Avec 3000 nouveaux cas par an en France, le glioblastome multiforme (GBM) est la tumeur primitive du cerveau la plus fréquente. C’est aussi la plus agressive, l’espérance de vie moyenne des patients au moment du diagnostic ne dépassant guère 15 mois. Depuis l’étude de Stupp et collaborateurs en 2005, l’association radiothérapie – temozolomide est le traitement de référence en première ligne des glioblastomes nouvellement diagnostiqués. La protéine de la translocation (translocator protein, TSPO), connu aussi sous le nom de récepteur périphérique des benzodiazépines ou PBR, joue un rôle dans la biosynthèse des stéroïdes et le transport du cholestérol. L’interaction de la TSPO avec VDAC (Voltage-Dependant Anion Channel) dans les pores de transition de la perméabilité mitochondrial suggère également un rôle dans la régulation de l’homéostasie cellulaire et de l’apoptose. Certains travaux ont rapporté une surexpression de la TSPO dans des tumeurs cérébrales, suggérant que cette protéine pourrait représenter une cible moléculaire pour la thérapie du GBM. En particulier, l’Erucylphosphohomocholine (ErPC3, erufosine), dont l’activité antitumorale semble dépendre de la TSPO, a été capable d’induire l'apoptose in vitro dans des lignées de cellules de gliome résistantes à la chimiothérapie. Un radioligand de la TSPO pour l’imagerie par tomographie par émission de positons (TEP), le [18F]DPA-714, a été mis au point au CEA et validé dans différents modèles de neuroinflammation. Les hypothèses qui ont sous-tendu ce travail de thèse sont 1°) que la surexpression de la TSPO dans le GBM pouvait être mise en évidence par imagerie TEP au [18F]DPA-714 et 2°) que la manipulation pharmacologique de la TSPO, via les ligands spécifiques de cette protéine ou via l’ErPC3,pourrait induire l’apoptose dans les GBM. Les objectifs de la thèse étaient : 1°) d’évaluer l’expression de la TSPO dans un panel de cellules de gliomes murin et humain ; 2°) de caractériser, in vitro et in vivo, l’effet sur la survie cellulaire de lignées de GBM de l’administration de ligands de la TSPO [(et RO5-4864)] d’une part, ou de l’ErPC3 d’autre part ; 3°) de développer un modèle préclinique d’imagerie in vivo utilisant le [18F]DPA-714 pour suivre l’effet thérapeutique du ou des ligands sélectionné(s). Ce travail de thèse a démontré la faisabilité de l'imagerie TEP par [18F]DPA-714 pour détecter les gliomes 9L dans un modèle préclinique de rat et évaluer l’efficacité du traitement par ErPC3, ce qui pourrait constituer une nouvelle approche d’imagerie moléculaire du GBM. Il a permis de confirmer l'effet pro-apoptotique de l’ErPC3sur le gliome de rat in vitro et in vivo où une infiltration de la zone de la tumeur par les cellules microgliales/macrophages (CD11b-positives) et les astrocytes (GFAP-positives) chez les animaux traités à l’ErPC3 a pu être mise en évidence. Ces résultats ouvrent des perspectives intéressantes pour la recherche clinique dans le traitement des GBM. / In France alone, there are 3000 new cases of glioblastoma multiforme (GBM) per year and therefore GBM is the most common and aggressive form of the primary tumor in the central nervous system (CNS). The clinical prognosis for glioblastoma patients is extremely poor with a median survival period that rarely exceeds 15 months post-diagnosis. Since the study performed by Stupp and colleagues in 2005, the standard treatment for newly diagnosed glioblastoma consists of surgical removal of the tumor, followed by radiotherapy and concomitant chemotherapy with temozolomide. The 18 kDa Translocator Protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR) is a mitochondrial membrane protein known to be implicated in cholesterol transport, protein import, transport of porphyrin, cell proliferation and apoptosis through its interaction with VDAC (Voltage-Dependent Anion Channel) in the mitochondrial permeability transition pore (PTPM). Previous studies have reported overexpression of TSPO in brain tumors, suggesting that this protein may represent a molecular target for the therapy of GBM. In particular, Erucylphosphohomocholine (ErPC3, erufosine), an alkylphosphocholine, seems to be a promising agent in the treatment of glioblastoma. Previous studies have reported its ability to induce apoptosisin otherwise highly apoptosis resistant glioma cell lines and ErPC3 induced apoptosis seems to require the presence TSPO. [18F]DPA-714, a new TSPO radioligand for positron emission tomography (PET) imaging, was developed at the CEA and validated in different models of neuroinflammation. The hypotheses underlying this thesis are: 1) that the overexpression of TSPO in GBM can be detected by PET imaging using [18F]DPA-714 and 2) that the targeting of TSPO, via specific ligands or via ErPC3, can induce apoptosis in GBM. The objectives of the thesis were: 1) to evaluate the expression of TSPO in a panel of rodent and human glioma cell lines and 2) to characterize, in vitro and in vivo, the anti-neoplastic effect of TSPO ligands (PK11195 and RO5-4864) or ErPC3 in glioma cell lines as well as 3) to develop a preclinical model for in vivo PET imaging using [18F]DPA-714 to monitor treatment efficacy of selected ligands. In this thesis, we demonstrated the feasibility of using PET imaging with [18F]DPA-714 to characterize 9L glioma in an orthotopic rat model and to evaluate the effect of ErPC3 treatment, which could provide a new approach to molecular imaging of GBM. We confirmed the pro-apoptotic effect of ErPC3 in the rat 9L glioma cells in vitro and in vivo, and found an infiltration of microglia/macrophages (CD11b-positive) and astrocytes (GFAP-positive) in the tumor area of animals treated with ErPC3. These results open interesting perspectives for clinical research in the treatment of GBM.

Le cancer de la prostate est le cancer le plus fréquent chez les hommes et la capacité des tumeurs à développer une résistance face aux thérapies anti-androgéniques vient souvent compromettre le pronostic des patients. Le développement de nouvelles approches thérapeutiques afin de circonvenir à la progression de ces tumeurs représente un besoin important la gestion de ce type de cancer. Plusieurs démonstrations récentes établissent l’implication de la famille des proprotéines convertases dans la progression tumorale. Ces enzymes ont pour fonctions biologiques de cliver une variété de précurseurs protéiques jouant des rôles importants dans la tumorigénèse. Dans le cancer de la prostate, la proprotéine convertase PACE4 est fortement surexprimée dans les cellules cancéreuses et joue un rôle dans la prolifération et la capacité à former des tumeurs, ce qui en fait une cible thérapeutique d’intérêt. En ce sens, des inhibiteurs peptidomimétiques ont été développés dans l’optique de la thérapie ciblée contre la PACE4. Toutefois, dans le but de développer une approche thérapeutique optimale, il convient néanmoins de comprendre le niveau de redondance fonctionnelle entre les différents membres de la famille des convertases, qui sont connus pour partager plusieurs de leurs substrats, ainsi que les mécanismes moléculaires régissant l’activité de la PACE4 et de ses substrats sous-jacents. L’utilisation d’une approche de répression génique stable envers les différentes convertases a permis de mettre en lumière les fonctions uniques de la PACE4 dans la progression tumorale. De plus, grâce à une approche de protéomique comparative, le premier substrat de la PACE4 dans le cancer de la prostate; le growth differenciation factor 15, a été découvert. Ce substrat permet de commencer à dresser l’implication de PACE4 dans le paysage moléculaire du cancer de la prostate. Grâce à des modalités d’imagerie moléculaire, l’emploi de versions radiomarquées des inhibiteurs peptidiques a également permis de démontrer que les composés s’accumulent dans les cellules cancéreuses en fonction des niveaux de PACE4 présents, et ce, tant in cellulo qu’in vivo. Ces données suggèrent un potentiel pour le développement d’un examen théranostique pour prédire la réponse tumorale à la pharmacothérapie anti-PACE4. Finalement, l’analyse de l’épissage alternatif de l’ARNm de PACE4 a permis l’élucidation des caractéristiques biochimiques et des fonctions spécifiques d’une nouvelle isoforme; la PACE4-altCT, qui est exprimée chez les cellules cancéreuses de la prostate, mais aussi d’autres types de cancer. Cette découverte a permis de redéfinir le modèle de travail en intégrant le concept de la rétention intracellulaire de cette isoforme qui semble médier la plupart de l’activité pro-proliférative reliée à l’activité PACE4, ce qui en fait la cible pharmacologique principale des inhibiteurs peptidiques dans le cancer de la prostate, mais aussi un biomarqueur potentiel. / Abstract: Prostate cancer is the most common cancer among men. The capabilities of tumors to adapt and overcome antiandrogenic therapy is persistently worsening patient’s prognostic and the development of novel therapeutic approaches to circumvent tumor progression therefore represents an unmet need. Many reports now demonstrate the implication of the enzymes from the proprotein convertase family in the progression of tumor from many cancer types. These enzymes are responsible for the processing of various protein precursors playing important roles in tumorigenesis. In prostate cancer, the proprotein convertase PACE4 is strongly overexpressed in cancer cells and plays a role in cell proliferation and tumor formation thus making a strong case for its use as a pharmacological target. For this reason, PACE4 peptidomimetic inhibitors were generated to develop PACE4-targeted therapies. However, to develop an optimal therapeutic approach regarding the inhibition of this enzyme, a complete understanding of the level of functional redundancy between the different convertases in prostate cancer is needed. Moreover, understanding the molecular mechanisms both upstream and downstream of PACE4 in prostate cancer cells would allow a better understanding of the considerations underneath such a therapeutic strategy. Using a stable gene silencing approach to knockdown all co-expressed member of the convertase family in prostate cancer cells, the roles of PACE4 in tumor progression were found to be unique and non-redundant among the other family member. Through a comparative proteomic approach, the first PACE4-specific substrate in prostate cancer; growth and differentiation factor 15, was identified. With this substrate growth factor, it is now possible to initiate the dissection of PACE4 biochemical functions in the prostate cancer molecular landscape. Using a radiolabelled version of the PACE4 peptide inhibitors, it was possible to demonstrate using molecular imaging that when applied in cellulo and in vivo, the compound is uptaken by cancer cells as well as by tissues according to their PACE4 expression levels. These data suggest that such PACE4 molecular imaging with pharmacological inhibitor could be developed as a theranostic assay to predict which tumor could be treated by PACE4-targetted therapy. Lastly, PACE4 mRNA alternative splicing analysis permitted the discovery of a new PACE4 isoform; named PACE4-altCT, which is strongly overexpressed by prostate cancer cells as well as other cancer types. As this isoform displays specific biochemical features and functions, notably being intracellularly retained and mediating most of the PACE4-associated cell growth capabilities, this discovery further redefined our working model, pointing to PACE4-altCT as the pharmacological target of inhibitory peptides in prostate cancer as well as a potential biomarker.

No description available.

The thesis argues that non-proliferation sanctions are effective primarily by their coercive effect, that is their power to change the target’s cost/benefit ratios. It does so by contrasting and comparing two key works in sanctions literature, authored by David Baldwin and the Targeted Sanctions Consortium respectively. In the case of the UN sanctions regime against the Democratic People’s Republic of Korea (DPRK), it concludes that the reason why no sufficient coercive effect has been apparent, is due to the lack of costs shouldered by the actors who have implemented the sanctions, as this reflects an apparent lack of commitment. This in turn does not sufficiently increase the possible cost of the DPRK, in continuing its nuclear weapons program. Therefore it is not incentivized to cancel its program.

Internetövervakning har under senaste tiden blivit ett aktuellt ämne i media. Det rapporteras bland annat om att företag som samlar in information om privatpersoner. Datan som samlas in kan innehålla information om användares beteende på internet såsom besökta sidor, sökhistorik, aktivitet på sociala medier med mera. Men hur mycket av detta vet en vanlig användare om? Uppsatsens syfte är att undersöka hur medvetna privatpersoner är om internetövervakning som bedrivs av företag och den datainsamling som den bygger på. Vad vet egentligen användarna om vad som samlas in, i vilket syfte, samt hur det samlas in? En studie utfördes bestående av intervjuer och enkäter som undersökte hur pass kunniga och upplysta privatpersoner var inom ämnet. Det undersöks också om användare aktivt försöker motverka detta. Därefter diskuteras vad kunskapsnivån kan bero på och vad de gör för att motverka datainsamling. Detta kopplas och jämförs mot tidigare forskning inom området. / Internet surveillance has recently become a hot topic in the media. It is reported among other things that companies collect information about individuals. The data collected may include information about the user's behavior on the internet such as pages visited, search history, activity on social media and more. But how much of this does a regular user know? The purpose of this study is to investigate how aware individuals are of internet surveillance conducted by companies and the data collection which it is based on. What do the users really know about what is collected, for what purpose, and how it is collected? A study was conducted consisting of interviews and questionnaires that examined how knowledge and education individuals have within the subject. It is also examined whether users are actively trying to counteract this. Thereafter it is discussed what this level of knowledge may depend on and what they are doing to counter data collection. This is linked and compared to previous research in the area.

Le carcinome hépatocellulaire est un grand problème de santé publique et la troisième de mortalité lié au cancer dans le monde. Il a été démontré qu'au sein des tumeurs se trouve un petite population des cellules cancéreuses avec des propriétés de cellules souches cancéreuses. Elles sont responsables de l'initiation des tumeurs ainsi que de la récidive post-traitement et résistance aux thérapies. Peu de choses sont connues par rapport à la biologie de ces cellules mais l'identification des facteurs favorisant leur existence pourrait conduire vers des nouvelles pistes thérapeutiques.Nous avons trouvé que le facteurs de transcription p73 est surexprimé chez les patients dans les tumeurs du carcinome hépatocellulaire sous forme de deux types d'isoformes, les isoformes complets et les isoformes tronqués. Les isoformes complets sont des suppresseurs de tumeurs et corrèlent avec un meilleur taux de survie alors que les isoformes tronqués agissent comme dominants négatifs de ces premiers et favorisent la récidive post-chirurgie.Les résultats in vitro ont montré que les isoformes tronqués de p73 sont surexprimés dans les cellules souches cancéreuses du carcinome hépatocellulaire et favorisent leur emergence / Hepatocellular carcinoma (HCC) is a major public health problem, being the second most lethal cancer with an increasing incidence around the world. The only approved systemic drug is the multikinase inhibitor Sorafenib, which prolongs patients’ survival by only three months. HCC is refractory to known chemotherapeutic drugs and more than 50% of patients relapse after surgical tumor removal. These phenomena are thought to be due to the existence of a population of poorly differentiated cancer cells, largely known as liver cancer stem cells (CSCs). Recent studies revealed that CSCs activate similar pathways as normal stem cells. They are therefore highly resistant to therapies and are thought to be capable of self-renewal and generation of tumor’s heterogeneous cell mass. The understanding of mechanisms proper to liver CSCs should allow the development of innovative drugs with original mechanism of action against liver CSC, likely to improve patients’ outcome. However, the development of new therapies against HCC is penalized by the limited number of experimental models.According to these current challenges in the field of HCC research, my PhD thesis project covers three main axes: Development of novel models of disease (IMODI consortium)The Innovative Models of Disease (IMODI) consortium is mainly dedicated to the development of innovative experimental models for 7 different types of cancer. Our participation to the project concerned 3 main objectives i) development of HCC patient-derived xenografts ii) development of new HCC cell lines and iii) set up a cryoconservation method of primary human hepatocytes (PHHs) in the aim to employ them in humanizing murine livers. 30 patients planned for HCC tumor resection were recruited and their clinicopathological data were collected. Fresh tumor specimens were subcutaneously xenografted in immune-deficient mice and dissociated for in-vitro tumor cell culture. One tumor led to the development of a moderately differentiated HCC PDX model, as confirmed by histological characterization. Several studies showed the importance of PDX models in drug discovery as they recapitulate the drug-sensitivity patterns seen in patients from which they derive but very few models have been described in the literature for HCC. In vitro, primary HCC cells could be maintained in culture for a limited period of time, in average 30 days. No HCC cell lines developed due to cells entering replicative senescence, as previously described

La thématique des maladies infectieuses représente un réel enjeu politique, économique et de santé publique. Les objectifs de mes travaux de thèse étaient de développer des approches protéomiques pour identifier, détecter, caractériser et/ou quantifier des protéines et de les appliquer à l’étude de maladies infectieuses pour lesquelles des données de protéomique pourraient ouvrir à de nouvelles approches thérapeutiques et/ou diagnostiques. Les stratégies d’identification et de détection des protéines développées pour l’étude de la maladie de Lyme ont permis de prouver la faisabilité du diagnostic par spectrométrie de masse et de proposer des protéines candidates-vaccin. Les stratégies de quantification mises en place pour l’étude de la toxoplasmose ont permis d’identifier et de quantifier un complexe protéique clef. Les stratégies de caractérisation du N-terminome pour l’étude du paludisme ont permis d’apporter des preuves expérimentales des processus de maturation et d’adressage des protéines. / Infectious diseases represent a real challenge in terms of politic, economic and public health. The purposes of my thesis works were to develop proteomic approaches able to identify, to detect, to characterize and/or to quantify proteins and to apply these approaches to the study of infectious diseases for which proteomic data cou Id open to new therapeutic and/or diagnostic strategies. Identification and specific detection strategies developed in the study of Lyme's disease allowed to prove the feasibility of diagnosis by mass spectrometry and to propose new vaccine-candidate proteins. Quantification strategies developed in the study of toxoplasmosis allowed us to identify and to quantify a key protein complex of the parasite. N-terminome characterization strategy developed in the study of malaria allowed us to bring experimental proofs of proteins processing and trafficking.

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common type of Non-Hodgkin´s Lymphoma (NHL). The standard treatment for DLBCL is R-CHOP chemoimmunotherapy (rituximab, cyclophosphamide, vincristine, doxorubicin and prednisone). About one -third of patients have refractory disease or the lymphoma relapses. Prognosis after relapse of refractory disease is poor. Fitter and younger patients are recommended new intensive salvage chemotherapy followed by autologous stem cell transplantation. Central nervous system (CNS) relapse is the most feared complication with dismal prognosis in DLBCL. High dose methotrexate intravenously administered concurrently with R-CHOP treatment has shown to be most promising to prevent CNS relapses. Primary CNS lymphoma (PCNSL) is a rare aggressive lymphoma limited to the CNS and eyes. PCNSL is a chemo-and radiosensitive disease, but long-term response is rare since the blood brain barrier (BBB) limits access of many drugs to the CNS. BBB disruption (BBBD) is a treatment modality where the BBB is opened by hypertonic mannitol infusion. Administration of chemotherapeutics will achieve over ten-fold concentrations in the CNS and eradicate microscopic disease involvement. This study retrospectively analyses patients who treated as first line with Bonn/Bonn-like treatment (study I), with BBBD treatment followed by high-dose treatment/autologous stem cell transplantation (HDT/ASCT) in first- or second-line (study II) or those treated with primary R-CHOP or its derivatives with or without concurrent CNS-targeted treatment (study III). HD-MTX-based multichemotherapy is an effective induction treatment in CNS lymphoma, but long-lasting responses are rare. BBBD-treatment is well-tolerated and a promising method to attain high drug concentrations in the CNS to eradicate microscopic disease involvement in first- and second-line. CNS-prophylaxis with HD-MTX prevents CNS events in high risk DLBCL. PCNSL is agressive disease despite excellent primary response with HD-MTX based multichemotherapy. BBBD-treatment is a promising method to eradicate microscopic disease in the CNS and achieve a long-term response and cure rate. Fatal CNS relapses can be avoided using CNS-targeted treatment. / Tiivistelmä Diffuusi suurisoluinen B-solulymfooma (DLBCL) on yleisin non-Hodgkin lymfooma (NHL), jonka standardihoitona toimii R-CHOP (rituksimabi, syklofosfamidi, vinkristiini, doksorubisiini, prednisoloni). Noin kolmasosalla potilaista tautii etenee hoidosta huolimatta tai uusii hoidon päätyttyä. Relapoituneen tai refraktaarin taudin ennuste on huono. Hyväkuntoisilla ja nuoremmilla potilailla pyritään etenemään uuteen induktiohoitoon ja korkea-annoshoitoon autologisen kantasolusiirteen turvin. Keskushermostouusiutuma on huonoennusteisin DLBCL:n komplikaatio. Suuriannosmetotreksaattihoito liitettynä R-CHOP-hoitoon estää keskushermostouusiutumia. Primaari aivolymfooma (PCNSL) on harvinainen keskushermoston ja silmien alueelle rajautuva lymfooma. PCNSL on herkkä sytostaatti-ja sädehoidolle, mutta pitkäkestoisia vasteita nähdään harvoin. Veriaivoeste estää useimpien tehokkaiden sytostaattien pääsyn keskushermostoon. Veriaivoesteen aukaisuhoidossa veriaivoeste avataan hypertonisella mannitoli-infuusiolla. Toimenpiteen jälkeisellä sytostaatti-infuusiolla saavutetaan kymmenkertaiset lääkeainepitoisuudet keskushermostossa ja voidaan hoitaa mikroskooppista veriaivoesteen takana sijaitsevaa tautia. Väitöskirjatyön tutkimukset ovat retrospektiivisiä. Ensimmäisessä osatyössä analysoitiin PCNSL potilaat, jotka saivat ensilinjassa Bonnin tai Bonnin kaltaista hoitoa. Toisessa osatyössä potilaat hoidettiin joko ensi- tai toisessa linjassa BBBD-hoidolla, päättyen konsolidaatiohoitona annettavaan korkea-annoshoitoon autologisen kantasolusiirteen turvin. Kolmannessa osatyössä analysoitiin suuren aivouusiutumariskin potilaita, joko yhdessä tai ilman keskushermostoon suunnattua hoitoa samanaikaisesti R-CHOP-hoidon kanssa. Suuriannosmetotreksaatti-pohjainen yhdistelmäsolunsalpaajahoito on tehokas induktiohoito aivolymfoomassa pitkäkestoisten vasteiden ollessa harvinaisia. BBBD-hoito on hyvin siedetty ja lupaava hoitomuoto, jolla keskushermostossa voidaan saavuttaa suuret lääkeainepitoisuudet, jotka riittävät hoitamaan mikroskooppisen taudin sekä ensi että toisessa linjassa. Keskushermostoprofylaksia suuriannosmetotreksaatilla estää keskushermosto-uusiutumia suuren riskin DLBCL-potilailla. PCNSL on agressiivinen tauti huolimatta erinomaisista metotreksaattipohjaisilla hoidoilla saavutetuista ensilinjan vasteista. BBBD-hoito on lupaava keino eradikoida mikroskooppinen tauti keskushermostosta ja saavuttaa pitkäaikaisia hoitovasteita, sekä pysyvä paraneminen aivolymfoomassa. Suuriannosmetotreksaattia sisältävällä sytostaattihoidolla voidaan estää fataaleja aivorelapseja DLBCL:ssä.

The field of early detection and intervention of hearing loss in neonates and infants has been marked by a growing international body of research investigating hearing screening programmes, protocols and outcomes of early detection for hearing loss. In South Africa, screening for neonates and infants in general and particularly for hearing loss is not common practice and is not meeting the needs of the South African population, with very few infants identified with hearing loss early in life. The Year 2002 Hearing Screening Position Statement recommends an intermediate step toward universal screening in the form of Targeted Newborn Hearing Screening (TNHS) as an option for developing countries with limited resources. The Neonatal Intensive Care Unit (NICU) provides a starting point for TNHS because it encompasses a number of risk factors for hearing loss. A combined descriptive and exploratory research methodology was followed to provide a comprehensive perspective on longitudinal hearing screening for NICU neonates and infants at a provincial hospital in South Africa. The quantitative methods included a structured interview to compile risk factor information. Immittance measurements used included acoustic reflex measurements, 226 Hz and 1000 Hz tympanometry. Automated Otoacoustic Emission (AOAE) as well as Automated Auditory Brainstem Response (AABR) screening was conducted. Routine follow-up visits at three month intervals were booked if a subject passed the screen and a follow-up screening for further testing was booked if a subject referred the screening. A total of 49 neonates and infants as well as mothers were enrolled in the first year and followed up for the second year of data collection period. The results indicated that the NICU had potential as platform for TNHS in South Africa. The high incidence of risk factors reported is more when compared with developed countries and highlights the importance of hearing screening in the at risk population for a developing country. The results confirmed reports that 226 Hz probe tone tympanometry produces erroneous responses in young infants. A high correspondence between high frequency tympanometry and AOAE results was found and underlines the need for differential diagnosis to accurately detect middle ear effusion and/or sensorineural hearing loss in neonates and infants. The unilateral AOAE refer rate (7%) was within range of the reported values for initial screening at discharge from the NICU. AABR results indicated a relatively high unilateral refer result (24%) and may be attributed to irritability and restlessness. The highest referral rates in the current study were recorded during the second and third visit and may be attributed to the presence of middle-ear pathology in older infants. The perceptions of mothers emphasized the lack of awareness regarding hearing and hearing loss in South Africa. Lack of knowledge may be a contributing actor to poor compliance with screening follow-up. Despite prevailing challenges, such as a low follow-up return rate, lack of awareness regarding the benefits of early detection of hearing loss, the effect of middle ear effusion on screening results, the cost of hearing screening and different priorities of the national healthcare system, such as Human Immunodeficiency Virus, demonstrated the NICU promise as platform for TNHS in South Africa. TNHS programmes may serve as starting point to direct universal neonatal hearing screening programmes in South Africa. / Dissertation (MCommunication Pathology)--University of Pretoria, 2008. / Speech-Language Pathology and Audiology / MComm Path / unrestricted

Brachydactyly type A1 (BDA1) is a rare autosomal dominant trait characterized by the shortening of the middle phalanges of digits 2-5 and of the proximal phalange of digit 1 in both hands and feet. Many of the brachymesophalangies including BDA1 have been associated with genetic perturbations along the BMP-SMAD signaling pathway. The goal of this thesis is to identify the molecular pathways that are associated with the BDA1 phenotype through the genetic assessment of BDA1-affected families. We identified four missense mutations that are clustered with other reported BDA1 mutations in the central region of the N-terminal signaling peptide of IHH. We also identified a missense mutation in GDF5 cosegregating with a semi-dominant form of BDA1. In two families we reported two novel BDA1-associated sequence variants in BMPR1B, the gene which codes for the receptor of GDF5. In 2002, we reported a BDA1 trait linked to chromosome 5p13.3 in a Canadian kindred (BDA1B; MIM %607004) but we did not discover a BDA1-causal variant in any of the protein coding genes within the 2.8 Mb critical region. To provide a higher sensitivity of detection, we performed a targeted enrichment of the BDA1B locus followed by high-throughput sequencing. We report the identification of a novel 9.5 Kb intergenic tandem duplication in two unrelated BDA1-affected families. In-vitro and in-vivo reporter assays demonstrated the enhancer activity of noncoding conserved sequence elements found within the microduplication. We also show an upregulation of the neighboring genes, NPR3 and PDZD2, in the patients' fibroblasts that suggests a gain-of-function through the duplication of cis-regulatory elements on dose sensitive genes. By expanding the repertoire of BDA1-causing mutations in IHH, GDF5, BMPR1B and at the BDA1B locus, we have begun to elucidate a common genetic pathway underlying phalangeal formation and elongation.