Student Number : 0002482E - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Proteins in the glutathione transferase family share a common fold. The close packing of secondary structures in the thioredoxin fold in domain 1 forms a compact hydrophobic core. This fold has a bababba topology and most proteins/domains with this fold have a topologically conserved isoleucine residue at the N-terminus of a-helix 3. Class Alpha glutathione transferases are one of 12 classes within the glutathione transferase family. To investigate the role of the conserved isoleucine residue in the structure, function and stability of glutathione transferases, homodimeric human glutathione transferase A1-1 (hGST A1-1) was used as a representative of the GST family. Ile71 was replaced with valine and the properties of I71V hGST A1-1 were compared with those of wildtype hGST A1-1. The spectral properties monitored using far-UV CD and tryptophan fluorescence indicated little change in secondary or tertiary structure confirming the absence of any gross structural changes in hGST A1-1 due to the incorporation of the mutation. Both wildtype and mutant dimeric proteins were determined to have a monomeric molecular mass of 26 kDa. The specific activity of I71V hGST A1-1 (130 mmol/min/mg) was three times that of wildtype hGST A1-1 (48 mmol/min/mg). I71V hGST A1-1 showed increased kinetic parameters compared to wildtype with a 10-fold increase in kcat/Km for CDNB. The increase in Km of I71V hGST A1-1 suggests the mutation had a negative effect on substrate binding. The DDG for transition state stabilisation was –5.82 kJ/mol which suggest the I71V mutation helps stabilise the transition state of the SNAR reaction involving the conjugation of reduced glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). A 2-fold increase in the IC50 value for I71V hGST A1-1 (11.3 mM) compared to wildtype (5.4 mM) suggests that the most noticeable change due to the mutation occurs at the H-site of the active site. Conformational stability studies were performed to determine the contribution of Ile71 to protein stability. The non-superimposability of I71V hGST A1-1 unfolding curves and the decreased m-value suggest the formation of an intermediate state. The conformational stability of I71V hGST A1-1 (16.5 kcal/mol) was reduced when compared to that of the wildtype (23 kcal/mol). ITC was used to dissect the binding energetics of Shexylglutathione to wildtype and I71V hGSTA1-1. The ligand binds 5-fold more tightly to wildtype hGST A1-1 (0.07 mM) than I71V hGST A1-1 (0.37 mM). The I71V mutant displays a larger negative DCp than wildtype hGST A1-1 (DDCp = -0.41 kJ/mol/K). This indicates that a larger solvent-exposed hydrophobic surface area is buried for I71V hGST A1-1 than for wildtype hGST A1-1 upon the binding of S-hexylglutathione. Overall the results suggest that Ile71 conservation is for the stability of the protein as well as playing a pivotal indirect role in catalysis and substrate binding.

Dengue é uma doença viral que atinge vários países, infectando milhares de pessoas anualmente. Sua transmissão ocorre pelo repasto sanguíneo feito pelas fêmeas dos mosquitos Aedes sp. infectadas e seus sintomas tem um amplo espectro de variação, que vão desde quadros assintomáticos até quadros graves com hipovolemia, podendo levar a óbito. A necessidade de adaptação do vírus a sistemas tão distintos (inseto e mamífero) restringe o espectro de variabilidade que o mesmo pode adquirir, permitindo que o vírus se adapte a ambos os hospedeiros com eficiência replicativa semelhante. Estudos com outros flavivírus já mostraram que a aquisição de variabilidade ocorre de maneiras distintas no hospedeiro mamífero e no inseto vetor, permitindo ao vírus máxima adaptação nas células infectadas. Levando em consideração estes estudos, este trabalho investigou a adaptação de duas cepas do vírus da dengue, a ACS-46, não neurovirulenta para camundongos adultos e a cepa JHA, neurovirulenta para camundongos adultos. O estudo foi feito utilizando cultura de células de mosquito (C6/36), cultura de células de mamífero (HepG2) e infecção em cérebro de camundongos neonatos. Utilizando sequenciamento de nova geração, foi possível verificar as mutações adquiridas ao longo de passagens em cada sistema de cultura. Foram realizadas 20 passagens seriadas em célula C6/36 e seis passagens alternadas entre C6/36 e HepG2. Os vírus obtidos na décima sexta e vigésima passagens do modelo seriado e na quarta e sexta passagens do modelo alternado foram completamente sequenciados utilizando a plataforma de sequenciamento em larga escala Ion Torrent. A análise das sequências revelou duas populações virais claramente distintas, já observadas a partir de décima sexta passagem, identificadas através da deleção de dois códons no sítio de glicosilação do domínio I da proteína de Envelope. Essa deleção induziu aumento na fusão do vírus à célula de mosquito, visto que a partir dessa passagem houve aumento no efeito citopático do vírus e na carga viral produzida durante infecções em células de mosquito. Contudo, essa mutação teve um perfil deletério em células humanas, fato deduzido pelo total desaparecimento da população com a deleção durante as passagens alternadas. Além desta deleção, foram detectadas sete mutações não sinônimas em região de proteínas não estruturais com porcentagens muito próximas, sugerindo que as mesmas fazem parte de uma mesma população. A cepa JHA teve seu caráter neurovirulento perdido após 29 passagens em C6/36, porém rapidamente recuperado após uma única passagem em cérebro de camundongo neonato. O sequenciamento dos vírus dessas passagens mostrou certa variabilidade em sítios distintos entre as variantes do vírus parental, vírus não neurovirulento e vírus neurovirulento passado em camundongo. Esse resultado indicou que aparentemente, não há sítios específicos determinantes de neurovirulência em DENV2, ao menos no nosso modelo, mas sim, um conjunto de mutações podem levar ao fenótipo neurovirulento, a depender das condições as quais o vírus é exposto. / Dengue is a viral disease that affects several countries, infecting thousands of people annually. It is transmitted by blood meal of the female Aedes sp mosquitoes infected. Symptoms have a wide range of variation, ranging from asymptomatic to severe symptoms, including hypovolemia and death. The need for adaptation in such distinct systems (insect and mammalian) restricts the variability that the viruses can acquire, allowing them to adapt to both hosts with similar replicative efficiency. Studies performed with other flaviviruses have shown that acquisition of variability occurs differently between the mammalian host and the insect vector, allowing the virus to adaptat in both systems. Therefore, this study investigated the adaptation of two strains of dengue virus, the ACS-46, not neurovirulent for adult mice and JHA strain, which is neurovirulent for adult mice. The study was done using mosquito cells culture (C6/36), mammalian cells (HepG2) and in vivo infection in newborn mouse brain. Using next-generation sequencing, we analyzed the mutations acquired over passages in each culture system. Twenty serial passages were conducted in C6/36 cells and six alternate passages between C6/36 and HepG2. Viruses obtained in the sixteenth and twenty passages from the serial experiment and the fourth and sixth passages from alternate model were completely sequenced using the next generation sequencing platform Ion Torrent. Sequence analysis revealed two clearly distinct viral populations observed from the sixteenth passage identified by deletion of two codons in the glycosylation site in the envelope protein domain I. This deletion led to increased fusion of the virus to mosquito cell, since the cytopathic effect increased from this passage to next as well as the viral load. However, this mutation had a deleterious profile in human cells since the mutated population was vanished during the alternate passages. It was also identified seven non-synonymous mutations in the region of nonstructural proteins with very similar percentages, suggesting that they belong to the same population. JHA strain lost the neurovirulent characteristic after 29 passages in C6/36, but quickly recovered it after a single passage in newborn mouse brain. The sequencing of the virus of these passages showed some variability in different sites among the parental virus, not neurovirulent viruses and neurovirulent virus after the single passage in mice. These data indicated that apparently there is no specific determinants of neurovirulence in DENV2, at least in our model, but rather a set of mutations can lead to neurovirulent phenotype, depending on the conditions in which the virus is exposed.

INTRODUÇÃO: a síndrome de Noonan apresenta herança autossômica dominante e é considerada uma doença relativamente frequente na população, com uma incidência estimada entre 1/1000 e 1/2500 nascidos vivos. Dentre os seus acometimentos destacam-se: dismorfismos faciais, baixa estatura, alterações cardíacas e criptorquia. A síndrome de Noonan é muito confundida com as síndromes Noonan-like devido à sobreposição dos achados clínicos. Estas, mais raras que a síndrome de Noonan, incluem as síndromes de LEOPARD, neurofibromatose-Noonan, cardiofaciocutânea e Costello. Atualmente sabe-se que tanto a síndrome de Noonan como as síndromes Noonan-like envolvem mutações em genes pertencentes à via de sinalização RAS-MAPK. Na síndrome de Noonan, pelo menos quatro genes desta via são responsáveis pelo fenótipo: PTPN11, SOS1, RAF1 e KRAS. Mutações no gene PTPN11, o primeiro gene descrito em associação com a síndrome, são encontradas em aproximadamente 40% dos casos. O segundo gene descrito, o gene KRAS, é responsável por cerca de 2% dos casos que não apresentam mutações no gene PTPN11. Mutações no gene KRAS estão presentes em pacientes com síndrome de Noonan com retardo mental e/ou atraso no desenvolvimento mais acentuados e em pacientes com a síndrome cardiofaciocutânea cujo envolvimento ectodérmico é mais sutil. OBJETIVO: devido à recente associação do gene KRAS com a síndrome de Noonan e outras síndromes Noonan-like é importante: (1) testar a frequência de mutação neste gene em pacientes que apresentam ou não mutações no gene PTPN11 e (2) tentar estabelecer uma correlação genótipo-fenótipo mais precisa, o que permitirá a realização de um aconselhamento genético mais adequado. MÉTODOS: foram avaliados 95 probandos com síndrome de Noonan e 30 com síndromes Noonan-like. O estudo molecular foi realizado através da reação em cadeia de polimerase, seguida das reações de purificação e sequenciamento bidirecional. RESULTADOS: foram encontradas mutações no gene PTPN11 em 20/46 (43%) pacientes com síndrome de Noonan, duas delas não descritas anteriormente. Relacionando o quadro clínico dos pacientes com síndrome de Noonan deste estudo, com e sem mutação no gene PTPN11, nota-se que os pacientes com mutação apresentam incidência significativamente maior de baixa estatura, de estenose pulmonar valvar e menor frequência de miocardiopatia hipertrófica. Uma mutação no gene KRAS foi encontrada em um paciente com síndrome de Costello, mutação esta ainda não relatada. Alterações gênicas em mais de um gene da via RAS-MAPK foram observadas em dois pacientes, sendo que uma delas em cada paciente não predizia um efeito fenotípico importante. Foram também encontrados três polimorfismos no gene KRAS, porém com mesma frequência no grupo controle. A fim de verificar a influência destes polimorfismos, as principais características da síndrome de Noonan foram relacionadas entre os pacientes com esta síndrome que apresentavam mutação no gene PTPN11 e comparadas quanto à presença ou ausência desses polimorfismos. Nenhuma diferença estatisticamente significante foi encontrada. CONCLUSÃO: Pacientes com síndrome de Noonan e mutações no gene PTPN11 apresentaram uma maior incidência de baixa estatura e de estenose pulmonar valvar e uma menor incidência de miocardiopatia hipertrófica. O gene KRAS, até então relacionado às síndromes de Noonan e cardiofaciocutânea, mostrou-se também responsável pela síndrome de Costello. Tanto as alterações gênicas consideradas não patogênicas como os polimorfismos encontrados no gene KRAS parecem não ter uma grande influência sobre a variabilidade fenotípica na síndrome de Noonan. Contudo, não é possível afastar totalmente que estas alterações apresentem um efeito sutil e que, em conjunto com outras variações genéticas e/ou ambientais, tenham um efeito modulador / INTRODUCTION: Noonan syndrome shows autosomal dominant inheritance, and is a relatively frequent disease in the population, with an estimated incidence between 1/1000 and 1/2500 live births. The main clinical features are: facial dysmorphisms, short stature, cryptorchidism and cardiac abnormalities. The differential diagnosis between Noonan syndrome and Noonan-like syndromes is not always easy, due to the overlap of the their clinicla findings. The Noonan-like syndromes, more rare that the Noonan syndrome, include the LEOPARD syndrome, neurofibromatosis-Noonan, cardiofaciocutaneous and Costello. Currently it is known that Noonan syndrome and Noonan-like syndromes involve mutations in genes belonging to the RAS-MAPK signaling pathway. In Noonan syndrome, at least four genes of this pathway are responsible for the phenotype: PTPN11, SOS1, RAF1 and KRAS. Mutations in PTPN11, the first gene described in association with this syndrome, are found in approximately 40% of cases. The second gene described, the KRAS gene, is responsible for about 2% of the cases that dont have mutations in the PTPN11 gene. Mutations in the KRAS gene are present in patients with Noonan syndrome with mental retardation and/or developmental delay more pronounced and in patients with cardiofaciocutaneous syndrome whose ectodermal involvement is more subtle. OBJECTIVE: Due to the recent association of the KRAS gene with Noonan and Noonan-like syndromes is important: (1) to test the frequency of mutation in this gene in patients with or without mutations in PTPN11 and (2) to estabilish a more precise genotype-phenotype correlation, allowing the realization of a more appropriate genetic counseling. METHODS: 95 probands with Noonan syndrome and 30 with Noonan-like syndromes were evaluated. The molecular analysis was performed by the polymerase chain reaction, followed by purification and bidirectional sequencing. RESULTS: PTPN11 gene mutation was found in 20/46 (43%) patients with Noonan syndrome, two of them not previously described. By correlating the clinical features of patients with Noonan syndrome in this study, with or without mutations in the PTPN11 gene, it was noted that patients with mutations have significantly higher incidence of short stature, pulmonary stenosis and lower incidence of hypertrophic cardiomyopathy. Mutations in KRAS gene were found in two patients a patient with Noonan syndrome ant the other with Costello syndrome. Gene alterations in more than one gene at the RASMAPK patway were observed in two patients, but one of the mutations in each patient didnt predict a significant phenotypic effect. Were also foud three polymorphisms in the KRAS gene, but with the same frequency in the control group. To check the influence of these polymorphisms, the main features of Noonan syndrome were related among patients with this syndrome who had mutations in the PTPN11 gene and compared of the presence or absence of these polymorphisms. No statistically significant difference was found. CONCLUSION: Patients with Noonan syndrome and PTPN11 gene mutation had a higher incidence of short stature and pulmonary valve stenosis and a lower incidence of hypertrophic cardiomyopathy. The KRAS gene, previously related to Noonan and cardiofaciocutaneous syndrome, was also responsible for Costello syndrome. Gene alterations considered as nonpathogenic and polymorphisms found in the KRAS gene seem to have a not great influence on the phenotypic variability in Noonan syndrome. However, it is not possible to completely rule out that these changes have a subtle effect and that, together with other genetic variations and/or environmental factors, may have a modulating effect

Thesis advisor: Julia E. Grigsby / The first part of this thesis is on properties of annular Khovanov homology. We prove a connection between the Euler characteristic of annular Khovanov homology and the classical Burau representation for closed braids. This yields a straightforward method for distinguishing, in some cases, the annular Khovanov homologies of two closed braids. As a corollary, we obtain the main result of the first project: that annular Khovanov homology is not invariant under a certain type of mutation on closed braids that we call axis-preserving. The second project is joint work with Adam Saltz. Plamenevskaya showed in 2006 that the homology class of a certain distinguished element in Khovanov homology is an invariant of transverse links. In this project we define an annular refinement of this element, kappa, and show that while kappa is not an invariant of transverse links, it is a conjugacy class invariant of braids. We first discuss examples that show that kappa is non-trivial. We then prove applications of kappa relating to braid stabilization and spectral sequences, and we prove that kappa provides a new solution to the word problem in the braid group. Finally, we discuss definitions and properties of kappa in the reduced setting. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Mathematics.

Thesis advisor: Michael J. Connolly / The Insular Celtic languages, such as Irish and Welsh, distinctively feature a morphophonemic process known as initial consonant mutation. Essentially the initial sound of a word changes due to certain grammatical contexts. Thus the word for 'car' may appear as carr, charr and gcarr in Irish and as car, gar, char and nghar in Welsh. Originally these mutations result from assimilatory phonological processes which have become grammaticalized and can convey morphological, semantic and syntactic information. This paper looks at the primary mutations in Irish and Welsh, showing the phonological changes involved and exemplifying their basic triggers with forms from the modern languages. Then it explores various topics related to initial consonant mutations including their historical development and impact on the grammatical structure of the Celtic languages. This examination helps to clarify the existence and operations of the initial mutations and displays how small sound changes can have a profound impact upon a language over time. / Thesis (BA) — Boston College, 2008. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Slavic and Eastern Languages. / Discipline: College Honors Program.

We aimed to characterize well-know PIDs, novel genetic defects and immunopathological mechanisms in Brazilian patients with susceptibility to mycobacterial diseases. The patients developed different mycobacterial diseases and M. tuberculosis was the most frequent species. Molecular and genetic analysis revealed mutations in different genes: RAG1 (P1), CD40LG (P2, P3, P4), NEMO (P5), NCF1 (P6), TLR2 (P7) IL-12Rb2 (P8), IL-12Rb1 (P9), TLR10 (P10), DKC1(P11), SOCS-1(P12) and IRAK2 (P13). Finally, MDMs from patients phagocytose normally but were unable to appropriately control intracellular M. tuberculosis growth in comparison to MDMs from healthy subjects. We concluded that the Brazilian patients have heterogeneous mutations previously associated with susceptibility to mycobacterial diseases and novel genetic variations were identified suggesting novel PIDs. In addition, the inability of MDMs to control the intracellular growth of M. tuberculosis indicates this contributes to patients´ susceptibility to mycobacterial infections. / Objetivamos identificar novos defeitos genéticos e mecanismos imunopatológicos em pacientes brasileiros com suscetibilidade a infecções por micobactérias. Os pacientes foram investigados se portadores de imunodeficiencias previamente caracterizadas tais como SCID, deficiência de CD40L, MSMD, defeitos na sinalização via TLRs e CGD. A análise genética foi realizada por sequenciamento Sanger e \'\'whole exome sequencing\'\' para identificar possíveis novas imunodeficiências primárias. Além disso a função dos macrófagos dos pacientes foi avaliada. Infecções por diferentes espécies de micobactérias foram apresentadas pelos pacientes, sendo M. tuberculosis a espécie mais frequentemente identificada. Mutações em diferentes genes foram encontradas: RAG1 (P1), CD40LG (P2, P3, P4), NEMO (P5), NCF1 (P6), TLR2 (P7), IL-12Rb2 (P8), IL-12Rb1 (P9), IRAK2 (P10), SOCS-1 (P11) e TLR10 (P12). MDMs dos pacientes fagocitaram normalmente M. tuberculosis, porém reduzida capacidade em inibir o crescimento da M. tuberculosis foi observada. Concluímos que os pacientes estudados possuem defeitos moleculares heterogêneos e que os MDMs desses indivíduos apresentam falhas no controle do crescimento da M. tuberculosis. Nossos dados sugerem que esses são fatores subjacentes à susceptibilidade a infecções por micobactérias nesses indivíduos.

The identification of alternative splicing in the human genome elucidated the potential to several enduring genomic questions. Not only could this phenomenon explain why organism complexity was not at all correlated with the genome size, or explain how an organisms could be affected by experience and environment at the molecular level, but it was perhaps the most flexible and adaptive regulatory mechanism identified to date. While the pathogenic aberrations of this mechanism have generally been readily investigated and identified as potential therapeutic targets, its meditative or advantageous instances have largely not been considered. Initiated exon skipping has been shown to have therapeutic effects in Muscular Dystrophy animal models and even in vitro human muscle cells (Aartsma-Rus, Annemieke, et al, Human Molecular Genetics 2003, McClorey, G., et al, Neuromuscular disorders, 2006). However, the consideration that this process may be occurring endogenously in human cells and contributing to other complex diseases has remained largely ignored. In this work, we have undertaken the first large-scale statistical examination of alternatively spliced variants between the tissues of diseased and normal patients. We hypothesize that there are endogenous alternative splicing events occurring in these tissues that purposefully mediate mutative damage and contribute to the differentiation between diseased and healthy phenotypes. By integrating data from several different sources and employing statistic and machine learning models, we have identified significant differences in gene characteristics between canonical and spliced variants correlated with changes in clinical outcomes. We conclude that this evidence supports our hypothesis that alternative splicing can be positively driven to mediate genetic damage. Expression of these genetically damaged and canonically spliced variants is clearly implicated in diseased tissue and poor clinical outcomes.

A síndrome de HIGM é uma imunodeficiência, caracterizada por níveis séricos normais ou elevados de IgM associados com baixos níveis de IgG, IgA e IgE. Neste estudo investigamos pacientes com HIGM autossômico recessivo. Selecionamos 15 pacientes com diagnóstico clínico sugestivo de AR-HIGM, 10 do sexo feminino, 05 do sexo masculino, onde onze são brasileiros, um é francês e três são turcos, com idades que variam de 2 a 40 anos. Todos os pacientes apresentam infecções recorrentes: 100% pneumonias, 80% otites médias agudas, 53% sinusites, 46% amigdalites, 40% diarréias 26% infecções urinárias, uma apresentou micobacteriose cutânea. Encontramos as seguintes mutações no gene AICDA: Pacientes EJ e GF, mutação missense na base 260 do cDNA (c.260G>C; p. Cys87Ser). Pacientes DA e RC apresentam defeito de splice, acarretando na deleção total do exon 4 do gene AICDA. Os pacientes estrangeiros foram previamente estudados para os genes AICDA, UNG e CD40, e nenhuma alteração foi encontrada. Neste caso, estudamos o gene INO80, e encontramos nos exons 04 (g.24012 G>A) e 26 (g.99976 G>T) do gene INO80, duas mutações missense em heterozigose no DNAg do paciente OD. O estudo molecular e genético é importante para a realização do diagnóstico diferencial, estratégia terapêutica e prognóstico dos casos. / HIGM syndrome is a rare immunodeficiency characterized by high or normal levels of serum IgM associated with low levels of IgG, IgA and IgE. We selected 15 patients with clinical diagnosis suggestive of AR-HIGM, 10 females, 05 males, where 11 are Brazilian, one is French and three are Turkish, with ages ranging from 2 to 40 years. All patients had recurrent infections: 100% pneumonia, 80% acute otitis media, 53% sinusitis, 46% tonsillitis, 40% recurrent diarrhea, 26% urinary tract infections, 20% stomatitis, and one patient presented a cutaneous mycobacteriosis. 20% of the patients had opportunistic infections: Mycobacterium marinum, Toxoplasma gondii, varicella-zoster virus, Pseudomonas aeruginosa, fungus, and Mycobacterium tuberculosis. As a result, we found two types of mutations in 4 diferent patients with no consanguinity. We found in AICDA gene the following mutations: Patients EJ and GF missense mutation in base 260 in cDNA, which results in a change of aminoacid (c.260G> C; Cys87Ser p.). Patients DA and RC showed a splice defect, resulting in a complete deletion of exon 4 in AICDA gene. All other patients were sequenced for AICDA, UNG and CD40 genes, and no changes were found. In this case, we studied the INO80 gene, and we found in exons 04 (g.24012 G> A) and 26 (g.99976 G> T) INO80 gene, two heterozygous missense mutations in DNAg of patient OD. Since these molecular genetic defects result in similar clinical features, molecular and genetic studies are important for the differential diagnosis, therapeutic strategy and prognosis of the cases.

Orientador: Rejane Maria Tommasini Grotto / Banca: Alexandre Naime Barbosa / Banca: André Martins / Resumo: A coinfecção HIV/VHC tornou-se um importante problema de saúde pública devido à possibilidade desses vírus agirem sinergicamente, acelerando a progressão da doença hepática relacionada ao VHC e podendo favorecer a proliferação do HIV. Apesar de estabelecido que a alta variabilidade genética do HIV gera mutações, conferindo ao vírus a capacidade de responder rapidamente as alterações da pressão seletiva exercida pelo sistema imunológico ou pela terapia antirretroviral (TARV), não é conhecido se a presença do VHC pode favorecer a emergência de variantes do HIV resistentes. Assim, este estudo teve como objetivo avaliar o perfil de mutações de resistência a inibidores de transcriptase reversa: Inibidores de Transcriptase Reversa análogo nucleosídeo ou nucleotídeo (ITRNs), Inibidores de Transcriptase Reversa não análogo nucleosídeo (ITRNNs); e inibidores de protease (IPs) em variantes de HIV circulantes em indivíduos coinfectados pelo VHC. Foram incluídos 19 pacientes coinfectados pelos vírus HIV/VHC, maiores de 18 anos, com carga viral plasmática do HIV de pelo menos 1.000 cópias de RNA/mL, atendidos no Ambulatório de Gastroenterologia da Faculdade de Medicina de Botucatu e, no Hospital dia "Domingos Alves Meira". A genotipagem e o sequenciamento foram realizados no Laboratório de Biologia Molecular do Hemocentro de Botucatu, Faculdade de Medicina, UNESP, utilizando o Trugene HIV-1 Genotyping Kit (Siemens HealthcareDiagnóstics, Inc. Tarrytown, NY, USA). Foram observados dois subtipos do HIV-1, B e F, sendo o subtipo B o mais freqüente, 94,74%. Para o VHC foram observados os genótipos 1 (94,74%) e 3 (5,26%). Todos os pacientes apresentaram mutações de resistência as classes avaliadas, tendo maior freqüência mutações associadas aos ITRNs como M184V em 57,89%. As mutações associadas aos ITRNNs mais freqüentes foram a K103N e G190A, presentes em 21,05% das amostras. As mutações principais ... / Abstract: HIV/HCV coinfection has become an important public health problem due to the possibility of these viruses act synergistically, which can accelerate the progression of liver disease related to HCV and may favor the spread of HIV. It is established that the high genetic variability of HIV generates mutations, giving the virus the ability to respond quickly to changes in selective pressure exerted by the immune system or by antiretroviral therapy (ART). It is not known whether the presence of HCV can promote the emergence of resistant variants of HIV. Thus, this study aimed to evaluate the profile of resistance mutations to classes of reverse transcriptase inhibitors: nucleoside and nucleotide analogue Reverse Transcriptase Inhibitors (NRTIs), non-nucleoside analogue Reverse Transcriptase Inhibitors (NNRTIs) and protease inhibitors (PIs), in circulating HIV variants in HCV coinfected individuals. In this study were included 19 HIV/HCV coinfected patients, over 18, with plasma HIV viral load of at least 1,000 RNA copies/mL. These patients were treated at the Ambulatory of Gastroenterology of the Faculty of Medicine of Botucatu and at the Day-Hospital "Domingos Alves Meira". Genotyping and sequencing were performed at the Laboratory of Molecular Biology of the Blood Center of Botucatu, Faculty of Medicine, UNESP, using the Trugene HIV-1 Genotyping Kit (Siemens HealthcareDiagnóstics, Inc. Tarrytown, NY, USA). Two subtypes of HIV-1, B and F, were observed, but the subtype B was observed more often, 94.74%. It was observed HCV genotypes 1 (94.74%) and 3 (5.26%). All patients presented resistance mutations to the evaluated classes, and the mutations associated to NRTIs as M184V were observed more often (57.89%). The most common mutations associated to NNRTIs were K103N and G190A, present in 21.05% of the samples. The main mutations associated to IPs more frequent were M46I, I54V and V82A, with 15.78% ... / Mestre

Mutations in FGFR2 are common in a subset of endometrial carcinomas. Given the emergence of small molecule inhibitors specific to this receptor tyrosine kinase, FGFR2 is an attractive therapeutic target. However, compensatory and adaptation mechanisms limit the clinical utility of compounds that target nodes in the receptor tyrosine kinase network. Here, we analysed the impact of FGFR inhibition in endometrial cancer cells and observed the emergence of a resistant population in an FGFR2-mutant cell line. To understand the mechanisms underlying this adaptation response, we used a phosphoproteomics approach to measure the kinase network in an unbiased manner. These experiments led to the identification of an AKT-related compensatory mechanism underpinning this resistance. Further dissection of this resistance mechanism utilising gene expression analysis showed PHLDA1, a negative regulator of AKT, was significantly down-regulated in resistant cells. This was further confirmed at the protein level. siRNA knockdown of PHLDA1 conferred immediate drug resistance in the FGFR2-mutant endometrial cancer cell line. Therefore, we identified PHLDA1 down-regulation as a mediator of drug resistance in FGFR2 mutant cancer cells, the first demonstration of the role of PHLDA1 in the acquisition and maintenance of drug resistance. Using a 3D physiomimetic model, we demonstrated that AKT inhibition alone also led to generation of a drug-resistant population. Most importantly, dual-drug therapy inhibited proliferation and induced cell death. Our data highlight how mass spectrometry and microarray gene expression analysis can complement each other in the identification of novel resistance mechanisms in cancer cells. These data suggest that combination treatment of FGFR2-mutant endometrial cancers, targeting both FGFR2 and AKT, represents a promising therapeutic approach.