The presence of pathogens in semen can compromise the outcome of assisted reproductive treatment, together with the possibility of the female partner or offspring becoming infected. This is cause for concern, especially in South Africa with a high prevalence of HIV-1. Most of these infected individuals are in their reproductive years with the desire to have their own genetically related children. Therefore, assisted reproductive treatment with effective risk reduction procedures, such as semen processing for the elimination of these pathogens is crucial. However, during sperm preparation by standard discontinuous density gradient centrifugation, the supernatant is aspirated to allow access to the purified sperm pellet. Pathogens from the upper layers can adhere to the inside surface of the test tube and flow down to re-infect the purified sperm sample. The use of a centrifuge tube insert may prevent the re-contamination of sperm samples after discontinuous density gradient centrifugation. Furthermore, seminal pathogens can bind specifically or non-specifically to spermatozoa, rendering semen decontamination procedures ineffective. Serine proteases, such as trypsin, have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could have a negative impact on sperm parameters. This research was therefore aimed towards the determination of: i) The effect of semen processing with trypsin and trypsin inhibitor on sperm parameters. ii) The prevalence of various bacteria in semen samples from men attending the Reproductive and Endocrine Unit at Steve Biko Academic Hospital. iii) The effectiveness of semen processing by discontinuous density gradient centrifugation with a centrifuge tube insert, for the elimination of some of the most prevalent bacteria, white blood cells and in vivo derived HIV-1. Evaluation of sperm parameters after semen processing indicated that trypsin and trypsin inhibitor did not have an impact on sperm mitochondrial membrane potential, vitality, motility and zona binding potential, or acrosin activity, respectively. Seminal bacteria were highly prevalent in patients wishing to participate in the Unit’s assisted reproductive program, with 49.5% of semen samples presenting with positive bacterial cultures. Semen processing by means of discontinuous density gradient centrifugation with the tube insert, eliminated significantly more in vitro derived (spiked) bacteria and white blood cells from semen compared to processing without the insert. Furthermore, the semen decontamination procedure was effective in removing HIV-1 RNA from 100% of samples and proviral DNA from 98.1% of semen samples from HIV-1 sero-positive patients. The effectiveness of discontinuous density gradient centrifugation for the elimination of seminal pathogens could, therefore, be improved by the addition of trypsin to the upper density layer, without supplementing the bottom layer with trypsin inhibitor. Additionally, semen decontamination efficiency could also be improved by the prevention of re-contamination of processed sperm samples by the utilization of a tube insert during discontinuous density gradient centrifugation. / Thesis (PhD)--University of Pretoria, 2014. / gm2014 / Obstetrics and Gynaecology / unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22148号 / 医博第4539号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 坂井 義治, 教授 小西 靖彦, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

LIMA, Glauber Pacelli Gomes de. Purificação, caracterização bioquímica e atividade biológica in vitro contra insetos praga de um novo inibidor de tripsina isolado de sementes de Sapindus saponaria L. (Sapindaceae). 119 f. : Dissertação (Mestrado) - Universidade Federal do Ceará, Centro de Ciências, Departamento de Bioquímica e Biologia Molecular, Programa de Pós-Graduação em Bioquímica. Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-05-20T13:23:01Z No. of bitstreams: 1 2012_dis_gpglima.pdf: 2370771 bytes, checksum: 509ae0531057d53f967b325467f88d5c (MD5) / Approved for entry into archive by demia Maia (demiamlm@gmail.com) on 2016-05-23T11:59:24Z (GMT) No. of bitstreams: 1 2012_dis_gpglima.pdf: 2370771 bytes, checksum: 509ae0531057d53f967b325467f88d5c (MD5) / Made available in DSpace on 2016-05-23T11:59:24Z (GMT). No. of bitstreams: 1 2012_dis_gpglima.pdf: 2370771 bytes, checksum: 509ae0531057d53f967b325467f88d5c (MD5) Previous issue date: 2012 / Environmental toxicity, low biodegradability and increased resistance in agricultural pest insects and in insect vectors of diseases to insecticides traditionally used for their control have encouraged the development of chemical alternatives with greater specificity and biodegradability especially from plants. In this way, the present study aimed the purification, characterization and evaluation of activity towards pest insect digestive enzymes of a new trypsin inhibitor obtained from Sapindus saponaria seeds. This new trypsin inhibitor obtained from S. saponaria seeds (SSTI2) belongs to Potato I inhibitor family and was purified by protein precipitation with trichloroacetic acid, affinity chromatography and reverse phase chromatography using UFLC system. The native inhibitor and its fragments generated by enzymatic treatment with trypsin and pepsin were analyzed and sequenced by MALDI-TOF and ESI-TOF mass spectrometry, determining its accurate molecular mass (MM = 7571, 976 Da) and primary structure (64 of 69 amino acids). The SSTI2 showed an IC50 of 8.3 x 10-2 μmol.L-1 against bovine trypsin, and a much lower inhibitory activity on the enzymes chymotrypsin (13.24 ± 0.28%) and papain (5.28 ± 0 , 42%), but was unable to inhibit proteolysis promoted by bromelain. In spite to show moderate inhibition of total digestive enzymes (4.74 ± 0.45% to 56.06 ± 1.41%), the inhibitor was very effective upon trypsin-like enzymes present in Aedes aegypti (92.44 ± 0.99%), Anthonomus grandis (77.93 ± 0.12%), Anticarsia gemmatalis ( 32.21 ± 0.57%) and Spodoptera frugiperda (71.44 ± 1.23%) guts. This strong inhibitory effect of SSTI2 on the catalytic activity of trypsin-like peptidases of insect’s midguts suggests a possible suppressive effect on development and survival of insects fed with diets containing the inhibitor. Thus, future in vivo assays and evaluation of other biochemical properties will be important to establish the potential biotechnological application of SSTI2, especially for the combat of Ae. aegypti, A. grandis and An. gemmatalis. Furthermore, the sequence of SSTI2 elucidated in this study allows the chemical synthesis, cloning of coding gene sequence and heterologous expression of this potential new biotechnological tool. / A toxicidade ao meio ambiente, a baixa biodegrabilidade e a crescente resistência de insetos praga da agricultura e de insetos vetores de doenças aos inseticidas tradicionalmente utilizados tem estimulado o desenvolvimento de alternativas com maior biodegradabilidade e especificidade, especialmente de origem vegetal. Nesse sentido, o presente estudo consistiu na purificação, caracterização e avaliação do efeito biológico in vitro de um novo inibidor de tripsina obtido das sementes de Sapindus saponaria sobre as enzimas digestivas de insetos. Através de precipitação com ácido tricloroacético (TCA), cromatografia de afinidade à tripsina e cromatografia de fase reversa em sistema UFLC, foi purificado um novo inibidor de tripsina (SSTI2) pertencente ao grupo de inibidores do tipo Batata I, uma categoria de inibidores de peptidases serínicas com reconhecido efeito tóxico sobre insetos. O inibidor nativo e fragmentos desse gerados por tratamento enzimático com tripsina e pepsina foram analisados e sequenciados por espectrometria de massa do tipo MALDI-TOF e ESI-TOF, determinando assim a sua massa molecular acurada (MM = 7571, 976 Da) e elucidando a sua estrutura primária (64/69 aminoácidos). O inibidor apresentou uma IC50 de 8,3 x 10-2 μmol.L-1 contra a tripsina bovina, mas apresentou baixa atividade inibitória sobre as enzimas quimotripsina (13,24 ± 0,28%) e papaína (5,28 ± 0,42%), e não foi capaz de inibir a proteólise promovida pela bromelaína. O SSTI2 inibiu moderadamente a atividade catalítica das enzimas digestivas totais dos insetos, com percentual de inibição variando entre 4,74 ± 0,45% a 56,06 ± 1,41%. No entanto, o inibidor foi eficaz em atuar sobre as enzimas digestivas do tipo tripsina presentes nos intestinos dos insetos Aedes aegypti (92,44 ± 0,99%), Anthonomus grandis (77,93 ± 0,12%), Anticarsia gemmatalis (32,21 ± 0,57%) e Spodoptera frugiperda (71,44 ± 1,23%). Esse expressivo efeito inibitório do SSTI2 sobre a atividade catalítica das peptidases do tipo tripsina provenientes do intestino médio dos insetos sugere um possível efeito supressor no desenvolvimento e sobrevivência de insetos alimentados com dietas contendo o inibidor. Assim, futuros estudos in vivo e avaliações de outras propriedades bioquímicas do inibidor serão importantes para determinar a potencial aplicação biotecnológica do SSTI2, especialmente para o combate de A. aegypti, A. grandis e A. gemmatalis. Além disso, a sequência do SSTI2 elucidada nesse estudo possibilita a síntese, clonagem do gene codificante e expressão heteróloga dessa potencial ferramenta biotecnológica.

Medical devices, such as catheters and heart valves, are an important part of patient care. However, blood-contacting devices can activate the blood coagulation cascade to produce factor (f) Xa, the clotting enzyme that induces thrombin generation. By activating platelets and converting soluble fibrinogen to fibrin, thrombin leads to blood clot formation. Blood clots that form on medical devices create problems because they may foul the device and/or serve as a nidus for infection. In addition, clots can break off from the device, travel through the circulation and lodge in distant organs; a process known as embolization. This is particularly problematic with central venous catheters because clots that form on them can break off and lodge in pulmonary arteries, thereby producing a pulmonary embolism. Similarly, clots that form on heart valves can break off and lodge in cerebral arteries, thereby producing a stroke. Therefore, anticoagulants, blood thinning drugs, are frequently used to prevent clotting on medical devices. Conventional anticoagulants, such as heparin and warfarin, target multiple clotting factors. Heparin binds to antithrombin in plasma and accelerates the rate at which it inhibits fXa, thrombin and many other clotting enzymes. Warfarin, which is a vitamin K antagonist, attenuates thrombin generation by interfering with the synthesis of the vitamin K-dependent clotting factors, which include fX and prothrombin, the precursor of thrombin. In contrast to heparin and warfarin, more recent anticoagulants inhibit only a single clotting enzyme. For example, fondaparinux, a synthetic heparin fragment, only inhibits fXa and dabigatran, an oral thrombin inhibitor, only targets thrombin. Although effective for many indications, fondaparinux was less effective than heparin for preventing clotting on catheters in patients undergoing heart interventions and dabigatran was less effective than warfarin for preventing strokes in patients with mechanical heart valves. The failure of these new anticoagulants highlights the need for a better understanding into the drivers of clotting on medical devices. Therefore, the overall purpose of this thesis is to gain this understanding so that more rational approaches to its prevention can be identified. In the classical model of blood coagulation, clotting is triggered via two distinct pathways; the tissue factor (TF) pathway or extrinsic pathway and the contact pathway or intrinsic pathway; pathways which are initiated by fVIIa and fXIIa, respectively. The mechanism by which medical devices initiate clotting is uncertain. Platelet and complement activation and microparticle formation have been implicated, which would drive clotting via the TF pathway. Alternatively, medical devices can bind and activate fXII, thereby initiating the contact pathway. We hypothesized that medical devices trigger clotting via the contact pathway and induce the local generation of fXa and thrombin in concentrations that exceed the capacity of fondaparinux and dabigatran to inhibit them. To test this hypothesis, we used catheters as a prototypical medical device and we used a combination of in vitro and rabbit models. Several lines of evidence indicate that catheters initiate clotting via the contact pathway. First, catheter segments shortened the clotting time of human plasma, and this activity was attenuated in fXII- or fXI-deficient plasma, which are key components of the contact pathway, but not in fVII-deficient plasma, which is the critical component of the extrinsic pathway. Second, corn trypsin inhibitor (CTI), a potent and specific inhibitor of fXIIa, attenuates catheter thrombosis. Third, selective knockdown of fXII or fXI with antisense oligonucleotides attenuated catheter-induced thrombosis in rabbits, whereas knockdown of fVII had no effect. Therefore, these results revealed the importance of the contact pathway in device-associated thrombosis, and identified CTI or fXII or fXI knockdown as novel strategies for preventing this problem. Focusing on fXIIa as the root cause of medical device associated clotting, we coated catheters with CTI using a polyethylene glycol (PEG) spacer. In addition to unmodified catheters, other controls included catheters coated with albumin via a PEG spacer or catheters coated with PEG alone. Compared with unmodified catheters or with the other controls, CTI-coated catheters attenuated clotting in buffer or plasma systems and were resistant to occlusion in rabbits. These findings support the concept that catheter-induced clotting is driven via the contact pathway and identify CTI coating as a viable strategy for its prevention. We next set out to test the hypothesis that fondaparinux and dabigatran, which inhibit fXa and thrombin, respectively, are less effective than heparin, which inhibits multiple clotting enzymes. Fondaparinux and dabigatran were less effective than heparin at preventing catheter induced clotting and thrombin generation, respectively. Likewise, in a rabbit model of catheter thrombosis, fondaparinux was less effective than heparin and dabigatran was only effective when administered at doses that yielded plasma dabigatran levels similar to those found at peak in human given the drug; at trough levels, dabigatran was no better than placebo. Finally, we also showed synergy between heparin and either fondaparinux or dabigatran. Thus, when co-administered to rabbits in doses that on their own had no effect, the combination of fondaparinux or dabigatran plus heparin extended the time to catheter thrombosis. These findings support the hypothesis that when catheters trigger clotting via the contact pathway, fXa and thrombin are generated in concentrations that overwhelm the capacity of fondaparinux or dabigatran to inhibit them. Furthermore, the synergy between heparin and fondaparinux or dabigatran has clinical implications because it explains why supplemental heparin attenuated the risk of catheter thrombosis in patients treated with fondaparinux who underwent cardiac procedures and it identifies the potential role of supplemental heparin in dabigatran-treated patients who require such interventions. In summary, we have shown that catheters trigger clotting via the contact pathway and have identified CTI coating or fXII or fXI knockdown as viable strategies for prevention of this problem. In addition, for prevention of catheter thrombosis, we also have shown that heparin, which inhibits multiple coagulation enzymes, is more effective than fondaparinux or dabigatran, which only inhibit fXa or thrombin, respectively; findings consistent with the clinical observations. Moreover, the synergy that we observed between fondaparinux or dabigatran and heparin identifies supplemental heparin as strategy for preventing catheter thrombosis in patients receiving these drugs. Taken together, these studies provide insight into the mechanisms of catheter thrombosis and potential strategies for its prevention. / Thesis / Doctor of Philosophy (PhD)