Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-05-30T13:47:23Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Colacao - GLEYDESZ.pdf: 1328379 bytes, checksum: bdbc54c225ef4bd1d59a4bd0b6bd2391 (MD5) / Made available in DSpace on 2017-05-30T13:47:23Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Colacao - GLEYDESZ.pdf: 1328379 bytes, checksum: bdbc54c225ef4bd1d59a4bd0b6bd2391 (MD5) Previous issue date: 2016-02-24 / A razão contagem de plaquetas/diâmetro longitudinal do baço (CP/DB) é um teste não invasivo desenvolvido para identificar varizes esofágicas em cirróticos. Hipertensão portal ocorre na forma hepatoesplênica da esquistossomose (EHE). Estes pacientes apresentam varizes esofágicas com risco de sangramento. Este estudo, de validação fase III, teve como objetivos: validar (artigo 1) a razão CP/DB e (artigo 2) um sistema de pontos como testes não invasivos para predição de varizes esofágicas na EHE. Oitenta e um pacientes com EHE realizaram endoscopia digestiva (EDA) para verificar a presença de varizes. Também realizaram ultrassonografia abdominal para aferição do diâmetro do baço, além de hemograma e testes hepáticos. Calculou-se a razão CP/DB para todos os pacientes. Foram observadas varizes em 63 pacientes (77,8%). Idade e sexo não foram significativamente diferentes entre pacientes com e sem varizes. Naqueles com varizes, a mediana da contagem de plaquetas (64.000/µL versus 129.500/µL, p=0,002) e a razão CP/DB (414,46 versus 878,16, p=0,003) foram significativamente menores, enquanto o diâmetro do baço (153,9 versus 147,1, p=0,026) foi significativamente maior. Utilizou-se uma curva ROC para avaliar o valor do ponto de corte da razão CP/DB. A razão CP/DB abaixo de 683,9 teve sensibilidade igual a 75,8% (IC 95%: 64,8 – 86,8), especificidade igual a 76,5% (IC 95%: 56,3 – 96,6), VPP igual a 92,2% (IC 95%: 84,8 – 99,5) e VPN igual a 46,5% (28 – 64,9). A acurácia da razão CP/DB, avaliada pela área sob a curva, foi igual a 0,74 (IC 95%: 0,622 – 0,827). O estudo revelou uma boa acurácia, porém o baixo VPN encontrado limita seu uso na prática clínica. O segundo artigo estudou 81 indivíduos suspeitos de apresentarem varizes esofágicas. Destes, 63 apresentaram varizes pelo padrão-ouro (endoscopia digestiva). As variáveis (categorizadas em “acima” e “abaixo” do normal) que apresentaram uma associação significante com o diagnóstico de varizes na análise univariada foram: contagem de plaquetas, diâmetro da veia esplênica, razão CP/DB, AST, APRI e INR. Em seguida, construiu-se um modelo de regressão logística multivariada. Permaneceram no modelo: AST, diâmetro da veia esplênica, contagem de plaquetas e INR. Criou-se um sistema de pontos, baseado nas variáveis que mostraram associação mais forte com varizes, para verificar em caráter exploratório o poder de discriminação. Para isso utilizou-se o coeficiente de regressão β (logaritmo do OR da variável) obtido das variáveis constantes no modelo multivariado final. O coeficiente β foi multiplicado por dez para obter-se uma pontuação de maior valor. Após o cálculo do escore total para cada paciente, aplicou-se a curva ROC. Foram encontrados sensibilidade de 83,02%, especificidade de 50,00%, VPP de 84,61% e VPN de 47,06%. O sistema mostrou boa sensibilidade, porém um baixo VPN. Isso torna o teste não adequado para o diagnóstico não invasivo das varizes, uma vez que mais da metade dos indivíduos com pontuação ˂ 12 poderia ter varizes que não seriam identificadas. Concluindo, ambos os testes mostraram-se promissores, no entanto a amostra foi pequena. Consideramos que se faz necessário realizar novos estudos com maior amostra para posterior validação a fim de estabelecer a eficácia dos testes na EHE. / The platelet count/spleen longitudinal diameter (PC/SD) ratio is a noninvasive test designed to identify esophageal varices in cirrhotic patients. Portal hypertension occurs in the hepatosplenic form of schistosomiasis (HSS). These patients present esophageal varices, with the risk of bleeding. The objectives of this phase III validation study were: to validate (Article 1) the PC/SD ratio, and (Article 2) a point system, as noninvasive tests to identify esophageal varices in HSS. A total of 81 patients with HSS underwent gastrointestinal endoscopy (GIE) to verify the presence of varicose veins. They also performed abdominal ultrasound in order to measure the diameter of the spleen, as well as a blood count and liver tests. The PC/SD ratio was calculated for all patients. Varices were observed in 63 patients (77.8%). Age and sex were not significantly different between patients with and without varices. Of those with varices, the median platelet count (64,000 / uL vs. 129,500 / uL, p = 0.002) and the PC/SD ratio (414.46 vs. 878.16, p = 0.003) were significantly lower, while the diameter of the spleen (153.9 versus 147.1, p = 0.026) was significantly higher. The ROC curve was used to evaluate the value of the cutoff point of the PC/SD ratio. A PC/SD ratio under 683.9 presented a sensitivity of 75.8% (95% CI: 64.8 to 86.8), a specificity of 76.5% (95% CI: 56.3 to 96, 6) a PPV of 92.2% (95% CI: 84.8 to 99.5) and an NPV of 46.5% (28 to 64.9). The accuracy of the PC/SD ratio, measured by the area under the curve, was 0.74 (95% CI: 0.622 to 0.827). While the study presented a high level of accuracy, the low NPV encountered would be a limiting factor for its use in clinical practice. The second article studied 81 individuals with suspected esophageal varices. Of these, 63 presented with varices using the gold standard method (gastrointestinal endoscopy). The variables (categorized as "above" and "below" normal) that demonstrated a significant association with the diagnosis of varices in the univariate analysis were: platelet count, splenic vein diameter, PC/SD ratio, AST, APRI and INR. A multivariate logistic regression model was then constructed, and the following items remained in the model: AST, splenic vein diameter, platelet count and INR. A point system was created, based on the variables that demonstrated the strongest association with varices, so as to verify an exploratory measure of the power of discrimination. For this we used the regression coefficient β (OR logarithm of the variable) obtained from the variables in the final multivariate model. The coefficient β is multiplied by ten to obtaina higher point score value. After calculating the total score for each patient, the ROC curve was applied. We encountered a sensitivity of 83.02%, a specificity of 50.00%, a PPV of 84.61% and an NPV of 47.06%. The system demonstrated good sensitivity, but a low NPV, which makes the test unsuitable for a noninvasive diagnosis of varices, since over half of the individuals with a score ˂12 could have varices, which were not identified. In conclusion, both tests have shown to be promising. However, the sample size was small. We believe that it is necessary to undertake further studies with a larger sample for further validation, in order to establish the efficacy of the tests in HSS.

As negociações apresentam uma zona de possível acordo sempre que o valor de reserva do comprador excede o valor de reserva do vendedor. Howard Raiffa permitiu uma formalização para analisar as negociações, ao representá-las por meio dessa zona de acordo. Neste estudo, propõe-se que esse modelo seja utilizado para análise das renegociações. Para tanto, foram incorporados alguns elementos da Economia dos Custos de Transação já que, entre a negociação e a renegociação, ocorre a deterioração do valor de reserva detido pelo agente que promoveu investimentos em ativos específicos à transação. Os elementos incorporados dessa teoria foram: racionalidade limitada, especificidade de ativos e comportamento oportunista. Em razão da racionalidade limitada, os acordos e contratos são incompletos, porque a previsão de todas as contingências é impossível ou, na melhor das hipóteses, demasiado dispendiosa. Com isso, muitas vezes as partes necessitam promover revisões contratuais, o que demanda o estabelecimento de renegociações. Porém, entre a negociação e a renegociação, sempre que uma das partes investir em ativos específicos à transação, o seu valor de reserva se torna menos favorável, reduzindo o seu poder relativo de negociação. Nessas condições, a contraparte pode agir oportunisticamente, expropriando quase-rendas que antes eram auferidas pelo agente responsável pelos investimentos específicos. Apesar de a Economia dos Custos de Transação adotar o comportamento oportunista como pressuposto comportamental, não afirma que todos os indivíduos agem oportunisticamente o tempo todo. A freqüência das transações e a reputação apresentam-se como restrições a esse comportamento. Além disso, alguns estudiosos entendem que os agentes podem não empregar o comportamento oportunista nas renegociações, já que muitas pessoas procurariam recompensar os indivíduos que no passado lhe fizeram alguma ação favorável. Para examinar que comportamento a contraparte emprega nessa situação, foi realizada uma pesquisa experimental com alunos da Universidade de São Paulo. Os participantes, agrupados em pares, deveriam negociar um determinado bem. A pesquisa envolveu dois estágios: negociação de preços para o primeiro ano, em que as partes tinham seus valores de reserva originais, pois os investimentos específicos ainda não haviam sido realizados; e negociação de preços para o segundo ano. Neste último estágio, denominado de renegociação, o valor de reserva de uma das partes havia se deteriorado, em razão dos investimentos específicos. A comparação entre os resultados da negociação e da renegociação permitiu constatar que alguns agentes empregaram o comportamento oportunista. Em 62,7% dos casos, houve alguma redução de preços entre a negociação e a renegociação. Em alguns casos, a pilhagem na renegociação foi tão intensa que o agente expropriado obteve um valor menor do que aquele proporcionado pelo valor de reserva original. A pesquisa revelou que o comportamento oportunista foi mais freqüente e ocorreu com maior intensidade com alunos que já se conheciam. Por fim, o comportamento oportunista não apresentou associações significativas com idade e sexo dos participantes, com o ano de ingresso na faculdade ou com a postura competitiva na primeira etapa das negociações. / A possible agreement zone appears in negotiations whenever the buyer's reservation value exceeds that of the seller. Howard Raiffa brought a fairly structured manner to the analysis of negotiations by representing them through this agreement zone. Our intention is for this model to be used in the analysis of renegotiations. To that end, some elements of Transaction Cost Economics (TCE) have been incorporated inasmuch as deterioration occurs, between the negotiation and the renegotiation, in the reservation value detained by the agent that made investments in transaction specific assets. The elements incorporated from the TCE were the following: limited rationality, asset specificity and opportunistic behavior. Limited rationality leads to incomplete agreements and contracts because forecasting all contingencies is impossible or, in the best of hypotheses, too expensive. For that reason, the parties often need to carry out contractual revisions, which require setting up renegotiations. Nevertheless, whenever one of the parties invests in transaction-specific assets between the negotiation and the renegotiation, its reservation value becomes less favorable thus reducing its relative negotiation power. Under those conditions, a counterpart can have an opportunistic behavior, thus expropriating the quasi rents that were before received by the agent responsible for the specific investments. Although TCE adopts opportunistic behavior as the behavior premise, it does not state that all individuals act opportunistically all of the time. Both the frequency of the transactions and the reputation limit this kind of behavior. Besides, some scholars understand that agents might not act opportunistically in negotiations, insofar as many people would seek to reward individuals who took a favorable action toward them. In order to examine which behavior becomes active in the counterpart in this situation, an experimental research was accomplished with students from the University of Sao Paulo, Brazil. Paired participants were instructed to negotiate a specific asset. The research involved two stages: price negotiation for the first year, in which the parties had their original reservation values, since specific investments had not yet been made; and price negotiation for the second year. In this last stage, called renegotiation, the reservation value of one of the parties had deteriorated due to specific investments. The compared outcomes of the negotiation and renegotiation allowed verifying that some agents did act opportunistically. In 62.7% of the cases there was a price reduction between the negotiation and the renegotiation. In some cases, hold-up in the renegotiation was so intense that the expropriated agent obtained a lower value than that of the original reservation value. The research revealed that opportunistic behavior was more frequent and more intense among students who already knew each other. Final conclusion was that the opportunistic behavior was not significantly associated with participants' age or gender, the year of college entrance or competitive stand in the first round of negotiations.

Polyketide secondary metabolites constitute a structurally-diverse and clinically-important family of natural products. The wide range of biological activities represented by these substrates have contributed to therapeutic agents with annual sales exceeding $20B USD. Large multi-domain proteins called polyketide synthases (PKSs) use simple building blocks to generate highly-oxygenated and stereochemically-rich frameworks with astonishing selectivity. These substrates often feature rigidifying biases imposed by macrocyclic lactones and substituted heterocycles, which can impact their bioactive conformation. The work of this dissertation combines synthetic chemistry and biochemistry to investigate chemoenzymatic production of macrocyclic polyketide natural products. Research focused on validating a transannular oxa-conjugate addition strategy to assembly 2,6-cis-tetrahydropyran (THP) ring systems, as demonstrated by synthesis of the macrocyclic core to neopeltolide. Ultimately, we wish to apply this chemistry to de novo PKS pathways for rapid, reliable, and sustainable production of THP-bearing products like neopeltolide, and toward building SAR libraries. Additionally, a second study probed the specificity of the macrolactonizing thioesterase (TE) domain from the 6-deoxyerythronolide B (DEBS) biosynthetic pathway. This pathway is the paradigm for type-I PKS systems, and is responsible for producing the macrolide core of erythromycin. Our on-going research evaluates the limits of promiscuity within this specific catalytic domain, to characterize the structural elements required to accurately predict macrolactonization. The long-term goal of this study is to assess the potential applicability of DEBS TE as a generalized cyclization biocatalyst for combinatorial biochemistry and chemoenzymatic research.

Detection of a brain tumour in medical images is always a challenging task. Factors like size, shape, and position of tumour vary from different patient’s brain. So, it's important to know the exact shape, size and position of a tumour in the brain making it a challenging task for detection. Some patients exhibit high glioma (HG) type tumor while others show low glioma (LG) type. So, knowing the detailed properties of a tumour to detect them in medical images is mandatory. So far many algorithms have been implemented on how to detect and extract the tumours in medical images, they used techniques such as hybrid approach with support vector machine (SVM), back propagation and dice coefficient. Among these algorithm which used back propagation as base classifier had a highest accuracy of 90%. In this work feature extraction of the medical images of patients’ tumors in database is extracted using Histogram of Oriented Gradient, later these images are classified into tumor and non tumor images using SVM. The detection of brain tumours in patient’s image is achieved by testing the performance of SVM based on Receiver Operating Characteristics (ROC). ROC include true positive rate, true negative rate, false positive rate and false negative rate. Using ROC we calculated accuracy, sensitivity and specificity values for all the medical images of the database. For image data folder of HG in vector form, SVM gave an accuracy of 97% for 95th slice of T1 modality with high true positive rate of 0.97 remaining highest among other modalities. Whereas SVM gave an accuracy of 87% for 135th slice of T1 modality with high true positive rate of 0.8 and low false positive rate of 0.06 among other image data folder of HG. For image data folder of LG, SVM gave an accuracy of 62% for the 90th slice of FLAIR modality with the high true positive rate of 0.5 and low false positive rate of 0.25 among all others. For synthetic data folder of HG, SVM gave an accuracy of 62% for a 100th slice of FLAIR modality with the high true positive rate of 0.5 and low false positive rate of 0.06 among all others. For synthetic data folder of LG, SVM gave an accuracy of 62% for a 100th slice of FLAIR modality with the high true positive rate of 0.5 and low false positive rate of 0.06 among all others.

Previous publications from this laboratory demonstrated that administration of leukemia inhibitory factor (LIF) (125 µg/kg) to young, male Sprague-Dawley rats at 6, 24, and 48 h after middle cerebral artery occlusion (MCAO) reduced infract volume, improved sensimotor skills, and alleviated damage to white matter at 72 h after the injury. In vitro studies using cultured oligodendrocytes (OLs) showed that LIF (200 ng/ml) also protects against 24 h of oxygen-glucose deprivation through activation of Akt signaling and upregulation of the antioxidant enzymes peroxiredoxin IV and metallothionein III. Other groups have demonstrated that LIF reduces neurodegeneration in animal models of disease, but the neuroprotective mechanisms of LIF during permanent ischemia have not yet been examined. The overall hypothesis to be tested in this project is whether LIF exerts similar protective mechanisms against neurons during ischemia through increased antioxidant enzyme expression in neurons. In the first set of experiments, superoxide dismutase (SOD) activity was significantly increased in the ipsilateral hemisphere of LIF-treated rats compared to rats that received PBS treatment at 72 h after MCAO. Western blot and immunohistochemical analysis revealed that SOD3 was upregulated in brain tissue and induced specifically in cortical neurons tissue at this time point. Neurons that expressed high levels of SOD3 at 72 h after MCAO also showed high levels of phosphorylated Akt (Ser473). LIF (200 ng/ml) reduced necrotic and apoptotic cell death against 24 h of OGD as measured by lactate dehydrogenase (LDH) release and caspase-3 activation. Quantitative real-time PCR analysis showed that LIF treatment upregulated SOD3 gene expression in vitro during OGD. Treatment with 10 µM Akt Inhibitor IV and transfection with SOD3 siRNA counteracted the neuroprotective effects of LIF in vitro, showing that upregulation of SOD3 and activation of Akt signaling are necessary for LIF-mediated neuroprotection. Several transcription factors that regulated Akt-inducible genes were previously identified by this lab, including myeloid zinc finger-1 (MZF-1) and specificity protein-1 (Sp1). The goal of the second set of experiments was to determine whether LIF exerted protective actions through MZF-1 and Sp1. According to analysis with Genomatix, MZF-1 and Sp1 have multiple binding sites in the promoter for the rat SOD3 gene. Western blot analysis showed that there was a trend towards increased MZF-1 protein expression in the brains of LIF-treated rats that approached significance. Immunohistochemical analysis and quantitative real-time PCR showed a significant in vitro upregulation in MZF-1 expression among LIF-treated neurons compared to PBS-treated neurons. Sp1 gene expression was not changed by LIF treatment, but there was a trend towards increased protein expression. In addition, there was a significant correlation between Sp1 and MZF-1 among brain samples from LIF-treated rats but not PBS-treated or sham rats at 72 h after MCAO. Immunohistochemical analysis revealed that Sp1 and MZF-1 co-localized with neuronal nuclei and SOD3 at 72 h after MCAO. Neurons that were transfected with MZF-1 or Sp1 siRNA following isolation did not show a significant decrease in LDH release after 24 h OGD that was observed among neurons transfected with scrambled siRNA. These data demonstrate that Sp1 and MZF-1 are involved with the neuroprotective signaling of LIF under ischemia. This laboratory has demonstrated that LIF activates transcription of protective genes and increases the activity of transcription factors through modulation of intracellular signaling. However, the upstream signaling mechanisms of LIF during ischemia had not previously been investigated. Previous investigators found that the LIF-specific subunit of the heterodimeric LIF receptor (LIFR) is induced by CNS injury. Western blot analysis was used to determine whether LIFR was induced in the brain and the spleen, which plays a role in the peripheral immune response, after MCAO. According to these results, LIF treatment significantly upregulates LIF in the brain compared to PBS treatment or sham injury at 72 h after MCAO. Genomatix analysis of the LIFR promoter region revealed a binding site for Sp1, which is one of the transcription factors responsible for neuroprotection by LIF. At this same time point, splenic LIFR expression is significantly reduced after MCAO compared to sham injury. LIF treatment did not significantly increase LIFR expression, but did significantly increase spleen size compared to PBS treatment at 72 h after MCAO. Although there was a trend towards increased LIFR expression in the spleen from 24 h to 72 h after MCAO, this increase was not statistically significant. However, there was a significant positive correlation between spleen weight and LIFR expression among rats euthanized 24-72 h after MCAO/sham injury. In addition, there was a significant negative correlation between LIFR expression in the brain and the spleen weight, thus showing that LIFR is upregulated following the splenic response. According to findings from other groups, JAK1 has been shown to associate with the heterodimeric LIF receptor (LIFR/gp130) and directly activate PI3K/Akt signaling. To test whether JAK1 contributes neuroprotection during ischemia, cultured neurons were treated with several concentrations (2.5-50 nM) of GLPG0634, a JAK1-specific inhibitor prior to 24 h of OGD. With the exception of the 2.5 nM concentration, all concentrations of GLPG0634 significantly decreased LDH release compared to DMSO treatment, with the 5 nM concentration having the most potent effect on reducing cytotoxicity. However, the 5 nM concentration had no significant did not significantly reduce LDH release compared to DMSO treatment under 24 h of normoxic conditions. These results indicate that JAK1 activity is primarily detrimental to neurons during ischemia. Although it is possible that LIF signaling activates JAK1, it is unlikely that JAK1 is responsible for LIF-mediated neuroprotection during ischemia. The results of these experiments allowed us to determine several molecular mechanisms for LIF-mediated neuroprotection. LIF, which binds to its heterodimeric receptor, activates Akt signaling during ischemia. The transcription factors Sp1 and MZF-1, which are located downstream of Akt, bind to the promoter of the SOD3 gene. In addition, Sp1 also regulates the LIFR gene. SOD3 upregulation increases total SOD activity, which decreases apoptotic and necrotic cell death during apoptosis. Due to its ability to promote antioxidant expression and survival signaling in multiple neural cell types, LIF shows promise as a novel treatment for permanent focal cerebral ischemia.

In this thesis, we focused on local structural features of the DNA backbone in protein-complexed DNA and non-complexed (naked) DNA, and its dependence on types of a base pairing in DNA, and on the base sequence. To reach this goal we analyzed about 1,400 crystal structures of DNA in complexes with proteins and more than 400 crystal structures of naked DNA. DNA local conformations were structurally classified into 38 dinucleotide conformers ntCs, which were described previously (Svozil et al. Nucleic Acids Res. 2008). The ntC were further clustered into 16 structural alphabet classes ntA to reduce the number of analyzed variables. We assembled base-paired dinucleotides from double helical DNA structures accord- ing to their assigned structural alphabet classes into so called Association matrices. Three basic Association matrices were analyzed; two compare ntA/ntA associations between dinucleotides forming only Watson-Crick base pairs in protein/DNA com- plexes and in naked DNA, respectively; the third one ntA/ntA associations between dinucleotides base-paired also by non-Watson-Crick pairs. We also analyzed As- sociation matrices of dinucleotides as a function of their sequences. The analyzes revealed differences in structural behavior of various ntA and their dependence on dinucleotide sequences.

An analysis of what has remained specific to the medium of painting in contemporary practice, as a response to observations from my own artistic practice and the publication of Painting Beyond Itself (2016) by Isabelle Graw and Ewa Lajer-Burcharth.

Abstract The collagens are a family of extracellular matrix proteins with a widespread tissue distribution. Collagen biosynthesis requires the hydroxylation of a number of proline residues by prolyl 4-hydroxylase. This posttranslational modification is essential for the synthesis of all collagens, as 4-hydroxyproline deficient collagens cannot form stable triple helices at body temperature. The genes for the human and mouse prolyl 4-hydroxylase α(II) subunits were cloned and characterized in this study. The human and mouse genes are 34.6 and 30.3 kb in size, respectively, consisting of 16 exons and 15 introns. The intron sizes vary from 48-49 bp to over 8 kb in both genes. The 5' flanking regions contain no TATA box, but there are several motifs that may act as transcription factor binding sites. A novel mutually exclusively spliced exon 12a was identified in both genes. Both variants of the α(II) subunit were found to be expressed in a variety of tissues and both formed a fully active recombinant tetramer with the β subunit when expressed in insect cells. Tissue distribution of the type I and type II prolyl 4-hydroxylase isoenzymes was studied in developing, mature, and malignant cells and tissues by immunofluorescence and Western blotting. The results indicate that the type I isoenzyme is the main form in many cell types. Skeletal myocytes and smooth muscle cells appeared to have the type I isoenzyme as their only prolyl 4-hydroxylase form, whereas the type II isoenzyme was clearly the main form in chondrocytes. A strong signal for the type II enzyme was detected in cultured umbilical and capillary endothelial cells, whereas the type I isoenzyme could not be detected in these cells by immunostaining or Western blotting. Similar studies on primary chondro- and osteosarcomas and benign bone tumours indicated that the type I isoenzyme is the predominant form in both types of bone sarcoma, whereas the type II isoenzyme was more abundantly expressed in benign tumours. In chondrosarcomas, the type II isoenzyme was expressed in the nonmalignant chondrocytes, whereas their malignant counterparts switched their expression pattern to that of the type I isoenzyme. Two isoforms of the catalytic prolyl 4-hydroxylase α subunit, PHY-1 and PHY-2, have previously been characterized from Caenorhabditis elegans. This study reports the cloning and characterization of a third C. elegans α subunit isoform, PHY-3, which is much shorter than the previously characterized vertebrate and C. elegans α subunits. Nematodes homozygous for a phy-3 deletion were phenotypically wild type and fertile, but the 4-hydroxyproline content of their early embryos was reduced by about 90%. The expression of PHY-3 was found to be restricted to spermatheca of late larvae and adult nematode, indicating that PHY-3 is likely to be involved in the synthesis of collagens of the early embryo egg shells.

Abstract In the cells, the last step of the beta-oxidation cycle, aiming at the degradation of fatty acids, is catalyzed by the enzyme named thiolase. It shortens the acyl chain of the acyl-CoA by two carbons. The reaction is reversible, it can proceed for both directions. Thiolases are divided into two categories, synthetic and degradative ones. These two classes of thiolases differ not only by their biological function, but also by their substrate specificity. Degradative thiolases accept substrates with various lengths but synthetic thiolases only accept short chain-acyl-CoAs as a substrate. In humans, at least six isozymes of thiolases are found. The mitochondrial biosynthetic thiolase, T2, differs from other thiolases by getting activated by potassium. In addition, it accepts branched acyl-CoA, namely 2-methyl-acetoacetyl-CoA, as a substrate. This molecule is an important reaction intermediate in the degradation of the amino acid isoleucine. Many human patients have been diagnosed to have a mutation in the gene of T2, and they are treated with a special diet. The results of this theses show that potassium ion rigidifies the groups of the T2 protein involved in the substrate binding. The presence of potassium increases the reaction rate and it also raises the affinity towards some of the substrates. The enzyme mechanistic studies with bacterial thiolase revealed that the oxyanion hole 1, formed by a water molecule and histidine side chain, is important for the synthetic reaction, not so much for the degradative direction. Binding studies showed that both the terminal sulfur of the substrate and the sulfur of the catalytic cysteine are important for the right positioning of the substrate. The electrostatics of the active site also have a significant role in the catalysis. These studies give a good basis for future studies aiming at drug development against this enzyme in pathogenic species.

Magister Scientiae (Biodiversity and Conservation Biology) / The expansion of the South African aquaculture industry coupled with the lack of effective parasite management strategies may potentially have negative effects on both the freshwater biodiversity and economics of the aquaculture sector. Koi and goldfish are notorious for the propagation of parasites worldwide, some of which have already infected indigenous fish in South Africa. Koi and goldfish have been released into rivers in South Africa since the 1800’s for food and sport fish and have since spread extensively. These fish are present in most of the river systems in South Africa and pose an additional threat the indigenous cyprinids in the Western Cape. Monogenean parasites of the genus Gyrodactylus are of particular concern, as their unique biology renders them a possible threat. Gyrodactylus kherulensis and G. kobayashii were identified from koi and goldfish respectively imported from Asia, Europe and locally bred fish. Morphometrics and the use of statistical classifiers, which includes univariate (ANOVA and Kruskal-Wallis), bivariate (Pearson’s correlation) and multivariate (Principal Component Analysis) placed the two species within their respective groups. There was some intraspecific variation among the different populations collected from the various locations, especially in the hamulus and ventral bar features, but the marginal hooklets, however, remained static for both helminth species. This illustrates again the importance of the minor variations in the marginal hook features in gyrodactylid taxonomy. Infection trials conducted by co-habitation of infected koi and goldfish with two indigenous redfin minnow species, Pseudobarbus burchelli and P. phlegethon showed that both G. kherulensis and G. kobayashii could successfully transfer and establish themselves on P. phlegethon, where the infection increased rapidly initially, but remained relatively constant thereafter. P. burchelli appeared to be inherently resistant as the parasite population growth rate initially remained steady, until the infection died off. The wild-caught indigenous fish were however not infected with any exotic Gyrodactylus species, but a new species, G. burchelli n. sp. described from the body surfaces of P. burchelli. / South Africa