Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Yeasts, like most organisms, have to survive in highly variable and hostile environments. Survival therefore requires adaptation to the changing external conditions. On the molecular level, specific adaptation to specific environmental conditions requires the yeast to be able: (i) to sense all relevant environmental parameters; (ii) to relay the perceived signals to the interior of the cell via signal transduction networks; and (iii) to implement a specific molecular response by modifying enzyme activities and by regulating transcription of the appropriate genes. The availability of nutrients is one of the major trophic factors for all unicellular organisms, including yeast. Saccharomyces cerevisiae senses the nutritional composition of the media and implements a specific developmental choice in response to the level of essential nutrients. In conditions in which ample nutrients are available, S. cerevisiae will divide mitotically and populate the growth environment. If the nutrients are exhausted, diploid S. cerevisiae cells can undergo meiosis, which produces four ascospores encased in an ascus. These ascospores are robust and provide the yeast with a means to survive adverse environmental conditions. The ascospores can lie dormant for extended periods of time until the onset of favourable growth conditions, upon which the spores will germinate, mate and give rise to a new yeast population. However, S. cerevisiae has a third developmental option, referred to as pseudohyphal and invasive growth. In growth conditions in which nutrients are limited, but not exhausted, the yeast can undergo a morphological switch, altering its budding pattern and forming chains of elongated cells that can penetrate the growth substrate to forage for nutrients. The focus of this study was on elements of the signal transduction networks regulating invasive growth in S. cerevisiae. Some components of the signal transduction pathways are well characterised, while several transcription factors that are regulated via these pathways remain poorly studied. In this study, the RMEt gene was identified for its ability to enhance starch degradation and invasive growth when present on a multiple copy plasmid. Rme1 p had previously been identified as a repressor of meiosis and, for this reason, the literature review focuses on the regulation of the meiotic process. In particular, the review focuses on the factors governing entry into meiosis in response to nutrient starvation and ploidy. Also, the transcriptional regulation of the master initiator of meiosis, IMEt, and the action of Ime1 p are included in the review. The experimental part of the study entailed a genetic analysis of the role of Rme1 p in invasive growth and starch metabolism. Epistasis analysis was conducted of Rme1 p and elements of the MAP Kinase module, as well as of the transcription factors, Mss11p, Msn1p/Mss10p, Tec1p, Phd1p and F108p. Rme1p is known to bind to the promoter of CLN2, a G1-cyclin, and enhances its expression. Therefore, the cell cyclins CLN1 and CLN2 were included in the study. The study revealed that Rme1 p functions independently or downstream of the MAP Kinase cascade and does not require Cln1 p or Cln2p to induce invasive growth. FL011/MUC1 encodes a cell wall protein that is required for invasive growth. Like the above-mentioned factors, Rme1 p requires FL011 to induce invasive growth. We identified an Rme1 p binding site in the promoter of FL011. Overexpression of Rme1p was able to induce FL01t expression, despite deletions of mss11, msn1, ttos, tee1 and phd1. In the inverse experiment, these factors were able to induce FL011 expression in an rme1 deleted strain. This would indicate that Rme1 p does not function in a hierarchical signalling system with these factors, but could function in a more general role to modify transcription. / AFRIKAANSE OPSOMMING: Die natuur is hoogs veranderlik en alle organismes, insluitende gis, moet by die omgewing kan aanpas om te kan oorleef. Baie eksterne faktore beïnvloed die ontwikkeling van die gissel. Vir die gis om by spesifieke omgewingstoestande aan te pas, moet die gis op 'n molekulêre vlak: (i) al die omgewingsparameters waarneem; (ii) die waargenome omgewingsparameters as seine na die selkern deur middel van seintransduksieweë gelei; en (iii) transkripsie van gene aktiveer of onderdruk en ensiemaktiwiteit reguleer om sodoende die gepaste molekulêre respons te implementeer. Die beskikbaarheid van voedingstowwe in die omgewing is een van die belangrikste omgewingseine wat eensellige organismes moet kan waarneem. Saccharomyces cerevisiae kan spesifieke ontwikkelingsopsies, na gelang van die voedingstowwe wat beskikbaar is, uitoefen. In groeiomstandighede waar daar 'n oorvloed van voedingstowwe is, verdeel S. cerevisiae d.m.v. mitose en vesprei dit deur die omgewing. Sodra die voedingstowwe uitgeput is, word mitose onderdruk. Diploïede S. cerevisiae inisieer meiose, wat aanleiding tot die vorming van vier spore gee. Hierdie spore bevat slegs die helfte van die ouer se chromosome en kan gevolglik met 'n ander spoor paar om weer 'n diploïede gissel te vorm. Die spore is bestand teen strawwe omgewingstoestande en kan vir lang tye oorleef. Wanneer die spoor aan gunstige groeitoestande blootgestel word, ontkiem dit om aan 'n nuwe giskolonie oorsprong te gee. S. cerevisiae het egter 'n derde ontwikkelingsopsie, naamlik pseudohife-differensiëring. Wanneer die beskikbaarheid van voedingstowwe in die omgewing afneem, maar nog nie uitgeput is nie, ondergaan die gis 'n morfologiese verandering. Hierdie verandering word gekenmerk deur selverlenging, nl. botselle wat slegs aan die een punt van die gissel vorm en dogterselle wat aan die moerderselle geheg bly. Dit lei tot die vorming van kettings van selle wat van die giskolonie af weggroei. Voorts kan die selkettings ook die groeisubstraat binnedring. Dit staan as penetrasie-groei bekend en laat die gis toe om na nuwe voedingsbronne te soek. Hierdie studie het op die elemente van seintransduksieweë, wat by penetrasiegroei betrokke is, gefokus. Sekere komponente van die seintransduksieweë is reeds goed gekarakteriseer, terwyl ander komponente nog grootliks onbekend is. In hierdie studie, word 'n rol vir RME1 in die verbetering van styselafbraak en penetrasiegroei geïdentifiseer. Aangesien Rme1 p voorheen as 'n onderdrukker van meiose geïdentifiseer is, is 'n litetaruurstudie oor die inisiasie van meiose saamgestel. Die faktore wat meiose induseer, naamlik 'n gebrek aan voedingstowwe en die sel se ploïedie, word bespreek. Die regulering van die meester inisieerder van meiosie, IME1, asook die proteïene waarmee Ime1p reageer, is ook in die studie ingesluit. Die eksperimentele deel van die studie behels die genetiese analise van Rme1 p tydens penetrasiegroei en styselhidroliese. 'n Epistase-analise tussen Rme1 p en elemente van die MAP-Kinasemodule, asook van die transkripsie faktore Mss11 p, Msn1p/Mss10p, Tec1p, Phd1p en F108p, is onderneem. Rme1p is bekend om aan die promotor van CLN2 te bind en transkripsie te induseer. Daarom is die selsikliene CLN1 en CLN2 in die studie ingesluit. Die studie dui daarop dat Rme1 ponafhanklik van die MAP-Kinasemodule funksioneer en nie Cln1 p en Cln2p benodig om penetrasiegroei te induseer nie. FL011/MUC1 kodeer vir 'n selwandproteïen wat noodsaaklik vir pentrasiegroei is. Soos in die geval van die bogenoemde faktore, benodig Rme1 p FL011 om penetrasiegroei te kan induseer. Ten spyte van mss11-, msn1-, ttos-, tec1- en phd1- delesies, kan ooruitdrukking van Rme1p die transkripsie van FL011 induseer. In die omgekeerde eksperiment kon die bogenoemde faktore FL011-transkripsie ten spyte van 'n rme1 delesie induseer. Die resultate dui daarop dat Rme1 p nie in 'n hiërargiese pad funksioneer nie, maar dat dit waarskynlik 'n meer algemene rol deur transkripsiemodifisering vervul.

O titânio comercialmente puro (Ti-Cp) e suas ligas destacam-se como biomateriais metálicos devido a sua biocompatibilidade sendo amplamente utilizados. Buscando aprimorar o sucesso clínico dos implantes de Ti-Cp em longo prazo, é necessário revestir a sua superfície, proporcionando uma eficiente ancoragem mecânica do implante com o tecido ósseo. Dentre os diversos revestimentos superficiais destacam-se os revestimentos híbridos orgânicos-inorgânicos à base de precursores alcóxidos de silício, obtidos via processo sol-gel. Para que ocorra uma satisfatória adesão do revestimento no substrato, precisa-se levar em consideração a natureza e a preparação da superfície metálica antes da aplicação deste revestimento. Na etapa inicial do trabalho, pré-tratamentos superficiais foram propostos previamente à aplicação dos revestimentos buscando aumentar a aderência entre o titânio e este revestimento híbrido. Utilizou-se três diferentes pré-tratamentos na superfície do Ti-Cp: o "piranha" (ácido sulfúrico e peróxido de hidrogênio), o "kroll" (ácido fluorídrico, ácido nítrico e água) e o "hidróxido de sódio". Em sequência, aplicou-se por processo de dip-coating sobre as superfícies tratadas, um revestimento híbrido produzido a partir dos precursores alcóxidos de silício tetraetoxisilano (TEOS) e metiltrietoxisilano (MTES), obtido pelo processo de sol-gel. Em uma segunda etapa, sobre a superfície do Ti-Cp com o pré-tratamento superficial que proporcionou uma maior aderência do revestimento híbrido ao titânio, aplicou-se por dip-coating, um revestimento híbrido à base de precursores alcóxidos de silício TEOS e MTES com adição de partículas de hidroxiapatita buscando aprimorar a diferenciação celular sobre o revestimento híbrido. O pré-tratamento com o hidróxido de sódio promove o melhor resultado dentre os pré-tratamentos, pois o revestimento híbrido aplicado posteriormente apresenta recobrimento regular e adesão ao substrato de Ti-Cp. Os resultados morfológicos por MEV-FEG mostraram um revestimento híbrido com boa dispersão da hidroxiapatita e um recobrimento regular e adesão ao substrato de Ti-Cp. Nos resultados biológicos Ti-Cp revestido com TEOS/MTES com a presença de partículas de hidroxiapatita obteve uma adesão celular semelhante ao Ti-Cp sem tratamento. Porém este mesmo revestimento não propiciou a proliferação e diferenciação celular. Os resultados indicaram que a combinação de fatores como a sua superfície hidrofóbica (91°) e a presença da hidroxiapatita no revestimento tornou a superfície desorganizada, acarretando em uma superfície com comportamento desfavorável para o desenvolvimento das células-tronco mesenquimais. / Commercially pure titanium (cp-Ti) and its alloys stand out among the metallic materials due to their biocompatibility being widely used in biomaterials. In order to improve the clinical success of cp-Ti implants in the long term, it is necessary to coat the surface, providing an efficient mechanical anchoring of the implant with the bone tissue. Among the various surface coatings are the hybrid coatings based on silicon alkoxide precursors, obtained by the sol-gel process, however, considering the nature and the preparation of the metal surface prior to the application of this coating. In the initial stage of the work, surface pre-treatments were proposed prior to the application of the coatings seeking to increase the adhesion between the titanium and this hybrid coating. Three different pretreatments were used on the cp-Ti surface: "piranha" (sulfuric acid and hydrogen peroxide), "kroll "(hydrofluoric acid, nitric acid and water) and "sodium hydroxide". Subsequently, a hybrid coating produced by the tetraethoxysilane silicon (TEOS) and methyltriethoxysilane (MTES) precursors obtained by the sol-gel process was applied by the dip-coating process onto the treated surfaces. In a second step, on the surface of the cp-Ti with the surface pretreatment that gave a greater adhesion of the hybrid coating to the titanium, dip-coating, a hybrid coating based on precursors silicon alkoxides TEOS and MTES with the addition of hydroxyapatite particles to enhance cell differentiation on the hybrid coating. Pretreatment with sodium hydroxide promotes the best result among the pre-treatments, since the hybrid coating applied afterwards presents regular coating and adhesion to the cp-Ti substrate. The morphological results by SEM-FEG showed a hybrid coating with good dispersion of the hydroxyapatite and a regular coating and adhesion to the cp-Ti substrate. In the biological results Ti-Cp coated with TEOS / MTES with the presence of hydroxyapatite particles obtained a cell adhesion similar to Ti-Cp without treatment. However, this same coating did not promote cell proliferation and differentiation. The results indicated that the combination of factors such as its hydrophobic surface (91°) and the presence of the hydroxyapatite encapsulated in the coating rendering the surface disorganized led to a surface with unfavorable behavior for the development of mesenchymal stem cells.

xi, 38 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / A critical question for developmental biology is the mechanism by which cells make fate decisions. In the hematopoietic system, stem cells differentiate into several different cell types, but the mechanisms that affect this process are incompletely known. Understanding these mechanisms is important because abnormal regulation of hematopoiesis can result in disease. STAT1 protein plays crucial roles in mediating innate immunity by transducing interferon signals, but recent results have also related STAT1 to hematopoietic cell differentiation. Here we cloned a previously uncharacterized zebrafish co-ortholog of the human STAT1 gene we call stat1b and investigated the functions of two zebrafish Stat1 proteins in hematopoiesis. The advantage of the zebrafish model is that, due to a whole genome duplication (WGD), some human genes have two co-orthologs in zebrafish. During evolution, co-orthologs have retained or acquired similar, complimentary, or new functions. Both stat1a and stat1b encode all four characteristic domains of the human STAT1 protein. Phylogenetic and conserved synteny analyses showed that stat1b and stat1a arose as duplicates in the teleost genome duplication event, and these analyses clarified the historical origin of the entire vertebrate STAT gene family. RT-PCR demonstrated maternal expression of both stat1a and stat1b . Expression of stat1b, but not stat1a, was detected in hematopoietic domains of embryos by in situ hybridization. Morpholino knockdown of stat1b , but not stat1a, mRNA expression resulted in a decrease in expression of the myeloid cell marker genes spi and mpx and an increase in expression of the hematopoietic progenitor marker gene scl and the erythrocyte marker gene gatal. These results show that in zebrafish, Stat1b protein functions in the commitment of hematopoietic cells to a myeloid cell fate. / Committee in charge: William Cresko, Chairperson, Biology; John Postlethwait, Advisor, Biology; Judith Eisen, Member, Biology; Jan Spitsbergen, Member, Not from U of O; J. Andrew Berglund, Outside Member, Chemistry

O titânio comercialmente puro (Ti-Cp) e suas ligas destacam-se como biomateriais metálicos devido a sua biocompatibilidade sendo amplamente utilizados. Buscando aprimorar o sucesso clínico dos implantes de Ti-Cp em longo prazo, é necessário revestir a sua superfície, proporcionando uma eficiente ancoragem mecânica do implante com o tecido ósseo. Dentre os diversos revestimentos superficiais destacam-se os revestimentos híbridos orgânicos-inorgânicos à base de precursores alcóxidos de silício, obtidos via processo sol-gel. Para que ocorra uma satisfatória adesão do revestimento no substrato, precisa-se levar em consideração a natureza e a preparação da superfície metálica antes da aplicação deste revestimento. Na etapa inicial do trabalho, pré-tratamentos superficiais foram propostos previamente à aplicação dos revestimentos buscando aumentar a aderência entre o titânio e este revestimento híbrido. Utilizou-se três diferentes pré-tratamentos na superfície do Ti-Cp: o "piranha" (ácido sulfúrico e peróxido de hidrogênio), o "kroll" (ácido fluorídrico, ácido nítrico e água) e o "hidróxido de sódio". Em sequência, aplicou-se por processo de dip-coating sobre as superfícies tratadas, um revestimento híbrido produzido a partir dos precursores alcóxidos de silício tetraetoxisilano (TEOS) e metiltrietoxisilano (MTES), obtido pelo processo de sol-gel. Em uma segunda etapa, sobre a superfície do Ti-Cp com o pré-tratamento superficial que proporcionou uma maior aderência do revestimento híbrido ao titânio, aplicou-se por dip-coating, um revestimento híbrido à base de precursores alcóxidos de silício TEOS e MTES com adição de partículas de hidroxiapatita buscando aprimorar a diferenciação celular sobre o revestimento híbrido. O pré-tratamento com o hidróxido de sódio promove o melhor resultado dentre os pré-tratamentos, pois o revestimento híbrido aplicado posteriormente apresenta recobrimento regular e adesão ao substrato de Ti-Cp. Os resultados morfológicos por MEV-FEG mostraram um revestimento híbrido com boa dispersão da hidroxiapatita e um recobrimento regular e adesão ao substrato de Ti-Cp. Nos resultados biológicos Ti-Cp revestido com TEOS/MTES com a presença de partículas de hidroxiapatita obteve uma adesão celular semelhante ao Ti-Cp sem tratamento. Porém este mesmo revestimento não propiciou a proliferação e diferenciação celular. Os resultados indicaram que a combinação de fatores como a sua superfície hidrofóbica (91°) e a presença da hidroxiapatita no revestimento tornou a superfície desorganizada, acarretando em uma superfície com comportamento desfavorável para o desenvolvimento das células-tronco mesenquimais. / Commercially pure titanium (cp-Ti) and its alloys stand out among the metallic materials due to their biocompatibility being widely used in biomaterials. In order to improve the clinical success of cp-Ti implants in the long term, it is necessary to coat the surface, providing an efficient mechanical anchoring of the implant with the bone tissue. Among the various surface coatings are the hybrid coatings based on silicon alkoxide precursors, obtained by the sol-gel process, however, considering the nature and the preparation of the metal surface prior to the application of this coating. In the initial stage of the work, surface pre-treatments were proposed prior to the application of the coatings seeking to increase the adhesion between the titanium and this hybrid coating. Three different pretreatments were used on the cp-Ti surface: "piranha" (sulfuric acid and hydrogen peroxide), "kroll "(hydrofluoric acid, nitric acid and water) and "sodium hydroxide". Subsequently, a hybrid coating produced by the tetraethoxysilane silicon (TEOS) and methyltriethoxysilane (MTES) precursors obtained by the sol-gel process was applied by the dip-coating process onto the treated surfaces. In a second step, on the surface of the cp-Ti with the surface pretreatment that gave a greater adhesion of the hybrid coating to the titanium, dip-coating, a hybrid coating based on precursors silicon alkoxides TEOS and MTES with the addition of hydroxyapatite particles to enhance cell differentiation on the hybrid coating. Pretreatment with sodium hydroxide promotes the best result among the pre-treatments, since the hybrid coating applied afterwards presents regular coating and adhesion to the cp-Ti substrate. The morphological results by SEM-FEG showed a hybrid coating with good dispersion of the hydroxyapatite and a regular coating and adhesion to the cp-Ti substrate. In the biological results Ti-Cp coated with TEOS / MTES with the presence of hydroxyapatite particles obtained a cell adhesion similar to Ti-Cp without treatment. However, this same coating did not promote cell proliferation and differentiation. The results indicated that the combination of factors such as its hydrophobic surface (91°) and the presence of the hydroxyapatite encapsulated in the coating rendering the surface disorganized led to a surface with unfavorable behavior for the development of mesenchymal stem cells.

Orientador: Prof. Dr. Daniel Carneiro Carrettiero / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2015. / A doenca de Alzheimer (AD) e a mais diagnosticada de todas as demencias e afeta aproximadamente 12,5% das pessoas com idade superior a 65 anos. A doenca piora progressivamente, causando um alarmante declinio na funcao cognitiva e eventualmente a morte. Duas caracteristicas histopatologicas da doenca sao presenca das placas do peptideo beta-amiloide (AB) e emaranhados neurofibrilares da proteina tau hyperphosphorilada. A hipotese amiloide da AD propoe que o beta-amiloide (AB) induz a fosforilacao da tau, resultando na formacao de agregados toxicos, perda da funcao neuronal e morte neuronal. Curiosamente, em neuronios nao-diferenciados AB e neuroprotetora em vez de neurotoxica. Em neuronios diferenciados, a neurotoxicidade de AB e dependente da expressao da proteina tau. A co-chaperona BAG2 foi identificada como capaz de estimular a degradacao da proteina tau fosforilada, sugerindo que pode reverter a eventual agregacao de tau hiperfosforilada citotoxica. A relevancia da BAG2 nos processos neurotoxicos induzidos pela AB, dentro de um contexto de maturac.o neuronal, nao foi anteriormente pesquisada e representa uma interessante hipotese no quanto possa ajudar a elucidar os mecanismos celular-moleculares responsaveis pela patologia da doenca de Alzheimer. O objetivo deste estudo foi caracterizar a funcao e regulacao de BAG2 no contexto da morte celular induzida por AB em neuronios nao-diferenciados e diferenciados. A expressao de BAG2 em celulas SH-SY5Y foi analisada sob condicoes de nao-diferenciacao e diferenciacao. O BAG2 foi superexpressada em celulas SH-SY5Y nao-diferenciadas, que foram depois tratadas 15 com o peptido AB. A morte celular foi avaliada por exclusao de azul de tripano. A regiao do promotor de BAG2 foi analisada para avaliar a frequencia e distribuicao de sitios-alvo de varios fatores de transcricao para identificar fatores que regulam a expressao do BAG2. Os niveis de expressao de BAG2 foram determinados utilizando RT-PCR. Nos mostramos que a expressao de BAG2 aumenta com a diferenciacao das celulas SH-SY5Y. AB e neurotrofico para as celulas SH-SY5Y nao-diferenciadas, porem e neurotoxica de celulas diferenciadas. A superexpressao da co-chaperona BAG2 foi suficiente para causar uma mudanca na sinalizacao de AB de neurotrofico para neurotoxico em celulas nao-diferenciadas. A superexpressao de BAG2, na ausencia de AB, nao teve efeito na morte celular ou no grau de diferenciacao em celulas SH-SY5Y nao-diferenciadas. Em seguida analisamos a regiao do promotor de BAG2 e descobrimos uma prevalencia de sitios do NF .kB, os quais co-localizavam com o sitio de iniciacao da transcricao do BAG2. O tratamento das celulas, tanto com ativadores e inibidor do NF-kB, revelou que o BAG2 e reprimido pela atividade do NF- kB. Em conjunto, estes dados sugerem que a ausencia de BAG2 em celulas nao-diferenciadas e suficiente para lhes conferir resistencia a morte induzida por AB. Assim, sugerimos que a inducao da expressao de BAG2 atraves da interrupcao ou alteracao de sinalizacao de NF-kB pode criar um ambiente celular em neuronios maduros que e sensivel, em vez de resistente, a morte celular induzida por AB. / Alzheimer¿s disease (AD) remains the most-diagnosed of all dementias, affecting approximately 12.5% of persons over 65 years of age. The disease progressively worsens causing an alarming decline in cognitive function, often culminating in death. Two histopathological hallmarks of Alzheimer¿s disease are beta-amyloid (AB) peptide plaques and neurofibrillary tangles of hyperphosphorylated tau protein. According to the amyloid hypothesis of AD, AB induces tau phosphorylation which results in the formation of toxic protein aggregates, loss of neuron function, and neuronal death. Interestingly, AB has been shown to be neurotrophic rather than neurotoxic to neural precursors in animal and cell culture models, and the neurotoxicity of AB in differentiated neurons has been found to be dependent upon tau protein expression. The co-chaperone BAG2 has recently been shown to stimulate the degradation of accumulated phosphorylated tau protein, suggesting that it may abrogate aggregation of tau into cytotoxic neurofibrillary tangles. The relevance of BAG2 to AB-induced neurotoxicity within the context of neuron maturation is not presently understood, and may help to further elucidate the cell-molecular mechanisms responsible for Alzheimer¿s disease pathology. The aim of this study was to characterize the function and regulation of BAG2 within AB-induced cell death in undifferentiated and differentiated neurons. BAG2 expression in SH-SY5Y cells was analyzed under undifferentiated and differentiated conditions. BAG2 was overexpressed in undifferentiated SH-SY5Y cells, which were then treated with AB peptide. 13 Cell death was evaluated by Trypan blue exclusion. The BAG2 promoter region was analyzed for the frequency and distribution of various transcription factor regulatory motifs to identify BAG2-regulating factors. BAG2 expression levels were determined using RT-PCR. We show that BAG2 expression increases on differentiation of SH-SY5Y cells. AB is neurotrophic to undifferentiated SH-SY5Y cells, while it is neurotoxic to differentiated cells. Overexpression of the co-chaperone BAG2 was sufficient to cause a switch in AB signaling from neurotrophic to neurotoxic in undifferentiated cells. BAG2 overexpression, in the absence of AB, had no effect on cell death or degree of differentiation in undifferentiated SH-SY5Y cells. We next analyzed to putative BAG2 promoter region and discovered a preponderance of NF-kB binding motifs that co-localized to the BAG2 transcription start site. Treatment of cells with NF-kB activators and inhibitor reagents revealed that BAG2 is repressed by NF-kB signaling. Together, these data suggest that the absence of BAG2 in undifferentiated cells is sufficient to render them refractive to AB-induced death. We thus suggest that the expression of BAG2 expression via the disruption or modification of NF-kB signaling may create a cellular environment in mature neurons that is sensitive rather than resistant to AB-induced cell death.

There exists a need to produce platelets in vitro for use in transfusion medicine, due to increased platelet demands and short shelf life. Our lab uses human induced pluripotent stem cells (iPSCs), as an attractive alternative supply, as iPSCs can be cultured indefinitely and differentiate into almost any cell type. Using a technique called forward programming, we over express three key haematological transcription factors (TFs), pushing iPSCs towards the megakaryocyte lineage, to produce mature megakaryocytes, the platelet precursor cell type. A major limitation of the forward programming technique is a reliance of lentiviral transduction to overexpress the three TFs, which leads to a number of issues including heterogeneity and high experimental costs. To overcome this, I have developed an inducible iPSC line by inserting the forward programming TFs into a genomic safe harbour, using genome editing techniques. TF expression is strictly controlled, with the TFs expressed only after chemical induction. Inducing forward programming is an efficient method for producing mature megakaryocytes and these cells maintain higher purity in long-term cultures, when compared to cells produced by the lentiviral method. Removing the requirement of lentiviral transduction is a major advancement, making forward programming more amenable to scaling-up, thus moving this technology closer towards our goal of producing in vitro platelets for use in transfusion medicine. I have also shown that forward programming generates a bi-potent progenitor population, from which erythroblasts can be generated, by altering only media conditions. As for megakaryocyte cultures, inducing forward programming improves the purity of erythroblasts produced, compared to the lentiviral method. I have developed single cell progenitor assays combined with index sorting of different cell surface markers, to allow retrospective analysis of cells which successfully generate colonies. The aim of this work is to better characterise the progenitor cells produced by forward programming, to allow further study of this cell type. Single cell RNA-seq of megakaryocytes revealed heterogeneity in long-term cultures and also identified novel candidate surface markers that may help to further characterise the progenitor cell population.

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-09-19T13:23:59Z No. of bitstreams: 1 DISS_2014_Heron Fernandes Vieira Torquato.pdf: 2253066 bytes, checksum: 04567ef3aaa1fd3c33944fec7ac526d6 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-09-26T12:32:53Z (GMT) No. of bitstreams: 1 DISS_2014_Heron Fernandes Vieira Torquato.pdf: 2253066 bytes, checksum: 04567ef3aaa1fd3c33944fec7ac526d6 (MD5) / Made available in DSpace on 2017-09-26T12:32:54Z (GMT). No. of bitstreams: 1 DISS_2014_Heron Fernandes Vieira Torquato.pdf: 2253066 bytes, checksum: 04567ef3aaa1fd3c33944fec7ac526d6 (MD5) Previous issue date: 2014-03-24 / CAPES / Torquato, H.F.V. Avaliação da atividade e do mecanismo de ação antileucêmica da cantinona. 2014. 73 f. Dissertação apresentada à Coordenação de Programas de Pós-Graduação em Ciências da Saúde da Faculdade de Medicina da Universidade Federal de Mato Grosso, como requisito parcial para a obtenção do título de Mestre em Ciências da Saúde, Área de Farmacologia. A leucemia mielóide aguda (LMA) é uma doença maligna de natureza proliferativa, em que as células leucêmicas não terminam o processo de diferenciação celular. Na terapia padrão da LMA é necessária intensa quimioterapia, a qual é acompanhada de baixas taxas de remissão, por longos períodos e toxicidade. Portanto, novas opções terapêuticas para o tratamento da leucemia são necessárias. A cantinona (Cant) representa uma subclasse de alcaloides beta-carbonílicos, isolada de plantas, especialmente das famílias Simaroubaceae e Rutaceae. São referidas atividades antiulcerogênica, antibacteriana e antifúngica para a Cant, e atividade citotóxica, antimalárica e antiviral para alguns análogos sintéticos. O objetivo desse trabalho foi investigar o potencial antitumoral e mecanismo de ação da Cant em linhagens celulares de LMA. Para tanto, a atividade citotóxica foi realizada nas linhagens Kasumi-1 e KG-1 por meio da marcação com anexina V e iodeto de propídeo após tratamento com Cant (28; 56; 113 e 227 μM) por 24 h. Verificou-se a ação do alcaloide sobre diferentes parâmetros celulares em células Kasumi-1 (45 μM), tais como integridade das membranas lisossomais e mitocondriais, ativação de caspases 3, 8 e 9, expressão de importantes proteínas relacionadas à sobrevivência das células (ERK 1/2 e Bcl-2). Adicionalmente, foi avaliada a capacidade da Cant (14 μM) em induzir a diferenciação mielóide em ambas linhagens celulares. Os resultados mostraram que a Cant apresentou potencial atividade antitumoral com CI50 de 38,9±1 μM para Kasumi-1 e 39,1±1 μM para KG-1. A Cant promoveu permeabilização lisossomal demonstrada pelo extravasamento do corante laranja de acridina dos lisossomos para o citosol e produziu queda em 54,6% (p<0,001) no potencial de membrana mitocondrial. A ativação das caspases 3 e 8 foram seguidas pelo aumento na fluorescência na ordem de 2,4 (p<0,01) e 2,5 (p<0,05) vezes, respectivamente, comparadas as células que não receberam tratamento e a caspase 9 (p<0,01) elevou o sinal em 2,1 vezes. A expressão proteica de ERK 1/2 e Bcl-2 foram reduzidas, sendo mais expressiva para 45 μM de Cant (43%, p< 0,01) no caso de ERK 1/2 e com 135 μM (53%, p< 0,01) para Bcl-2. Na diferenciação celular, verificou-se aumento na expressão de CD15+ e CD 11b+, na ordem de 2,5 vezes (p<0,001), nas células Kasumi-1 tratadas com 14 μM de Cant, quando comparado às células não tratadas. Contudo, em células tronco leucêmicas (CD34+CD38-Lin) houve redução na expressão destas em aproximadamente 7 vezes (p<0,001), em relação às células sem tratamento (controle). Em KG-1 tratadas com 14 μM de Cant, a expressão de CD15+ foi elevada na ordem de 2,5 vezes (p<0,001), porém nenhuma diferença foi observado para CD11b+, enquanto a expressão de células tronco leucêmicas dobrou (p<0,001). Esses dados em conjunto demonstram que a Cant apresenta atividade antitumoral em células LMA, sendo a morte celular mediada pelas ativações das vias extrínseca e intrínseca da apoptose, possivelmente, com maior contribuição da via mitocondrial, com participação importante dos lisossomos neste mecanismo. Em adição aos seus efeitos sobre a proliferação de células leucêmicas, Cant apresentou importante ação na diferenciação celular, o que a credencia como importante candidato a fármaco antitumoral na LMA, uma vez que a doença é passível de sofrer terapias baseadas na diferenciação, o que atualmente fornece melhor prognóstico ao paciente. / Torquato, H.F.V. Evaluation of the activity and mechanism of action antileukemic of canthinone. 2014. 73 f. Dissertation submitted to the Health Science Post Graduate Programs Coordination of the School of Medicine of the Federal University of Mato Grosso, as a partial requirement for the degree of Master in Health Sciences. Pharmacology Area. Acute myeloid leukemia (AML) is a malignant proliferative disease, where the leukemic cells do not complete the cell differentiation process. The standard therapy of AML includes intensive chemotherapy despite of its association with a low rate of remission afterward, it is also has been considered as long-term treatment with a significant toxicity. Therefore, a development of new therapeutic approaches might impact on treatment of AML. The canthinone (Cant) is a beta-carbonyl subclass of alkaloids isolated from plants, specifically Simaroubaceae and Rutaceae families. Certain activities such as antiulcer, antibacterial and antifungal were referred to Cant, whereas other activities [cytotoxic, antimalarial and antiviral] referred to some forms of its synthetic analogues. The aim of this study is to investigate the antitumor potential of Cant in AML cell lines. For this, the cytotoxic activity was performed in the lines Kasumi-1 and KG-1 marked by annexin V and PI after treatment with Cant (28, 56, 113 and 227 μM) for 24 h., where the action of its antitumor mechanism it action was investigated in Kasumi-1 cells (45 μM). The action of the alkaloid on different cell parameters was evaluated, such as the integrity of lysosomal and mitochondrial membranes, caspases 3, 8 and 9 activation, expression of important proteins related to cell survival (ERK1/2 and Bcl-2). In addition, we evaluated the ability of Cant (14 μM) to induce myeloid differentiation in both cell lines. The results showed that Cant has a potential antitumor activity with IC50 of 38.9 ± 1 μM for Kasumi-1 and 39.1±1 μM for KG-1. As well as a lysosomal permeabilization effect demonstrated by leakage of acridine orange dye from the lysosomes to cytosol, causing a drop of 54.6% (p< 0.001) in the mitochondrial membrane potential. The activation of caspases 3 and 8 were followed by an increase in fluorescence at 2.4 (p< 0.01) and 2.5 (p< 0.05) times respectively, compared to untreated cells, and caspase 9 (p< 0.01) increased 2.1 times the signal. The protein expression of ERK 1/2 and Bcl-2 was reduced, more expressively from 45 μM of Cant (43%, p<0.01) on ERK 1/2 and 135 μM of Cant (53 %, p<0.01) on Bcl-2. In the evaluation of cell differentiation, an increase of 2.5 times (p<0.001) noticed with the expression of CD15+ and CD 11b+ in Kasumi-1 cells treated with Cant (14 μM) compared to untreated cells. However, the results shows a decrease in the expression of leukemic stem cell markers (CD34+ CD38-Lin-) with approximately 7-fold (p <0.001) in comparison with control. In KG-1 cell line treated with Cant (14 μM), CD15+ expression rose up to 2.5 times (p<0.001). The expression of leukemic stem cells markers was doubled (p<0.001) with no difference in CD11b+. All these data has been shown, that Cant has antitumor activity in AML cells via cell death mechanism through activation of the extrinsic and intrinsic pathways of apoptosis, possibly with a greater contribution of the mitochondrial pathway with a significant lysosomal role. In addition to its effects on the proliferation of leukemic cells, Cant showed a significant action on cell differentiation, which qualifies it as a candidate for antitumor drug for AML treatment, where the disease therapy can be based on differentiation.

Orientador: Andreia Bufalino / Resumo: O carcinoma espinocelular (CEC) é a sexta malignidade mais comum do mundo, e na região de cabeça e pescoço representa mais de 90% das malignidades. Mesmo com os recentes avanços nos protocolos de tratamento cirúrgico, radioterápico e quimioterápico, a taxa de sobrevida de 5 anos para pacientes com carcinoma espinocelular de cabeça e pescoço (CECCP) permanece reduzida. Diversos fatores podem contribuir para esse baixo índice de sobrevida e, somado a isso, ainda não existem marcadores biológicos que possam contribuir na orientação da melhor opção terapêutica dos pacientes e na previsão do seu diagnóstico. Estudos desta natureza representam uma valiosa oportunidade para esclarecer os mecanismos moleculares envolvidos na patogênese do CECCP. Dentre estes eventos moleculares, o processo da diferenciação celular é capaz de regular a expressão de genes ligados a importantes funções celulares, incluindo o controle da proliferação celular. Neste contexto, o objetivo deste estudo foi avaliar a relação de marcadores de proliferação e diferenciação celular com parâmetros clínico-patológicos de amostras de CECCP por meio da imuno-histoquimica (IQ). Diante disto, nosso estudo avaliou um total de 46 amostras de CECCP. Quanto a expressão dos marcadores de proliferação celular, avaliamos Ki-67 (n=46) e Ciclina D1 (n=46). Para os marcadores de diferenciação celular, avaliamos CD10 (n=42) e Vimentina (n=41). A expressão aumentada de Ki- 67 foi significantemente associada com pior prognóstico (p... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Squamous cell carcinoma (SCC) is the sixth most common malignancy in the world, and in the head and neck region it represents more than 90% of malignancies. Even with recent advances in surgical, radiotherapeutic and chemotherapeutic treatment protocols, the 5-year survival rate for patients with squamous cell carcinoma of the head and neck (HNSCC) remains low. Several factors may contribute to this low survival rate and, in addition, there are still no biological markers that can contribute to the orientation of the best therapeutic option of the patients and the prediction of their diagnosis. Studies of this nature represent a valuable opportunity to clarify the molecular mechanisms involved in the pathogenesis of HNSCC. Among these molecular events, the process of cell differentiation is able to regulate the expression of genes linked to important cellular functions, including the control of cell proliferation. In this context, the objective of this study was to evaluate the relationship of proliferation and cellular differentiation markers with clinical-pathological parameters of HNSCC samples by means of immunohistochemistry (IQ). In view of this, our study evaluated a total of 46 HNSCC samples. Regarding the expression of the cellular proliferation markers, we evaluated Ki-67 (n = 46) and Cyclin D1 (n = 46). For cell differentiation markers, we evaluated CD10 (n = 42) and Vimentin (n = 41). Increased Ki-67 expression was significantly associated with poorer prognosis (p... (Complete abstract click electronic access below) / Mestre

O Diabetes Mellitus tipo I (DM1) é causado pela destruição auto-imune das células &#946; pancreáticas, encontradas na porção endócrina do pâncreas, constituída pelas ilhotas pancreáticas. As células &#946; são responsáveis pela produção e liberação de insulina, um hormônio que promove a internalização da glicose pelas células. Junto com outros hormônios, a insulina é um dos principais reguladores do nível de glicose sanguinea (glicemia). Uma das terapias utilizadas para o tratamento do DM1 é o transplante de ilhotas pancreáticas. Entretanto, um dos maiores problemas em relação a esta terapia é a falta de massa celular adequada para ser infundida no paciente. Uma tentativa para solucionar este problema, é o desenvolvimento de fontes alternativas de células produtoras de insulina, como as células-tronco, que possuem a capacidade de se diferenciarem em diversos tipos de células, inclusive nas produtoras de insulina. Pax4 é um dos fatores de transcrição responsáveis pela diferenciação de células &#946; , sendo essencial para o apropriado desenvolvimento e maturação destas, constitui um bom candidato para induzir a diferenciação de células-tronco em células produtoras de insulina in vitro. Para introduzir o Pax4 nas células-tronco, sem provocar alterações no genoma das células diferenciadas, em virtude dos potenciais efeitos indesejáveis de vetores que se integram ao genoma celular, recorreu-se às proteínas contendo domínio de transdução (PTDs), que são capazes de carregar a proteína Pax4, através da membrana, diretamente para o interior das células. As PTDs são pequenas sequências peptídicas que permitem a translocação de proteínas através de membranas celulares e sua internalização em células-alvo. Uma das PTDs mais comumente estudadas é a VP22, produto do gene UL49 do Herpes Simplex vírus tipo I. Portanto, a proteína de fusão VP22.Pax4 permitiria que o Pax4 fosse inserido em células-tronco, possibilitando que este fator de transcrição ative a transcrição de certos genes que aumentariam a eficiência de diferenciação das células-tronco em células produtoras de insulina. Para tal, amplificamos e clonamos o cDNA do Pax4 a partir do RNA das células RINm5f de insulinoma murino, construímos o vetor pVP22.Pax4, o qual foi transfectado em células CHO, que passaram a produzir a proteína de fusão VP22.Pax4. Após o tratamento de células-tronco com a proteína de fusão VP22.eGFP e análise por microscopia confocal, comprovamos que a VP22 é capaz de tranduzir a proteína de fusão também neste tipo celular. Portanto, incorporamos a um dos passos do protocolo de diferenciação de células-tronco em células produtoras de insulina, utilizado em nosso laboratório, a co-cultura com células CHO produtoras de VP22.Pax4. Observamos que a introdução do Pax4 leva a formação de um número maior de agregados celulares (clusters) produtores de insulina. Concluímos, então, que a utilização da VP22 como ferramenta para internalização de proteínas em células-tronco é viável e que a adição do Pax4 pode trazer melhorias para protocolos que busquem a produção de células produtoras de insulina. / Diabetes Mellitus type 1 (DM1) is caused by an auto-imunne destruction of the pancreatic &#946; cells, found in the endocrine portion of the pancreas, known as pancreatic islets. These &#946; cells are responsible production and release of insulin, a hormone which promotes glucose internalization by cells. Along with other hormones, insulin is a major regulator of blood glucose levels (glycemia). One of the therapeutical strategies used to treat DM1 is pancreatic islet transplantation. One of the major problem related to this therapy is the lack of adequate cell mass to be infused into the pacients. An attempt to solve this problem is the development of an alternative source of insulin-producing cells by differentiation of stem cells, which display this differentiating potential. Pax4 is one of the transcription factors responsibles for &#946; cell differentiation, being essential for its proper development and maturation, therefore being a good candidate to induce stem cell differentiation into insulin producing cells in vitro. A promising alternative to avoid the alterations of the differentiated cells genome due to its undesirable effects of integrating vectors, but yet allowing the Pax4 to act in diferentiation within the cells are the proteins with a transduction domain (PTDs), which would have the ability to lead the Pax4 protein directly into the cells. The Pax4 could thus act in the nucleus and generate specific transcriptional responses. The PTDs are small peptide sequences which allow translocation of proteins across cell membranes and their internalization into target cells. One of the most commonly studied PTDs is the VP22, a product of the UL49 gene from Herpes Simplex vírus type I. Therefore, the VP22.Pax4 fusion protein would transduce Pax4 into the stem cells, thus allowing the transcription activation of certain genes by Pax4, leading to improvement in the process of stem cells differentiation into insulin-producing cells. To this end, we cloned the Pax4 cDNA from RINm5f murine insulinoma cells, constructed the pVP22.Pax4 vector and transfected this construct into CHO cells, which then produced the VP22.Pax4 fusion protein. Upon verifying that VP22 was also able to transduce proteins into stem cells, by confocal microscopy analysis, after the treatment of these cells with the fusion protein VP22.eGFP, we incorporated the fusion protein VP22.Pax4 to one of the steps of the protocol used for stem cells diferentiation into insulin producing cells in our lab, by co-culturing with CHO cells producing VP22.Pax4. We observed that the addition of Pax4 led to the formation of a higher number of insulin producing cell clusters, therefore we conclude that VP22 may be used as a tool to internalize proteins into stem cells, and that the addition of Pax4 may improves protocols seeking the production of insulin-producing cell

O objetivo deste trabalho foi avaliar a influência das características química e de rugosidade da superfície de titânio sobre a adesão, proliferação e diferenciação de células semelhantes aos osteoblastos de rato (Osteo-1), cultivados em meio de cultura adicionado de BMP-7. MATERIAL E MÉTODO: Células Osteo-1 foram cultivadas sobre discos de titânio com superfícies:1) lisa, 2) jateada por areia de grânulos grandes e atacada por ácido (SLA) e 3) rugosa SLA e quimicamente modificada e hidrofílica (SLAactive) na presença ou ausência de 20ng/ml de rhBMP- 7 no meio de cultura. A adesão e viabilidade das células Osteo-1 foram analisadas após 24 horas de contato com as superfícies em estudo. A diferenciação celular foi avaliada através da análise do conteúdo de proteína total (PT), conteúdo de colágeno, atividade de fosfatase alcalina (ALPase), em 7, 14 e 21 dias, e da formação de matriz mineralizada, em 21 dias. Os resultados foram comparados pela análise de variância (ANOVA) e teste de Tukey. RESULTADOS: A adesão (p=0.3485) e a viabilidade (p=0.5516) celular, o conteúdo de colágeno (p=0.1165) e a formação de matriz mineralizada (p=0.5319) não foram afetados pelas diferentes superfícies ou pela adição de rhBMP-7 ao meio. Células Osteo-1 cultivadas sobre superfície SLA apresentaram um aumento significativo no conteúdo de proteína total aos 21 dias. A relação atividade de ALPase/PT (p=0.0000) foi afetada pelos tratamento e tempo. CONCLUSÃO: Os resultados sugerem que a adição de rhBMP- 7 ao meio de cultura não promoveu efeito sobre a adesão, proliferação e diferenciação de células semelhantes a osteoblastos nas diferentes superfícies testadas. Todas as superfícies de titânio testadas permitiram uma completa expressão do fenótipo de osteoblasto como a mineralização da matriz pela célula Osteo-1. / The aim of the present study was to assess the influence of the chemical characteristics and roughness of titanium surfaces on the attachment, proliferation and differentiation of osteoblast-like cells cultured in medium supplemented with bone morphogenetic protein-7 (BMP-7). METHODS: Osteo-1 cells were grown on titanium discs presenting the following surfaces: 1) machined surface, 2) coarse gritblasted and acid-etched (SLA), and 3) modified SLA (SLAactive) in the absence or presence of 20 ng/ml rhBMP-7 in culture medium. The attachment and viability of osteo-1 cells were evaluated after 24 h. Cell differentiation was evaluated by analysis of total protein content (TP), collagen content and alkaline phosphatase (ALPase) activity at 7, 14 and 21 days and of mineralized matrix formation at 21 days. The results were compared by analysis of variance (ANOVA) and Tukey\'s test. RESULTS: Cell attachment (p=0.3485), cell viability (p=0.5516), collagen content (p=0.1165) and mineralized matrix formation (p=0.5319) were not affected by the different surfaces or by the addition of rhBMP-7 to the medium. Osteo-1 cells cultured on SLA surface presented a significant increase in TP at 21 days. The ALPase/TP ratio (p=0.0000) was affected by treatment and time. CONCLUSION: The results suggest that the addition of rhBMP-7 to the culture medium did not promote any effect on the adhesion, proliferation or differentiation of osteoblast-like cells grown on the different surfaces tested. All titanium surfaces analyzed permitted the complete expression of the osteoblast phenotype such as matrix mineralization by osteo-1 cells.