Chemical-induced models have revealed the crucial role of oxidative stress and mito-chondrial dysfunction in the development of Parkinson’s Disease. In this project, firstly, we in-vestigated the mechanism of action of rotenone, a commercialized pesticide that was previously described to reproduce the bradykinetic dopaminergic neurodegeneration symptoms of Parkin-son’s Disease in zebrafish by inhibition of the mitochondrial complex I. We found out that rote-none caused change in the morphology of the zebrafish dopaminergic mitochondrial network. We also observed the altered expression of various genes involves in mitochondrial fusion and fission in response to rotenone exposure. Secondly, to develop the use of adult zebrafish as a toxin-based model for Parkinson’s Disease, we sought to minimize any off-target effects by exposure of rotenone specifically to the brain. We demonstrated that microinjection of rotenone into the forebrain ventricular zone of adult zebrafish decreases the number of dopaminergic neurons. However, behavioural tests suggested that did not translate into locomotor impairment in these fish. Taken together, these results gave us more information about the potential use of zebrafish to study the physiological mechanism leading to dopaminergic degeneration and allow for the development of therapeutic strategies for Parkinson’s Disease.

INTRODUCTION: Immune checkpoint inhibitors are a new form of immunotherapy that has transformed the treatment landscape for an ever-increasing number of malignancies. While these medications utilize and enhance the immune system to treat malignancies, they can also have significant side effects, termed immune related adverse events, that in many ways resemble autoimmune disease states. One such example is inflammatory arthritis, which has been found to resemble a number of different presentations, including rheumatoid arthritis and seronegative spondyloarthropathies. In addition to these traditional inflammatory arthritis phenotypes, worsening of pre-existing arthritis is another subgroup of inflammatory arthritis that has previously not been considered in this population. Furthermore, while the effects of these autoimmune arthropathies on functionality is well-documented, it is not known whether there is a significant effect on functionality in patients that experience immune checkpoint inhibitor-induced arthritis. Given that patient reported outcomes are a validated and routinely utilized measure of functionality and quality of life, the Health Assessment Questionnaire, pain visual analogue scale and Patient Global Assessment were used to measure these outcomes following diagnosis. Our aim here is to explore the subtypes of inflammatory arthritis that result from this type of treatment and its overall effect on functionality and quality of life. METHODS: This study was a retrospective review of patients at one academic center who experienced an inflammatory arthritis resulting from immune checkpoint inhibition and required a referral to a rheumatologist for further work-up. Patients were evaluated in clinic at which time they also completed a Health Assessment Questionnaire as part of standard of care. Once patients were evaluated, their inflammatory arthritis was classified based on which clinical arthritis that it matched most closely, including polymyalgia rheumatica, rheumatoid arthritis, seronegative spondyloarthritis, or an exacerbation or osteoarthritis. Other demographic information such as gender, age, and race were also collected. Patient questionnaires were scored and compared to the type of inflammatory arthritis to assess for any correlations. RESULTS: We found 30 patients that had an inflammatory arthritis resulting from immune checkpoint inhibition, with 12 having a polyarthritis similar to rheumatoid arthritis, 11 patients having osteoarthritis exacerbation, 4 patients with a polymyalgia rheumatica arthritis phenotype and 3 patients with a spondyloarthopathy. In terms of the patient reported outcomes, the overall score was 0.57 ± 0.47, indicating that there was little effect of these arthropathies on overall functionality. The pain visual analogue scale had an average score of 41.8 ± 31.4 mm and the Patient Global Assessment had an overall score of 25.6 ± 26.7 mm. DISCUSSION: The overall results suggest that the inflammatory arthritis phenotypes do not significantly impact the functionality or quality of life of most patients who experience this side effect. Given that the use of immune checkpoint inhibitors will continue to grow, the overall need to better understand the resulting arthritis presentations is key. This is perhaps most true for those with pre-existing osteoarthritis, given the widespread nature of the disease in the general population and the prominence of the exacerbation as seen in our cohort.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21960号 / 医博第4502号 / 新制||医||1037(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 平井 豊博, 教授 松原 和夫, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22326号 / 医博第4567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 滝田 順子, 教授 小川 誠司, 教授 遊佐 宏介 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23079号 / 医博第4706号 / 新制||医||1049(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 松田 道行, 教授 稲垣 暢也, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>For this dissertation, four experiments were conducted to evaluate the effect of dietary addition of exogenous protease and amylase enzymes on growth performance and nutrient utilization in broiler chickens. An additional fifth experiment was designed to determine the role of central and peripheral appetite regulators in birds fed diets deficient in dietary phosphorus (P). This arose from consistent reports in literature of a direct effect of dietary P concentration on feeding response in broiler chickens. </p><p>Experiment 1 examined the growth performance and protein utilization responses of broiler chickens to purified trypsin inhibitors (TI) and exogenous protease additions. Experimental diets were arranged as a 2 × 2 factorial with factors being dietary TI (1,033 or 10,033 TIU/g) and exogenous protease (0 or 15,000 PROT/kg). Protease supplementation improved BW gain (<i>P < </i>0.01) and gain to feed ratio (<i>P < </i>0.05) of birds. The relative weight of pancreas increased (<i>P < </i>0.05) with added TI on d 14 and 21 but was reduced (<i>P < </i>0.001) with protease supplementation. Apparent ileal digestibility (AID) of all amino acids (AA), except methionine, decreased (<i>P < </i>0.001) with added TI, but increased (<i>P < </i>0.05) with protease supplementation. Duodenal trypsin and chymotrypsin activities were reduced (<i>P < </i>0.05) with added TI but increased <i>(P < </i>0.01) with protease supplementation. It was concluded that dietary addition of purified TI negatively affects nutrient utilization by broiler chickens and that the efficacy of the exogenous protease might be independent of dietary TI concentration. A follow-up experiment was conducted (Experiment 2) to evaluate the impact of TI and exogenous protease supplementation on endogenous AA loss in broiler chickens. Four diets were arranged as a 2 × 2 factorial with factors being dietary TI (0 or 8,000 TIU/g) and exogenous protease (0 or 15,000 PROT/kg). There was no effect of TI, exogenous protease, or their interaction on growth performance of birds. Endogenous nitrogen (N) loss and all AA (except Cys) increased (P < 0.05) due to added dietary TI. Exogenous protease had no effect on endogenous loss of N and all AA. The AID of Ca, Fe, Mg, Mn, and Cu was reduced (P < 0.05) by added dietary TI. Protease supplementation improved the AID of Cu (P < 0.01) and K (P < 0.05). Secretion of crude mucin and sialic acid (g/kg DM intake) increased (P < 0.05) with increased dietary TI and was not recovered by protease supplementation. It was concluded from this study that TI increases the endogenous loss of AA, reduces the digestibility of minerals in broiler chickens, and that exogenous protease had no effect on endogenous AA flow, irrespective of added dietary TI. </p><p>In Experiment 3, the responses of broiler chickens fed corn-soybean meal-based diets to dietary α-amylase supplementation during 4 growth phases were evaluated. Birds were assigned to 8 treatment diet in a 2 × 4 factorial arrangement of 2 dietary levels of α-amylase supplementation (0 or 80 kilo-Novo alpha amylase units (KNU) per kg diet) and 4 post hatching growth phases (d 0 to 11, d 11 to 21, d 21 to 42, or d 42 to 56). Body weight gain and feed efficiency of birds improved (<i>P</i> < 0.01) with α-amylase supplementation. There were main effects of α-amylase, growth phase and interaction (<i>P</i> < 0.01) on AID of starch. The total tract retention (TTR) of starch increased (<i>P</i> < 0.05) with amylase supplementation but was not different across growth phases. Amylase supplementation improved (<i>P</i> < 0.05) gross energy utilization in birds, and specifically, during d 11 to 21 post hatching, the viscosity of jejunal digesta and pancreatic amylase activity increased (<i>P</i> < 0.01) with amylase supplementation. The conclusion from the study was that the growth phase of birds may affect the response to exogenous amylase. Following the result of this study, Experiment 4 was conducted to evaluate the effect of amylase supplementation on starch and energy digestibility at various intestinal sites in broiler chickens. Experimental diets comprised 3 concentrations of α-amylase supplementation (0, 80, or 160 KNU/kg diet) and sampling was done on 4 intestinal sites: anterior jejunum (AJ), posterior jejunum (PJ), anterior ileum (AI) and posterior ileum (PI). There were linear and quadratic (<i>P</i> < 0.01) responses of increasing α- amylase supplementation on starch and energy digestibility at the PJ and AI, with only linear effects on TTR of starch (<i>P</i> < 0.05). A linear increase in starch disappearance and digestible energy (kcal/kg) was observed (<i>P</i> < 0.01) with digesta flow from AJ to PJ with increasing amylase supplementation, which may be related to the observed decrease in the viscosity of the jejunal digesta (<i>P</i> < 0.05). Results from this experiment demonstrate the efficacy of exogenous amylase to improve starch, and energy digestibility in broiler chickens, with the highest impact observed in the posterior jejunum.</p><p>A final study (Experiment 5) was conducted to evaluate the impact of dietary phosphorus (P) concentration on hypothalamic molecular regulation of appetite by broiler chickens. Birds were randomly assigned to 3 experimental diets which contained 1.2 (P-deficient), 2.8 (P-marginal) or 4.4 (P-adequate) g/kg non-phytate P (nPP). A decrease in feed intake and BW gain was observed (P < 0.001) in birds fed the P-deficient diet. There was upregulation (P < 0.05) in the mRNA expression of Sodium-phosphate cotransporter (NaPi-IIb), anorexia-related hypothalamic cholecystokinin receptor (CCKAR) and melanocortin receptors (MC3R and MC4R) in birds fed P-deficient diets, whereas cholecystokinin (CCK) mRNA was downregulated (P < 0.01). It may be concluded that a deficiency in dietary P decreases feed intake in broiler chickens by altering the expression of anorexigenic genes in the gut and hypothalamus.</p>

There is currently no standardized treatment of COVID-19. Despite several vaccines being deployed, the need for an effective treatment in hospitalized patients is heavily sought after. In this systematic review, six clinical trials were analyzed for their findings on the effect of commercially available anti-viral protease inhibitors in treatment of COVID-19. The aim of the study was to evaluate the effect on the hospitalization period and mortality of patients suffering from COVID-19 and if HIVprotease inhibitors could be repositioned to treat COVID-19 in combinational therapy. The findings suggest that protease inhibitors targeting the hepatitis C virus (HCV) proteases could potentially beeffective at reducing the hospitalization period and that the effect can be further enhanced by using them in combination with drugs targeting the RNA polymerases. Further studies are needed confirm these findings. Lopinavir/ritonavir which was the most common protease inhibitor included in the study did not reduce the hospitalization period or the mortality significantly. It is unclear whether the HCV protease inhibitors could reduce mortality. Further studies regarding this outcome are warranted.

Les b-lactamines, grâce à leur efficacité clinique, sont parmi les antibiotiques les plus prescrits pour traiter des infections bactériennes. Cependant, leur utilité est compromise par la prolifération des b-lactamases (BLs) avec des profils d’hydrolyse de substrats très larges. La résistance induite par les BLs compromet également les b-lactamines les plus puissantes (c-à-d les carbapénèmes). OXA-48, une carbapénèmase de classe D (CHDL), a été initialement identifiée dans une souche de K. pneumoniae de Turquie en 2001. OXA-48 hydrolysent fortement les pénicillines, faiblement les carbapénèmes, et quasiment pas les céphalosporines de troisième génération (C3G). Cependant certains variants comme OXA-163 ou OXA-405 hydrolysent les C3G et pas les carbapénèmes. La comparaison de la structure tri-dimensionnelle d’OXA-48 avec d’autres CHDLs a révélé de petites différences principalement localisées dans les boucles qui relient des éléments de structure. Notamment, la boucle localisée entre les feuillets b5 et b6 semble jouer un rôle majeur dans l’hydrolyse des carbapénèmes.Afin de mieux comprendre la contribution de la boucle b5-b6 dans l’hydrolyse des carbapénèmes, nous avons étudié, grâce à des outils biochimiques et structuraux, des variants naturels ou synthétiques d’OXA-48 présentant des modifications dans la boucle: le rôle du remplacement de chaque AA de la boucle par une alanine, des délétions croissantes ou de l’augmentation de la taille de la boucle. Nous avons également réalisé l’échange de boucle entre OXA-48 et OXA-18, une oxacillinase inhibée par l’acide clavulanique et hydrolysant fortement les C3G mais pas les carbapénèmes. La protéine recombinante OXA-48loop18 hydrolysait les C3G, et conservait une activité significative d’hydrolyse des carbapénèmes. L’échange de boucle a permis l’élargissement du site actif, permettant l’accès à des b-lactamines possédant un radical volumineux (e.g. ceftazidime). De plus, le remplacement de chaque AA de la boucle par une alanine a relevé de faibles changements hydrolytiques. En réalisant des délétions croissantes d’AA soit en partant de la gauche de la boucle (Tyr-211 vers Pro-217) ou de la droite (Pro-217 vers Tyr-211), nous avons montré que l’activité d’hydrolyse des carbapénèmes diminuait avec la taille des délétions, alors que celle des C3G augmentait. Les délétions de 4 AA présentent les plus fortes activités hydrolytiques des C3G et une perte totale de l’activité carbapénèmase, excepté pour le simple mutant, OXA-48∆P217, qui présentait un profile d’hydrolyse avec une forte activité carbapénèmase et C3G. La cristallographie et la modélisation moléculaire ont montré une grande flexibilité de la boucle, permettant l’entrée de b-lactamines de tailles variables. De plus, l’étude de nouveaux variants d’OXA-48 a permis d’identifier des déterminants structuraux importants dans le profil d’hydrolyse observé. Ainsi, la délétion I215-E216 associée à la substitution R214K dans la boucle b5-b6 de OXA-517 permet une forte hydrolyse des carbapénèmes et des C3G. De même, dans OXA-519, une substitution V120L située à proximité de la boucle b5-b6, a pour conséquence une diminution de l’affinité pour tous les substrats. La chaine latérale plus encombrante de la L120 empêche l’insertion des b-lactamines, diminuant l’affinité de l’enzyme. Finalement, nous avons caractérisé OXA-535, la b-lactamase naturelle et chromosomique de Shewanella bicestrii JAB-1 qui, n’ayant uniquement 91,5% d’identité en AA avec OXA-48, présente le même profil d’hydrolyse. OXA-535 présentait 98.9% d’identité en AA avec OXA-436, codé par un gène plasmidique, suggérant ainsi que S. bicestrii portant le gène blaOXA-535 pourrait être le progéniteur du gène plasmidique blaOXA-436.Nos travaux ont montré le formidable pouvoir d’adaptation de OXA-48 à évoluer par mutation afin d’accommoder différents substrats, et comment la nature et la longueur de la boucle b5-b6 pouvait influencer sur la spécificité de substrat. / Antimicrobial resistance is the most alarming emerging problem in infectious diseases. b-Lactams, due to their safety, reliable killing properties and clinical efficacy, are among the most frequently prescribed antibiotics used to treat bacterial infections. However, their utility is being threatened by the worldwide proliferation of b-lactamases (BLs). BL-mediated resistance does not spare even most powerful b-lactams, carbapenems, whose activity is challenged by carbapenemases. OXA-48, a carbapenem-hydrolyzing class D b-lactamase (CHDL) initially identified from a Klebsiella pneumoniae isolate from Turkey in 2001, has since spread globally with the isolation of more than 30 variants. Most OXA-48-like enzymes hydrolyze penicillins at high level, carbapenems at low level and lack significant expanded-spectrum cephalosporin (3GC) hydrolysis, others such as OXA-163 hydrolyze expanded-spectrum cephalosporins and poorly carbapenems. Comparison of OXA-48 tertiary structure with those of other CHDLs revealed small differences located mainly in the loops connecting secondary structure elements, which may vary in length and orientation. The loop located between the b5 and b6 strands (Tyr211 to Pro217) has been suggested to play a major role in carbapenem hydrolysis.To better understand the contribution of the b5-b6 loop in the carbapenem hydrolysis of OXA-48-like carbapenemases, we investigated, using biochemistry and structural biology, natural OXA-48 variants with changes in different loops, replaced each AA of the loop b5-b6 by alanines, performed increasing deletions or increased the size of this loop by replacing it with that of OXA-18, a clavulanic acid inhibited class D b-lactamase that presents activity against expanded-spectrum cephalosporins and none against carbapenems. The resulting OXA-48loop18 was able to hydrolyze expanded-spectrum cephalosporins and conserved partial carbapenem hydrolysis. Structural analysis demonstrated that the loop swap produced an opening of the active site, being now accessible to b-lactams with bulky sidechains e.g. ceftazidime. Additionally, by performing alanine replacements in the b5-b6 loop we could show reduced hydrolysis of carbapenems, mostly reflected by changes in kcat. By increasing deletions in the b5-b6 loop, starting from Tyr211 to Pro217 and from the Pro217 to Tyr211, the activity against carbapenems decreased with the size of the deletion whereas the activity against ceftazidime increased. 4 AA deletions revealed the highest 3GC activity, except for one single AA mutant, OXA-48∆P217, with high level carbapenem and ceftazidime hydrolysis. Crystallography along with molecular modelling showed an increased flexibility of this loop allowing different sized b-lactams to enter the active site. Moreover, the characterization of three novel natural OXA-48 variants revealed structural features important in the observed hydrolysis profile. Thus, the I215-E216 deletion and R214K substitution in the b5-b6 loop of OXA-517 induced the hydrolysis of carbapenems and C3G at high level. In OXA-519, the V120L substitution is located at the bottom of the binding site, in the close vicinity of the active Ser70 and the b5-b6 loop, and therefore overall higher Km values were observed compared to OXA-48. The bulkier side chain of L120 in OXA-519 hampers the approach of b-lactam substrate, resulting in a decrease of the substrate affinity. Finally, we have characterized the chromosomally-encoded OXA-535 that is more distantly related to OXA-48 (91.5% AA identity), despite similar hydrolysis profiles. Interestingly, OXA-535 presented 98.9% of AA identity with the plasmid-mediated OXA-436 suggesting that the blaOXA-535 gene might be the progenitor of the plasmid-encoded blaOXA-436 gene.Taken together, our work illustrates the propensity of OXA-48 to evolve through mutations to accommodate different substrates in its active site and how the b5–b6 loop determines the specificity of the enzyme.

Sodium-Glucose Linked Transporter 2 (SGLT2) inhibitors block renal glucose reabsorption and have shown marked cardiac protection in type 2 diabetics, and surprisingly, also in non-diabetics. However, the mechanism by which these drugs improve cardiovascular outcomes is unknown. Metabolic heart disease, which is characterized by cardiac hypertrophy and diastolic dysfunction, is associated with obesity and insulin resistance and leads to adverse cardiovascular outcomes including heart failure with a preserved ejection fraction. A high fat, high sucrose “Western” diet can induce metabolic syndrome, an aggregate of obesity-driven clinical phenotypes including insulin resistance, elevated triglycerides, hypertension, and abnormal cholesterol. Using a mouse model of metabolic syndrome and adult rat ventricular myocytes (ARVMs) in vitro, we aim to determine if the SGLT2 inhibitor Ertugliflozin (ERTU) can prevent metabolic syndrome-induced cardiac pathophysiology and whether ERTU can exert a direct action on cardiomyocytes, a cell type lacking SGLT2. SGLT2 inhibitors have been proposed to act directly on the Sodium-Hydrogen Exchanger 1 (NHE1) and thus could have direct action on cardiomyocytes that may mediate cardioprotective effects. Mice were fed either a control diet (CD) or a high fat high sucrose (HFHS) diet ± ERTU for 16 weeks. Echocardiography was performed and heart weights were obtained. ARVMs were used to assess ERTU’s effect on insulin sensitivity in vitro in a high-palmitate, insulin resistance model, and to test the efficacy of the known NHE1 inhibitor Cariporide (NHEi). A NHE1 activity ammonium chloride pulse assay was performed in HEK293 cells over-expressing either wild-type (WT) or a known NHEi-insensitive point mutant NHE1 ± NHEi or ERTU. In HFHS-fed mice, ERTU attenuated weight gain and restored blood glucose, insulin, hemoglobin A1c, and HOMA-IR to CD levels. HFHS-induced cardiac hypertrophy and diastolic dysfunction were prevented with ERTU. In vitro, high palmitate media decreased insulin stimulated AKT signaling compared to low palmitate media and was rescued by either ERTU or NHEi treatment. ERTU inhibited WT NHE1 activity in HEK293 cells by 67%, whereas activity of the NHEi-insensitive mutant NHE1 was unaffected by ERTU treatment. ERTU prevented the hallmarks of diet-induced metabolic heart disease (cardiac hypertrophy and diastolic dysfunction) in mice. These benefits exceed the expected consequences of glucose control alone. The actions of ERTU on ARVMs in vitro suggest the favorable effects on cardiac structure and function may be due, at least in part, to the direct action of the drug on cardiomyocytes. Furthermore, mutational overexpression studies show that ERTU can directly affect NHE1 in cardiac myocytes. Taken together, this thesis provides evidence that the direct cardioprotective effects of ERTU could be via inhibition of NHE1, a critical modulator of intracellular pH and sodium in the cardiomyocyte, with known implications in the pathophysiology of diabetes and heart failure. / 2022-06-04T00:00:00Z

Ph.D. THESIS TITLE Nutritional Biology of Synanthropic Mites (Acari: Acaridida) ABSTRACT Several attempts to describe the nutritional biology of acaridid mites were undertaken, however full understanding of these processes remains incomplete. The objective of this Ph.D. thesis was to expand our knowledge concerning digestive physiology of stored product and house dust mites and to apply this knowledge to their nutritional biology. The research approach adopted in this Ph.D. thesis includes in vitro characterization of enzymatic activity in whole mite extracts (WME) and spent growth medium extracts (SGME), evaluation of the enzyme activities with respect to the gut physiological pH, enzyme inhibition experiments, in vivo localization of enzyme activities in the mite gut, determination of effects of nutrient or antifeedant additives in experimental diets on mite population growth and determination of the feeding preferences of synanthropic mites as assessed by in vitro and in vivo analyses. The gut contents of twelve species of synanthropic acaridid mites were determined to be within a pH range of 4 to 7 and showed a pH gradient from the anterior to the posterior midgut. The pH in digestive tract of synanthropic acaridid mites corresponds to the activity of proteases, α-glucosidases, α-amylases and...