Nonalcoholic fatty liver disease (NAFLD) is categorized into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) and has emerged as a risk factor for more critical clinical conditions. However, the underlying mechanisms of NAFLD pathogenesis are not fully understood. In this study, expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis and autophagy were analyzed in normal, NAFL and NASH human livers by western blotting. Levels of some ER stress-transducing transcription factors, including cleaved activating transcription factor 6, were higher in NASH than in the normal tissues. However, the expression of a majority of the ER chaperones and foldases analyzed, including glucose-regulated protein 78 and ER protein 44, was lower in NASH than in the normal tissues. Levels of apoptosis markers, such as cleaved poly (ADP-ribose) polymerase, were also lower in NASH tissues, in which expression of some B-cell lymphoma-2 family proteins was up-or down-regulated compared to the normal tissues. The level of the autophagy substrate p62 was not different in NASH and normal tissues, although some autophagy regulators were up-or down-regulated in the NASH tissues compared to the normal tissues. Levels of most of the proteins analyzed in NAFL tissues were either similar to those in one of the other two types, NASH and normal, or were somewhere in between. Together, these findings suggest that regulation of certain important tissues processes involved in protein quality control and cell survival were broadly compromised in the NAFLD tissues.

Abstract Leptin is a hormone secreted by adipose tissue. It is involved in the regulation of appetite and energy expenditure. Leptin binds to its receptor (LEPR) that is expressed in the central nervous system as well as in other tissues including adipocytes and endothelial cells. Plasma leptin level reflects the amount of adipose tissue and previously, it has been shown to be associated with the risk for coronary artery disease. Two LEPR polymorphisms, Lys109Arg and Gln223Arg, have been extensively studied and they have been associated with several risk factors of atherosclerosis. Earlier studies have also shown that the risk for developing atherosclerosis and various other diseases might already be determined during the fetal period or immediately after birth. It seems that intrauterine undernourishment might cause changes on epigenetic level and result in alterations in gene expression. It has been suggested that the impaired fetal growth could affect plasma leptin level and leptin messenger RNA expression from adipose tissue. Long-term fructose consumption has also been shown to result in leptin resistance. Recently, leptin has been observed to be associated with autophagy. Autophagy has been demonstrated to act in several interesting processes such as fat storage in adipocytes and liver. Autophagy and the leptin system might also regulate each another. The aim of this thesis was to investigate the association of LEPR polymorphisms with thickness of the wall of carotid artery as well as with fatal and nonfatal cardiovascular disease events. In addition, we aimed to clarify the effects of fetal undernourishment and fructose consumption on the leptin system and autophagy. We were also interested in studying the role of the leptin system and autophagy in elevated triglycerides and liver fat accumulation seen as a result of high-fructose diet. In our studies, we observed that LEPR polymorphisms, Lys109Arg and Gln223Arg, are associated with intima-media thickness of carotid artery. Moreover, 19-year follow-up study showed that 109Arg homozygotes display lower incidence of cardiovascular events and lower total mortality. In our animal experiments, we were able to detect that fructose diet affects both LEPR isoform and autophagy gene expression. It seems that these changes might partly explain the mechanism behind the rise in blood triglyceride levels and liver fat accumulation caused by fructose diet. In conclusion, the results of this study clarify the role of leptin receptor in cardiovascular diseases. In addition, they offer new information especially about the effects of fructose diet on the leptin system, the dysfunction of which might predispose to the development of diseases. / Tiivistelmä Leptiini on rasvakudoksen tuottama hormoni. Se osallistuu ruokahalun ja energiankulutuksen säätelyyn. Leptiini sitoutuu reseptoriinsa (LEPR), joita on sekä keskushermostossa että muissakin kudoksissa, myös adiposyyteissä ja endoteelisoluissa. Plasman leptiinitaso heijastaa rasvakudoksen määrää ja sen on aiemmin osoitettu olevan yhteydessä sepelvaltimotaudin riskiin. Erityisesti kahta LEPR:n polymorfiaa, Lys109Arg ja Gln223Arg, on tutkittu aiemmin ja niiden on osoitettu olevan yhteydessä useisiin ateroskleroosin riskitekijöihin. Aiemmat tutkimukset ovat myös osoittaneet, että ateroskleroosiin ja useisiin muihin sairauksiin sairastumisen riski voi osittain määräytyä jo sikiöaikana tai varhain syntymänjälkeisen kehityksen aikana. Vaikuttaa siltä, että sikiöaikainen aliravitsemus voi aikaansaada muutoksia epigeneettisellä tasolla ja aiheuttaa näin muutoksia geeniekspressiossa. On ehdotettu, että sikiön heikentynyt kasvu vaikuttaisi plasman leptiinitasoon ja rasvakudoksen leptiinin lähetti-RNA:n ilmentymiseen. Pitkäaikaisen fruktoosinkulutuksen on myös osoitettu aiheuttavan leptiiniresistenssiä. Hiljattain leptiinin on havaittu olevan yhteydessä myös autofagiaan. Autofagian on osoitettu vaikuttavan useisiin kiinnostaviin prosesseihin, kuten rasvan varastoitumiseen adiposyytteihin sekä maksaan. Autofagia ja leptiinijärjestelmä mahdollisesti myös säätelevät toisiaan. Tämän väitöskirjan tavoitteena oli tutkia LEPR-polymorfioiden yhteyttä kaulavaltimon seinämän paksuuteen sekä sydän- ja verisuonitautitapahtumiin ja kuolleisuuteen. Pyrimme lisäksi selvittämään sikiöaikaisen aliravitsemuksen ja fruktoosin käytön vaikutusta leptiinijärjestelmään sekä autofagiaan ja olimme kiinnostuneita tutkimaan näiden osuutta fruktoosin kulutuksen seurauksena nähtävien metabolisten muutosten, kuten kohonneiden triglyseridien sekä maksan rasvoittumisen, synnyssä. Tutkimuksessamme havaittiin yhteys LEPR polymorfioiden Lys109Arg ja Gln223Arg sekä kaulavaltimon paksuuden välillä. Lisäksi 19-vuoden seurantatutkimus osoitti 109Arg-homotsygotian liittyvän pienentyneeseen sydän- ja verisuonitapahtumien ilmaantuvuuteen sekä matalampaan kokonaiskuolleisuuteen. Eläinmallissamme havaitsimme sekä LEPR-muotojen että autofagiageenien ilmentymisen muuttuneen fruktoosidieetin vaikutuksesta. Vaikuttaa siltä, että nämä muutokset voisivat osaltaan selittää esimerkiksi fruktoosiruokavalion aiheuttaman veren triglyseriditasojen nousun sekä maksan rasvoittumisen rotilla. Tutkimuksen tulokset selventävät leptiinireseptorin roolia sydän- ja verisuonitautien taustalla. Lisäksi ne tarjoavat uutta tietoa erityisesti fruktoosinkulutuksen vaikutuksesta leptiinijärjestelmään, jonka häiriöt altistavat sairauksien kehittymiselle.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that localises to focal adhesions. FAK is crucial for many cellular processes that are disturbed in malignancy, including proliferation, cell cycle, cell survival, adhesion, and migration. Mouse models have shown that FAK is involved in tumour formation and progression. Other studies demonstrated a functional correlation between FAK expression, tumour progression and malignancy in human cancer, making FAK a potentially important therapeutic target. Several FAK inhibitors have been developed most of which target the FAK kinase function. However, FAK may predominantly act as a scaffolding molecule rather than as a kinase, therefore, disruption of FAK’s interaction with protein binding partners could be a good strategy to inhibit some cancer processes. The identification and characterisation of novel FAK interactions may help to uncover important molecular mechanisms that, in turn, regulate key cellular processes involved in tumour formation and/or progression. Disruption of their function, or inhibition of their binding to FAK, will define their roles and identify whether they are good anti-cancer targets. In this thesis work, I set out to identify novel binding partners of FAK, and study the role of a sub-set of these in tumour biology by impairing them in squamous cell carcinoma cancer cells in vitro. To do this I employed protein microarray and phage display methodologies using FAKΔ375 and FAK-FERM recombinant proteins as bait, respectively. I identified a number of novel proteins that interact directly with FAK. Then I set out to characterise some of these proteins. The first of these, Axl, is a protein receptor tyrosine kinase that has previously been linked with tumour progression and metastasis in number of human cancers. I confirmed the interaction between FAK and Axl in SCC cells and showed that the FAK-Axl interaction is predominantly a scaffolding function of FAK, which seems to be unregulated, at least by any of the major phosphorylation events characterised for FAK. I also found that Axl controls cell spreading, cell polarisation and invasive migration in this cancer cell lines. The second protein I characterise is the autophagy protein Ambra1. I found that Ambra1 is required for selective targeting of active Src to the autophagy pathway – a process that SCC cancer cells use when they are under adhesion stress, such as when FAK is deleted. Thus, Axl and Ambra1 are potentially important proteins in SCC biology. They bind to FAK and function at cell adhesions to promote cancer-associated cellular processes. Analysis of FAK binding proteins may be a useful strategy to discover proteins that function in various aspects of cancer cell behaviour.

Tauopathies are neurodegenerative diseases that affect millions of people around the world. Tauopathies include more than 20 neurodegenerative diseases. Some of the most common tauopathies are Alzheimer’s disease (AD), frontotemporal dementia (FTD), chronic traumatic encephalopathy (CTE), Pick’s disease, corticobasal degeneration, progressive supranuclear palsy (PSP), agyrophillic grain disease, and amyotrophic lateral sclerosis (ALS). These diseases can cause significant memory loss, behavioral changes, motor deficits and speech impairments. Tauopathies stem from accumulation of the microtubule associated protein tau (MAPT). Tau stabilizes microtubules and helps with axonal transport. In a disease state tau becomes hyperphosphorylated and truncated leading to its aggregation. More recently tau has been shown to propagate from cell to cell potentially acting as a signaling molecule that contributes to disease progression. In addition during disease, tau mislocalizes to dendrites leading to synaptic dysfunction. This mislocalization may also lead to subsequent neurodegeneration. Today, many strategies have been implemented to treat tauopathies. Some of these strategies include kinase inhibitors, immunotherapy, tau aggregation inhibitors, and microtubule-stabilizing compounds. However none these strategies have been effective in stopping tau pathology nor do they address tau degradation pathways. Therefore we hypothesized that utilizing small molecules that target degradation pathways such as autophagy or proteasomal degradation would improve clearance of aberrant tau. We previously showed that a natural product (+)-aR,11S-myricanol (1) from Myrica cerifica (bayberry/southern wax myrtle) root bark reduced levels of tau. In this study we discovered that 1 is composed of two enantiomers and two possible atropisomers. We found that one enantiomer (-)-aS,11R-myricanol (3) was responsible for the anti-tau activity of 1 in multiple models of tauopathy. We also found that 3 selectively targets and lowers specific tau species. To better understand how these tau species were being reduced we took a non-biased approach and subjected 3 treated samples to stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry (MS) proteomic analysis. We found that autophagy pathways were most affected by 3 and that 3 was predicted to mimic the drug rapamycin, a well-established macroautophagy activator. In addition we confirmed our MS findings by simultaneously giving 3 treated cells an autophagy inhibitor which blocked 3’s tau reductions. Moreover we created a tetralin derivative of 1, 13, that produced the same effects on tau as 3 but did not rely upon stereochemistry for its activity. This work supports targeting the autophagy degradation pathway as a viable approach to improving aberrant tau accumulation. In order to further support our hypothesis, we collected and screened several known heat shock protein 70 (Hsp70) inhibitors and tau aggregation inhibitors for cellular anti-tau activity. While it is known that Hsp70 inhibition facilitates tau clearance through proteasomal degradation, it is not known what role tau aggregation inhibition plays in the cellular degradation of tau. Moreover understanding which inhibitory activity contributes most to tau degradation would lead to the creation of better drug scaffolds. In this study, we found that several Hsp70 inhibitors from different scaffold backbones had varying effects on tau degradation. The rhodacyanine and phenothiazine compounds were most effective at lowering cellular tau while the adenosine analog, sulfonamide, dihyropyrimidine, piperidine-3-carboxamide, phenoxy-N-arylacetamide, and flavonol, were not as effective. We also examined the effects of several tau aggregation inhibitor scaffolds such as the carbocyanine, oleuropein, anthraquinone, aminothienopyridazine, hydroxytyrosol and rhodanine on tau expression reduction. We found that none had effective tau reductions except the carbocyanine. However when we performed a lactate dehydrogenase (LDH) assay, carbocyanine was shown to be extremely toxic. These results lead us to further investigate if the tau expression reducing Hsp70 inhibitors had anti-tau aggregation activity and if the tau aggregation inhibitors had any Hsp70 inhibitory activity. We discovered that many of the Hsp70 inhibitors also had anti-tau aggregation activity while none of the aggregation inhibitors had Hsp70 inhibitory activity. We found a positive correlation between tau expression reductions and anti-tau aggregation activity for the Hsp70 inhibitors. Our work demonstrates that both Hsp70 activity and tau aggregation in vitro best predicts anti-tau activity of small molecules. Also these dual acting Hsp70 inhibitors support our hypothesis that targeting the degradation pathways can improve tau clearance. Overall, this work indicates the importance of targeting degradation pathways to improve tau clearance. Utilizing small molecules that have dual activities against tau could prove beneficial as a novel therapeutic approach to treat tauopathies. In addition using small molecules that target different degradation pathways simultaneously could be another viable therapeutic strategy for treatment of tauopathies.

Glaucoma is a neurodegenerative protein misfolding disorder classified by increases in IOP, damage to retinal ganglion cells (RGCs), optic nerve (ON) head damage, and progressive irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form of glaucoma, constituting over 90% of clinical cases. POAG is observed in patients where normal outflow channels, mainly the trabecular meshwork (TM), are exposed at the angle formed by the iris and cornea. However, due to TM cellular dysfunction, aqueous outflow resistance is increased preventing normal circulation of aqueous humor. Recent studies have shown that in 2-4% of POAG cases, increased intracellular levels of a secreted glycoprotein, called myocilin, are present in the TM. Myocilin is a 504aa glycoprotein, with an unknown precise function. Recent studies have postulated the importance of myocilin in oligodendrocyte differentiation, axonal myelination, and early apoptosis of retinal cells in development, but exact function is still widely debated. However, it is important to note that pathology associated with myocilin is only seen during POAG. Also, only cells of the TM exhibit toxicity when overexpressing mutant myocilin. In the normal eye, myocilin is secreted from the ER of TM cells, however, mutations in the MYOC gene lead to an aggregation-prone form of the protein, which is inefficiently processed and degraded from the ER, leading to build-up and associated toxicity. There are over 70 known MYOC mutations associated with glaucoma, with over 90% occurring in the C-terminal OLF domain. Some of the more common, pathological mutations are: I477N, Y437H, P370L, W286R, N480K. All of these mutations have been observed in patients with glaucoma, and all lead to build-up and aggregation of the mutant protein within the ER of TM cells. Recently, work out of our lab has discovered an interaction between mutant forms of myocilin and the chaperone Grp94. Grp94 is the resident Hsp90 isoform of the ER. Grp94 is an important chaperone in ER quality control, aiding in the output of properly folded secretory and membrane-bound proteins. Besides protein folding, other roles of Grp94 in the ER include: calcium buffering, roles in ER quality control (including targeting misfolded proteins for ERAD), peptide binding, and roles in ER stress. Generally, terminally misfolded proteins in the ER are degraded through ERAD; the Grp94 mediated shuttling of misfolded proteins to the ER trans-membrane machinery for ubiquitination and subsequent translocation to the cytosol for proteasomal degradation. However, in the case of POAG, ERAD is inefficient in mutant myocilin degradation, causing protein accumulation within the ER. In this study, we demonstrate that specific Grp94 inhibition of interaction with mutant or misfolded myocilin leads to myocilin degradation and subsequent lowering of protein accumulation in the TM, thus reducing downfield pathology associated with POAG. Grp94 preserves mutant myocilin in the ER of TM cells leading to protein accumulation and aggregation precipitating TM cellular dysfunction. We showed in various in vitro cell assays that Grp94 inhibition leads to a reduction in intracellular protein levels, while alleviating TM cellular toxicity. Furthermore, in the Tg-MYOCY437H mouse model of POAG, we showed that topical ocular administration of a specific Grp94 inhibitor alleviated glaucomatous pathologies, including elevated IOP, myocilin accumulation in the TM, reduced scotopic/photopic visual responses, and RGC health and viability. Finally, we have proven the importance of ER-stress pathway malfunction in the development of POAG pathology, while also discovering the involvement of the autophagy mechanism for myocilin degradation following Grp94 inhibition. Overall, this work proves that Grp94 is an important regulator of myocilin pathology during POAG. While there are no current therapeutics on the market that directly target the underlying POAG disease mechanism, specific Grp94 inhibition shows great promise and should be considered for human clinical trials. If successful, specific targeted Grp94 inhibition could be the first curative therapeutic options for patients suffering from myocilin-associated POAG.

Amyotrophic Lateral Sclerosis (ALS) is a rare and fatal neurodegenerative disorder resulting in the loss of motor neurons from the spinal cord and frontal cortex. The patterns of neurodegeneration, affected regions, age of onset, and time course of disease progression are all highly variable between and within variants of the disease. Familial ALS (fALS), inherited versions of ALS due to genetic changes, accounts for between 5-20% of all ALS cases, while the rest are sporadic, with either no causative mutation identified or no familial history of ALS. Recently, the discovery of C9orf72 hexanucleotide repeat expansions have been identified as one of the most common causes of familial ALS, with some patients presenting with dual phenotypes of ALS and frontotemporal dementia, leading to new hypotheses about the nature of neurodegenerative diseases. Despite the continued discovery of new ALS causative genes, little is known about the pathogenesis of the disease. While almost all variants include the presence of intracellular protein inclusions, the site of the protein plaques and involved proteins varies between genetic and phenotypic variants of this disease. Due to the lack of clear pathogenic mechanisms, several hypotheses have been developed to explain the process of neurodegeneration. Autophagy, the process of self-eating, leading to destruction of damaged or excess proteins and organelles, has been implicated as being altered in ALS. Multiple variants have demonstrated altered mitochondrial morphology and cellular energetic dynamics, which could explain previous observations that implicate the process of apoptosis in cellular death. Many of the involved proteins in ALS have functional roles for intracellular, nucleocytoplasmic, and axonal transport of various proteins or RNA. These three competing hypotheses are currently the most prominent hypotheses in the pathogenesis of ALS, and have largely been considered as separate and competing in past research. Is there a chance that the true pathogenesis leading to neuronal destruction via apoptosis involve all three hypotheses? Altered protein and RNA transport dynamics could lead to changes in cellular stress responses or overload autophagy pathways, leading to exacerbated cellular stress responses, leading to alterations in mitochondrial morphology and eventually cell death via apoptosis.

Philosophiae Doctor - PhD / Breast cancer is one of the most common cancers among women in South Africa and the second leading cause of cancer death after lung cancer. According to the American Cancer Society 2015, women have a 12% chance of developing invasive breast cancer and a 3% chance of dying from it. Despite the wide variety of breast cancers e.g. lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS), many share the same etiology and target tissue. Estrogen related carcinogenesis with regard to breast cancer typically results from the activation of distinct signalling pathways. These pathways are not mutually exclusive and are often constituted by receptor mediated stimulation of cell proliferation caused by specific transcriptional gene activation, reactive oxygen species (ROS) formation causing DNA damage and consequently mutations. The molecular pathways that cause drug resistance are not fully understood and the search continues to find novel targets for treatment. The effects of non-toxic triterpenes, oleanolic acid and ursolic acid and the role of autophagy and apoptosis as mechanisms to overcome drug resistance in breast cancer were studied in vitro in MCF-7 breast cancer cells and MCF10A breast cells. In this study the first aim was to establish the influence of OA and UA on cell growth and to see if opposing proliferation patterns could observed between the presumably ERɑ negative (ERɑ/ß -/+) MCF-10A and ERɑ positive (ERɑ/ß +/+) MCF-7 cells. This was followed by morphology studies to establish the possible presence of cytotoxicity and examination of molecular pathways contributing to the anti-cancerous properties of UA and OA and their validity as therapeutic agents. The MCF-7 breast cancer cell line and the immortalized normal mammary cell line, MCF-10A were treated with different concentrations of UA and OA for 6hrs, 12hrs, 24hrs, 48hrs, and 72hrs respectively. Cell morphology was studied in hematoxylin and eosin as well as Hoechst and acridine orange stained cells and viability was measured using crystal violet staining. Molecular techniques employed included the Tali® Apoptosis - and the cellROX assays, flow cytometry and western blotting. Morphological, viability and apoptotic studies have shown that at their lowest concentration, both UA and OA have anti-proliferative and apoptotic effects on MCF-7 and to a lesser extent on MCF-10A. Flow cytometric analysis of treated cells has demonstrated cell arrest in the S- and G2/M phase. The MCF-7 and MCF-10A cells growth inhibition effect may be due to increased autophagy and apoptosis as an alternative to decreased proliferation in MCF-7 cells. This possibility should be evaluated in further studies. The results showed that UA was more effective OA in decreasing cell numbers and it may be applied as treatment for breast cancer. Our observation has shown the treatment with OA and UA increased cell death in MCF-7 cells.The opposing proliferation patterns observed between the presumably ERɑ negative (ERɑ/ß -/+) MCF-10A and ERɑ positive (ERɑ/ß +/+) MCF-7 cells could possibly be ascribed to ERß forming homodimers that may facilitate proliferation, whereas ERɑ/ß heterodimers (expressed in 59% of breast cancers) are frequently associated with the ERɑ antagonising actions of ERß. The results indicate a trend towards biphasic and anti- proliferative effects of the reactants in breast cancer cells which may contribute towards the development of anti- cancer therapies. However, further work is must be done to identify the OA and UA mechanism(s) responsible for anticancer activity. / Libyan Embassy

La maladie de Paget est caractérisée par un remodelage osseux anarchique débutant par une phase de résorption excessive suivie d’une phase de formation désordonnée. Comme les ostéoclastes sont recrutés en plus grand nombre et sont hyperactifs aux sites affectés par la maladie, cette cellule a été incriminée dans ce désordre osseux. Le phénotype de l’ostéoclaste pagétique comporte de plus un défaut du processus autophagique, de même qu'une résistance à l'apoptose, dont les mécanismes restent mal connus. Certains facteurs génétiques et environnementaux contribuent en partie au phénotype, mais d'autres facteurs pourraient y être associés. Des travaux du laboratoire ont mis en évidence six événements de l’épissage alternatif associés à la maladie de Paget. Parmi les gènes identifiés, le gène TBC1D25 et ses deux isoformes connus n’ont jamais été étudiés dans l’ostéoclaste. Le gène TBC1D25 possède un domaine hautement conservé TBC régulant l’activité des petites GTPases Rabs dans le transport vésiculaire et un domaine LIR liant la protéine LC3 durant le processus de l’autophagie. Ces domaines fonctionnels se retrouvent seulement dans l’isoforme long. L’hypothèse de recherche est que l’altération de l’épissage alternatif du gène TBC1D25 dans les ostéoclastes pagétiques explique en partie le phénotype de la cellule. Le changement dans le ratio de l’expression des isoformes affecterait le processus de l’autophagie, en plus d’affecter la principale fonction de l’ostéoclaste, la résorption osseuse. L’objectif principal de l’étude est de caractériser l’expression et la fonction de TBC1D25 dans les ostéoclastes humains. Des ostéoclastes humains différenciés à partir de monocytes fœtaux ont été utilisés pour l’étude de la fonction de TBC1D25 dans l’autophagie, l’apoptose et la résorption osseuse. Les travaux ont permis de localiser les protéines dans l’ostéoclaste dans des conditions maintenant un niveau basal de l’autophagie et dans des conditions induisant l’autophagie. L’interaction entre TBC1D25 et Rab34 a été confirmée pour la première fois dans les ostéoclastes. De plus, une variation de cette interaction a été observée dans les différentes conditions modulant le niveau d’induction de l'autophagie. Les résultats préliminaires montrent une augmentation du ratio LC3II/LC3I lors de la diminution de l’expression de TBC1D25 dans des conditions augmentant l’induction de l'autophagie. Par contre, aucun effet a été observé sur la résorption osseuse ou sur l'apoptose lors de la diminution de l’expression de TBC1D25. En conclusion, les résultats préliminaires montrent que TBC1D25 préviendrait l’augmentation du ratio LC3II/LC3I dans l’ostéoclaste soit en inhibant l’induction de l’autophagie ou en favorisant la dégradation des autophagosomes par l’entremise de son action sur Rab34. / Abstract : Paget’s disease of bone (PDB) is characterized by increases in bone turnover starting with excessive resorption followed by disorganized bone formation. Because the initial phase of PDB involves excessive bone resorption, osteoclasts have been identified as the cells primarily affected in PDB. Pagetic osteoclasts are overactive, resistant to apoptosis and exhibit defects in autophagy, but the mechanisms involved are still unclear. While genetic and environmental factors associated with PDB may partially account for the osteoclast phenotype, other genetic contributors have been identified. Recent work from our laboratory has identified six alternative splicing events associated with PDB. Among those genes, TBC1D25 and its two known isoforms have never been studied in osteoclasts. The two functional domains of TBC1D25 (TBC and LIR) are only present in the long isoform. The highly conserved TBC domain regulates small Rab GTPases in vesicular transport and the LIR domain interacts with LC3 during autophagy. Our research hypothesis is that altered alternative splicing of TBC1D25 in pagetic osteoclasts could contribute to phenotype. Differential isoform expression could affect osteoclast autophagy and bone resorption. The aim of the study is to characterize the expression and function of TBC1D25 proteins in human osteoclasts. Osteoclasts differentiated from cord blood monocytes were used to investigate the function of TBC1D25 in autophagy, apoptosis and bone resorption. First, the localization of the protein has been characterized in conditions maintaining basal autophagy and in rapamycin-induced autophagy. Interactions between TBC1D25 and Rab34 have been observed for the first time in osteoclasts. Moreover, changes in the interaction were observed with autophagy induction. Preliminary results suggest increases in LC3II/LC3I ratio with decreasing TBC1D25 expression when autophagy induction is stimulated. On the other hand, preliminary results showed that decreased expression of TBC1D25 did not affect bone resorption, nor apoptosis. In conclusion, preliminary results show that in osteoclasts, TBC1D25 could prevent the increase of LC3II/LC3I ratio by inhibiting autophagy induction or by promoting the clearance of autophagosomes through its action on Rab34.

Le gène BAG3 a été identifié comme étant un nouveau gène responsable de cardiomyopathie dilatée (CMD), première cause d'insuffisance cardiaque (IC). La protéine BAG3 est une co-chaperonne qui participe au contrôle de l'homéostasie protéique via son rôle dans l'autophagie, protégeant ainsi les cellules contre la protéotoxicité induite par les protéines dégradées ou mal repliées. L'hypothèse qu'une inactivation de la voie autophagique contrôlée par BAG3 induirait une protéotoxicité cardiomyocytaire à l'origine de la CMD apparait particulièrement attractive et constitue l'hypothèse centrale de ce travail. Nos résultats indiquent que les mutations de BAG3 abolissent l'interaction avec la chaperonne HSP70, une protéine centrale du contrôle qualité des protéines. Nous avons observé une cytotoxicité des mutants BAG3, une altération de la fonction chaperonne HSP70-dépendante et une absence de réponse autophagique en condition de stress (jeun, choc thermique, expression d'une protéine pro-agrégante). In vivo (modèle poisson-zèbre) l'extinction de l'expression de BAG3 ou la surexpression des mutants conduisent à l'apparition d'un phénotype d'insuffisance cardiaque (¿dème péricardique) chez les embryons injectés. Par édition génomique, nous développons également un modèle de cardiomyocytes dérivés de cellules iPS porteurs ou non de la mutation afin d'explorer plus en avant la fonction contractile de ces cellules. Nos résultats confirment donc le rôle de BAG3 dans la CMD et indiquent que l'altération de la fonction protéostasique serait à l'origine de la maladie. Cette nouvelle voie physiopathologique dans la CMD pourrait s'avérer être, plus généralement, une voie centrale dans l'IC. / The BAG3 gene was identified as a novel gene responsible for dilated cardiomyopathy (DCM), a major cause of heart failure (HF). The BAG3 protein is a co-chaperone that participates in the control of protein homeostasis via its role in autophagy, protecting cells against the proteotoxicity induced by degraded or misfolded proteins. The hypothesis that inactivation of the autophagic pathway controlled by BAG3 would induce cardiomyocyte proteotoxicity behind the CMD appears particularly attractive and is the central hypothesis of this work. Our results indicate that BAG3 mutations abolish the interaction with the chaperone HSP70, a central actor of the protein quality control. We observed cytotoxicity of BAG3 mutants, an impaired HSP70-dependent chaperone function and absence of autophagic response under stress conditions (starvation, heat shock, expression of a pro-aggregating protein). In vivo (zebrafish model) the extinction of BAG3 expression or mutants overexpression lead to the occurrence of a heart failure phenotype (pericardial edema) in injected embryos. Through genomic edition, we also develop a model of iPS-derived cardiomyocytes carrying or not the mutation in order to further explore the contractile function of these cells. Our results confirm the role of BAG3 in DCM and indicate that the alteration of the proteostasis function is the cause of the disease. This new pathophysiological pathway in DCM may prove to be more generally, a central line in the IC.

Cervical cancer is reported by the World Health Organisation to be the second most common type of cancer to affect women in poorer socioeconomic countries. Treatment of this pathology remains sub-optimal at advanced stages and continues to be of importance on the research agenda. Previous studies have reported cytotoxic and antiproliferative effects of 2-methoxyestradiol (2-ME) in vitro on a HeLa cervical cancer cell line. These results were promising but use of 2-ME itself is limited due to pharmacodynamic constraints. In an attempt to overcome these, a sulphamoylated analogue of 2-ME, namely 2-ethyl-3-O-sulphamoyl-estra- 1,3,5(10)16-tetraene or compound 19 (C19), was synthesised. In this in vitro study, the induction of a block in mitosis with subsequent culmination of apoptosis and autophagy as types of cells death was investigated after HeLa cells were exposed for 24 hours to a 0.5 μM C19 solution. This was achieved by morphological assessment (fluorescent, Polarization-optical transmitted light differential interference contrast microscopy (PlasDIC) and transmission electron microscopy (TEM)) and flow cytometry (cell cycle progression, cyclin B1 analysis, phosphatidylserine (PS) flip and aggresome formation). Spectrophotometric quantification of the apoptotic initiator and executioner caspases 8 and 3 respectively was done to determine their involvement in the crosstalk between apoptosis and autophagy. Results included the following: (i) PlasDIC microscopy illustrated the appearance of an increased number of cells blocked in metaphase, stress signaling, premature cell shrinkage, hypercondensed chromatin and the presence of apoptotic bodies after C19 exposure. The presence of ghost cells, cell debris and decreased cell density of the treated cells correlated with the autophagy control. (ii) Fluorescence microscopy employing triple staining highlighted an increased lysosomal activity and staining of C19-exposed cells when compared to the control, as well as evidence of apoptotic and metaphase-blocked cells. This is indicative of both the autophagic and apoptotic cell death process. (iii) TEM allowed for examination of the ultrastructure of the intracellular processes, and revealed that apoptotic cells have hallmarks of both autophagy and apoptosis, confirming the results of light microscopy. (iv) Cell cycle analysis demonstrated more cells present in the sub-G1 and G2/M populations, indicating the induction of apoptosis (confirmed with PS fip flow cytometric quantification) and a metaphase block (corroborated by an increased cyclin B1 fluorescence). (v) The increase in autophagosome formation seen on fluorescence- and transmission electron microscopy was confirmed by flow cytometry demonstrating an upregulation of aggresome formation in C19-exposed cells. This investigation demonstrated induction of both types of cells death by this novel compound. (vi) The upregulation of caspases 8 and 3 was demonstrated in the C19-treated cells, indicating apoptosis induction via the extrinsic pathway. (vii) Confocal microscopy demonstrated complete microtubule disintegration in the C19-exposed HeLa cells. Both apoptotic and autophagic cell death mechanisms were induced in C19-treated HeLa cells after spindle abrogation kept the cells in metaphase block. Insight gained into the molecular effect of C19 on HeLa cells may be used as a springboard for in vivo studies, furthering the development of this promising anticancer agent toward clinical application. / Dissertation (MSc)--University of Pretoria, 2012. / Physiology / MSc / Unrestricted