published_or_final_version / Chinese Medicine / Master / Master of Philosophy

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Introduction Human CD36 is a class B scavenger receptor expressed in a variety of cell types including macrophages and endothelial cells. This heavily glycosylated membrane protein has a large ectodomain (ED) responsible for binding a variety of ligands. These include oxidised LDL and long chain fatty acids, which link CD36 to the development of atherosclerosis and insulin resistance, respectively. CD36 has also been implicated in the phagocyte-uptake of apoptotic cells, anti-angiogenic effects in endothelial cells and development of a variety of fibrotic diseases, through interaction with thrombospondin-1. The main objective of this study was to express, purify and characterise the ligand binding ectodomain of CD36 with a view to better understanding how CD36 binds multiple ligands. Methods A baculovirus expression system was used to express and secrete CD36 ED with a 12-His tag from insect cells using an N-terminal secretion sequence. Subsequent purification was performed using nickel affinity chromatography with functionality of the ED assessed through the ability to bind modified LDL in a solid phase binding assay. The N-glycosylation status of the purified ED was explored through use of the N-glycosidase PNGase F and mass spectrometric analysis. Initial studies to measure binding kinetics involved use of surface plasmon resonance (SPR) and binding of commercially available antibodies, mAb1955, mAb1258 and mAbFA6-152, to purified CD36 ED immobilised on an NTA SPR sensor chip. Results The N-glycosylation status of CD36 ED produced in Sf21 and Hi5 insect cells was different with heterogeneous N-glycosylation being identified at individual glycosylation sites. Solid-phase ligand binding revealed that both glycosylated and deglycosylated ED retain affinity for modified LDL. The purified CD36 ED was found to homo-oligomerise particularly at higher concentrations. MAb1955 and mAb1258 were found to bind to the nickel on the sensor chip precluding microkinetic analysis of binding to CD36. However, mAbFA6-152 was found to bind to the immobilised CD36 ED with high avidity and two-step binding kinetics. Conclusions and future direction This study shows for the first time that N-glycosylation is not important for the binding of modified LDL to CD36. Furthermore it was demonstrated that native CD36 ED produced in Sf21 insect cells can be deglycosylated without denaturation, which may be critical for the success of future crystallisation trials. The characterisation of mAbFA6-152 binding by SPR is proof-of-principle that CD36 ED can be studied using this technique paving the way for future biophysical analysis of ligand binding.

Organ‐transplant recipients (OTRs) on a new class of immunosuppressants, rapamycin and its analogues, have reduced cutaneous Squamous Cell Carcinomas (cSCCs). Rapamycin, an mTORC1 inhibitor, is also a known autophagy inducer in experimental models. Autophagy, which literally means self‐eating, is a cell survival mechanism but can also lead to cell death. Therefore, the main hypothesis behind this work is that rapamycin prevents epidermal tumourigenesis by either affecting epidermal mTOR regulation of autophagy and/or selectively affecting epidermal AKT isoform activity. Epidermal keratinocytes move from the proliferating basal layer upwards to the granular layers where they terminally differentiate, forming a layer of flattened, anucleate cells or squames of the cornified layer which provides an essential environmental barrier. However, epidermal terminal differentiation, a specialised form of cell death involving organelle degradation, is poorly understood. The work presented in this thesis shows that analysis of the autophagy marker expression profile during foetal epidermal development, indicates autophagy is constitutively active in the terminally differentiating granular layer of epidermis. Therefore, I hypothesize that autophagy is a mechanism of organelle degradation during terminal differentiation of granular layer keratinocytes. In monolayer keratinocytes, activation of terminal differentiation is accompanied by autophagic degradation of nuclear material, nucleophagy. This suggests that constitutive autophagy is a pro‐death mechanism required for terminal differentiation. In cultured keratinocytes and in epidermal cultures, rapamycinmediated mTORC1 inhibition strongly increases AKT1 activity as well as up‐regulates constitutive granular layer autophagy promoting terminal differentiation. Therefore, autophagy is an important fundamental process in keratinocytes which may be the mechanism by which terminally differentiating keratinocytes of the epidermal granular layer degrade their organelles required for barrier formation. This may have implications for the treatment of patients with barrier defects like psoriasis. In immunosuppressed OTRs, rapamycin may promote epidermal autophagy and AKT1 activity adding to its anti‐tumourigenic properties.

Comprehensive in vitro modelling of inflammatory human skin conditions is an essential first step in the development and assessment of potential therapeutic approaches. Mouse models or monolayer keratinocyte cultures come with distinct limitations which might be complimented or overcome by the use of human-specific organotypic 3D culture models. Over the course of this thesis, an organotypic culture system, based on patientderived immortalised keratinocyte cell lines on a dermal equivalent collagen 1 gel, was established and used to recapitulate phenotypical features for two hereditary skin diseases, Harlequin ichthyosis and Tylosis with oesophageal cancer. Small molecular compounds, supplied via the medium, or RNA interference were used to modulate disease-specific changes in histology and marker expression of the skin equivalent. Since hyperproliferative skin conditions can be associated with an aberrant wound healing phenotype, the organotypic system was manipulated to obtain a basic in vitro wound healing model. This model displays typical features of re-epithelialisation over time (both normal and disease-specific) which can further be manipulated via shRNAmediated knockdown or the exogenous supply of compounds. In parallel, a non-disease model was used to assess the topical application of novel nanopolymeric drug delivery systems in regard to their ability to penetrate across the permeability barrier. Penetrance profiles for the organotypic model (in dependence of co-application with chemical enhancers) showed a similar pattern as for topical applications performed in parallel on explant skin. In conclusion, a highly adaptable human organotypic keratinocyte culture model was developed and used to recapitulate (and manipulate) skin disease phenotypes and epidermal wound healing in vitro, as well as perform first essential assessments of novel drug delivery systems.

Senescence is classically defined as an irreversible cell cycle arrest. There is now convincing evidence that senescent cells accumulate during human ageing, potentially driving age-related dysfunction through depletion of mitotically active cells and stimulation of chronic inflammation. Recently, a landmark paper demonstrated that removal of p16INK4A (p16)-positive senescent cells in mice prolonged healthy lifespan, suggesting a direct link between senescence and age-related dysfunction. As such, restraining the senescent pool or slowing their rate of accumulation presents an attractive therapeutic strategy for extending healthspan. Previously, our laboratory has demonstrated that siRNA transfection can reverse deep senescence in p16-positive primary adult human mammary epithelial cells using a panel of senescence markers. Subsequent siRNA screening revealed 28 hits which strongly induced reversal in the deeply senescent HMECs. In this project, siRNA knockdown of p16 combined with p21WAF1/CIP1 (p21) was found to reverse deep replicative senescence in primary adult human mammary fibroblasts, as defined by a panel of senescence markers. This discovery provided the opportunity to screen for novel siRNAs which induce reversal in both cell types. Screening in the deeply senescent HMFs of the 28 shortlisted candidates and 33 protein interactors identified using bioinformatics revealed 45 siRNAs which significantly increased cell number compared to the negative siRNA control. Subsequent immunofluorescence staining and high content analysis of the top 14 candidates identified 10 hit siRNAs which induced senescence reversal as defined by a panel of markers. Interestingly, these 10 hits were enriched for cytoskeletal and cell adhesion processes, suggesting an interplay between external forces and senescence induction. The top siRNA hit, early growth response 2 or EGR2, a transcription factor, was validated and selected for further exploration as a novel driver of senescence. Bioinformatics analysis revealed an enrichment for EGR2 binding sites within genes dynamically expressed in HMEC senescence, including p16, p21, and nine hits identified to reverse senescence in HMFs and HMECs. Furthermore, deeply senescent HMFs and HMECs were found to have a significantly increased nuclear EGR2 foci number compared to proliferating cells, and this was significantly decreased in reversed HMFs. In conclusion, it is proposed that EGR2 may represent a novel driver of both HMF and HMEC senescence.

nÃo hà / A leishmaniose cutÃnea à um importante problema de saÃde no Brasil, existindo como doenÃa autÃctone em todos os estados brasileiros. No perÃodo de 1980 a 2005, foram registrados 613.644 casos no paÃs. No CearÃ, à uma doenÃa endÃmica em vÃrias regiÃes e em muitos municÃpios faz centenas de vÃtimas todos os anos. Em Ãpocas de epidemias, o nÃmero de registros tem chegado a mais de 4000 notificaÃÃes por ano. Nos Ãltimos 28 anos (1980-2008) foram diagnosticados 55.925 pessoas acometidas da doenÃa, o que reflete uma mÃdia de 2000 vÃtimas de leishmaniose cutÃnea por ano. Sabe-se que os primeiros registros da doenÃa no Cearà datam de 1925. Apesar de ter todo este impacto na saÃde pÃblica do estado, existem muitas lacunas no conhecimento desta infecÃÃo, quer seja do ponto de vista histÃrico; do ponto de vista clÃnico e relacionadas ao tratamento. Objetivo: Fazer um levantamento sobre os aspectos histÃricos e as descobertas mais relevantes relacionadas Ãs leishmanioses; investigar quando e como foi introduzida no estado do CearÃ, bem como seus descobridores; descrever de modo detalhado as manifestaÃÃes clÃnicas da leishmaniose cutÃnea no CearÃ; relatar a evoluÃÃo histÃrica do tratamento, bem como apresentar uma alternativa de tratamento para a leishmaniose cutÃnea. Metodologia: Foram consultadas fontes primÃrias e fontes secundÃrias. Fontes SecundÃrias 1. Livros textos clÃssicos sobre leishmaniose; livros de escritores cearenses sobre tema envolvendo saÃde do inÃcio do sÃculo passado; teses; artigos de revistas cientÃficas. 2. Acervo de bibliotecas em Fortaleza (CE), Salvador (BA), Rio de Janeiro (RJ), SÃo Paulo(SP), Estados Unidos (Universidade de VirgÃnia, Universidade de Miami) e Inglaterra (London School of Tropical Medicine & Hygiene e FundaÃÃo Wellcome). 3. Pesquisas no Pubmed, Lilacs e Medline; 4. Pesquisa de microfilmes de jornais e de RelatÃrios da Provincia do CearÃ, do perÃodo de 1850 a 1930, do Acervo da Biblioteca PÃblica Menezes Pimentel (Fortaleza-CE). 5. Pesquisa nas pÃginas da OMS e do yahoo e google, em especial para as biografias. Fontes PrimÃrias: 1. Entrevistas com pesquisadores que viveram a histÃria da leishmaniose, com familiares de pesquisadores que fizeram a histÃria; 2. Dados e fotografias de lesÃes de pacientes de acervo pessoal. Resultados: Os primeiros registros da doenÃa (em livros) no Cearà sÃo de 1909 e registros oficiais (documentos de governo) somente existem a partir de 1917. O surgimento da leishmaniose cutanea ocorreu apÃs a grande seca e a epidemia de varÃola (1877-1879), que forÃaram a migraÃÃo de milhares de cearenses para a regiÃo AmazÃnica, para trabalhar na exploraÃÃo da borracha. Ao retornarem para o Cearà trouxeram a infecÃÃo, que aqui encontrou condiÃÃes propÃcias para se transmitir. Os primeiros registros da leishmaniose na AmazÃnia datam de 1820. Do ponto de vista clÃnico observamos que a infecÃÃo pela Leishmania braziliensis causa febre, adinamia e linfadenopatia satÃlite e alguns pacientes apresentam hepatoesplenomegalia. O aspirado dos linfonodos mostrou, na maioria dos casos, a presenÃa de leishmania e a cultura do creme leucocitÃrio em um paciente isolou o parasita. Do ponto de vista terapÃutico, de 21 pacientes tratados com fluconazol na dose de 7mg/kg/dia, durante uma mÃdia de 7 semanas, 18 tiveram as Ãlceras totalmente cicatrizadas e dois pacientes dos trÃs que inicialmente nÃo responderam, obtendo a cura com associaÃÃes de medicamentos que incluÃram o fluconazol como uma das drogas administradas. ConclusÃo: A leishmaniose cutÃnea do Cearà à uma doenÃa importada da AmazÃnia, onde a enfermidade jà existia desde o inÃcio do sÃculo XIX e que deve ter chegado no Cearà antes do inicio do sÃculo XX; a infecÃÃo pela Leishmania braziliensis causa uma doenÃa sistÃmica devido ao parasitismo e à provavelmente nesta fase que a leishmania se localiza nas mucosas onde poderà causar doenÃa mais tarde; o fluconazol à uma alternativa para o tratmento da leishmaniose cutÃnea. / Cutaneous leishmaniasis is an important health problem in Brazil.The disease has spread to all regions and autochtonous cases have been reported from all states. The total number of cases reported in the country from 1980 to 2005, was 613, 644. In CearÃ, cutaneous leishmaniasis is an endemic disease in all regions of the state and in many municipalities hundreds of cases are reported every year. In times of epidemics the number of cases has reached more than 4000 per year. In the 28 years from 1980 to 2008, 55.925 new cases were reported in the state. The first well documented cases of cutaneous leishmaniasis in Cearà date back to 1925. Even though it causes a great impact in public health, many informations related to the knowledge of the disease are lacking in its historical, clinical and therapeutics aspect. Objective:1.To study all relevant historical aspects related to the leishmaniasis;2. To investigate when and how it emerged in CearÃ, as well as who studied it; 3. To describe in a detailed form the clinical manifestations of cutaneous leishmaniasis; 4. To describe the historical evolution of therapy as well as to propose a therapeutic alternative for the disease. Methods:We consulted primary and secondary sources. Secondary sources. 1. Old books (classics) on leishmaniais; Books and articles from the beginning of the XX century by writers from Cearà as well as thesis and articles from scientific journals. 2. Archives of Libraries in Fortaleza(Ce), Salvador(BA), Rio de Janeiro(RJ), SÃo Paulo (SP), EUA( Universidade de Virginia, Universidade de Miami) and England (London School of Tropical Medicine & Hygiene and Wellcome Foundation); 3. Search in pubmed, Medline and lilacs; 4. To search information from microfilms of newspapers and reports from the Government Province of Cearà from 1850 to 1930 in the archives of Menezes Pimentel Public Library in Fortaleza (Ce); 5. WHO home page, yahoo.com and google.com, specially for scientists biographies. Primary sources: 1. Interview with researchers, family members of old researchers that made the history. 2. Author personal archives of photographs of cutaneous leishmaniasis patients skin and mucosal lesions as well as lymph nodes. Results: The first references to the existence of the disease (in books) in Cearà are form 1909 and in government reports are from 1917. The emergence of the disease in Cearà occurred after the great drought and the smallpox epidemic (1877-1879). These phenomenons forced migration of dozens thousands of people to the Amazon, to work on rubber production. In their return they brought with them the infection and the disease found here the appropriate conditions to develop. The first documented information on the disease in the Amazon region date back to 1820. From the clinical point of view, we documented that the infection by Leishmania braziliensis causes fever, malaise and satellite lymph nodes enlargement and in some patients liver and spleen enlargement is seen. Lymph nodes aspirates demonstrated Leishmania in most patients and Leishmania was cultured from Buffy coat of one patient. Concerning therapy, 21 patients were treated with fluconazole 7mg/kg/day for an average of 7 weeks, 18 were cured, and 2 of the three that did not responded to fluconazole initially, were cured with drugs associations that included fluconazole in the therapeutic scheme. Conclusion: 1. Cutaneous leishmaniasis in Cearà is an imported disease from the Amazon region, where the condition exists since the beginning of the XIX century and had its arrival in Cearà at the end of the XIX century; 2. Leishmania braziliensis infection causes a systemic disease due to parasite dissemination and it is probable in this phase, that leishmania locates in the mucosa, where it may causes disease in a later phase; 3. Fluconazole is a therapeutic alternative for cutaneous leishmaniasis.

Anfíbios possuem uma microbiota cutânea que os protege contra patógenos. Essa proteção se dá pela produção de moléculas antimicrobianas e pela competição por espaço e nutrientes contra patógenos. Alterações na composição da microbiota, causadas por fatores bióticos e abióticos do ambiente e por fatores ecofisiológicos do hospedeiro, podem afetar a resistência dos anfíbios à doenças. Assim, é possível que ambientes artificiais, por conter condições ambientais diferentes dos naturais e por alterarem aspectos ecofisiológicos dos indivíduos, devem modular a microbiota cutânea de animais mantidos e nascidos em cativeiro. Nós avaliamos diferenças inter e intra-populacionais no perfil da comunidade bacteriana de Scinax alcatraz entre três grupos: indivíduos selvagens; indivíduos nascido em cativeiro; e indivíduos mantidos em cativeiro por dois anos. Também verificamos o efeito temporal de ambientes artificiais no perfil da microbiota cutânea entre e dentre indivíduos selvagens mantidos em cativeiro ao longo de 312 dias. Os parâmetros microbiológicos utilizados foram riqueza de morfotipos bacterianos e abundância de colônias bacterianas. As diferenças encontradas entre populações apontam para o ambiente como um importante modulador da microbiota cutânea. No entanto, as diferenças encontradas entre indivíduos de uma mesma população apontam para a importância de aspectos fisiológicos do hospedeiro na modulação. Por fim, a avaliação temporal foi importante para mostrar que tanto aspectos ambientais quanto aspectos ecofisiológicos atuam juntos na modulação da comunidade bacteriana cutânea de anfíbios mantidos em cativeiro / Amphibians harbor a skin microbiota that provides protection against pathogens. This protection happens by production of antimicrobial substances and by competition for space and nutrients against pathogens. Changes in the microbiota composition, caused by biotic and abiotic factors of the environment and ecophysiology factors of the host, may affect disease resistance of amphibians. As artificial environments contain different environmental conditions compared to natural ones and may alter physiological aspects of individuals, it may modulate the cutaneous microbiota of captive animals. Our study evaluated inter- and intra-population differences in the profile of bacterial community of Scinax alcatraz from three distinct groups: wild individuals; individuals born in captivity and individuals kept in captivity for two years. We also investigated the temporal effects of artificial environments on the cutaneous microbiota profile between and within wild individuals kept in captivity over 312 days. Microbiological parameters analyzed were richness of bacterial morphotypes and abundance of bacterial colonies. The differences found between populations show that the environment may be an important modulator of the microbial community. However the differences between individuals within a population demonstrate the importance of physiological aspects of the host for the composition of the microbiota. Finally, the temporal evaluation performed was important to show that both environmental and ecophysiological aspects act together in modulating the cutaneous microbiota community of amphibians kept in captivity

CONTEXT: Nitric Oxide (NO) is an endothelial-derived vasoactive molecule that causes an increase in blood flow and oxygen delivery to tissue. A reduction in NO bioavailability has been found to occur in adults over the age of 60 and can be reversed pharmacologically by improving NO synthase (NOS) activity. Reversing these age-related changes with alternative interventions, such as aerobic exercise, has shown some promising results. OBJECTIVE: To quantify blood NO-bioavailability (as measured by blood nitrite levels) in a population of aerobically trained elderly men and compare these data to a group of age-matched, inactive individuals. In addition, we measured the cutaneous vasodilator response to local skin heating as a bioassay for NO-mediated cutaneous dilation. SETTING: BYU Human Performance Research Center (HPRC). PARTICIPANTS: 16 healthy elderly men (age = 66 ± 7.07 years) were divided into two groups based on physical fitness levels and estimated VO2max in ml O2•kg-1•min-1 (Trained = 39.1 ± 1.21, Untrained = 29.0 ± 2.70). INTERVENTIONS: A blood sample was collected and analyzed for NO. A microdialysis study was performed and dialysate was collected at 32°C and at 42°C. During the heating process, skin blood flow (skin vasomotor activity) was monitored and reported as cutaneous vascular conductance (CVC). MAIN OUTCOME MEASURES: Whole blood nitrite concentrations, pre- and post-heat nitrite concentrations, and CVCmax were compared between trained and untrained groups. RESULTS: Whole blood nitrite concentration was similar in trained subjects and untrained subjects averaging 25.77 ± 6.75 and 21.43 ± 7.20 µM, respectively (F1,13 = 0.19; P = 0.6671]. Local skin heating had no impact on the concentration of nitrite in dialysate samples ([NOx]dialysate F1,26 = 0.01; P = 0.7567). In addition, the plateau in % CVCmax following 30 minutes of local heating was similar for trained and untrained subjects averaging 67.7 ± 5.8 and 68.0 ± 6.2 % CVCmax, respectively (F1,13 = 0.00; P = 0.9673). CONCLUSIONS: The results of this study indicate that age-mediated reductions in whole blood NO-bioavailability and decrements in NO-mediated cutaneous vasodilation during local heating were similar in aerobically fit and sedentary adults 60 years old or older. We conclude that a commitment to aerobic fitness was unable to overcome the age-related dysfunction of the NOS system.

Coordinated movements are regulated by the brain, spinal cord and sensory feedback. The interaction between the spinal cord and sensory feedback also play a significant role in facilitating plasticity and functional recovery after neural trauma. Cross-education describes training one side of the limb to enhance the strength of the homologous muscle on the contralateral side. Previous study with chronic stroke participants found significant strength gains in the more affected leg following unilateral dorsiflexion training on the less affected side, which suggested cross-education can be used to boost strength gain when training the more affected side is hard to initiate. However, there is lack of evidence showing cross-education in the arm muscles after stroke and the neural pathways mediating strength cross-education in stroke participants require further study. The modulatory role of sensory feedback in movement control has been studied by using cutaneous stimulation as a proxy of the sensory input from skin. Mechanistic studies on neurological intact participants show that cutaneous reflex pathways are widespread in the cervical and lumbar spinal cord and have a global effect on the muscles in the non-stimulated limbs. In rehabilitation training, sensory enhancement from prolonged electrical stimulation has been used to facilitate training outcomes for those had stroke and other neurological disorders. Therefore, cutaneous pathways may be important in regulating cross-education training-induced strength gain. The purpose of this dissertation was to explore the effects of upper limb cross-education strength training in chronic stroke participants and the role of sensory inputs in regulating intra- and interlimb neural excitability in neurologically intact participants. In the first project (Chapter 2), we explored the efficacy of cross-education strength training in wrist extensor muscles of chronic stroke participants. Strength improvements were found bilaterally with altered excitabilities in the cutaneous pathways on the untrained side. These results show the potential role of cutaneous pathways in mediating strength transfer after unilateral strength training which led us to further explore the factors may affect the cutaneous modulation. In neurologically intact participants, we investigated the effects forearm position (Chapter 4), stimulation trigger mode and parameters (Chapter 5) on the cutaneous reflexes in the stimulated limb. Following the findings from Chapter 3, 4, and 5, the interlimb effects of self-induced sensory enhancement on the cutaneous reflexes were examined in Chapter 6. Taken together, data from this thesis confirms the clinical application of cross-education in strength training after stroke. It addresses that exaggerated bilateral strength gains and neural plasticity can be induced following unilateral strength training on the less affected side. In addition, sensory enhancement may be applied to amplify cross-education effects in strength training. / Graduate / 2020-09-12