Identification d'auto-antigènes et caractérisation d'auto-anticorps dans les hépatites auto-immunes

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

AltitudeOmics: Red Blood Cell metabolic adaptation to high altitude hypoxia

Red blood cells (RBCs) are key players in systemic oxygen transport. RBCs respond to in vitro hypoxia  through  the so-called  oxygen-dependent  metabolic  regulation,  which  involves  the competitive  binding  of  deoxyhemoglobin  and  glycolytic  enzymes  to  the  N-terminal  cytosolic domain  of  band  3.  This  mechanism  promotes  the  accumulation  of  2,3-DPG,  stabilizing  the deoxygenated state of hemoglobin, and cytosol acidification, triggering oxygen off-loading through the  Bohr  effect.  Despite  in  vitro  studies,  in  vivo adaptations  to  hypoxia  have  not  yet  been completely elucidated. Within  the  framework  of  the AltitudeOmics  study,  erythrocytes  were  collected  from  21 healthy volunteers at sea level, after exposure to high altitude (5260m) for 1, 7 and 16days, and following  reascent  after  7days  at 1525m.  UHPLC-MS  metabolomics  results  were  correlated  to physiological and athletic performance parameters. Immediate  metabolic  adaptations  were  noted as early as a few hours from ascending  to >5000m, and maintained for 16 days at high altitude.  Consistent with the mechanisms elucidated in vitro, hypoxia promoted glycolysis and deregulated the pentose phosphate pathway, as well purine catabolism, glutathione homeostasis, arginine/nitric oxide and sulphur/H2S metabolism. Metabolic adaptations were preserved one week after descent, consistently with improved physical performances in comparison to the first ascendance, suggesting a mechanism of metabolic memory.

Dynamic Compartmentalization of Persistent UPEC in the Superficial Bladder Epithelium

Urinary tract infections (UTIs) are typically caused by bacteria that colonize different regions of the urinary tract, mainly the bladder and the kidney. Approximately 25% of women that suffer from UTIs experience a recurrent infection within 6 months of the initial bout, making UTIs a serious economic burden resulting in more than 10 million hospital visits and $3.5 billion in healthcare costs in the United States alone. Type-1 fimbriated Uropathogenic E. coli (UPEC) is the major causative agent of UTIs, accounting for almost 90 % of bacterial UTIs. The unique ability of UPEC to bind and invade the superficial bladder epithelium allows the bacteria to persist inside epithelial niches and survive antibiotic treatment. Persistent, intracellular UPEC are retained in the bladder epithelium for long periods, making them a source of recurrent UTIs. Hence, the ability of UPEC to persist in the bladder is a matter of major health and economic concern, making studies exploring the underlying mechanism of UPEC persistence highly relevant.

In my thesis, I will describe how intracellular Uropathogenic E.coli (UPEC) evade host defense mechanisms in the superficial bladder epithelium. I will also describe some of the unique traits of persistent UPEC and explore strategies to induce their clearance from the bladder. I have discovered that the UPEC virulence factor Alpha-hemolysin (HlyA) plays a key role in the survival and persistence of UPEC in the superficial bladder epithelium. In-vitro and in-vivo studies comparing intracellular survival of wild type (WT) and hemolysin deficient UPEC suggested that HlyA is vital for UPEC persistence in the superficial bladder epithelium. Further in-vitro studies revealed that hemolysin helped UPEC persist intracellularly by evading the bacterial expulsion actions of the bladder cells and remarkably, this virulence factor also helped bacteria avoid t degradation in lysosomes.

To elucidate the mechanistic basis for how hemolysin promotes UPEC persistence in the urothelium, we initially focused on how hemolysin facilitates the evasion of UPEC expulsion from bladder cells. We found that upon entry, UPEC were encased in “exocytic vesicles” but as a result of HlyA expression these bacteria escaped these vesicles and entered the cytosol. Consequently, these bacteria were able to avoid expulsion by the cellular export machinery.

Since bacteria found in the cytosol of host cells are typically recognized by the cellular autophagy pathway and transported to the lysosomes where they are degraded, we explored why this was not the case here. We observed that although cytosolic HlyA expressing UPEC were recognized and encased by the autophagy system and transported to lysosomes, the bacteria appeared to avoid degradation in these normally degradative compartments. A closer examination of the bacteria containing lysosomes revealed that they lacked V-ATPase. V-ATPase is a well-known proton pump essential for the acidification of mammalian intracellular degradative compartments, allowing for the proper functioning of degradative proteases. The absence of V-ATPase appeared to be due to hemolysin mediated alteration of the bladder cell F-actin network. From these studies, it is clear that UPEC hemolysin facilitates UPEC persistence in the superficial bladder epithelium by helping bacteria avoid expulsion by the exocytic machinery of the cell and at the same time enabling the bacteria avoid degradation when the bacteria are shuttled into the lysosomes.

Interestingly even though UPEC appear to avoid elimination from the bladder cell their ability to multiple in bladder cells seem limited.. Indeed, our in-vitro and in-vivo experiments reveal that UPEC survive in superficial bladder epithelium for extended periods of time without a significantly change in CFU numbers. Indeed, we observed these bacteria appeared quiescent in nature. This observation was supported by the observation that UPEC genetically unable to enter a quiescence phase exhibited limited ability to persist in bladder cells in vitro and in vivo, in the mouse bladder.

The studies elucidated in this thesis reveal how UPEC toxin, Alpha-hemolysin plays a significant role in promoting UPEC persistence via the modulation of the vesicular compartmentalization of UPEC at two different stages of the infection in the superficial bladder epithelium. These results highlight the importance of UPEC Alpha-hemolysin as an essential determinant of UPEC persistence in the urinary bladder.

Étude du rôle pathogénique de la formiminotransférase-cyclodésaminase dans l'hépatite auto-immune de type 2

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Identification d'auto-antigènes et caractérisation d'auto-anticorps dans les hépatites auto-immunes

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Spatial and temporal specificity of Ca2+signalling inChlamydomonas reinhardtiiin response to osmotic stress

Ca2+-dependent signalling processes enable plants to perceive and respond to diverse environmental stressors, such as osmotic stress. A clear understanding of the role of spatiotemporal Ca2+ signalling in green algal lineages is necessary in order to understand how the Ca2+ signalling machinery has evolved in land plants. We used single-cell imaging of Ca2+-responsive fluorescent dyes in the unicellular green alga Chlamydomonas reinhardtii to examine the specificity of spatial and temporal dynamics of Ca2+ elevations in the cytosol and flagella in response to salinity and osmotic stress. We found that salt stress induced a single Ca2+ elevation that was modulated by the strength of the stimulus and originated in the apex of the cell, spreading as a fast Ca2+ wave. By contrast, hypo-osmotic stress induced a series of repetitive Ca2+ elevations in the cytosol that were spatially uniform. Hypo-osmotic stimuli also induced Ca2+ elevations in the flagella that occurred independently from those in the cytosol. Our results indicate that the requirement for Ca2+ signalling in response to osmotic stress is conserved between land plants and green algae, but the distinct spatial and temporal dynamics of osmotic Ca2+ elevations in C. reinhardtii suggest important mechanistic differences between the two lineages.

Spatial and temporal specificity of Ca2+signalling inChlamydomonas reinhardtiiin response to osmotic stress

Ca2+-dependent signalling processes enable plants to perceive and respond to diverse environmental stressors, such as osmotic stress. A clear understanding of the role of spatiotemporal Ca2+ signalling in green algal lineages is necessary in order to understand how the Ca2+ signalling machinery has evolved in land plants. We used single-cell imaging of Ca2+-responsive fluorescent dyes in the unicellular green alga Chlamydomonas reinhardtii to examine the specificity of spatial and temporal dynamics of Ca2+ elevations in the cytosol and flagella in response to salinity and osmotic stress. We found that salt stress induced a single Ca2+ elevation that was modulated by the strength of the stimulus and originated in the apex of the cell, spreading as a fast Ca2+ wave. By contrast, hypo-osmotic stress induced a series of repetitive Ca2+ elevations in the cytosol that were spatially uniform. Hypo-osmotic stimuli also induced Ca2+ elevations in the flagella that occurred independently from those in the cytosol. Our results indicate that the requirement for Ca2+ signalling in response to osmotic stress is conserved between land plants and green algae, but the distinct spatial and temporal dynamics of osmotic Ca2+ elevations in C. reinhardtii suggest important mechanistic differences between the two lineages.

NleH effectors interact with Bax inhibitor-1 to block apoptosis during enteropathogenic Escherichia coli infection

The human pathogens enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli and the related mouse pathogen Citrobacter rodentium subvert a variety of host cell signaling pathways via their plethora of type III secreted effectors, including triggering of an early apoptotic response. EPEC-infected cells do not develop late apoptotic symptoms, however. In this study we demonstrate that the NleH family effectors, homologs of the Shigella effector kinase OspG, blocks apoptosis. During EPEC infection, NleH effectors inhibit elevation of cytosolic Ca(2+) concentrations, nuclear condensation, caspase-3 activation, and membrane blebbing and promote cell survival. NleH1 alone is sufficient to prevent procaspase-3 cleavage induced by the proapoptotic compounds staurosporine, brefeldin A, and tunicamycin. Using C. rodentium, we found that NleH inhibits procaspase-3 cleavage at the bacterial attachment sites in vivo. A yeast two-hybrid screen identified the endoplasmic reticulum six-transmembrane protein Bax inhibitor-1 (BI-1) as an NleH-interacting partner. We mapped the NleH-binding site to the N-terminal 40 amino acids of BI-1. Knockdown of BI-1 resulted in the loss of NleH's antiapoptotic activity. These results indicate that NleH effectors are inhibitors of apoptosis that may act through BI-1 to carry out their cytoprotective function.

Shapes and Dynamics of Blood Cells in Poiseuille and Shear Flows

The dynamics, shape, deformation, and orientation of red blood cells in microcirculation affect the rheology, flow resistance and transport properties of whole blood. This leads to important correlations of cellular and continuum scales. Furthermore, the dynamics of RBCs subject to different flow conditions and vessel geometries is relevant for both fundamental research and biomedical applications (e.g drug delivery). In this thesis, the behaviour of RBCs is investigated for different flow conditions via computer simulations. We use a combination of two mesoscopic particle-based simulation techniques, dissipative particle dynamics and smoothed dissipative particle dynamics. We focus on the microcapillary scale of several μm. At this scale, blood cannot be considered at the continuum but has to be studied at the cellular level. The connection between cellular motion and overall blood rheology will be investigated. Red blood cells are modelled as viscoelastic objects interacting hydrodynamically with a viscous fluid environment. The properties of the membrane, such as resistance against bending or shearing, are set to correspond to experimental values. Furthermore, thermal fluctuations are considered via random forces. Analyses corresponding to light scattering measurements are performed in order to compare to experiments and suggest for which situations this method is suitable. Static light scattering by red blood cells characterises their shape and allows comparison to objects such as spheres or cylinders, whose scattering signals have analytical solutions, in contrast to those of red blood cells. Dynamic light scattering by red blood cells is studied concerning its suitability to detect and analyse motion, deformation and membrane fluctuations. Dynamic light scattering analysis is performed for both diffusing and flowing cells. We find that scattering signals depend on various cell properties, thus allowing to distinguish different cells. The scattering of diffusing cells allows to draw conclusions on their bending rigidity via the effective diffusion coefficient. The scattering of flowing cells allows to draw conclusions on the shear rate via the scattering amplitude correlation. In flow, a RBC shows different shapes and dynamic states, depending on conditions such as confinement, physiological/pathological state and cell age. Here, two essential flow conditions are studied: simple shear flow and tube flow. Simple shear flow as a basic flow condition is part of any more complex flow. The velocity profile is linear and shear stress is homogeneous. In simple shear flow, we find a sequence of different cell shapes by increasing the shear rate. With increasing shear rate, we find rolling cells with cup shapes, trilobe shapes and quadrulobe shapes. This agrees with recent experiments. Furthermore, the impact of the initial orientation on the dynamics is studied. To study crowding and collective effects, systems with higher haematocrit are set up. Tube flow is an idealised model for the flow through cylindric microvessels. Without cell, a parabolic flow profile prevails. A single red blood cell is placed into the tube and subject to a Poiseuille profile. In tube flow, we find different cell shapes and dynamics depending on confinement, shear rate and cell properties. For strong confinements and high shear rates, we find parachute-like shapes. Although not perfectly symmetric, they are adjusted to the flow profile and maintain a stationary shape and orientation. For weak confinements and low shear rates, we find tumbling slippers that rotate and moderately change their shape. For weak confinements and high shear rates, we find tank-treading slippers that oscillate in a limited range of inclination angles and strongly change their shape. For the lowest shear rates, we find cells performing a snaking motion. Due to cell properties and resultant deformations, all shapes differ from hitherto descriptions, such as steady tank-treading or symmetric parachutes. We introduce phase diagrams to identify flow regimes for the different shapes and dynamics. Changing cell properties, the regime borders in the phase diagrams change. In both flow types, both the viscosity contrast and the choice of stress-free shape are important. For in vitro experiments, the solvent viscosity has often been higher than the cytosol viscosity, leading to a different pattern of dynamics, such as steady tank-treading. The stress-free state of a RBC, which is the state at zero shear stress, is still controversial, and computer simulations enable direct comparisons of possible candidates in equivalent flow conditions.

La valeur prédictive et pronostique d’une faible expression des récepteurs d’oestrogènes en cancer du sein

L’objectif de ce projet fut d’évaluer le bénéfice associé à la thérapie antihormonale (TA) pour les cancers du sein avec des récepteurs d’estrogènes (ER) faiblement positifs (<10 fmol/mg de cytosol). Nous avons identifié 2221 patientes avec cancer du sein dont ER ont été évalués par méthode biochimique (Ligand-Based Assay ou LBA) de 1976 à 1995 et suivies jusqu’en 2008. Des modèles à risques proportionnels de Cox ont été utilisés pour évaluer l’impact des différents niveaux de ER sur la survie au cancer du sein chez les patientes ayant reçu ou non une TA. Parmi les 2221 patientes incluses dans l’étude, 661 (29,8%) ont reçu une TA. Chez celles avec TA, une diminution significative de la mortalité spécifique au cancer du sein n’a été démontrée que pour les niveaux de ER ≥10 fmol/mg de cytosol. Ainsi, ceci ne supporte pas une TA pour les cancers du sein faiblement positif pour ER testés en LBA.

Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation

Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants.

Unravelling metabolism of Leishmania by metabolomics

The leishmaniases are neglected tropical diseases with an urgent need for effective drugs. Better understanding of the metabolism of the causative parasites will hopefully lead to development of new compounds targeted at critical points of the parasite’s biochemical pathways. In my work I focused on the pentose phosphate pathway of Leishmania, specifically on transketolase, sugar utilisation, and comparison between insect and mammalian infective stages of the parasites. The pentose phosphate pathway (PPP) is the major cellular source of NADPH, an agent critical for oxidative stress defence. The PPP uses glucose, reduces the NADP+ cofactor and produces various sugar phosphates by mutual interconversions. One of the enzymes involved in this latter part is transketolase (TKT). A Leishmania mexicana cell line deleted in transketolase (Δtkt) was assessed regarding viability, sensitivity to a range of drugs, changes in metabolism, and infectivity. The Δtkt cell line had no obvious growth defect in the promastigote stage, but it was more sensitive to an oxidative stress inducing agent and most of the drugs tested. Most importantly, the Δtkt cells were not infective to mice, establishing TKT as a new potential drug target. Metabolomic analyses revealed multiple changes as a consequence of TKT deletion. Levels of the PPP intermediates upstream of TKT increased substantially, and were diverted into additional reactions. The perturbation triggered further changes in metabolism, resembling the ‘stringent metabolic response’ of amastigotes. The Δtkt cells consumed less glucose and glycolytic intermediates were decreased indicating a decrease in flux, and metabolic end products were diminished in production. The decrease in glycolysis was possibly caused by inhibition of fructose-1,6-bisphosphate aldolase by accumulation of the PPP intermediates 6-phosphogluconate and ribose 5-phosphate. The TCA cycle was fuelled by alternative carbon sources, most likely amino acids, instead of glucose. It remains unclear why deletion of TKT is lethal for amastigotes, increased sensitivity to oxidative stress or drop in mannogen levels may contribute, but no definite conclusions can be made. TKT localisation indicated interesting trends too. The WT enzyme is present in the cytosol and glycosomes, whereas a mutant version, truncated by ten amino acids, but retaining a C-terminal targeting sequence, localised solely to glycosomes. Surprisingly, cells expressing purely cytosolic or glycosomal TKT did not have different phenotypes regarding growth, oxidative stress sensitivity or any detected changes in metabolism. Hence, control of the subcellular localisation remains unclear as well as its function. However, these data are in agreement with the presumed semipermeable nature of the glycosome. Further, L. mexicana promastigote cultures were grown in media with different combinations of labelled glucose and ribose and their incorporation into metabolism was followed. Glucose was the preferred carbon source, but when not available, it could be fully replaced with ribose. I also compared metabolic profiles from splenic amastigotes, axenic amastigotes and promastigotes of L. donovani. Metabolomic analysis revealed a substantial drop in amino acids and other indications coherent with a stringent metabolic response in amastigotes. Despite some notable differences, axenic and splenic amastigotes demonstrated fairly similar results both regarding the total metabolic profile and specific metabolites of interest.

Effects of feeding carnitine and ractopamine on rainbow trout (Oncorhynchus mykiss, Walbaum 1972)

L-carnitine is required for the transfer of long-chain fatty acids from the cytosol to the mitochondrial matrix for 13-oxidation of them and ractopamine, beta adrenergic agonists, have potential stimulating lipolysis and altering rates of protein degradation and synthesis. Present study was carried out to improve lipid body oxidation and protein-sparing action of fish through addition of L-carnitine and ractopamine to diet of rainbow trout, Oncorhynchus mykiss, Walbaum 1972. An eight-week feeding trial was carried out to evaluate the effects of supplementation of tree levels of L-carnitine tartrate (0, 1 and 2 g/kg) and two levels of ractopamine hydrochloride (0 and 10 ppm) on growth performance, fillet muscle fatty acid compositions and blood biochemical parameters in 288 juvenile rainbow trout (130 g) at 3X2 factorial experimental design. Ractopamine and 1 g/kg carnitine improved the specific growth rate, feed conversion ratio, protein efficiency ratio and weight gain at the end of experiment. The protein and lipid contents of fillet muscle were affected by the inclusion of 10 mg/kg ractopamine in the diet, increasing crude protein and reducing crude fat (P<0.05) of fish fillet muscle. The highest protein and lowest fat contents of fish fillet were observed in diet that contains 2 g/kg carnitine plus ractopamine. Ractopamine and carnitine increased levels of albumin, total protein and globulin in fish blood serum, but carnitine increased triacylglycerol and cholesterol. Fatty acids compositions of fish fillet were also affected by ractopamine and carnitine. All fatty acids except for eicosapentaenoic acid and docosahexaenoic acid, were increased by dietary supplementation of ractopamine. Total saturated fatty acids were not affected by carnitine. Supplementation (P>0.05). However, total n-3 poly unsaturated fatty acids were reduced by carnitine supplementation. A significant interaction was observed between ractopamine and carnitine supplementation regarding the saturated (P<0.01) and n-3 poly unsaturated fatty acid (P<0.001) of fish fillet. This study shows that supplementation of 1 g/kg carnitine and 10 ppm ractopamine could improve performance of juvenile rainbow trout and their combination in diet results in protein increment, fat reduction and change in profile of fatty acids in fillet muscle.

Phospholipases and the network of auxin signal transduction with ABP1 and TIR1 as two receptors: a comprehensive and provocative model

Three types of phospholipases, phospholipase D, secreted phospholipase A2, and patatin-related phospholipase A (pPLA) have functions in auxin signal transduction. Potential linkage to auxin receptors ABP1 or TIR1, their rapid activation or post-translational activation mechanisms, and downstream functions regulated by these phospholipases is reviewed and discussed. Only for pPLA all aspects are known at least to some detail. Evidence is gathered that all these signal reactions are located in the cytosol and seem to merge on regulation of PIN-catalyzed auxin efflux transport proteins. As a consequence, auxin concentration in the nucleus is also affected and this regulates the E3 activity of this auxin receptor. We showed that ABP1, PIN2, and pPLA, all outside the nucleus, have an impact on regulation of auxin-induced genes within 30 min. We propose that regulation of PIN protein activities and of auxin efflux transport are the means to coordinate ABP1 and TIR1 activity and that no physical contact between components of the ABP1-triggered cytosolic pathways and TIR1-triggered nuclear pathways of signaling is necessary to perform this.

A biochemical analysis into the co-translational folding of a G protein-coupled receptor; GPR35

The folding and targeting of membrane proteins poses a major challenge to the cell, as they must remain insertion competent while their highly hydrophobic transmembrane (TM) domains are transferred from the ribosome, through the aqueous cytosol and into the lipid bilayer. The biogenesis of a mature membrane protein takes place through the insertion and integration into the lipid bilayer. A number of TM proteins have been shown to gain some degree of secondary structure within the ribosome tunnel and to retain this conformation throughout maturation. Although studies into the folding and targeting of a number of membrane proteins have been carried out to date, there is little information on one of the largest class of eukaryotic membrane proteins; the G-protein-coupled receptors (GPCRs). This project studies the early folding events of the human ortholog of GPR35. To analyse the structure of the 1st TM domain, intermediates were generated and assessed by the biochemical method of pegylation (PEG-MAL). A structurally-similar microbial opsin (Bacterioopsin) was also used to investigate the differences in the early protein folding within eukaryotic and prokaryotic translation systems. Results showed that neither the 1st TM domain of GPR35 nor Bacterioopsin were capable of compacting in the ribosome tunnel before their N-terminus reached the ribosome exit point. The results for this assay remained consistent whether the proteins were translated in a eukaryotic or prokaryotic translation system. To examine the communication mechanism between the ribosome, the nascent chain and the protein targeting pathway, crosslinking experiments were carried out using the homobifunctional lysine cross-linker BS3. Specifically, the data generated here show that the nascent chain of GPR35 reaches the ribosomal protein uL23 in an extended conformation and interacts with the SRP protein as it exits the ribosome tunnel. This confirms the role of SRP in the co-translational targeting of GPR35. Using these methods insights into the early folding of GPCRs has been obtained. Further experiments using site-directed mutagenesis to reduce hydrophobicity in the 1st TM domain of GPR35, highlighted the mechanisms by which GPCRs are targeted to the endoplasmic reticulum. Confirming that hydrophobicity within the signal anchor sequence is essential of SRP-dependent targeting. Following the successful interaction of the nascent GPR35 and SRP, GPR35 is successfully targeted to ER membranes, shown here as dog pancreas microsomes (DPMs). Glycosylation of the GPR35 N-terminus was used to determine nascent chain structure as it is inserted into the ER membrane. These glycosylation experiments confirm that TM1 has obtained its compacted state whilst residing in the translocon. Finally, a site-specific cross-linking approach using the homobifunctional cysteine cross-linker, BMH, was used to study the lateral integration of GPR35 into the ER. Cross-linking of GPR35 TM1 and TM2 could be detected adjacent to a protein of ~45kDa, believed to be Sec61α. The loss of this adduct, as the nascent chain extends, showed the lateral movement of GPR35 TM1 from the translocon was dependent on the subsequent synthesis of TM2.