Self-renewal of mouse embryonic stem (mES) cells is dependent upon the presence of leukemia inhibitory factor (LIF). LIF induces tyrosine phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3) which is thought to promote self-renewal by inducing key target genes. The molecular chaperone heat shock protein 90 (Hsp90) is involved in signal transduction pathways and regulates STAT3 activity in different cell types. However, the role of Hsp90 in regulating STAT3 activity in mES cells has not previously been investigated. The aim of this study was to investigate if Hsp90 interacts with STAT3 in mES cells and to determine if this interaction is important for the maintenance of self-renewal. It was found that when mES cells were cultured for 24.0 hours in the absence of LIF, the expression levels of total STAT3, tyrosine-phosphorylated STAT3 (pYSTAT3), and the pluripotency marker, Nanog, were down regulated. However, the expression level of Hsp90 was found to be slightly up-regulated over the same period. Significantly, it was found that the amount of STAT3 in differentiating mES cells available for binding to Hsp90 was decreased upon down-regulation of STAT3 by LIF withdrawal. Therefore, STAT3-Hsp90 interactions in mES cells were dependent on the presence of LIF, which suggested that the reduction in STAT3-Hsp90 interaction may have resulted from the low levels of STAT3. Despite a dramatic reduction in the expression levels of pYSTAT3 upon 24.0 hours of culture of mES cells in the presence of the STAT3 tyrosine phosphorylation inhibitor, cucurbitanin I, there was no obvious reduction in the levels of total STAT3, Oct-3/4 or Nanog. These results suggested that the levels of unphosphorylated STAT3 rather than pYSTAT3, maybe more important in the maintenance of mES cells self-renewal.

Part I Inhibition of Gastric Acid Secretion Fat in the small bowel is a powerful inhibitor of gastric acid secretion. The gastric inhibitory agent(s) liberated from intestinal mucosa by the presence of fat has been named enterogastrone. Gastric inhibitory polypeptide (GIP), has been considered a candidate for enterogastrone. GIP is released into the circulation by infusion of fat into the proximal small bowel and inhibits gastric acid secretion under select experimental conditions. It has been proposed that the release of somatostatin, a potent inhibitor of acid secretion, may mediate the gastric inhibitory action of GIP. Recently, monoclonal antibodies raised to both GIP and somatostatin have been produced. The suitability of these antibodies for the study of the physiological roles proposed for their respective peptides is not known. This study examined the inhibitory action of GIP and somatostatin on gastric acid secretion in the rat and in man. GIP was found to be a weak inhibitor of meal-stimulated gastric acid secretion in man when given in supraphysiological doses. When administered at a dose which produces less than the normal maximal physiological plasma level, GIP had little effect on the acid secretory response to the meal and no effect on either plasma gastrin or plasma SLI concentrations. In the rat, infusion of GIP produced a 60% reduction of meal-stimulated acid secretion, independent of changes in serum gastrin release. Intraduodenal infusion of oleic acid in the rat reduced the gastric acid secretory response to a liver extract meal by 80% without affecting serum gastrin levels. A humoral gastric inhibitory agent, or "enterogastrone", was demonstrated in the portal blood of the rat following fat infusion. Intravenous infusion of portal serum, which had been collected during an intraduodenal infusion of fat, reduced meal-stimulated acid secretion in a second animal. A comparison of the inhibition of gastric acid secretion produced by intraduodenal infusion of either glucose or oleic acid with the release of IR-GIP in the portal serum was performed. The inhibitory effect of an intraduodenal fat infusion could not be explained by plasma IR-GIP. The release of GIP was not found to play a significant role in the mechanism for gastric inhibition by intestinal fat. Part II Monoclonal antibodies as Probes of Humoral Inhibitors of Gastric acid secretion The ability of recently produced monoclonal antibodies to block in vivo the inhibitory action of exogenous GIP and somatostatin on gastric acid secretion was examined. Anti-GIP monoclonal antibody demonstrated a high affinity for GIP when compared to the polyclonal rabbit antiserum R07 in the ELISA. When administered either as an intravenous bolus, or after incubation with GIP for 1 hour at 37°C, the antibody was unable to block the inhibitory effect of a GIP infusion on meal-stimulated gastric acid secretion in the rat. Monoclonal antibody 3.65H may not be suitable for the study of the role of endogenously released GIP. Two anti-somatostatin monoclonal antibody clones 58 and 510, when given as intravenous boluses, blocked the inhibitory action of exogenous somatostatin on meal-stimulated gastric acid secretion in the rat. The antibody clone S10 however, had no effect on the inhibitory action of exogenous GIP on gastric acid secretion. Although both monoclonal antibodies S8 and SIO effectively prevented the gastric inhibitory effect of infused somatostatin, the ability to block the physiological action of endogenously released gastric somatostatin remains to be determined. / Surgery, Department of / Medicine, Faculty of / Graduate

Parce qu’ils sont la première cause virale d’infections des voies respiratoires supérieures et inférieures, les rhinovirus humains (RVH) constituent un problème majeur de santé publique. À ce jour, aucun vaccin ni antiviral n’est disponible pour lutter contre ces agents pathogènes. Un crible en doublehybride chez la levure nous a permis d’identifier un nouveau partenaire peptidique de la protéase virale à cystéine 2A (2Apro), l’hexapeptide LVLQTM. Ce dernier agit comme un véritable pseudosubstrat de la 2Apro et inhibe son activité. Ce peptide a été modifié chimiquement à son extrémité C-terminale avec un groupement réactif électrophile fluorométhylcétone pour former une liaison covalente avec le groupement thiol nucléophile du site actif de l'enzyme viral. Des expériences réalisées ex vivo et in vivo ont montré que le peptide LVLQTM modifié était un puissant inhibiteur de la réplication du RVH dans les cellules A549 et chez la souris. La structure 3D déjà connue de la 2Apro du RVH-2 a ensuite permis de modéliser la fixation de LVLQTM dans la poche de liaison du substrat de la protéase et la comparaison des séquences des 2Apro des espèces RVH-A, -B et -C a révélé que les résidus impliqués dans l'interaction avec le peptide LVLQTM sont relativement bien conservés. Si le peptide inhibiteur semblait donc agir contre tous les sérotypes de RVH, son utilisation à des fins thérapeutiques pouvait être étendue à d'autres entérovirus puisqu’il inhibait également la 2Apro de l’entérovirus 71 (EV-71) et par conséquent la réplication virale. De plus, la comparaison de la séquence des protéases 2A de l’EV-71 avec celle du RVH-A2 n’a révélé aucune différence majeure. Par conséquent, cette étude ouvre de nouvelles perspectives dans la mise au point d’un antiviral à large spectre d’action contre tous les entérovirus / Human rhinoviruses (HRV) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. To date no vaccine or antiviral are available against these pathogens. Using a high-throughput yeast two-hybrid screening, we identified a six amino acid “hit” peptide, LVLQTM, which acted as a pseudo-substrate of the viral 2A cysteine protease (2Apro) and inhibited its activity. This peptide was chemically modified at its C-terminus with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. Based on HRV-2 2Apro crystallographic data, a virtual docking model was then set up to predict the inhibitor binding mode into the ligand binding pocket of the enzyme. Sequence comparison between different 2Apro from HRV-A, -B and –C species revealed that the aminoacid residues involved in the interaction with the inhibitor are relatively well conserved. If our peptide inhibitor seemed to be of general use against all HRV serotypes, its use for therapeutic purposes could be extended to other enterovirus-associated diseases since it was also active against Human Enterovirus 71 (EV-71) 2A proteases and EV-71 replication. Moreover, comparison of the sequence of these proteases with the one of HRV-A2 revealed only minor differences in the residues involved in the interaction with LVLQTM. Therefore, this study opens new doors in the development of an antiviral against a wide range of enteroviruses

Peptídeos com ação inibitória sobre a enzima conversora de angiotensina I (ECA) e com o potencial de reduzir a pressão arterial têm sido obtidos a partir de diferentes tipos de alimentos ou matérias-primas, sendo grande o interesse em aproveitar resíduos da indústria alimentícia como fontes desses peptídeos. Neste aspecto, as amêndoas de cupuaçu (Theobroma grandiflorum S.), que são em sua maioria descartadas pela indústria, apresentam um teor considerável de proteínas e poderiam ser aproveitadas como fontes de peptídeos inibidores da ECA. Assim, o objetivo deste trabalho foi verificar se o concentrado proteico obtido a partir de amêndoas fermentadas de cupuaçu após ser submetido à hidrólise com a enzima pepsina poderia gerar peptídeos com ação inibitória sobre a ECA in vitro. Foi observado que após a hidrólise do concentrado proteico com pepsina por 1h foi obtido um efeito de 50 % de inibição da ECA, em ensaio realizado com o substrato Abz-FRK(Dnp)-P-OH. Posteriormente, esse hidrolisado foi submetido ao fracionamento por cromatografia em fase reversa (RP-HPLC) e resultou em cinco frações (F1-F5), das quais a terceira teve uma subfração (F3.1) com quatro novos peptídeos identificados por LC-MS/ MS com potencial em inibir a ECA. Esses quatro peptídeos (FWVAM, YRLAF, LGYFK, VTTVVTGLTF) foram sintetizados e submetidos aos ensaios para a determinação do IC50 e Ki. Os peptídeos YRLAF e LGYFK, que apresentaram mecanismo de inibição do tipo competitivo e acompetitivo, respectivamente, tiveram valores de IC50 de 4.73 e 11.11µM, e de Ki de 9.14 e 8.15 µM. Dentre os peptídeos identificados merece destaque VTTVVTGLTF que demonstrou ser um inibidor do tipo acompetitivo e apresentou as menores IC50 (0.70 µM) e Ki (2.79 µM). Em contraste, FWVAM atuou como substrato da ECA e não peptídeo inibidor. A partir dos resultados obtidos neste estudo fica demonstrado que as amêndoas fermentadas de cupuaçu podem ser fonte de peptídeos com ação inibitória da ECA, com potencial efeito anti-hipertensivo a ser, futuramente, investigado a partir de estudos in vivo. / Angiotensin converting enzyme (ACE) inhibitory peptides with the potential to reduce blood pressure have been obtained from different types of food or raw materials, and there is a great interest in utilize residues from the food industry as sources of peptides. In this regard, cupuassu almonds (Theobroma grandiflorum S.), which are mostly discarded by the industry, has a considerable protein content and could be used as source of ACE-inhibiting peptides. Thus, the objective of this work was to verify if the protein concentrate obtained from fermented almonds of cupuassu after being submitted to the hydrolysis with the enzyme pepsin could generate peptides with inhibitory activity on ACE in vitro. In this study it was observed that after hydrolysis of the protein concentrate with pepsin for 1 h, a 50% effect of ACE inhibition was obtained in an assay performed with the Abz-FRK (Dnp) -P-OH substrate. Posteriorly, the hydrolyzate was subjected to fractionation by reverse phase chromatography (RP-HPLC) and resulted in five fractions (F1-F5), of which the third had a subfraction (F3.1) with four new peptides identified by LC-MS / MS with the potential to inhibit ACE. These four peptides (FWVAM, YRLAF, LGYFK, VTTVVTGLTF) were synthesized and assayed for IC50 and Ki. The YRLAF and LGYFK peptides, which showed a competitive and uncompetitive type inhibition mechanism respectively, presented IC50 values of 4.73 and 11.11µM, and the values for Ki were 9.14 and 8.15 µM. Among the peptides identified, it is possible to highlight VTTVVTGLTF, which was shown to be an inhibitor of the uncompetitive type and presented the lowest value for IC50 (0.70 µM) and Ki (2.79 µM). While FWVAM acted as a substrate of the ACE and not as an inhibitory peptide. From the results obtained in this study it is demonstrated that cupuassu fermented almonds can be a source of peptides with ACE inhibitory activity with potential antihypertensive effect to be further investigated from in vivo studies.

O esquecimento intencional pode cumprir uma função estratégica no sistema cognitivo, que permite aos indivíduos não pensar sobre acontecimentos indesejados do passado, tais como eventos traumáticos, dolorosos e violentos, dos quais se prefere não recordar. Enquanto esquecer involuntariamente é uma falha da lembrança, por outro lado, esquecer intencionalmente parece ser uma função estratégica da memória. A presente dissertação teve por objetivo investigar os mecanismos neurocognitivos que contribuem para o esquecimento de memórias. A dissertação se organizou em dois estudos. O primeiro estudo consiste em uma revisão sistemática de artigos empíricos publicados nos últimos dez anos sobre a supressão de memórias indesejadas. O segundo estudo é um ensaio empírico, no qual foi executado um experimento adaptado do paradigma Think/No-Think com a utilização de marcadores eletrofisiológicos de eletroencefalograma. Participaram do experimento 22 sujeitos, alocados aleatoriamente em dois grupos com estratégias distintas de esquecimento: Supressão de memória e substituição de pensamentos. Durante toda a tarefa experimental, os participantes tiveram dados de EEG continuamente gravados. Os resultados decorrentes do ensaio empírico estão de acordo com os achados da literatura, indicando que a positividade parietal em torno de 400-800ms após a apresentação do estímulo é um marcador de lembrança consciente durante a recuperação de memória. Apenas na estratégia de supressão de memória houve uma redução da positividade centro-parietal durante o esquecimento, entre 450 e 700ms após apresentação do estímulo. Além disso, uma maior deflexão no componente N2 durante a supressão é um preditor de esquecimento induzido. Os achados indicam que é possível mapear o sistema neurocognitivo subjacente à supressão de memórias. / Intentional forgetting can be characterized as a strategic function of the cognitive system that allows us not to think about unwanted memories from our past, as for example emotional events or traumatic experiences that we would prefer not to remember. While forgetting involuntarily is a failure of recollection, on the other hand, forgetting intentionally seems to be a strategic function of memory. The aim of this dissertation was to investigate the neurocognitive mechanisms that contribute to forgetting memories. The dissertation was organized in two studies. The first study consists of a systematic review of empirical articles published in the last ten years on the suppression of unwanted memories. The second study is an empirical essay, in which an experiment adapted from the Think/No-Think paradigm was performed, with the use of electrophysiological markers of electroencephalogram. Twenty-two subjects participated in the experiment, randomly assigned to two groups with distinct strategies of forgetting: Memory suppression and thought substitution. Throughout the experimental task, participants had continuously recorded EEG data. The results of the empirical essay are in agreement with the literature findings, indicating that the parietal positivity around 400-800 ms after the presentation of the stimulus is a marker of conscious memory during memory recovery. Only direct memory suppression reduced centro-parietal positivity during forgetting, between 450 and 700 ms post-stimulus. Also, a greater deflection in the N2 component during suppression is an induced forgetting predictor. The findings indicate that it is possible to map the neurocognitive system underlying memory suppression.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22321号 / 医博第4562号 / 新制||医||1041(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 川上 浩司, 教授 浅野 雅秀, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The objective of this research was to study the impact of ripening inhibitor level and type on the formation, stability, and activity of antimicrobial thyme oil nanoemulsions formed by spontaneous emulsification. Oil-in-water antimicrobial nanoemulsions (10 wt%) were formed by titrating a mixture of essential oil, ripening inhibitor, and surfactant (Tween 80) into 5mM sodium citrate buffer (pH 3.5). Stable nanoemulsions containing small droplets (d < 70 nm) were formed. The antimicrobial activity of the nanoemulsions decreased with increasing ripening inhibitor concentration, which was attributed to a reduction in the amount of hydrophobic antimicrobial constituents transferred to the separated hydrophobic domain, mimicking bacterial cell membranes, by using dialysis and chromatography. The antimicrobial activity of the nanoemulsions also depended on the nature of the ripening inhibitor used: palm ≈ corn > canola > coconut which also depended on their ability to transfer hydrophobic antimicrobial constituents to the separated hydrophobic domain.

BACKGROUND: Breast cancer is the most common cancer type in women, with 276,480 women diagnosed with breast cancer in the United States in 2020.1 Death from breast cancer is usually caused by metastasis to distant sites, rather than from the primary tumor itself.2 Regional spread of breast cancer to the tumor draining lymph nodes (TDLN) often precedes further dissemination to the rest of the body, and as such is an important prognostic tool during cancer diagnosis and staging.3 During tumor growth, multiple immune cells and stromal cells in both the primary tumor microenvironment (pTME) and the TDLN undergo changes that promote tumor growth, metastasis and immune evasion in the TDLN and to the rest of the body. Among breast cancer subtypes, triple negative breast cancer (TNBC) is one of the most aggressive, and is most likely to be resistant to traditional chemotherapies, has the worst 5-year survival percentage, regardless of stage.4 Additionally, human growth factor receptor-2 (Her2) positive breast cancers are aggressive breast cancers as well, and Her2+ breast cancers are most likely to metastasize to the TDLN.5 PROBLEM: Little is known about how breast cancer cells induce genetic or transcriptomic changes in immune cells and stromal cells in the lymph node microenvironment and pTME as cancer cells metastasize to the TDLN. OBJECTIVE: To elucidate changes observed in immune and stromal cells in the TDLN via single cell sequencing, and to quantify changes in exhaustion status of lymphocytes in the metastatic TDLN microenvironment that may be causal of lymph node metastasis. METHODS: With a bioinformatics approach on single cell RNA sequencing, and with flow cytometry, we will measure changes in the transcriptomes of cells in the primary tumor microenvironment and the TDLN and compare changes in the primary site, tumor draining lymph node, and a normal lymph node. We measure changes in the expression of canonical exhaustion markers and inhibitory receptors: Tim-3, Lag-3, Tigit, CTLA-4, PD-1, and CD160 in lymphocytes in the metastatic TDLN and nonmetastatic spleens in Balb/c mice with flow cytometry. Additionally, we measured changes in myeloid and lymphoid populations in the spleens and lymph nodes with flow cytometry. RESULTS: B cell accumulation was observed in both the single cell RNA sequencing analysis and via flow cytometry in both the metastatic TDLN itself and in the spleens of mice with metastasis to the TDLN. Increased RNA of exhaustion markers in CD8+ T cells was observed in the primary tumor samples. Additionally, a trend of increased Natural Killer cells, B cells, naive and memory CD4+ and CD8+ T cells expressing canonical exhaustion markers was observed in both the metastatic TDLNs and the spleens of mice with cancer, indicating systemic immune suppression may occur as TNBCs and Her2+ breast cancers metastasize to the TDLN. / 2022-11-24T00:00:00Z

>Magister Scientiae - MSc / The Northern Cape province has not been able to achieve the 90% immunization target recommended for South Africa’s expanded programme on immunisation (EPI). The situation has been attributed to the lack of access to EPI in the rural community. The Northern Cape’s poor infrastructure renders the provision of equitable preventive care service to rural communities a complex and costly task. The province is predominantly a rural setting consisting of farmland, with low population densities, and many residents have poor access to public transport to receive primary health care services from surrounding fixed or satellite clinics. Consequently, mothers often do not adhere to the immunization schedules, and lack awareness of the risks associated with the failure to have their infants vaccinated against communicable diseases. EPI services delivered via mobile clinics serve the primary health care needs for rural communities, but their sustainability remains a challenge. Additional barriers are found in literature which highlighted the shortage of health professionals, unreliable funding, limited transportation within rural areas and deficiencies in maintenance and suitability of mobile clinic vehicles.

The smartphone has quickly become the most used device to access the internet. Academic and public concern has been raised if overuse of smartphone technology can have detrimental effects on brain and behavior. Preliminary results suggest that excessive smartphone usage may be linked to impaired inhibitory control. The present study investigates whether such a relationship is present in a sample of healthy individuals with varying degrees of usage. To investigate the proposed relationship, the Stroop color and word task and the stop-signal task was utilized to measure inhibitory control, while screen time was utilized to measure duration of smartphone usage. A Pearson‘s correlation analysis and an independent t-test/Mann Whitney-U test analyzed the results, which did not yield statistical significance.