Dissertations / Theses on the topic 'Compound mutation'

Objective: Description of the clinical, biochemical and genetic features of a Polish patient with familial glucocorticoid deficiency. Methods: Detailed clinical investigation, hormonal analysis and sequencing of the coding region of the melanocortin 2 receptor (MC2R) gene in this patient. Results: We report on a 3-month-old boy with familial glucocorticoid deficiency who presented at the age of 3 months with skin hyperpigmentation, muscle weakness, mild jaundice and constipation. Hormonal analyses revealed high ACTH and TSH serum concentrations, low serum cortisol concentration along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered completely. His physical and mental development progresses normally. Genetic analysis disclosed a novel compound heterozygous MC2R mutation p.Leu46fs and p.Val49Met. Conclusion: The heterozygous p.Leu46fs mutation adds to the small number of MC2R nonsense mutations and is the first frameshift mutation within the first transmembrane domain of the receptor. According to molecular modeling the Val49Met mutation results in a structural change of the first transmembrane domain and in a potential novel interaction of the transmembrane domains I and VII<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Objective: Description of the clinical, biochemical and genetic features of a Polish patient with familial glucocorticoid deficiency. Methods: Detailed clinical investigation, hormonal analysis and sequencing of the coding region of the melanocortin 2 receptor (MC2R) gene in this patient. Results: We report on a 3-month-old boy with familial glucocorticoid deficiency who presented at the age of 3 months with skin hyperpigmentation, muscle weakness, mild jaundice and constipation. Hormonal analyses revealed high ACTH and TSH serum concentrations, low serum cortisol concentration along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered completely. His physical and mental development progresses normally. Genetic analysis disclosed a novel compound heterozygous MC2R mutation p.Leu46fs and p.Val49Met. Conclusion: The heterozygous p.Leu46fs mutation adds to the small number of MC2R nonsense mutations and is the first frameshift mutation within the first transmembrane domain of the receptor. According to molecular modeling the Val49Met mutation results in a structural change of the first transmembrane domain and in a potential novel interaction of the transmembrane domains I and VII.<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Genetic analysis of Neurofibromatosis type 1 (NF1) may facilitate the identification of patients in early phases of the disease. Here, we present an overview of our diagnostic research spanning the last eleven years, with a focus on the description of 225 NF1 mutations, 126 of which are novel, found in a series of 605 patients (513 unrelated) in Italy. Between 2003 and 2013, 443 unrelated patients were profiled by DHPLC analysis of 60 amplicons derived from genomic NF1 DNA and subsequent sequencing of heterozygotic PCR products. In addition, a subset of patients was studied by MLPA to identify any duplications, large deletions or microdeletions present at the locus. Over the last year, 70 unrelated patients were investigated by MLPA and sequencing of 22 amplicons spanning the entire NF1 cDNA. Mutations were found in 70% of the 293 patients studied by DHPLC, thereby fulfilling the NIH criterion for the clinical diagnosis of NF1 (detection rate: 70%); furthermore, 87% of the patients studied by RNA sequencing were genetically characterized. Mutations were also found in 36 of the 159 patients not fulfilling the NIH clinical criteria. These data support the use of RNA-based methods for genetic analysis and provide novel information for relevant for improving the management of symptoms in oligosymptomatic patients.

Abstract: During my Ph.D. internship at the ‘PaceLab’, I have been involved in several scientific projects that, although involving different experimental models and with different aims, share the common background of cardiac pacemaker modulation and ion channels activity. In this sec-tion are briefly described the three main research lines in which I took part; a more complete discussion of the data will be presented later in this thesis. Notably, by the time I am writing this proposition, two of these studies have been submitted to scientific journals. - Identification of the bradycardic molecule contained in the traditional Chinese medi-cine drug Tongmai Yangxin: The identification of new bradycardic agents able to specifically bind HCN channels, rises great interest in the scientific community since a decreased cardiac pacemaker cur-rent would lead to an general heart rate lowering without side-effects. To date, despite a long and intensive investigation, only one pure bradycardic agent (Ivabradine)1-3 is used in the clinical setting. Ivabradine is considered virtually free of negative side-effects even though, in some rare cases, it can elicit minor optical limitations. A few years ago, our research group started a collaboration with a traditional Chinese medicine (TMC) drug producer (Le Ren Tang Pharmaceutical Factory, Tianjin, China) with the aim of unravelling the molecular mechanism of one of their products: Tongmai Yangxin (TMYX). This drug is currently used in China for the treatment of several car-diac diseases like coronary artery disease, palpitation, heart failure and angina4 and, like other TCM compounds, it is a mixture of botanical and animal products. Remarkably, recent investigations highlighted its ability to reduce cardiac metabolic disorders, oxida-tive stress and inflammation on stable angina patients4-6. Our previous studies explored the bradycardic action of this drug; in particular, its ef-fects were evaluated on freshly isolated rabbit SAN myocytes. TMYX displayed a dose dependent and fully reversible rate slowing of the spontaneous action potential (AP) ac-tivity, specifically acting on the pacemaker current by shifting the activation curve of HCN channels towards more negative potentials; furthermore, its efficacy appeared to be strictly correlated to the intracellular cAMP concentration. Detailed analysis demon-strate that this drug acts as a functional cAMP surmountable competitive antagonist, competing for the CNBD of f-channels according to a mode of action never observed before for the regulation of this current. Still, the nature of the bio-active molecule is un-known. With the aim of identify this principle, TMYX was divided into 4 fractions (F1-4) by our Chinese colleagues at the Pharmaceutical Informatics Institute of Zhejiang University (Hangzhou, China) according to the solubility of its components, and the ability of each preparation to modulate spontaneous rate and the If current was evaluated by patch-clamp experiments in rabbit SAN myocytes. Data clearly demonstrate that only the most hydrophilic fraction (F1) displayed features similar to the total drug, decreasing the AP rate up to ~20% specifically acting on the pacemaker current. To further narrow down the number of molecules to examine, F1 was subsequently di-vided into 4 sub-fractions (F1.1-1.4) and, as for the previous preparations, their effects were examined on rabbit SAN myocytes. The results pointed out the presence of the target molecule both in F1.1 and F1.2 since they were able to reduce the spontaneous AP firing by ~25 and ~20%, respectively, with a specific action on If current. Eventually, HPLC data revealed the presence of uridine in both these sub-fraction, sug-gesting that this molecule (or one of its derivates) could be involved in the bradycardic process. Therefore, a preliminary experiment was carried out to analyze its possible car-diac rate regulation properties on rabbit SAN myocytes. Surprisingly, the perfusion of uridine (1 μM) generated a small increase in the AP rate (+3.45%) but a decrease in the pacemaker current at -65mV. Additional experiment are requested in order to shed more light on the properties of this molecule, however, given that uridine, and in partic-ular its cyclic nuclidic form (cUMP), have been reported to interact with some isoforms of the HCN channels family7-9, it appears to be a good starting point for the identifica-tion of the active principle of TMYX. - Age-related changes in cardiac autonomic modulation and heart rate variability in mice: The incidence of mortality caused by age-associated cardiovascular diseases is increas-ing dramatically and it will represent a serious clinical issue in the next decades10,11. In humans, the natural process of aging is associated with progressive changes in cardi-ac autonomic nervous system (ANS) regulation that may predispose to higher cardiac risks. Animal models of aging are needed to gain insights into the relation between the aging of the ANS and cardiac pathophysiology. Specifically, the aim of this study is to verify the translational relevance of mouse models for further in-depth evaluation of the link between cardiac ANS regulation and increased arrhythmic risk with advancing age. Therefore, heart rate and time- and frequency-domain indexes of HRV were calculated from ECG recordings in two groups of conscious C57BL6/J male mice of different ages (4- and 19-months-old) during daily undisturbed conditions following peripheral β‐adrenergic (atenolol), muscarinic (methylscopolamine), and β‐adrenergic + muscarinic blockades and β‐adrenergic (isoprenaline) stimulation. Eventually, vulnerability to ar-rhythmias was evaluated during daily, undisturbed conditions and following β‐adrenergic stimulation. HRV analysis and heart rate responses to autonomic blockades revealed that 19-month-old mice had a lower vagal modulation of cardiac function compared with 4-month-old mice. This age-related autonomic effect did not however affect basal heart rate, since it compensated for the lower intrinsic heart rate observed in 19-month-old compared with 4-month-old mice. Both time- and frequency-domain indexes of HRV were reduced fol-lowing muscarinic, but not β‐adrenergic, blockade, suggesting that HRV is largely modulated by vagal tone in mice. Finally, 19-month-old mice showed a larger vulnera-bility to both spontaneous and isoprenaline-induced arrhythmias. These results reveal the presence of a reduced cardiac vagal modulation and HRV asso-ciated with an increased vulnerability to cardiac arrhythmias in older mice, which is consistent with the human condition. Given their short life span, mice could be further exploited as an aged model for studying the trajectory of vagal decline with advancing age using HRV measures, and the mechanisms underlying its association with proarrhythmic remodeling of the senescent heart. - Electrophysiological characterization of a SCN5A compound mutation (K1578N-G1866fs) discovered in a young patient affected by sinus node disfunction, atrial flut-ters and drug-induced long QT syndrome: Given its high relevance in the generation of the cardiac AP event, alterations in the ge-netic sequence encoding for NaV1.5 channel (SCN5A) are often related to severe dis-function in the heart function12. In this situation, a compound mutation (K1578N/G1866fs) has been reported in a child affected by severe bradycardia, atrial flutter and drug-induced QT prolongation. Nota-bly, the parents, who present a heterozygous mutation each, did not suffer of any cardi-ac problem and their ECG signals were unremarkable. Following clinical examinations, the diagnosis was sinus node disfunction and the patient was implanted with a pace-maker13. With this study we intended to characterize the electrical properties of the Na+ current carried by the mutated NaV1.5 channels in order to better understand the impact of these alteration and explore whether the patient will benefit from a specific pharmaco-logical treatment. According to the literature14,15, SCN5A gene can undergo a series of alternative splicing events, among which the inclusion of different exon 6 sequences that identify the neona-tal and the adult NaV1.5 isoform. Consequently, the electrical difference between these two isoforms was assessed by patch-clamp experiments on HEK-293 cells transfected with a vector containing their genetic sequence. The results displayed no differences in the current density compared to the adult isoform (HP: -120 mV) while a positive shift of both activation and inactivation curves were detected (5.1 mV and 8.8 mV, respec-tively). The impacts of the mutations on the Na+ current were then evaluated on the same mod-el both in the patient (compound mutation K1578N/G1866fs in the neonatal isoform) and in the parents (heterozygous expression in the adult isoform) conditions. For what concerns the parents, the data collected are reasonably in agreement with the clinical in-vestigation indicating no pathologic conditions. On the other hand, the transfection of both the mutations in the neonatal SCN5A isoform caused a dramatic reduction in the current density associated to a rightward shift of the activation curve (6.5 mV), com-pared to the corresponding WT isoform. In addition, since the time at which the neonatal to adult isoform switch occurs has still not been clearly identify, the compound mutation was also inserted in the WT adult vector. However, no changes were detected in the current density while a leftward shift of the activation curve (7.5 mV), which can be ascribed to the isoform change, suggested that the alterations have a similar effect on the activation kinetics regarding of the iso-form in which are expressed. Eventually, no changes in the recovery from inactivation process were found between all the conditions investigated. All in all, these data show an important loss-of-function of NaV1.5 channels in the pa-tient’s condition, suggesting that the two mutations (K1578N–G1866fs), when ex-pressed together, generate a far worst phenotype than their single heterozygous expres-sion. Furthermore, even when incorporated in the adult SCN5A isoform, the effects caused by the alterations did not change suggesting that the neonatal to adult isoform switch will probably grant no benefits for the patient.

La terapia personalizzata ha cambiato lo scenario clinico dei tumori ALK-positivi. Tuttavia, la resistenza farmacologica rimane un ostacolo importante cui far fronte. Lorlatinib è un farmaco di terza generazione che inibisce la maggior parte dei mutanti di ALK resistenti agli attuali inibitori. In questo studio sono state utilizzate in vitro e in vivo linee cellulari resistenti a lorlatinib derivanti da linfoma anaplastico a grandi cellule (ALCL), carcinoma polmonare non a piccole cellule (NSCLC) e neuroblastoma, allo scopo di studiare i meccanismi che guidano l'acquisizione della resistenza. In vitro, cellule di ALCL trattate con alte concentrazioni di lorlatinib hanno acquisito le seguenti mutazioni composte di ALK: G1202R/G1269A e C1156F/L1198F. In vivo, Le cellule di ALCL xenotrapiantate in topi immunocompromessi hanno acquisito le seguenti mutazioni ricorrenti: N1178H (5 topi su 10) e G1269A (4 topi su 10). È interessante notare che nelle cellule umane, dove l’espressione di NPM/ALK è tipicamente nucleare, la presenza della mutazione induce la localizzazione citoplasmatica di NPM/ALKN1178H, probabilmente mimandone l’overespressione. Nelle cellule resistenti si è osservata una significativa alterazione dei pathway di PI3K/AKT e RAS/MAPK, come dimostrato dall’analisi dell’espressione genica tramite RNA-seq. Il coinvolgimento di questi pathway nella resistenza a lorlatinib è stato confermato dalla validazione funzionale con inibitori. Le cellule di NSCLC trattate in vitro con lorlatinib hanno acquisito iperattivazione di EGFR. Il trattamento con un inibitore di EGFR, erlotinib, , è stato in grado di ripristinare la sensibilità a lorlatinib. In cellule di neuroblastoma resistenti a lorlatinib, il sequenziamento dell'intero esoma e l’analisi proteomica hanno rivelato una forma tronca di NF1 e l’iperattivazione di EGFR e ErbB4. Questi dati forniscono un'ampia caratterizzazione dei meccanismi di resistenza che possono insorgere in diversi tumori ALK-positivi dopo il trattamento con lorlatinib.<br>Targeted therapy changed the standard of care in ALK-dependent tumors. However, resistance remains a major challenge. Lorlatinib is a third-generation ALK inhibitor that inhibits most ALK mutants resistant to current ALK inhibitors. In this study, we utilize lorlatinib-resistant anaplastic large cell lymphoma (ALCL), non–small cell lung cancer (NSCLC), and neuroblastoma cell lines in vitro and in vivo to investigate the acquisition of resistance and its underlying mechanisms. ALCL cells acquired compound ALK mutations G1202R/G1269A and C1156F/L1198F in vitro at high drug concentrations. ALCL xenografts selected in vivo showed recurrent N1178H (5/10 mice) and G1269A (4/10 mice) mutations. Interestingly, intracellular localization of NPM/ALKN1178H skewed toward the cytoplasm in human cells, possibly mimicking overexpression. RNA sequencing of resistant cells showed significant alteration of PI3K/AKT and RAS/MAPK pathways. Functional validation by small-molecule inhibitors confirmed the involvement of these pathways in resistance to lorlatinib. NSCLC cells exposed in vitro to lorlatinib acquired hyperactivation of EGFR, which was blocked by erlotinib to restore sensitivity to lorlatinib. In neuroblastoma, whole-exome sequencing and proteomic profiling of lorlatinib-resistant cells revealed a truncating NF1 mutation and hyperactivation of EGFR and ErbB4. These data provide an extensive characterization of resistance mechanisms that may arise in different ALK-positive cancers following lorlatinib treatment.

The widespread expression of polypeptide growth factors from the earliest stages of embryonic development through to mature issues in the adult organism suggests an involvement in a reiterated developmental process affecting the underlying cellular growth and differentiation of many tissues. The hair follicle has taken on increased significance with the observation that many genetic mutations in these peptide growth factor genes affect its development. The targeted disruption of genes encoding members of the EpidermalGrowth Factor (EGF) and Fibroblast Growth Factor (FGF) families in the mouse has revealed a functional role for these proteins in the regulation of hair follicle growth. Experimental data and other factors are examined and results given. A second experimental system was used to determine if a functional relationship between certain peptide growth factors was conserved in the Merino sheep. The induction of a catagen-like state in the wool follicle and other epidermal changes associated with EGF treatment may be related to the transciptional induction of these peptide growth factors<br>Doctor of Philosophy (PhD)

La terapia antiretrovirale purtroppo può indurre la selezione di ceppi virali resistenti ai farmaci, che possono essere trasmessi ad altre persone. In questo studio è stato messo a punto un saggio fenotipico per comprendere la dinamica evolutiva delle resistenze trasmesse, confrontando la capacità replicativa (RC), e quindi il potenziale epidemico, conferiti dai geni della proteasi (PR) e della trascrittasi inversa (RT) di ceppi virali provenienti da pazienti con infezione recente. Inoltre il saggio fenotipico è stato impiegato per studiare l'influenza del background genetico del gene dell’integrasi (IN) nella selezione di uno specifico percorso di resistenza. I geni dei pazienti sono stati clonati in specifici cloni molecolari appartenenti alla famiglia dei pNL4-3mod dotati di gene reporter (Green Fluorescent Protein) e cassetta di clonaggio per i geni menzionati, provenienti dal virus dei pazienti. La RC è stata valutata misurando la fluorescenza emessa da cellule infettate in coltura. Nelle infezioni recenti (quindi con resistenze trasmesse), gli isolati con mutazioni di resistenza nella RT hanno mostrato una RC paragonabile agli isolati non mutati e potrebbero avere lo stesso potenziale epidemico. È interessante notare che gli isolati in tutte le infezioni recenti hanno mostrato una RC significativamente inferiore rispetto agli isolati nelle infezioni non trattate di vecchia data, suggerendo un ruolo attivo del sistema immunitario, in maggiore salute nelle infezioni recenti, nella selezione di virus con una RC inferiore. I dati inoltre hanno rilevato che l’impatto sulla RC delle mutazioni di resistenza nella PR è maggiore rispetto a quello delle mutazioni nella RT. Per quanto concerne l’IN, la combinazione G140S/Q148H in tutti i cloni ha determinato una RC più alta rispetto agli altri percorsi di resistenza, suggerendo che il background genetico del gene IN non è determinante per la selezione di specifici percorsi di resistenza osservati in vivo.<br>Antiretroviral therapy induces the selection of HIV drug resistance strains, which occasionally are transmitted to other patients. A recombinant phenotypic assay was developed to understand the evolutionary dynamic of transmitted drug resistance, analyzing the replicative capacity (RC) conferred by protease (PR) and reverse transcriptase (RT) of viral strains from recently infected patients. In addition, it was used to investigate the influence of the viral genetic background of the integrase (IN) gene in selecting a specific resistance pathway, comparing the RC of the resistance mutations spontaneously selected in vivo to those of alternative resistance pathways introduced by site-direct mutagenesis. Selected genes were cloned in the pNL4-3mod HIV molecular clone, containing the GFP reporter gene and a cloning cassettes for the mentioned genes, which allow the cloning of the respective genes from clinical HIV strains. The RC was evaluated by measuring the fluorescence in cells infected in vitro. In recent infections, isolates bearing RT resistance mutations showed a RC comparable to that of wild type isolates and might have the same epidemic potential. Interestingly, wild type isolates from recent infections displayed a significantly lower RC than isolates from non-recent infections, suggesting an active role of the immune system (in greater health in recent infections) in selecting virus with lower RC. The data also revealed that the impact of PR resistance mutations on RC is much deeper than that of RT resistance mutations. Concerning the IN gene, clones with the G140S/Q148H combination showed a higher RC, compared to alternative mutational pathways, suggesting that the genetic background of HIV IN gene at baseline is not determinant for the selection of the naturally observed INSTI mutations. The results also confirmed that dolutegravir (DTG) has a high barrier against the development of resistance providing an explanation for its higher clinical efficacy.

Dissertação de mestrado em Biotecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra<br>Usher syndrome is an autosomal recessive disease characterized by the association of retinitis pigmentosa and sensorineural hearing loss with or without vestibular dysfunction. Prevalence for this disease was estimated to be 3-4 per 100,000 individuals. Distinguished by clinical features, Usher syndrome can be divided in three types, where Usher syndrome type 1 is the most severe form. Causing disease mutations were reported in 10 genes, 6 of them associated to Usher syndrome type I. MYO7A gene is the most commonly mutated gene for this type, representing approximately 50% of the cases. MYO7A gene codes for myosin VIIa protein, previously described as a motor transport protein and participating in the establishment of cell-cell adhesions. This work aimed to evaluate the possibility of two novel MYO7A variants, identified in compound heterozygosity in a Usher syndrome type 1 Portuguese patient using a Targeted resequencing approach to 9 of the Usher syndrome associated genes, be responsible for the phenotype. Accordingly, the segregation analysis of these variants (c.3503+1delG and c.5561dupT) in the available patient relatives was performed, as well as the analysis of other five variants located between the two variant loci. Furthermore, 250 samples from normal individuals were analysed for the two novel variants and all of them presented the normal genotypes. Additionally, an in silico study was performed aiming to evaluate the evolutionary conservation of the two variant loci, revealing that the first is highly conserved among the mammalian analysed species while the second is highly conserved among the vertebrate analysed species. Since MYO7A c.3503+1delG variant is located at the donor splice site of MYO7A exon 27, alteration of splice site analysis was performed with in silico Splice site prediction and the complete absence of the normal donor splice site at MYO7A exon 27 was observed. Altogether, these results support the hypothesis that the two variants (c.3503+1delG and c.5561dupT) can be a compound heterozygous mutation responsible for Usher syndrome type I phenotype in the studied patient. In order to confirm the presence of the two variants in the MYO7A transcript nucleotide sequences, nasal epithelium samples from the patient and two normal individuals were studied and both wt and c.5561dupT alleles were expressed, while c.3503+1delG was only identified as the wt allele. However, it is predictable that a complex phenomenon be occurring at this splice site due to c.3503+1delG mutation. Finally, considering that it is x predictable that c.5561dupT mutation causes a MYO7A frameshift leading to a truncated myosin VIIa protein with a partially abnormal second MyTH4 domain and completely without the second FERM domain and the protein C-terminus, it seemed relevant to understand the effect of such mutation at the cellular and molecular level. Therefore, protein-protein interaction studies were performed with two constructions of C-terminal MyTH4-FERM (wt and mut), tagged with GFP. These studies suggested that the previously reported interaction of myosin VIIa with myrip was preserved in the presence of the MyTH4-FERM mut, and a gain of myosin VIIa function was achieved with the MyTH4-FERM mut, which seems to interact with munc13-4 protein, an interaction that was not seen with the MyTH4-FERM wt. Co-localization of both myosin VIIa with MyTH4-FERM mut and munc13-4 proteins was observed near vesicles structures when both were simultaneously expressed in HEK293a cells. This study reveals that MYO7A c.3503+1delG and c.5561dupT mutations are a compound heterozygous mutation causing USH1 in a Portuguese patient, and elucidate some functional myo7a protein alterations that may be important to understand the molecular mechanisms of this disease. However, further studies are required to clarify these implications at the cellular level.<br>O Síndrome de Usher é uma doença autossómica recessiva caracterizada pela associação de duas patologias, retinite pigmentosa e da perda auditiva sensoneural, às quais poderá estar também associada uma disfunção vestibular. Estima-se que esta doença afecta 3 a 4 indivíduos em cada 100 000. Distinguidos pelas características clínicas, o Síndrome de Usher pode ser dividido em três tipos, de entre os quais o Síndrome de Usher do tipo 1 é a forma mais severa. Até à data foram identificados 10 genes que possuem mutações causadoras desta doença, 6 dos quais foram associados ao Síndrome de Usher do tipo 1. O gene MYO7A é o gene onde foram encontradas mais mutações em doentes com este tipo de síndrome, representando cerca de 50% dos casos. O gene MYO7A codifica uma proteína designada de miosina VIIa, a qual já foi previamente descrita como uma proteína transportadora que também está envolvida no estabelecimento de adesões intercelulares. Este trabalho tem por objectivo avaliar a possibilidade de duas novas variantes do gene MYO7A serem responsáveis pelo fenótipo do Síndrome de Usher do tipo 1. Estas duas novas variantes foram identificadas em heterozigotia composta num doente português com Síndrome de Usher do tipo 1, através de uma abordagem de Targeted resequecing para 9 dos genes associados ao Síndrome de Usher. Assim sendo, foi feita a análise não só destas variantes (c.3503+1delG e c.5561dupT) mas também de outras cinco variantes localizadas entre entes loci, nos familiares em que foi possível. Além disso, as duas novas variantes foram analisadas em 250 amostras provenientes de indivíduos saudáveis, sendo que não foram encontradas em nenhuma destas amostras. Para além disso, com o objectivo de avaliar a conservação evolutiva destes loci foram realizados estudos in silico que demonstraram que ambos os loci são extremamente conservados entre as espécies de mamíferos analisadas. Uma vez que a variante c.3503+1delG do gene MYO7A está localizada no donor splice site do seu exão 27, foi feita uma previsão da alteração do local de splicing com um softweare online (splice site prediction), previsão esta que revelou uma completa destruição deste local de splicing na presença desta variante. Todos estes resultados sustentam a hipótese de que estas duas variantes (c.3503+1delG e c.5561dupT) possam constituir uma mutação heterozigótica composta responsável pelo fenótipo do Síndrome de Usher do tipo 1 no doente estudado. Com o intuito de perceber se estas duas novas variantes estariam alteradas na sequência de nucleótidos do transcrito do gene MYO7A, foram utilizadas uma amostra do epitélio nasal do doente e duas amostras do epitélio nasal de dois indivíduos normais, tendo xii sido verificada a expressão quer do alelo normal quer do alelo com a variante c.5561 na amostra do doente. No que diz respeito à variante c.3503+1delG, apenas foi possível identificar o alelo selvagem. Contudo, é espectável que na presença desta mutação possa estar a ocorrer um fenómeno complexo. Por último, considerando a probabilidade que a mutação c.5561dupT cause uma frameshift no transcrito do gene MYO7A, originando uma miosina VIIa truncada com um segundo domínio MyTH4 parcialmente anómalo que leva à destruição completa do segundo domínio FERM e do C-terminal da proteína, é relevante elucidar quais os efeitos desta mutação ao nível celular. Posto isto, foram realizados estudos de interacção proteína-proteína com duas construções dos domínios MyTH4-FERM do terminal carboxílico (selvagem e mutada) com uma tag de GFP. Estes estudos sugerem que a interacção já descrita entre a miosina VIIa e a myrip foi preservada na presença da mutação. Para além disso, foi identificado aquilo que parece ser um ganho de função por parte destes domínios do terminal carboxílico da miosina VII. Na presença da mutação, observou-se interacção com a munc13-4, o que não é detectado na presença destes domínios na sua forma selvagem. A ocorrência desta interacção e de um possível ganho de função na presença da mutação por parte da miosina VIIa é suportado pela observação da co-localização da muc13-4 e dos domínios do terminal carboxílico da miosina VIIa mutada perto de estruturas que se assemelham a vesículas, quando ambas são expressas nas células HEK293a. Este estudo revela que as mutações c.3503+1delG e c.5561dupT do gene MYO7A constituem uma mutação heterozigótica composta que é causa do fenótipo de Síndrome de Usher do tipo 1 num doente Português. Para além disso, elucida algumas alterações da miosina VIIa que podem ser importantes para elucidar os mecanismos moleculares desta doença. Contudo, mais estudos serão necessários para clarificar estas implicações ao nível celular

Planar heterocycles such as benzoxazoles are biologically active and often used as a scaffold for drug discovery. However, due to some toxicities associated with this group of compounds, analogues of benzoxazoles such as benzothiazoles, benzimidazoles and more recently, the oxazolopyridines have been developed in medicinal chemistry programs. The oxazolopyridine is an easy and versatile scaffold to synthesise as it is stable and has many sites for the addition of functional groups. The oxazolopyridine scaffold is also highly polar, contributing to improved target interaction in biological systems. This results in the anti-bacterial, anti-fungal, anti-parasitic and anti-inflammatory activities, and in sirtuin modulation of the oxazolopyridines. In fact, oxazolopyridines are shown to be non-toxic in vitro and in vivo in some studies, making it a very suitable scaffold for drug candidates. However, the full toxicity profile of the oxazolopyridine compounds, which include mutagenicity, is unknown and needs to be investigated to determine any unwanted effects. The oxazolopyridines and imidazopyridines are structurally similar and have some similar biological activities, but more concerningly, the carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) 46 also belongs to the imidazopyridine family. In the current study, 2-(3-aminophenyl)oxazolopyridine, 60 was chosen as the lead compound given its structural similarities to PhIP 46. It was therefore hypothesised that compound 60 may be genotoxic (DNA damaging) and mutagenic, where its mode of action is similar to PhIP 46 via the amino group attached. Toxicology screens were conducted on compound 60 in vitro. Consistent with previous studies, compound 60 was found to be non-cytotoxic in the colony forming assay. However, compound 60 was genotoxic in cells in the γH2AX assay. The non-cytotoxic but genotoxic nature of compound 60 was found to be attributed by the ability of the cells to repair the DNA damage to maintain survival after 24 hours. Compound 60 was later found to be mutagenic in the Ames test indicating that the cellular repair of the DNA damage is error-prone. In fact, compound 60 is a pro-mutagen which is dependent on oxidation and enzymatic bio-activation by liver enzymes for activity. More importantly, compound 60 was also found to transform cells in the soft agar invasion assay. Further studies were carried out to determine the genotoxic and mutagenic mode of action of compound 60. Through quantitative structural-activity relationship (QSAR) determination, the amino group of compound 60 was not the factor for activity. This was supported by the generated Hammett plot using analogues of 2-aryloxazolopyridine where it was understood that the activity of compound 60 was driven by molecular electron density and not solely by the presence of the amino group as hypothesised. This was an important turning point for this study where the focus was shifted towards studying the electron density on the oxazolopyridine core. Given that activation is oxidation dependent, selected 2-aryloxazolopyridine analogues were found to produce N-oxides at the pyridine ring of the oxazolopyridine core. The 2-phenyloxazolopyridine-N-oxides were also found to be genotoxic in cells and was thought to be the active species. However, it was found that the 2-aryloxazolopyridine-N-oxides still required further bio-activation by liver enzymes to be mutagenic in cells. This has led to further QSAR determination of the roles of the nitrogen atoms in the oxazolopyridine core. It was therefore proposed that upon the N-oxidation of the pyridine nitrogen, a second oxidation event on the oxazole nitrogen may occur to generate the active mutagen. Identification of the mutagenic species by enzymatic bio-activation of a 2-aryloxazolopyridine analogue was attempted using ultra pressure liquid chromatography and mass spectrometry (UPLC-MS). There was evidence that the 2-aryloxazolopyridine-N-oxide was formed under these conditions but more work was required to identify the mutagenic species. Therefore, the evidence presented in this thesis is the first detailing the mutagenic mode of action and liability of the oxazolopyridine compounds which has been widespread in drug development. In fact, this may have significant ramifications for the use of the oxazolopyridine scaffold in the future.

碩士<br>國立成功大學<br>環境工程學系碩博士班<br>91<br>Seven airborne samples with varying degrees of contamination. The airborne samples of both particle phase and gas phase of SVOCs were collected using high-volume samplers (PS-1) to asses the genotoxicity of airbone SVOCs. Air samples at seven sites / environments, including a motorcycle tunnel, a gasoline engine tunnel, and a diesel bus parking lot, an urban site in Tainan, plus three incense sticks, were collelcted for genotoxicity assessment using Mutatox test both with and without activation with S-9. Ambient air at traffic sites, i.e. the gasoline engine tunnel and the motorcycle vehicle tunnel were found to have positive genotoxicity with or without S-9 activation. Extracts from diesel parking lot were found to have only suspected genotoxicity with or without S9 activation. Finally, the urban site was found to be negative genotoxic with or without S-9 activation. As to the smoke from incense burning, all the SOF of tested incense were found to be positive genotoxic after S-9 activation. The XOC samples of tested incense were found to have suspect genotoxic after S-9 activation. The genotoxicity of particle-associated (SOF) samples was generally higher than those in the vapor phase (XOC) extacts in all tested air pollutions. This study shows Mutatox assay is an excellent assessment tool for monitoring toxicity of air samples. This in vitro bioassay is fast, convenient, and cost-effective, and can be widely used as for preliminary toxicity screening of environmental samples.

The widespread expression of polypeptide growth factors from the earliest stages of embryonic development through to mature issues in the adult organism suggests an involvement in a reiterated developmental process affecting the underlying cellular growth and differentiation of many tissues. The hair follicle has taken on increased significance with the observation that many genetic mutations in these peptide growth factor genes affect its development. The targeted disruption of genes encoding members of the EpidermalGrowth Factor (EGF) and Fibroblast Growth Factor (FGF) families in the mouse has revealed a functional role for these proteins in the regulation of hair follicle growth. Experimental data and other factors are examined and results given. A second experimental system was used to determine if a functional relationship between certain peptide growth factors was conserved in the Merino sheep. The induction of a catagen-like state in the wool follicle and other epidermal changes associated with EGF treatment may be related to the transciptional induction of these peptide growth factors

博士<br>國立中山大學<br>生物科學系研究所<br>98<br>Missense, nonsense, and frameshift mutations in the human anion exchanger 1 (AE1) have been associated with inherited distal renal tubular acidosis and hereditary spherocytosis. These two disorders are almost always mutually exclusive. However, we have recently found an unusual exception, i.e, a patient with complete distal renal tubular acidosis and severe hereditary spherocytosis. DNA sequencing revealed a novel mutation AE1 E522K (Band 3 Kaohsiung) combined with AE1 G701D mutation in this patient. We hypothesize these AE1 mutations cause these two disorders because of trafficking defect. To elucidate this hypothesis, we analyzed protein trafficking and subcellular location of AE1 and these mutants transfected into MDCK cells. Our results showed that they formed homodimers or heterodimers with each other. Homodimers of the wild-type and E522K mutant were localized at the plasma membrane, whereas the G701D mutant largely remained in the cytoplasm. On the other hand, heterodimers of either E522K or G701D and the wild-type AE1 were located in the plasma membrane, whereas E522K/G701D heterodimers remained in the cytoplasm. As for erythroid isoform of anion exchanger 1, analysis of protein trafficking and subcellular localization of the wild-type erythroid isoform of human anion exchanger 1 and these mutants transfected into k562 cells also showed that they can form homodimers or heterodimers with each other. Erythroid AE1 E522K/G701D cell-surface expression was significantly lower compared with WT homodimer expression. This result coincided with that erythroid AE1 of the patient’s red cell membrane can be detected 28% that of normal control in immunoblotting. Our study shows that the compound E522K/G701D mutation of human anion exchanger 1 causes trafficking defects in kidney and red blood cell lines, and these may explain the complete distal renal tubular acidosis and hereditary spherocytosis of the patient.

碩士<br>國立交通大學<br>生物資訊及系統生物研究所<br>105<br>Drug resistance has become bottleneck in clinical cancer therapy of non-small cell lung carcinoma (NSCLC) patients. The mutations of epidermal growth factor receptor (EGFR) on exon 19 deletion and exon 21 point mutation are respond well to the first and second generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) such as Gefitinib and Erlotinib for NSCLC therapy; however, patients acquired the T790M mutation of EGFR will induce drug resistance. In addition, cancer stemness properties are also a pivotal factor of drug resistance in NSCLC. Accordingly, development of novel compounds for overcoming drug resistance of NSCLC is highly desired. In this study, we show a novel synthetic compound PT262 that derived from 5,8-quinolinedione has potential in overcoming drug resistance of EGFR (T790M) and cancerous stemness in NSCLC. This new compound can induce cell death and apoptosis in the drug-resistant H1975 cells, which contained EGFR (T790M). This compound inhibited Survivin protein expression and conversely increased the active caspase 3 and cleaved PARP for apoptosis induction. In addition, this compound can inhibit the stemness protein expression of Oct4 in lung cancer cells. Moreover, it can also decrease the cell viability in drug resistance NSCLC cells, which were separated from pleural effusion of NSCLC. Taken together, we develop a novel compound, which has potential to further investigate for overcoming drug resistance in NSCLC.

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2016<br>Among the 20 amino acids incorporated into a polypeptide chain during protein synthesis, arginine (Arg; R) is the one with the highest relative mutability. In genetic diseases, almost 15% of missense mutations occur in Arg residues and more than half of these missense mutations are Arg to cysteine (Cys; C) substitutions. The Arg residue is more prone to mutations due to the fact that this residue can be coded by six different codons, four of them containing CpG dinucleotides, a preferential site for cytosine methylation with further spontaneous deamination to thymine. The effect of the aminothiol compounds cysteamine (CySH) and mercaptoethylguanidine (MEG) in the specific rescue of R to C (R>C) mutations have already been studied in our group through the characterization of the most common variant (p.R336C) found in cystathionine β-synthase (CBS) deficiency. It was postulated that those compounds would bind to the mutant residue C, forming a structure resembling the wild-type (WT) residue R, and thus restoring the enzyme activity. Cystathionine-β-synthase is a cytoplasmatic homotetrameric enzyme. It catalyses the condensation of the amino acids L-Homocysteine (L-Hcy) and L-Serine (L-Ser) to form L-Cystathionine (L-Cth). Each subunit (63 kDa) binds to the cofactors pyridoxal 5'-phosphate (PLP) and heme and comprises a N-terminal heme-binding domain, a catalytic core domain which binds PLP, and a C-terminal regulatory domain. The CBS protein contains 28 Arg residues, localized all over its sequence. In order to understand if the rescue of the activity of R>C CBS variants by aminothiol compounds, such as CySH and MEG, depends on the localization of the affected residue, six residues presenting different accessible surface area (ASA) values and domain localization were selected for mutagenesis, namely the R18, R121, R164, R336, R369 and R491 residues. Additionally, a control variant corresponding to an Arg residue to histidine (His; H) substitution (R>H) was also studied. The CBS proteins (WT and variants) were produced in a prokaryotic expression system and further purified by affinity chromatography. To evaluate the impact of aminothiol compounds on the structure and function of CBS R>C variants, the purified recombinant proteins were characterized in the absence and presence of these compounds in respect to their thermostability by differential scanning fluorimetry (DSF), and to their conformational flexibility by susceptibility to limited proteolysis. The enzyme activity and the quantitative assessment of free Cys residues were also evaluated. The obtained data strongly suggest that MEG, but not CySH, is able to increase the resistance to proteolysis of the majority of the studied R>C variants. However, this did not result in a higher enzyme activity for all of them. Since the observed effect was independent from ASA and domain localization, we anticipate that rescuing of R>C variants by aminothiols will be residue-specific and should always be investigated in a case-by-case basis.<br>Entre os 20 aminoácidos incorporados numa cadeia polipeptídica durante a síntese proteica, a arginina (Arg; R) é o aminoácido com maior mutabilidade relativa. Nas doenças genéticas, quase 15% das mutações missense são substituições de arginina para cisteína (Cys; C). O resíduo Arg é altamente susceptível a mutagénese devido ao facto de este aminoácido poder ser codificado por seis codões diferentes, contendo quatro deles dinucleótidos CpG, um local preferencial para a metilação de citosina com posterior desaminação espontânea para timina. O efeito dos compostos aminotiol cisteamina (CySH) e mercaptoetilguanidina (MEG) na recuperação específica de mutações R para C (R>C) foi já estudado no nosso grupo através da caracterização da variante mais comum (p.R336C) da deficiência na enzima cistationina β-sintase (CBS). Foi então postulado que estes compostos ligar-se-iam ao resíduo Cys mutante, formando uma estrutura semelhante ao resíduo Arg selvagem, restaurando assim a actividade enzimática. A proteína CBS é uma enzima homotetramérica citoplasmática. Cada uma das suas subunidades (63 KDa) liga-se aos cofatores piridoxal 5'-fosfato (PLP) e heme. Esta enzima catalisa a condensação dos aminoácidos L-homocisteína (L-Hcy) e L-serina (L-Ser) para formar L-cistationina (L-Cth). Cada monómero da CBS contém três domínios: um domínio N-terminal de ligação ao heme, que inclui os primeiros 70 aminoácidos; um domínio catalítico, contendo os 340 aminoácidos centrais; e um domínio regulador C-terminal com 140 resíduos. A CBS contém, na sua sequência primária, 28 resíduos de Arg igualmente distribuídos pelos diferentes domínios. De modo a compreender se a recuperação da atividade das variantes R>C da CBS, por compostos aminotiol como CySH e MEG, depende da localização do resíduo afetado, foram selecionados para mutagénese seis resíduos com valores de área de superfície acessível (ASA) e diferentes localizações, nomeadamente os resíduos R18, R121, R164, R336, R369 e R491. De entre estes, os resíduos R18 e R491 são resíduos expostos ao solvente e estão localizados no domínio N-terminal e C-terminal, respetivamente. O resíduo R121 apresenta um ASA de apenas 6% e localiza-se no domínio catalítico. Os resíduos R164, R336 e R369 são representativos de resíduos parcialmente acessíveis ao solvente, localizando-se na sua totalidade no domínio catalítico. Adicionalmente, foi estudada, como controlo, uma substituição equivalente de Arg para Histidina (His; H) (R>H). As proteínas CBS, selvagem (WT) e variantes, foram produzidas num sistema de expressão procariótico e purificadas por cromatografia de afinidade. Para avaliar o impacto dos compostos aminotiol na estrutura e função das mutações R>C da CBS, estas variantes foram caracterizadas na ausência e presença destes compostos relativamente à sua termostabilidade por fluorimetria de varrimento diferencial (DSF), e à sua suscetibilidade à proteólise limitada. A atividade enzimática e a aferição quantitativa dos resíduos de Cys livres foram também avaliadas. Os dados obtidos sugerem que o MEG, mas não a CySH, tem capacidade para modular a flexibilidade conformacional (maior resistência à proteólise limitada) das variantes e de aumentar a atividade das variantes R>C da CBS. Uma vez que o efeito observado é independente do ASA e da localização do resíduo, antecipamos que a recuperação das variantes R>C por compostos aminotiol possa ser alargada a outras proteínas com mutações R>C, devendo no entanto este efeito sempre ser avaliado especificamente para cada variante.