Awareness of detoxification, nowadays known as biotransformation, has become an integral part of our daily lives. It is a modern buzz word that is used to promote anything from health food to enhancement of performance in sports. Another lesser known application for detoxification is as a therapy for alleviating symptoms of inborn errors of metabolism. Detoxification is the process where endogenous and xenobiotic metabolites are transformed to less harmful products, in the liver and kidneys, in two phases. Phase 1 detoxification includes oxidation, hydroxylation, dehydrogenation metabolic reduction and hydrolysis. Phase 2 detoxification uses conjugation reactions to increase hydrophillicty of metabolites for excretion in bile and urine. Glycine N-acyltransferse (GLYAT; EC 2.3.1.13) is one of the amino acid conjugation enzymes. There are two variants of human GLYAT. I focused on the full-length mRNA human GLYAT isoform a, with a long term view of using it as a viable therapeutic enzyme for enhanced detoxification of harmful metabolites. I investigated if it is possible to clone and express a biologically active GLYAT. To achieve this goal I used three expression systems: traditional bacterial expression using the pET system; second generation cold shock bacterial expression using the pCOLDTF expression vector to improve solubility of the recombinant protein; and baculovirus expression in insect cells since therein some form of post translation glycosylation of the recombinant protein can occur which might improve solubility and ensure biological activity. The recombinant GLYAT expressed well in all three expression systems but was aggregated and no enzyme activity could be detected. A denature and renature system was also used to collect aggregated recombinant GLYAT and used to try to refold the recombinant protein in appropriate refolding buffers to improve solubility and obtain biological activity. The solubility of the recombinant GLYAT was improved but it remained biologically inactive. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

Awareness of detoxification, nowadays known as biotransformation, has become an integral part of our daily lives. It is a modern buzz word that is used to promote anything from health food to enhancement of performance in sports. Another lesser known application for detoxification is as a therapy for alleviating symptoms of inborn errors of metabolism. Detoxification is the process where endogenous and xenobiotic metabolites are transformed to less harmful products, in the liver and kidneys, in two phases. Phase 1 detoxification includes oxidation, hydroxylation, dehydrogenation metabolic reduction and hydrolysis. Phase 2 detoxification uses conjugation reactions to increase hydrophillicty of metabolites for excretion in bile and urine. Glycine N-acyltransferse (GLYAT; EC 2.3.1.13) is one of the amino acid conjugation enzymes. There are two variants of human GLYAT. I focused on the full-length mRNA human GLYAT isoform a, with a long term view of using it as a viable therapeutic enzyme for enhanced detoxification of harmful metabolites. I investigated if it is possible to clone and express a biologically active GLYAT. To achieve this goal I used three expression systems: traditional bacterial expression using the pET system; second generation cold shock bacterial expression using the pCOLDTF expression vector to improve solubility of the recombinant protein; and baculovirus expression in insect cells since therein some form of post translation glycosylation of the recombinant protein can occur which might improve solubility and ensure biological activity. The recombinant GLYAT expressed well in all three expression systems but was aggregated and no enzyme activity could be detected. A denature and renature system was also used to collect aggregated recombinant GLYAT and used to try to refold the recombinant protein in appropriate refolding buffers to improve solubility and obtain biological activity. The solubility of the recombinant GLYAT was improved but it remained biologically inactive. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

The androgen receptor (AR) fulfils important roles for both sexes. By mediating the biological function of androgens, the AR has remained the target for endocrine therapies treating prostate cancer. The AR also determines the effectiveness of medroxyprogesterone acetate (MPA) in treating AR positive breast cancer. Every man will be affected by prostate cancer if he lives long enough. Prostate cancer continues to be a leading cause of death for males despite research into this cancer covering more than 60 years since Huggins' seminal 1941 study showing that androgens play a key role in this cancer. Unfortunately, significant advances have not been forthcoming and the effect of treatment has remained largely the same over past decades, whereby initial treatment provides temporary remission but eventually advanced cases become refractory to further intervention and the disease recurs in a more aggressive form. A plethora of factors are exquisitely sensitive to minute changes in the AR's structural profile, which can be altered by a single mutation, resulting in aberrant activity. A principal feature of these variant ARs associated with prostate cancer, is enhanced capacity to bind a number of molecules other than its cognate ligand, dihydrotestosterone (DHT). The promiscuous activity of this receptor leads to continued AR signalling and stimulus for the cancer despite low androgen levels induced by treatment regimes. A key question is whether mutations occurring within the AR occur as a result of cancer or contribute to the propagation of the cancer. Recent research has demonstrated that treatments incorporating anti-androgens such as flutamide, which are designed to impede prostate cancer progression by inhibiting AR activity, may actually provide selective pressure favouring somatic mutation of the receptor to take place. The specific changes to the AR which are responsible for gains of function have not been resolved as their crystal structures, which are used to provide conformational analysis of proteins, are tremendously problematic to produce with little success found in literature. Generating representative crystals of the AR protein involves producing soluble recombinant protein. Unfortunately the AR is prone to aggregation and is highly unstable, especially in the presence of antagonistic molecules or absence of a stabilising ligand, preventing the protein from being maintained in the soluble state required for crystallization. In order to produce sufficient quantities of soluble material for crystallization, the androgen receptor's ligand binding domain (LBD) was produced as a recombinant protein in Escherichia coli bacteria strain BL21 (DE3) in the presence of DHT, flutamide, as well as in the absence of ligand. Since soluble unbound AR-LBD has not been produced until now, the bacterial culture containing no ligand was further processed to the stage of cleaving the purification tag from the recombinant protein and represents considerable progress into producing soluble material for crystallizing the troublesome yet considerably important AR in the absence of ligand. Although distinct from prostate cancer in males, AR activity in breast tissue is also a factor determining the action of drugs, such as MPA, included in therapies aimed at breast cancer. The use of MPA has declined primarily due to its adverse effects including unsuccessful generation of a biological response, as well as the advent of other drugs administered for hormonal therapies treating breast cancer. Alternative drugs are needed when breast cancer therapies fail as tumours develop resistance to primary drugs. Although there are a number of drugs on the market, success would be maximised if the determined therapy is matched with the patient, based for example, on their genetic makeup. There is a conundrum whereby some patients with an AR do not respond to MPA, a drug normally recognised by the receptor. In clinical trials it was discovered that an AR with threonine instead of methionine at residue 780 (M780T) fails to activate in response to MPA, but the exact mechanism has remained elusive and needs to be answered at the molecular level. The X-ray crystallographic studies that generate 3D images of macromolecules and wet chemistry, which have traditionally been used to provide insight into science in these dimensions, are incorporated with computer based molecular simulation. This is both complementary and distinct to traditional scientific methodologies, enabling further elucidation of protein-protein interactions, and the influence applied to such inter-relations by natural and drug ligands. This approach has been used, and is continually developed, to understand the binding mechanisms of current drugs as well as designing new drugs. In order to produce a receptor representing the M780T variant, the crystal structure representing the AR-LBD was mutated in silico, into which MPA was then docked. It was found that MPA binds into the M780T AR-LBD with considerably more spatial displacement compared to the position of DHT in the crystal structure, and is predicted to be the primary reason for the inability of MPA to activate this variant AR. The clarification of MPA binding and failure to elicit a response from the variant AR is significant for a cohort of breast cancer patients, as not only does the presence of an AR in the tumour determine the effectiveness of MPA, but correct composition of the AR, specifically, the absence of a M780T mutation. In the absence of this AR mutation, MPA could effectively be used either as an alternative to primary drugs, or in secondary therapies when primary therapies fail. Aberrant activity of variant ARs in response to MPA should also be taken into consideration when analysing drug studies about the effectiveness of MPA. The findings on the loss of response to MPA by the M780T variant AR have been included in the journal article &quotDecreased Androgen Receptor Levels and Receptor Function in Breast Cancer Contribute to the Failure of Response to Medroxyprogesterone Acetate" appearing in the September 2005 issue of Cancer Research journal.

Snake venoms are a complex mixture of polypeptide and other molecules that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Amongst the most potently toxic venoms in the world are those of the Australian venomous snakes, which belong almost exclusively to the elapid family. Their venoms posses a number of unique properties by which they target the mammalian cardiovascular and neuromuscular systems and are the focus for the identification of novel pharmacologically interesting compounds which may be of diagnostic or therapeutic benefit. Although much is known about the biochemical properties of Australia snake venoms as a whole, little research attention has focused upon individual components at the molecular level. This thesis describes the cloning, characterisation and comparative analysis of a number of unique toxins from the venom gland of the coastal taipan (Oxyuranus scutellatus) and a total of seven other related Australian snakes. These include the factor X- and factor V-like components of a prothrombin activator that causes a highly coagulable state in mammals. Comparative analysis of the sequences identified in this study, along with recombinant expression of an active form of the factor X-like component, provides important information on the structural, functional and evolutionary relationships of these molecules. Numerous other toxins were similarly identified and characterised including a pseudechetoxin-like protein, multiple phospholipase A2 enzymes and neurotoxin isoforms as well as vasoactive venom natriuretic peptides. Identified transcripts included not only toxin sequences but also other cellular peptides implicated in toxin processing, including a calglandulin-like protein. This thesis is the first description of the majority of these molecules at either the cDNA or protein level, and provides a means to study the activity of individual components from snake venoms and probe their function within the systems they specifically target. This study represents the most detailed and comprehensive description to date of the cloning and characterisation of different genes associated with envenomation from Australian snakes.

The venom of cone snails (predatory marine molluscs of the genus Conus) has yielded a rich source of novel neuroactive peptides or “conotoxins”. Conotoxins are bioactive peptides found in the venom duct of Conus spp. Like other neuropeptides, conotoxins are expressed as propeptides that undergo posttranslational proteolytic processing. Peptides derived from propeptides are typically cleaved at a pair of dibasic residues (Lys-Arg, Arg-Arg, Lys-Lys or Arg-Lys) by proteases found in secretory vesicles. However, many precursor peptides contain multiple sets of basic residues, suggesting that highly substrate specific or differentially expressed proteases can determine processing outcomes. As many of the substrate-specific proteases remain unidentified, predicting new bioactive peptides from cDNA sequences is presently difficult, if not impossible. In order to understand more about the substrate specificity of conotoxin substrate-specific proteases a characterisation study of one such endoprotease isolated from the venom duct of Conus textile was undertaken. The C. textile mollusc was chosen as a good source from which to isolate the endoprotease for two reasons; firstly, these cone shells are found in great abundance on the Great Barrier Reef (Queensland, Australia) and are readily obtainable and secondly, a number of conotoxin precursors and their cleavage products have been previously identified in the venom duct. In order to purify the endoprotease an activity-guided fractionation protocol that included a para-nitroanilide (p-NA) substrate assay was developed. The p-NA substrate mimicked the cleavage site of the conotoxin TxVIA, a member of the C. textile O-superfamily of toxins. The protocol included a number of chromatographic techniques including ion exchange, size-exclusion and reverse-phased HPLC and resulted in isolation of an active protease, termed Tex31, to >95% purity. The purification of microgram quantities of Tex31 made it possible to characterise the proteolytic nature of Tex31 and to further characterise the O-superfamily conopeptide propeptide cleavage site specificity. Specificity experiments showed Tex31 requires a minimum of four residues including a leucine in the P4 position (LNKR↓) for efficient substrate processing. The complete sequence of Tex31 was determined from cDNA. A BLAST search revealed Tex31 to have high amino acid sequence similarity to the CAP (abbreviated from CRISP (Cysteine-rich secretory protein), Antigen 5 and PR-1 (pathogenesis-related protein)) superfamily and most closely related to the CRISP family of mammalian and venom proteins that, like Tex31, have a cysteine-rich C-terminal domain. The CAP superfamily is widely distributed in the animal, plant and fungal kingdoms, and is implicated in processes as diverse as human brain tumour growth and plant pathogenesis. This is the first report of a biological role for the N-terminal domain of CAP proteins. A homology model of Tex31 constructed from two PR-1 proteins, Antigen 5 and P14a, revealed the highly conserved and likely catalytic residues, His78, Ser99 and Glu115. These three amino acids fall within a structurally conserved N-terminal domain found in all CAP proteins. It is possible that other CAP proteins are also substrate-specific proteases. With no homology to any known proteases, Tex31 may belong to a new class of protease. The sequence alignment of five Tex31-like proteins cloned from C. marmoreus, C. litteratus, C. arentus, C. planboris, and C. omaria show very high sequence similarity to Tex31 (~80%), but only one weakly conserved serine residue was identified when the conserved residues of the new Tex31-like protein sequences were aligned with members of the CAP superfamily. Future work to identify members of catalytic diad or triad, e.g. by site-directed mutagenesis, will rely on the expression of active recombinant Tex31. In this study neither Escherichia coli nor Pichia pastoris expression systems yielded active recombinant Tex31 protein, possibly due to the number of cysteine residues hindering the expression of correctly folded active Tex31. This study has shown Tex31 to be highly sequence specific in its cleavage site and it is likely that this high substrate specificity has confounded previous attempts to identify the proteolytic nature of other CAP proteins. With the proteolytic nature of one member of the CAP protein family confirmed, it is hoped this important discovery may lead the way to discovering the role of other CAP family members.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Prédire la capacité de développement et d'implantation des embryons reste un enjeu majeur pour l’Assistance Médicale à la Procréation (AMP). L’AMP doit répondre au désir du couple d’avoir un enfant en limitant les risques encourus par la mère et l’enfant en cas de grossesse multiple. Tous les laboratoires d’AMP utilisent des critères morphologiques pour évaluer la compétence au développement des embryons en dépit de la faible valeur prédictive de cette analyse. L'interaction ovocyte-cumulus participe à l’acquisition par l'ovocyte de sa compétence au développement. Cette interaction met en jeu l’expression de gènes spécifiques dans les cellules de cumulus (CCs). Notre objectif était d'identifier des marqueurs non invasifs de la compétence ovocytaire au développement. Ainsi nous avons recherché au niveau des CCs des gènes et des protéines exprimés en fonction de l’aptitude de l’ovocyte fécondé à atteindre le stade de blastocyste. L'expression des gènes des CCs a été étudiée par puce à ADN et qPCR haut débit. Après avoir tenu compte de la variabilité des patientes, nous avons identifié les gènes RGS2, POLR3K et CUL4B comme biomarqueurs. L'expression des protéines des CCs a été étudiée par puce à protéines et après validation des anticorps ciblant les protéines d'intérêt, les protéines RGS2, POLR3K et MERTK ont été identifiées comme biomarqueurs de la compétence au développement de l'ovocyte. Ces résultats permettent d’envisager la création d’un modèle prédictif multicritère incluant la morphologie de l’embryon à J2, les gènes et protéines marqueurs. / The ability to predict the developmental and implantation ability of embryos remains a major goal in human assisted reproductive technology (ART).ART should allow couple to become parents while limiting the risks to the mother and the child in case of multiple pregnancy. ART laboratories use morphological criteria to evaluate the oocyte competence despite the poor predictive value of this analysis. The oocyte-cumulus interaction helps the oocyte to acquire its developmental competence partly through the expression of specific genes at the cumulus level. Therefore our aim was to identify at the level of cumulus cells (CCs) genes and proteins related to oocyte developmental competence as non-invasive marker. Gene expression of CCs was studied using microarray and high throughput qPCR according to the developmental competence of the oocyte (ability to reach the blastocyst stage after fertilization). While taking into account the patient variability we identified RGS2, POLR3K and CUL4B as biomarkers at RNA level. Then protein expression of CCs was studied using Reverse Phase Protein Array. After validation of the antibodies targeting the proteins of interests, RGS2, POLR3K and MERTK were identified as protein biomarkers of the developmental competence of the oocyte. These results lead us to consider a multi variables predictive model including the morphology of the embryo at J2, genes and protein markers.

Main purpose of this study was to increase protein expression in Chinese hamster ovary (CHO) cells by improving protein secretion of translated proteins. The goal was to find signal peptides from the screening of signal peptide libraries for improvement of protein secretion using a CHO-cell express selection system. Biopharmaceutical products, proteins such as monoclonal antibodies (mAbs), are most commonly produced using mammalian expression systems such as the expression in CHO cells. The posttranslational modifications of the proteins being expressed in CHO cells are similar to the expressional modifications in human cells, why the CHO cells are suitable for production of proteins used for human therapy. The expression of proteins in the cell is a complex mechanism, fundamentally depending on the DNA sequences in the cell nucleus. Secretion of translated proteins has been showed to be a bottleneck when improving expression. Secretion is initiated by the signal peptide, a n-terminal prolongation of the protein that is recognized by a signal recognition particle (SRP) when being translated by the ribosome. The sequence and structure of the signal peptide has been proved to affect secretion and altering the signal peptide could improve secretion even when changing signal peptide between different species. Designing variants of the signal peptides and analyzing protein expression might lead to improvements of the construct design and more protein produced from the cells, which would save time, money and material for the producer. To construct plasmids containing the gene of interest (GOI) and different signal peptides, several gene cloning methods were used. The plasmids were amplified using Escherichia coli (E. coli) transformation. The constructs were expressed by transfection into the CHO cell genome, and expression were analyzed using flow cytometry. When analyzing expression of a Fc-fusion protein with 5 different signal peptides, the signal peptide Azurocidin is the one showing highest expression levels in this study. In addition, IgG kapa and Albumin signal peptides did not show as high protein expression levels, even if they were better than the L1d and H5b signal peptides. Since signal peptides are exchangeable between proteins and species, it might be that Azurocidin is improving secretion and protein expression with other proteins than Fc-fusion proteins which would be an interesting aspect for further studies. When altering signal peptides with library sequences, the experimental challenges were crucial for the protein expression results and due to these issues, no library sequence could be seen to conquer others when it comes to protein expression levels. Transfection and cultivation procedures needs to be studied and improved before being able to draw conclusions about which signal peptide library sequences that might improve secretion and increase the protein expression.