and 21.8% had moderate and high risk. When glycosylated hemoglobin was associated with the risk of developing type 2 diabetes Mellitus, no association was found p=0.218. Conclusion: No statistically significant relationship was found when glycosylated hemoglobin was associated with the risks of developing type 2 diabetes mellitus in the study population. / Trabajo de investigación

A crossover design study was conducted to determine if reducing pain, by using alternative sites off the finger tip, would increase testing frequency and improve clinical outcome as measured by glycosylated hemoglobin. Subjects with type I and type 2 diabetes tested with the FreeStyle alternative-site meter (group I) or tested with their original meter (group 2). After 3 months the subjects used the alternate meter. Testing frequency and blood glucose concentrations were recorded for the month before the study began and monthly thereafter. Glycosylated hemoglobin was tested initially, at the crossover point, and at study conclusion. Insulin users increased testing frequency from 2.4 to 3.0 tests per day. Testing frequency for non-insulin users remained the same at 1.5 tests per day. Testing frequency was essentially the same with the FreeStyle and the original meters. The average hemoglobin A1c was 7.4% (standard deviation 1.5%) initially, 7.3% (standard deviation 1.5%) at the crossover point, and 6.9% (standard deviation 1.1%) after 6 months. There was no significant difference in hemoglobin A1c measurements between meter types after 6 months. Thirteen months later a final hemoglobin A1c, testing frequency, and a questionnaire regarding meter preference and pain rating were obtained. Seventy-four percent of participants preferred the alternative-site meter, which was rated as significantly (p < .05) less painful. Testing frequency significantly improved (p = .001) while free strips were being provided. Testing frequency 13 months later was not significantly different from the baseline (p = .101). Hemoglobin A 1 c was significantly lower 6 months after the study began (p = .000) and 13 months later (p = .008) at baseline.

Thesis (MScEng)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Recombinant glycoproteins are important biopharmaceuticals, providing solutions for numerous previously untreatable illnesses, in everything from cancer to infertility. Most recombinant biopharmaceuticals are produced in mammalian cells due to their ability to provide the correct post-translational processing for use in humans. The post-translation processing influences many of the protein’s properties including pharmacokinetics, bioactivity, secretion, half-life, solubility, recognition and antigenicity. The aim of this thesis is to further study the upstream production of a glycosylated recombinant protein produced by Chinese hamster ovary (CHO) cells on production scale within the confines of an existing process. The process in question uses adherent CHO cells to produce a glycosylated recombinant hormone. As with most recombinant protein production processes, this process has two sections to the upstream production: a seed train to grow enough cells to inoculate production, and a production section, which focuses on the production of a recombinant protein. The seed train is predominantly conducted in roller bottles, while the production section takes place in perfusion bioreactors, where the cells are attached to microcarriers, with spin-filters for cell retention. The whole process uses medium with serum. There are two process challenges regarding an existing recombinant-protein production process: 1. The gradual increase, over the past several campaigns, of the final population doubling level of the cells (which must remain within certain specified limits) at the end of the seed train. 2. The low glycosylation levels of the product seen in certain campaigns, which meant that a certain number of final product batches were below the specified acceptable glycosylation limits. Following a literature survey several controlled process variables were chosen for investigation and hypotheses made on their effect on the seed train or glycosylation. To investigate their effect on the PDL and cell growth in the seed train: - Medium volume: decreasing the medium volume will yield a lower PDL due to slower cell growth caused by lower glucose availability. - Seeding density: if cells obtain confluence by the time they are harvested, decreasing the seeding density will yield a higher PDL. - Cultivation temperature: decreasing the temperature ought to decrease the growth rate. - Medium feed temperature: there will be no significant difference to the cell culture when pre-heated or cold medium is used. Aeration: using vent caps will increase the oxygen content of the medium in the roller bottles and the cell growth, yielding a higher PDL. To investigate their effect on glycosylation during production: - pH: better glycosylation will be seen at pH 6.9, than at pH 6.7. - Perfusion rate: a higher perfusion rate will lead to better glycosylation due to increased glucose and glutamine concentrations. In the seed train, the only factor that significantly influenced the final PDL was the seeding density. Cell growth was inhibited once cells reached confluence, so lowering the seeding density lead to a higher PDL. It is recommended to use a high seeding density to ensure a lower PDL. Historic data indicated that the seeding density was not the cause of the apparent increase of the final PDL, as all previous campaigns had been seeded with a high seeding density. What then became apparent was that the final PDL remained relatively constant during a campaign and that the increase in final PDL occurred between campaigns. It appears that the apparent increase in the final PDL is due to differences in cell counting between operators as each new campaign was managed by different operators. It is recommended that a mechanical cell counter be used to verify cells counts and to maintain a standard between campaigns. In the bioreactors, varying the pH proved to have no significant effect on the glycosylation levels. However, both the initial perfusion rate and the specific perfusion rate proved to be important from both historical data and the data generated during these experiments. Lower levels of the initial perfusion rate lead to better glycosylation and it is recommended that an initial perfusion rate of 1.0 volumes/day be used. The relationship between the specific perfusion rate and the glycosylation appears to be non-linear and requires further study, for now it is recommended that the specific perfusion rate be kept below 0.3 volumes/day/109 cells. Probable reasons for the unsatisfactory glycosylation seen in certain runs could also be proposed from these two factors: • RP33-133 : Very high specific perfusion rate • RP32-135 : High initial perfusion rate and very high specific perfusion rate • RP32-138 : High initial perfusion rate • RP33-139 : High initial perfusion rate Further research is recommended into the effect of the specific perfusion rate as well as the specific glucose consumption rate and the specific glutamine concentration on the glycosylation. / AFRIKAANSE OPSOMMING: Rekombinante glikoproteïene is baie belangrike biofarmaseutiese produkte wat oplossings bied vir talle voorheen ongeneeslike siektes in alles van kanker tot onvrugbaarheid. Meeste rekombinante farmaseutiese produkte word gemaak deur diere-selle as gevolg van hulle bevoegtheid om die korrekte na-translasie stappe te volg sodat die produkte in mense gebruik kan word. Die na-translasie stappe beïnvloed baie van die proteïene se karaktertreke insluitende die farmakokinetika, bioaktiwiteit, uitskeiding, half-leeftyd, oplosbaarheid, herkenbaarheid and antigeniciteit. Die doel van hierdie tesis is om die stroomop produksie van ‘n rekombinante glikoproteïene vervaardig deur Chinese hamster ovariale (CHO) selle verder te bestudeer binne die grense van ‘n bestaande proses op grootskaalse vlak. Die huidige proses gebruik CHO selle om ‘n rekombinante glikohormoon te produseer. Soos meeste prosesse wat rekombinante proteïene produseer bestaan die stroomop gedeelte van die proses uit twee dele: ‘n saad trein wat genoeg selle maak vir produksie en ‘n produksie gedeelte wat fokus op die vervaardiging van die glikoproteïen. Die saad trein bestaan hoofsaaklik uit roller bottels terwyl produksie plaasvind in perfusie bioreaktors waar die selle op “microcarriers” groei, met spin-filters om die selle binne die bioreaktors te hou; die hele proses gebruik medium met serum. Daar is twee probleme in die stroomop gedeelte van die bestaande proses: 1. Die geleidelike toename oor die afgelope paar jaar van die finale verdubbelingsvlak van die selle aan die einde van die saad trein 2. Die lae glukosilering van die eindproduk wat veroorsaak dat sekere lotnommers buite spesifikasie is Na ‘n literatuur studie, was seker beheerde proses parameters gekies om verder te bestudeer en hipotesisse gemaak oor hulle effek op die saad trein of die vlak van glukosilering. Die volgende faktore is bestudeer vir hulle effek op die finale verdubbelingsvlak van die selle in die saad trein: - Medium volume: ‘n laer medium volume sal lei tot a laer verdubbelingsvlak van die selle as gevolg van stadige groei - Konsentrasie van selle vir inokulasie: as die selle konfluent is teen die tyd wat hulle versamel word sal ‘n laer konsentrasie selle lei tot ’n hoër verdubellingsvlak. - Temperatuur: laer temperatuur behoort te lei tot ‘n stadiger groei koers van die selle - Medium voer-temperatuur: die voer-temperatuur van die medium sal geen beduidende verskil maak - Belugting: die gebruik van “vent-caps” sal die suurstof inhoud van die roller bottels verhoog Die volgende faktore is bestudeer vir hulle effek op die glukosilering tydens produksie: - pH: beter glukosilering word verwag by by pH 6.9 dan by pH 6.7 - Perfusie koers: ‘n hoër perfusie koers sal lei tot beter glukosilering as gevolg van hoër glukose en glutamien konsentrasies Die konsentrasie van die selle wat gebruik word vir inokulasie blyk die enigste faktor te wees wat die finale verdubbelingsvlak van die selle en die groei van die selle in die saad trein beïnvloed het. Die groei van die selle was beprek wanneer die selle konfluent geraak het en dus het ‘n laër sel konsentrasie by inokulasie gelei tot ‘n hoër sel verdubbelingsvlak. Dit word aanbeveel dat ‘n hoë sel konsentrasie by inokulasie gebruik word. Die geleidelike toename van die finale verdubbelingsvlak van die selle in die saad trein is waarskynlik as gevolg van die variasie in sel tellings tussen verskillende operateurs eerder as as gevolg van die beheerde proses parameters. Dit word aanbeveel dat ‘n meganiese sel-teller gebruik word om die verskil in sel tellings tussen operateurs te kontroleer en om ‘n standaard te handhaaf tussen produksie lotte. In die bioreaktors, het die pH geen beduidende invloed gehad op die glukosilering maar uit historiese data en die huidige data van hierdie eksperimente blyk albei die begin perfusie koers en die spesifieke perfusie koers ‘n belangrike invloed te hê op die glukosilering. Laër vlakke van die begin perfusie koers lei tot beter glikosilsie en dit word aanbeveel dat elke produksielot ‘n begin perfusie koers het van 1.0 volume/dag. Die verhouding tussen die glukosilering en die spesifieke perfusie koers blyk om nie-liniêr te wees nie. Nog navorsing hieroor word aanbeveel, maar vir nou word dit aanbeveel dat die spesifieke perfusie koers onder 0.3 volumes/dag/109 selle gehou word. Hierde twee faktore blyk die oorsaak te wees vir die lae glukosilering wat in sekere produksielopies gevind was: • RP33-133 : baie hoë spesifieke perfusie koers • RP32-135 : hoë begin perfusie koers en baie hoe spesifieke perfusie koers • RP32-138 : hoë begin perfusie koers • RP33-139 : hoë begin perfusie koers Dit word aanbeveel dat verdere navorsing gedoen word op die effek van die spesifieke perfusie koers asook die spesifieke koers van glukose verbruik en die spesifieke glutamien konsentrasie op die glukosilering van die produk.

The purpose of this study was to determine whether adults with type 2 diabetes participating in American Diabetes Association (ADA) diabetes self-management education (DSME) randomly assigned to an intensive follow-up group (IFG), utilizing continuous glucose monitoring system (CGMS), or a standard follow-up group (SFG) have any significant differences in mean HgbA1c values and health belief scores over time. Baseline HgbA1c values and health beliefs were measured using the revised Expanded Health Belief Model (HBM) questionnaire. The questionnaire measured the 8 HBM domains: perceived susceptibility; severity; treatment benefit; cues to action; motivation; barriers; self-efficacy and structural elements. Twelve weeks after DSME, patients returned for follow-up based on random assignment. The SFG received routine follow-up care: HgbA1c measurements; behavioral goals and education assessments. The IFG received routine follow-up and CGMS. Patients wore the CSMS for 72 hours and recorded their daily food, blood glucose values, medications and physical activities. Results were analyzed and reviewed with patients. Both groups returned in 24 weeks for HgbA1c measurements and to complete the HBM questionnaire. A repeated measure ANOVA analysis showed a statistically significant reduction in mean HgbA1c at each time period (F=86.75. p>.0001 ) from week 1 to week 12 (SFG 8.6-7.1; IFG 8.5 --7.1,) and from week 12 to week 24 ( SFG 7.1 to 6.9; IFG 7.1 -- 7.0). There were no significant differences found between the groups. (F = 0.17 p > 0.87). Following DMSE and follow-up intervention some health belief scores improved but no significant differences were found between groups except for severity scores. (SFG 27.05, IFG 25.00, p=0.03). The power of the study to detect small differences between the groups was affected by the higher than anticipated attrition and the significant lowering of HgbA1c in the education arm of the study. Both groups achieved a high success rate (58% IFG; 55% SFG) to lower the HgbA1c to the ADA goal of less than 7. DSME and follow-up care (both standard follow-up and more intensive follow-up) achieved a significant lowering of HgbA1c (1.6%), which has been shown to reduce diabetes related morbidity and health costs.