L’objectif était de mieux définir les relations entre l’exposition à certaines fibres minérales et les anomalies génétiques somatiques associées à la transformation tumorale de cellules de l’appareil respiratoire. Deux études indépendantes ont été conduites à partir du modèle murin Nf2+/- de mésothéliome malin (MM) développé dans le laboratoire, exposé par inoculation intrapéritonéale à des fibres d’amiante crocidolite (souris abs-Nf2+/- et abs-Nf2+/+) et à des fibres céramiques réfractaires (FCR) (souris ceram-Nf2+/-).Ce choix a été fondé selon une stratégie raisonnée à partir de la connaissance des anomalies génétiques observées dans le MM. La validité de ce modèle a été vérifiée en comparant les MM murins et humains. Des MM ont été induits par le crocidolite chez des souris abs-Nf2+/- et abs-Nf2+/+. Les souris abs-Nf2+/- ont significativement développé plus de MM que les souris abs-Nf2+/+. Les caractéristiques histologiques des MM murins sont analogues aux MM humains, avec des altérations génétiques similaires, en terme de fréquence et de mécanismes d’inactivation (mutations ponctuelles pour le gène TP53, délétions pour les gènes NF2 et P16/CDKN2A). Ce modèle murin a été ensuite utilisé pour évaluer la toxicité de FCR. Les souris ceram-Nf2+/- ont développé des MM similaires aux MM humains du point de vue histologique et moléculaire. Ainsi, le profil des altérations génétiques des MM murins ceram-Nf2+/- est semblable à celui des MM murins abs-Nf2+/- et abs-Nf2+/+, et humains. Les cellules mésotheliales des souris exposées aux FCR et à l’amiante semblent suivre les mêmes voies de transformation néoplasique. Une étude à la recherche d’altérations génétiques de ces 3 gènes et des gènes Ki-ras et EGFr fréquemment mutés dans le cancer broncho-pulmonaire (CBP) a été conduite à partir de cas de CBP sélectionnés (50 sujets exposés à l’amiante (E+) et 50 sujets non exposés (E-) appariés sur l’âge, le sexe, le type histologique et le tabagisme) provenant d’une série de cas opérés. A l’instar du MM, l’analyse du gène P16/CDKN2A suggère que le mécanisme d’inactivation pourrait être différent chez les sujets E+ (par délétion) par rapport aux sujets E- (par hyperméthylation du promoteur), indépendamment de l’âge et du tabagisme. Deux SNP (rs12947788 et rs12951053) du gène TP53 sont plus fréquemment retrouvés chez les sujets E+. En revanche, aucune différence significative n’est retrouvée pour les autres gènes entre ces 2 groupes. Ainsi, les mutations du gène NF2 dans le MM seraient plus liées à une spécificité cellulaire et à une fonction particulière de la protéine nf2 dans ces cellules / Résumé en anglais non communiqué

Meningioma and glioma are the most common primary brain tumors, but their etiologies are largely unknown. Although meningioma is usually benign, their intracranial location can lead to lethal consequences, and despite progress in surgery, radiotherapy, and chemotherapy the prognosis for patients with glioma remains poor. The only well-established environmental risk factor for meningioma and glioma is ionizing radiation. Evidence for inherited predisposition to meningioma and glioma is provided by a number of rare inherited syndromes where collectively these diseases account for only a small proportion of the twofold increased risk of brain tumors seen in first-degree relatives for meningioma and glioma patients. It is very possible that much of the excess familial risk is a consequence of co-inheritance of multiple low-risk genetic variations. With this in mind, the aims of the studies in this thesis were to discover genetic risk variants influencing the probability of acquiring the disease and to identify the association between risk variants on the tumor phenotype. To identify genetic variants influencing meningioma risk, a comprehensive tagging of the selected genes in a case-control study was performed. We identified nine risk variants in EGF, ERBB2, and LRIG2 genes. However, these findings could not be confirmed in another larger independent dataset. In addition, the study identified a correlation between LRIG2 protein expression and ER status when analyzed with different parameters. In a separate study with a larger sample of meningioma patients, the same correlation between LRIG2 and ER status was observed. To explore the potential association between reported germline risk variants and somatic genetic events, matched tumor and blood samples from glioma patients were analyzed by SNP array. The results identified correlations between EGFR gene variants and somatic aberrations within the EGFR locus and CDKN2A/B locus. To further study the relationship between germline risk variants and tumor phenotype, the same patient material was used and analyzed by three different techniques: SNP array, IHC, and FISH. The results revealed EGFR risk variants effecting copy number variation of the EGFR gene and the expression of the IDH1 and p53. Further comparison between different techniques such as SNP array and FISH analysis revealed the difficulty in achieving consistent results with different techniques. To summarize, the glioma studies show a link between genotype and phenotype where genetic risk variants in the EGFR gene were found to be associated with specific somatic aberrations. These associations are biologically interesting because EGFR is involved in multiple cellular processes. Additional studies of the direct functional role of these observations need to be conducted to elucidate the molecular mechanisms underlying the identified association between germline gene variants and somatic aberrations. For the meningioma studies, no significant risk variants influencing the disease were found but a correlation between LRIG2 and ER status was observed. This result suggests a potential role for the LRIG protein in the pathogenesis of meningioma, but more studies are needed to confirm this hypothesizes. / <p>Cancer research foundation in northern Sweden and Lions cancer research foundation at Umeå university</p>

O processo de carcinogênese na cavidade bucal ocorre em etapas, apresentando alterações sobre o genoma celular, sendo muitas vezes precedido por lesões denominadas potencialmente malignas. Os principais fatores de risco para o desenvolvimento do Câncer bucal são o fumo e o álcool. O desafio atual é identificar os pacientes com maior risco para o desenvolvimento do câncer bucal através da utilização de métodos não invasivos e eficazes. O objetivo deste trabalho foi avaliar a frequência de perda de heterozigosidade (LOH) no lócus 9p21, a atividade proliferativa celular, o padrão de descamação e relação núcleo/citoplasma na carcinogênese bucal. Para tal finalidade foi realizada a coleta citopatológica de indivíduos que foram divididos nos seguintes grupos: Controle (n=26), Álcool-Fumo (n=32), Leucoplasia (n=38) e grupo Carcinoma Espinocelular (CEC n=35). A partir do raspado citológico foi confeccionada uma lâmina para impregnação por prata e análise de AgNOR (velocidade de proliferação celular) e realização da técnica de Papanicolau para a análise do padrão de descamação e relação núcleo/citoplasma das células. O restante das células foi utilizado para extração do DNA para amplificação por PCR e sequenciamento. Observamos que a frequência de LOH foi maior no grupo Leucoplasia. A velocidade de proliferação celular foi maior no grupo Leucoplasia em relação ao grupo controle Na análise de padrão de descamação, observamos maior quantidade de escamas anucleadas no grupo Leucoplasia, de superficiais no grupo controle e de células parabasais no grupo CEC. A relação núcleo/citoplasma foi maior no grupo CEC em comparação aos outros. Concluímos que a LOH e o AgNOR são métodos promissores para o rastreamento e monitoramento dos pacientes com maior risco para o desenvolvimento do Câncer bucal. / The carcinogenesis process in the bucal cavity occurs in stages, appearing on the cellular genome, and is often preceded by potentially malignant lesions. The main risk factors for the development of bucal cancer are smoking and alcohol intake. The current challenge is to identify patients at greatest risk for the development of bucal cancer through the use of non-invasive and effective methods. The objective of this work is to evaluate the loss of heterozygosity (LOH) in the 9p21 locus, the cell proliferative activity, the pattern of epithelial desquamation and the nucleus/cytoplasm ratio of the epithelial cells. For this purpose a cytopathological sample was collected from individuals of 4 groups: control (n = 26), alcohol-smoking (n = 32), leukoplakia (n = 38) and the squamous cell carcinoma group (SCC n = 35). From the cytological scraping, a slide was silver impregnate for AgNOR analysis (cell proliferation velocity), another slide was stained by the Papanicolau technique for the analysis of the desquamation pattern and nucleus/cytoplasm ratio of the cells. The remaining cells were used for DNA extraction, followed by PCR amplification and fragments sequencing. We observed that the cell proliferation velocity rate was higher in the Leukoplakia group compared to the Control group. The LOH frequency was higher in the Alcohol-smoking and Leukoplakia groups. We observed increased anucleated cells in the leukoplakia group, while the nucleated superficial predominated in the control group and the parabasal cells in the SCC group. An increased nucleus/cytoplasm was detected only in the CEC group. We conclude that LOH and AgNOR methods are promising for the screening and monitoring of individuals at higher risk for the development of bucal cancer.

O processo de carcinogênese na cavidade bucal ocorre em etapas, apresentando alterações sobre o genoma celular, sendo muitas vezes precedido por lesões denominadas potencialmente malignas. Os principais fatores de risco para o desenvolvimento do Câncer bucal são o fumo e o álcool. O desafio atual é identificar os pacientes com maior risco para o desenvolvimento do câncer bucal através da utilização de métodos não invasivos e eficazes. O objetivo deste trabalho foi avaliar a frequência de perda de heterozigosidade (LOH) no lócus 9p21, a atividade proliferativa celular, o padrão de descamação e relação núcleo/citoplasma na carcinogênese bucal. Para tal finalidade foi realizada a coleta citopatológica de indivíduos que foram divididos nos seguintes grupos: Controle (n=26), Álcool-Fumo (n=32), Leucoplasia (n=38) e grupo Carcinoma Espinocelular (CEC n=35). A partir do raspado citológico foi confeccionada uma lâmina para impregnação por prata e análise de AgNOR (velocidade de proliferação celular) e realização da técnica de Papanicolau para a análise do padrão de descamação e relação núcleo/citoplasma das células. O restante das células foi utilizado para extração do DNA para amplificação por PCR e sequenciamento. Observamos que a frequência de LOH foi maior no grupo Leucoplasia. A velocidade de proliferação celular foi maior no grupo Leucoplasia em relação ao grupo controle Na análise de padrão de descamação, observamos maior quantidade de escamas anucleadas no grupo Leucoplasia, de superficiais no grupo controle e de células parabasais no grupo CEC. A relação núcleo/citoplasma foi maior no grupo CEC em comparação aos outros. Concluímos que a LOH e o AgNOR são métodos promissores para o rastreamento e monitoramento dos pacientes com maior risco para o desenvolvimento do Câncer bucal. / The carcinogenesis process in the bucal cavity occurs in stages, appearing on the cellular genome, and is often preceded by potentially malignant lesions. The main risk factors for the development of bucal cancer are smoking and alcohol intake. The current challenge is to identify patients at greatest risk for the development of bucal cancer through the use of non-invasive and effective methods. The objective of this work is to evaluate the loss of heterozygosity (LOH) in the 9p21 locus, the cell proliferative activity, the pattern of epithelial desquamation and the nucleus/cytoplasm ratio of the epithelial cells. For this purpose a cytopathological sample was collected from individuals of 4 groups: control (n = 26), alcohol-smoking (n = 32), leukoplakia (n = 38) and the squamous cell carcinoma group (SCC n = 35). From the cytological scraping, a slide was silver impregnate for AgNOR analysis (cell proliferation velocity), another slide was stained by the Papanicolau technique for the analysis of the desquamation pattern and nucleus/cytoplasm ratio of the cells. The remaining cells were used for DNA extraction, followed by PCR amplification and fragments sequencing. We observed that the cell proliferation velocity rate was higher in the Leukoplakia group compared to the Control group. The LOH frequency was higher in the Alcohol-smoking and Leukoplakia groups. We observed increased anucleated cells in the leukoplakia group, while the nucleated superficial predominated in the control group and the parabasal cells in the SCC group. An increased nucleus/cytoplasm was detected only in the CEC group. We conclude that LOH and AgNOR methods are promising for the screening and monitoring of individuals at higher risk for the development of bucal cancer.

O processo de carcinogênese na cavidade bucal ocorre em etapas, apresentando alterações sobre o genoma celular, sendo muitas vezes precedido por lesões denominadas potencialmente malignas. Os principais fatores de risco para o desenvolvimento do Câncer bucal são o fumo e o álcool. O desafio atual é identificar os pacientes com maior risco para o desenvolvimento do câncer bucal através da utilização de métodos não invasivos e eficazes. O objetivo deste trabalho foi avaliar a frequência de perda de heterozigosidade (LOH) no lócus 9p21, a atividade proliferativa celular, o padrão de descamação e relação núcleo/citoplasma na carcinogênese bucal. Para tal finalidade foi realizada a coleta citopatológica de indivíduos que foram divididos nos seguintes grupos: Controle (n=26), Álcool-Fumo (n=32), Leucoplasia (n=38) e grupo Carcinoma Espinocelular (CEC n=35). A partir do raspado citológico foi confeccionada uma lâmina para impregnação por prata e análise de AgNOR (velocidade de proliferação celular) e realização da técnica de Papanicolau para a análise do padrão de descamação e relação núcleo/citoplasma das células. O restante das células foi utilizado para extração do DNA para amplificação por PCR e sequenciamento. Observamos que a frequência de LOH foi maior no grupo Leucoplasia. A velocidade de proliferação celular foi maior no grupo Leucoplasia em relação ao grupo controle Na análise de padrão de descamação, observamos maior quantidade de escamas anucleadas no grupo Leucoplasia, de superficiais no grupo controle e de células parabasais no grupo CEC. A relação núcleo/citoplasma foi maior no grupo CEC em comparação aos outros. Concluímos que a LOH e o AgNOR são métodos promissores para o rastreamento e monitoramento dos pacientes com maior risco para o desenvolvimento do Câncer bucal. / The carcinogenesis process in the bucal cavity occurs in stages, appearing on the cellular genome, and is often preceded by potentially malignant lesions. The main risk factors for the development of bucal cancer are smoking and alcohol intake. The current challenge is to identify patients at greatest risk for the development of bucal cancer through the use of non-invasive and effective methods. The objective of this work is to evaluate the loss of heterozygosity (LOH) in the 9p21 locus, the cell proliferative activity, the pattern of epithelial desquamation and the nucleus/cytoplasm ratio of the epithelial cells. For this purpose a cytopathological sample was collected from individuals of 4 groups: control (n = 26), alcohol-smoking (n = 32), leukoplakia (n = 38) and the squamous cell carcinoma group (SCC n = 35). From the cytological scraping, a slide was silver impregnate for AgNOR analysis (cell proliferation velocity), another slide was stained by the Papanicolau technique for the analysis of the desquamation pattern and nucleus/cytoplasm ratio of the cells. The remaining cells were used for DNA extraction, followed by PCR amplification and fragments sequencing. We observed that the cell proliferation velocity rate was higher in the Leukoplakia group compared to the Control group. The LOH frequency was higher in the Alcohol-smoking and Leukoplakia groups. We observed increased anucleated cells in the leukoplakia group, while the nucleated superficial predominated in the control group and the parabasal cells in the SCC group. An increased nucleus/cytoplasm was detected only in the CEC group. We conclude that LOH and AgNOR methods are promising for the screening and monitoring of individuals at higher risk for the development of bucal cancer.

Le mésothéliome est une tumeur maligne rare des séreuses, au pronostic sombre, dont le facteur de risque principal connu est l’exposition à l’amiante. Il se déclare avec un temps de latence d’environ 30 ans après le début de l’exposition, atteint principalement la plèvre et touche majoritairement les hommes. L’analyse de cette pathologie, reconnue comme une maladie professionnelle et pour laquelle une Déclaration Obligatoire de maladie a été instituée en janvier 2012, s’est focalisée jusqu’alors essentiellement sur l’homme, tant sur le plan épidémiologique que sur le plan biologique. Il apparaît pourtant dans la littérature internationale des différences notables par sexe.Ces constations posent la question d’investiguer d’autres facteurs de risque, en particulier l’hypothèse d’une prédisposition génétique, les mécanismes biologiques intervenant dans les voies de la carcinogenèse du mésothéliome étant partiellement connus. Ceci incite à étudier les facteurs épidémiologiques, cliniques, biologiques et immunohistochimiques, prédisposant chez la femme au développement du mésothéliome pleural et péritonéal, et d’évaluer leurs impacts pronostiques, à partir des cas diagnostiqués entre 1998 et 2013, issus de la plus importante base de données française. Elle incite également à établir un état des lieux épidémiologique actualisé par sexe en France pour le mésothéliome pleural et péritonéal ; ces données étant inexistantes pour la France pour le péritoine. / Mesothelioma is a rare malignant serous tumor with a poor prognosis of which asbestos exposure is the major known risk factor. It occurs with a latent period about 30 years after exposure, reaches mainly the pleura and affects mainly the men. The study of this pathology, recognized as an occupationnal disease and for which a Mandatory Declaration of Disease was introduced in January 2012, focused until then mainly on men, both epidemiologically and biologically. However, there are significant gender differences in the international literature.These results raise the question of investigating other risk factors, in particular the hypothesis of genetic predisposition, the biological mechanisms involved in mesothelioma carcinogenesis pathways being partially known. This encourages the study of epidemiological, clinical, biological and immunohistochemical factors predisposing women to the development of pleural and peritoneal mesothelioma, and to evaluate their prognostic impacts, based on cases diagnosed between 1980 and 2015, from the largest French database. It also encourages the establishment of an updated epidemiological inventory by gender in France for pleural and peritoneal mesothelioma; these data are non-existent for peritoneum.

Pancreatic ductal adenocarcinoma (PDAC) is a serious oncological disease, which ranks among cancers with the worst prognosis and a three-year life expectancy of 10%. Ex-vivo organoid cultures derived from cancer tissue are popular and reliable research models, which reflect the morphology and histology of the original tissue. Genetic background leading to development PDAC confer typical alterations in genes KRAS, TP53, SMAD4 a CDKN2A. The aim of this thesis was to determine mutations present in organoid cultures derived from human PDAC. We used online genomic databases to estimate specific mutations typical for PDAC. Based on that research we designed protocols for the detection of PDAC genetic alterations and optimized those methods using cultured cells. We applied the approach on primary ex- vivo organoids derived from surgical cancer specimens and detected mutations in KRAS, TP53, SMAD4, or deletion of exons in CDKN2A. Alternatively, we proposed improvements for the analysis of genetic background in PDAC. The data obtained within this thesis will be used for the stratification of metabolomics and biochemical analyses further in the project.

No description available.

Ce travail avait deux objectifs: 1) définir des groupes de patients (pts) susceptibles de bénéficier d’un conseil génétique par l’identification de facteurs prédictifs de l’existence d’une mutation du gène CDKN2A, un des gènes majeurs de prédisposition au mélanome, dans les familles ne comportant que deux cas (Fam_2 cas). 2) la caractérisation épidémiologique et clinique d’entités particulières du mélanome dans l’objectif secondaire de contribuer à l’identification de gènes de prédisposition à ces entités. Les 2 entités étudiées étaient le mélanome cutané (MC) associé au cancer du rein (CR) et les mélanomes muqueux de la sphère ano-génitale (MMAG).Les populations d’étude sont une collection de 293 pts atteints de MC recrutés de façon consécutive sans connaissance à priori de l'histoire familiale et la collection française MELARISK qui comprend ≥ 3000 sujets prélevés appartenant à des familles à cas multiples de mélanomes ou ayant un MC survenant dans un contexte particulier (association à un autre cancer, topographie rare, survenue avant l’âge de 20 ans, MC multiples sporadiques). Nous avons étudié l'effet de 3 facteurs prédictifs potentiels sur la présence d’une mutation de CDKN2A dans une famille en fonction du nombre de pts atteints dans une famille (2 pts versus ≥3 pts). L’étude a été menée dans 483 familles françaises comprenant 387 Fam_2 cas, et 96 familles avec ≥3 pts atteints de mélanome (Fam_3+ cas). Les facteurs étudiés dans la famille un à un puis conjointement étaient : l’âge médian <50 ans au diagnostic de MC, la survenue de ≥1 cas de MC primitifs multiples (MPM) et la survenue de ≥1 cas de cancer du pancréas (CPCP). La fréquence des mutations était plus élevée dans les Fam_3+ cas (32%) que dans les Fam_2 cas (13%). Alors qu’un âge jeune au diagnostic et la survenue de ≥ 1 MPM étaient associés à la présence de mutations de CDKN2A dans les Fam_2 cas, un âge jeune au diagnostic ainsi que la présence de ≥1 cas de CP était associé significativement aux mutations de CDKN2A dans les Fam_3+ cas. L’étude a montré que les caractéristiques cliniques associées aux mutations de CDKN2A varient, en France, pays de faible incidence de mélanome, en fonction du degré d’agrégation familiale. L’identification de facteurs prédictifs de mutations de CDKN2A dans les Fam_2 cas a contribué à définir des sous-groupes de familles (âge jeune au diagnostic, survenue de MPM) dans lesquels la fréquence des mutations de CDKN2A est supérieure à 20% et auxquels il est légitime de proposer un test génétique. L’analyse des deux séries de pts MM+CR et MMAG a permis d’identifier, en les comparant à la série de MC recrutés de manière consécutive, leurs caractéristiques cliniques et histologiques. Dans ces deux séries, nos résultats ont mis en évidence un contexte de prédisposition héréditaire en partie indépendant de CDKN2A. L’étude de l’association MC et CR chez un même patient a eu deux conséquences pratiques: pour les cliniciens ces résultats suggèrent l’intérêt d’un examen dermatologique en cas de CR et l’intérêt de l’échographie abdominale dans le bilan initial d’un MC pour le dépistage du CR; pour la recherche en génétique, cette série a contribué à l’identification d’une mutation germinale dans le gène MITF qui augmente le risque de développer un MC, un CR ou l’association des deux cancers et qui a des propriétés biologiques intéressantes. L’étude des MMAG a montré que ces mélanomes pouvaient être associés à des MC chez un même malade et/ou survenir dans un contexte familial de mélanome. Le corollaire clinique de ces résultats est que l’examen dermatologique de dépistage ou de surveillance doit être à la fois cutané et muqueux dans un contexte familial de mélanome et qu’en cas de MMAG un examen dermatologique des apparentés doit être proposé comme c’est la règle dans les MC. L’absence de mutation de CDKN2A dans ces localisations muqueuses incite à entreprendre des études génétiques pour identifier les gènes impliqués. / This thesis had two main objectives: 1) To define groups of patients which may benefit from genetic counseling by identifying predictors of mutations of the CDKN2A gene, a major gene predisposing to cutaneous melanoma (CM) in families with only two cases. 2) Epidemiological and clinical characterization of specific entities of melanoma with the secondary objective of contributing to the identification of susceptibility genes for these entities. Coexistence of CM with renal cell carcinoma and mucosal anogenital melanomas were studied.The study populations are a collection of 293 melanoma patients that were ascertained systematically and the French collection MELARISK which is a collection including over 3000 subjects drawn from families with multiple cases of melanoma or melanoma occurring in a particular context (association with another cancer, rare locations, occurrence before the age of 20, multiple sporadic melanomas).We investigated association of three clinical features with the presence of a CDKN2A mutation in a family by extent of CM family clustering (2 versus ≥3 CM patients among first-degree relatives in a family).The study was conducted in 483 French families including 387 families with two melanoma patients, and 96 families with three or more patients with melanoma. The factors examined individually and in a joint analysis in a family were: median age at diagnosis <50 years, ≥1 patient in a family with multiple primary melanomas (MPM) or with pancreatic cancer. The frequency of CDKN2A mutations was higher in F3+ families (32%) than in F2 families (13%). While early age at melanoma diagnosis and occurrence of MPM in ≥1 patient were significantly associated with the risk of a CDKN2A mutation in F2 families, early age at melanoma diagnosis and occurrence of pancreatic cancer in a family were significantly associated with CDKN2A mutations in F3+ families. Thus this study showed that clinical features associated with CDKN2A mutations vary, in France, a country of low incidence of melanoma, according to the degree of familial clustering. Identifying predictors of CDKN2A mutations in families with two melanoma cases has helped to define subgroups of families (early age at CM diagnosis, and/or ≥1 MPM patient) in which the frequency of CDKN2A mutations is above 20% such that these subgroups of F2 families should be offered genetic testing.The analysis of two series of patients, either patients with melanoma coexisting with renal cell carcinoma or patients with anogenital mucosal melanoma identified their clinical and histological features by comparing them to a series of melanomas that were ascertained systematically. In both series, our results suggested a genetic predisposition at least partly independent of CDKN2A. The study of the c renal cell carcinoma; coexistence of CM and renal cancer in the same patient had two practical consequences for clinicians: it suggests the interest of a dermatologic screening visit in patients with renal cell carcinoma and that abdominal ultrasonography or computed tomography scanning performed at the initial workup and during the follow-up of patients with CM may be of value for the early detection of renal cancer. Regarding genetic research, this series has contributed to the identification of a germline mutation in the MITF gene that increases the risk of developing melanoma, renal cancer or both cancers and has interesting biological properties. The study of anogenital melanoma has shown that these melanomas could be associated with cutaneous melanoma in the same patient and it has also shown a high frequency of family history of melanoma associating mucosal and CM suggesting a shared genetic predisposition. Consequently dermatological screening or monitoring must include examination of both skin and mucosa in families with multiple cases of CM; and in case of a mucosal melanoma, a dermatological examination should be offered to relatives. The genetic mechanism has to be identified

The tumour suppressor protein p16INK4a (p16) is a cyclin-dependent kinase (CDK) inhibitor that plays a key role in the regulation of the cell cycle by controlling the progression of cells through the G1 to S phase transition. Dysregulation of the protein through deletion, silencing or mutation of the gene encoding p16 is implicated in a range of different cancers including melanoma, cervical and oesophageal to name a few. p16 is composed of four ankyrin repeats and it has a very low thermodynamic and kinetic stability and rapidly unfolds even in the absence of denaturants. This low stability means that the protein is highly vulnerable to point mutations, which can result in functional inactivation through a range of different mechanisms such as deletion of key binding contacts, disruption of secondary or tertiary structure and consequent destabilisation leading to unfolding or aggregation. Heavy-chain antibodies are a unique form of antibody devoid of light chains found in the serum of the Camelid family (camels and llamas). Despite the absence of light chains, heavy-chain antibodies have evolved to complement traditional antibodies and retain the full binding capacity seen in canonical IgG antibodies. The single variable domain, known as a nanobody, is, at 15 kDa, the smallest antigen binding fragment, a tenth the size of a standard IgG antibody. The small size and relative ease of production, coupled with an unusually high stability, makes nanobodies useful tools as biological reagents, crystallography chaperones and therapeutics. The research contained within this PhD looks at the development of nanobodies to target p16. By leveraging the high stability of selected nanobodies, the aim was to obtain binders that could stabilise and reactivate a range of unstable cancer-associated mutants. The initial stages of the project focused on generating and optimising the expression and purification of p16 constructs prior to immunisation of animals to raise nanobodies. A high-throughput approach was taken to generate forty-five different p16 constructs with a range of different solubility and purification tags. These constructs were assessed in a multi-factorial expression screen, which resulted in the identification of a p16 construct with a ten-fold improvement in soluble expression levels compared with previous studies. A range of biophysical techniques, including circular dichroism and chemical denaturation, were performed to characterise this protein fully prior to immunisation. The second part of this project utilised a phage display library of two immune nanobody libraries generated against p16 and a p16 variant stabilised by previously published second-site mutations. This process yielded a large number of diverse nanobodies. Biophysical characterisation of these nanobodies was first performed, and they were found to have a range of chemical and thermal stabilities. Assays were then developed to test the ability of the nanobodies to stabilise p16. Two nanobodies were found to dramatically stabilise wild-type p16, with an increase in stability of approximately 44 % and 60 %, respectively. Furthermore, these nanobodies were also able to stabilise a subset of cancer-associated point mutants. Although there are NMR structures of p16, as well as a crystal structure of p16 bound to CDK6, the resolution of is very low, most likely due to the high backbone flexibility of p16. The last part of the project aimed to obtain a higher-resolution structure of p16 by using the two stabilising nanobodies as crystallisation chaperones. The more stabilising of the two nanobodies resulted in crystals that diffracted to a resolution of less than 2 $\AA$, a significant improvement compared with the previously published structure. In conclusion, a number of nanobodies were generated against tumour-associated p16 and shown to be capable of stabilising p16, allowing structure determination to high resolution and restoration of the stability of cancer-associated mutants to wild-type levels. In the project, a range of different approaches for nanobody production were explored, and these will be important for future applications. Moreover, the crystal structure of the p16-nanobody complex showed that the nanobody binds on the opposite face of p16, to the face involved in binding to CDKs; thus, this nanobody could potentially be exploited as a pharmacological chaperone to stabilise and restore the activity of cancer-associated mutant p16 in the cell.