Diverse Peptide Hormones Affecting Root Growth Identified in the Medicago truncatula Secreted Peptidome.

Multigene families encoding diverse secreted peptide hormones play important roles in plant development. A need exists to efficiently elucidate the structures and post-translational-modifications of these difficult-to-isolate peptide hormones in planta so that their biological functions can be determined. A mass spectrometry and bioinformatics approach was developed to comprehensively analyze the secreted peptidome of Medicago hairy root cultures and xylem sap. We identified 759 spectra corresponding to the secreted products of twelve peptide hormones including four CEP (C-TERMINALLY ENCODED PEPTIDE), two CLE (CLV3/ENDOSPERM SURROUNDING REGION RELATED) and six XAP (XYLEM SAP ASSOCIATED PEPTIDE) peptides. The MtCEP1, MtCEP2, MtCEP5 and MtCEP8 peptides identified differed in post-translational-modifications. Most were hydroxylated at conserved proline residues but some MtCEP1 derivatives were tri-arabinosylated. In addition, many CEP peptides possessed unexpected N- and C-terminal extensions. The pattern of these extensions suggested roles for endo- and exoproteases in CEP peptide maturation. Longer than expected, hydroxylated and homogeneously modified mono- and tri-arabinosylated CEP peptides corresponding to their in vivo structures were chemically synthesized to probe the effect of these post-translational-modifications on function. The ability of CEP peptides to elevate root nodule number was increased by hydroxylation at key positions. MtCEP1 peptides with N-terminal extensions or with tri-arabinosylation modification, however, were unable to impart increased nodulation. The MtCLE5 and MtCLE17 peptides identified were of precise size, and inhibited main root growth and increased lateral root number. Six XAP peptides, each beginning with a conserved DY sulfation motif, were identified including MtXAP1a, MtXAP1b, MtXAP1c, MtXAP3, MtXAP5 and MtXAP7. MtXAP1a and MtXAP5 inhibited lateral root emergence. Transcriptional analyses demonstrated peptide hormone gene expression in the root vasculature and tip. Since hairy roots can be induced on many plants, their corresponding root cultures may represent ideal source materials to efficiently identify diverse peptide hormones in vivo in a broad range of species.

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP3-4 kinase activity.

Inositol tris/tetrakis phosphate kinases (IP3-4K) in the human fungal priority pathogens, Cryptococcus neoformans (CnArg1) and Candida albicans (CaIpk2), convey numerous virulence functions, yet it is not known whether the IP3-4K catalytic activity or a scaffolding role is responsible. We therefore generated a C. neoformans strain with a non-functional kinase, referred to as the dead-kinase (dk) CnArg1 strain (dkArg1). We verified that, although dkARG1 cDNA cloned from this strain produced a protein with the expected molecular weight, dkArg1 was catalytically inactive with no IP3-4K activity. Using recombinant CnArg1 and CaIpk2, we confirmed that, unlike the IP3-4K homologs in humans and Saccharomyces cerevisiae, CnArg1 and CaIpk2 do not phosphorylate the lipid-based substrate, phosphatidylinositol 4,5-bisphosphate, and therefore do not function as class I PI3Ks. Inositol polyphosphate profiling using capillary electrophoresis-electrospray ionization-mass spectrometry revealed that IP3 conversion is blocked in the dkArg1 and ARG1 deletion (Cnarg1Δ) strains and that 1-IP7 and a recently discovered isomer (4/6-IP7) are made by wild-type C. neoformans. Importantly, the dkArg1 and Cnarg1Δ strains had similar virulence defects, including suppressed growth at 37°C, melanization, capsule production, and phosphate starvation response, and were avirulent in an insect model, confirming that virulence is dependent on IP3-4K catalytic activity. Our data also implicate the dkArg1 scaffold in transcriptional regulation of arginine metabolism but via a different mechanism to S. cerevisiae since CnArg1 is dispensable for growth on different nitrogen sources. IP3-4K catalytic activity therefore plays a dominant role in fungal virulence, and IPK pathway function has diverged in fungal pathogens.IMPORTANCEThe World Health Organization has emphasized the urgent need for global action in tackling the high morbidity and mortality rates stemming from invasive fungal infections, which are exacerbated by the limited variety and compromised effectiveness of available drug classes. Fungal IP3-4K is a promising target for new therapy, as it is critical for promoting virulence of the human fungal priority pathogens, Cryptococcus neoformans and Candida albicans, and impacts numerous functions, including cell wall integrity. This contrasts to current therapies, which only target a single function. IP3-4K enzymes exert their effect through their inositol polyphosphate products or via the protein scaffold. Here, we confirm that the IP3-4K catalytic activity of CnArg1 promotes all virulence traits in C. neoformans that are attenuated by ARG1 deletion, reinforcing our ongoing efforts to find inositol polyphosphate effector proteins and to create inhibitors targeting the IP3-4K catalytic site, as a new antifungal drug class.

Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, higher energy collisional dissociation, and electron transfer dissociation MS applied to the N-linked glycoproteome of Campylobacter jejuni.

Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or ß-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence.

The molecular details of a novel phosphorylation-dependent interaction between MRN and the SOSS complex.

The repair of double-strand DNA breaks (DSBs) by homologous recombination is crucial in the maintenance of genome integrity. While the key role of the Mre11-Rad50-Nbs1 (MRN) complex in repair is well known, hSSB1 (SOSSB and OBFC2B), one of the main components of the sensor of single-stranded DNA (SOSS) protein complex, has also been shown to rapidly localize to DSB breaks and promote repair. We have previously demonstrated that hSSB1 binds directly to Nbs1, a component of the MRN complex, in a DNA damage-independent manner. However, recruitment of the MRN complex has also been demonstrated by an interaction between Integrator Complex Subunit 3 (INTS3; also known as SOSSA), another member of the SOSS complex, and Nbs1. In this study, we utilize a combined approach of in silico, biochemical, and functional experiments to uncover the molecular details of INTS3 binding to Nbs1. We demonstrate that the forkhead-associated domain of Nbs1 interacts with INTS3 via phosphorylation-dependent binding to INTS3 at Threonine 592, with contributions from Serine 590. Based on these data, we propose a model of MRN recruitment to a DSB via INTS3.

Molecular Analysis of pSK1 par: A Novel Plasmid Partitioning System Encoded by Staphylococcal Multiresistance Plasmids.

The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.

Proteomics of the oxidative stress response induced by hydrogen peroxide and paraquat reveals a novel AhpC-like protein in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous pathogen most typically associated with wound infections, but also the main cause of mortality in patients suffering from cystic fibrosis (CF). The ability to adapt to oxidative stress associated with host immune defense may be one mechanism by which P. aeruginosa establishes infection in the cystic fibrosis lung and eventually out-competes other pathogenic bacteria to persist into chronic infection. We utilized a proteomics approach to identify the proteins associated with the oxidative stress response of P. aeruginosa PAO1 to hydrogen peroxide and superoxide-inducing paraquat. 2-DE and MS allowed for the identification of 59 and 58 protein spots that were statistically significantly altered following H(2) O(2) and paraquat treatment, respectively. We observed a unique mass and pI pattern for alkylhydroperoxide reductase C (AhpC) that was replicated by hypothetical protein PA3529 following treatment with 10 mM H(2) O(2) . AhpC belongs to the 2-Cys peroxiredoxin family and is a redox enzyme responsible for removing peroxides in bacterial cells. MS analysis showed that PA3529 was altered by the formation of a dimer via a disulfide bond in a manner analogous to that known for AhpC, and by cysteine overoxidation to Cys-sulfonic acid (SO(3) H) postoxidative stress. PA3529 is therefore a functional AhpC paralog expressed under H(2) O(2) stress. Following paraquat-induced oxidative stress, we also observed the overabundance and likely oxidative modification of a second hypothetical antioxidant protein (PA3450) that shares sequence similarity with 1-Cys peroxiredoxins. Other induced proteins included known oxidative stress proteins (superoxide dismutase and catalase), as well as those involved in iron acquisition (siderophore biosynthesis and receptor proteins FpvA and FptA) and hypothetical proteins, including others predicted to be antioxidants (PA0848). These data suggest that P. aeruginosa contains a plethora of novel antioxidant proteins that contribute to its increased resistance against oxidative stress.

Mhp493 (P216) is a proteolytically processed, cilium and heparin binding protein of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface-accessible, heparin-binding proteins P120 and P85. Antiphosphoserine antibodies recognized P85 in 2-D immunoblotting studies and TiO(2) chromatography of trypsin digests of P85 isolated a single peptide with an m/z of 917.3. A phosphoserine residue in the tryptic peptide (90)VSELpSFR(96) (position 94 in P85) was identified by MALDI-MS/MS. Polyhistidine fusion proteins (F1(P216), F2(P216), F3(P216)) spanning Mhp493 bound heparin with biologically significant Kd values, and heparin, fucoidan and mucin inhibited this interaction. Latex beads coated with F1(P216), F2(P216) and F3(P216) adhered to and entered porcine kidney epithelial-like (PK15) cell monolayers. Microtitre plate-based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae. Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium-binding domain.

Improving the Identification and Coverage of Plant Transmembrane Proteins in Medicago Using Bottom-Up Proteomics.

Plant transmembrane proteins (TMPs) are essential for normal cellular homeostasis, nutrient exchange, and responses to environmental cues. Commonly used bottom-up proteomic approaches fail to identify a broad coverage of peptide fragments derived from TMPs. Here, we used mass spectrometry (MS) to compare the effectiveness of two solubilization and protein cleavage methods to identify shoot-derived TMPs from the legume Medicago. We compared a urea solubilization, trypsin Lys-C (UR-TLC) cleavage method to a formic acid solubilization, cyanogen bromide and trypsin Lys-C (FA-CTLC) cleavage method. We assessed the effectiveness of these methods by (i) comparing total protein identifications, (ii) determining how many TMPs were identified, and (iii) defining how many peptides incorporate all, or part, of transmembrane domains (TMD) sequences. The results show that the FA-CTLC method identified nine-fold more TMDs, and enriched more hydrophobic TMPs than the UR-TLC method. FA-CTLC identified more TMPs, particularly transporters, whereas UR-TLC preferentially identified TMPs with one TMD, particularly signaling proteins. The results suggest that combining plant membrane purification techniques with both the FA-CTLC and UR-TLC methods will achieve a more complete identification and coverage of TMPs.

Altered serum protein levels in frontotemporal dementia and amyotrophic lateral sclerosis indicate calcium and immunity dysregulation.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases that are considered to be on the same disease spectrum because of overlapping genetic, pathological and clinical traits. Changes in serum proteins in FTD and ALS are poorly understood, and currently no definitive biomarkers exist for diagnosing or monitoring disease progression for either disease. Here we applied quantitative discovery proteomics to analyze protein changes in FTD (N = 72) and ALS (N = 28) patient serum compared to controls (N = 22). Twenty three proteins were significantly altered in FTD compared to controls (increased-APOL1, C3, CTSH, EIF5A, MYH2, S100A8, SUSD5, WDR1; decreased-C1S, C7, CILP2, COMP, CRTAC1, EFEMP1, FBLN1, GSN, HSPG2, IGHV1, ITIH2, PROS1, SHBG, UMOD, VASN) and 14 proteins were significantly altered in ALS compared to controls (increased-APOL1, CKM, CTSH, IGHG1, IGKC, MYH2; decreased-C7, COMP, CRTAC1, EFEMP1, FBLN1, GSN, HSPG2, SHBG). There was substantial overlap in the proteins that were altered in FTD and ALS. These results were validated using western blotting. Gene ontology tools were used to assess functional pathways potentially dysregulated in the two diseases, and calcium ion binding and innate immunity pathways were altered in both diseases. When put together, these results suggest significant overlap in pathophysiological peripheral changes in FTD and ALS. This study represents the first proteomics side-by-side comparison of serum changes in FTD and ALS, providing new insights into under-recognized perturbed pathways and an avenue for biomarker development for FTD and ALS.

Mass spectrometric characterization of the surface-associated 42 kDa lipoprotein JlpA as a glycosylated antigen in strains of Campylobacter jejuni.

Campylobacter jejuni is the most common cause of bacterial gastroenteritis in the developed world. Immunoproteomics highlighted a 42-45 kDa antigen that comigrated on two-dimensional (2-DE) gels with the C. jejuni major outer membrane protein (MOMP). Predictive analysis revealed two candidates for the identity of the antigen, the most likely of which was the surface-associated lipoprotein, JlpA. Recombinant JlpA (rJlpA) reacted with patient sera, confirming that JlpA is antigenic. Polyclonal antibodies raised against rJlpA reacted against 3 JlpA mass variants from multiple C. jejuni. These variants differed by approximately 1.5 kDa, suggesting the presence of the N-linked C. jejuni glycan on two sites. Soybean agglutinin affinity and 2-DE purified 2 JlpA glycoforms (43.5 and 45 kDa). Their identities were confirmed using mass spectrometry following trypsin digest. Glycopeptides within JlpA variants were identified by proteinase-K digestion, graphite micropurification and MS-MS. Sites of glycosylation were confirmed as asparagines 107 and 146, both of which are flanked by the N-linked sequon. Sequence analysis confirmed that the N146 sequon is conserved in all C. jejuni genomes examined to date, while the N107 sequon is absent in the reference strain NCTC 11168. Western blotting confirmed the presence of only a single JlpA glycoform in both virulent (O) and avirulent (GS) isolates of NCTC 11168. MS analysis showed that JlpA exists as 3 discrete forms, unmodified, glycosylated at N146, and glycosylated at both N(146/107), suggesting glycan addition at N146 is necessary for N107 glycosylation. Glycine extracts and Western blotting revealed that doubly glycosylated JlpA was the predominant form on the C. jejuni JHH1 surface; however, glycosylation is not required for antigenicity. This is the first study to identify N-linked glycosylation of a surface-exposed C. jejuni virulence factor and to show strain variation in glycosylation sites.

Some brief considerations on the relationship between theory and practice.

The present crisis in models of training and in psychoanalytical education in general can be linked to the gulf that has come to be created between analytical theory and clinical practice. The paper(1) examines the historical facts that have led to this split and suggests the need to return to the models of Freud and Jung. Both these fathers of depth psychology stressed the dangers inherent in the dogmatic use of theory and both insisted that theory must always spring from and be able to account for clinical practice rather than vice versa, as is so often the case today. The paper also looks at how theory should be taught in our analytical institutes in order to ensure that what we transmit to our candidates is not knowledge in the form of dogma but rather a way of proceeding that will enable them to think creatively about their clinical practice and thus produce new knowledge, essential if depth psychology is to remain relevant to our post-modern culture.

Through the Iron Curtain: analytical space in post-Soviet Russia.

This paper discusses the experience of working as an analyst in post-totalitarian Russia in order to explore some of the general theoretical and clinical issues involved in working in a different cultural and linguistic context, and the particular problems encountered in the Russian cultural context. It describes how the Soviet regime worked actively to create a new totally collective mentality through the destruction of individual differences and the collectivization of private space, and the effects this produced in the individual and collective psyche. It examines the difficulties encountered when working with Russian analysands in creating and maintaining the setting, in preserving boundaries, in creating analytical space, and in working with certain particular transference-countertransference dynamics. It focuses on the contrast between my own Western experience of space and the spatial experience of the analysands, and describes the process of helping them use analytical space to interiorize and create a new experience of psychic space. The paper uses dreams to illustrate some of these dynamics, and the particular psychic problems associated with the traumas created by totalitarian regimes.

On Murray Jackson's 1961 'Chair, couch and countertransference'.

One of the problems facing psychoanalysts of all schools is that theory has evolved at a much faster pace than practice. Whereas there has been an explosion of theory, practice has remained, at least officially, static and unchanging. It is in this sense that Murray Jackson's 1961 paper is still relevant today. Despite the rise of the new relational and intersubjective paradigms, most psychoanalysts, and not a few Jungian analysts, still seem to feel that the couch is an essential component of the analytical setting and process. If the use of the couch is usually justified by the argument that it favours regression, facilitates analytical reverie and protects the patient from the influence of the analyst, over time many important psychoanalysts have come to challenge this position. Increasingly these analysts suggest that the use of the couch may actually be incompatible with the newer theoretical models. This contention is strengthened by some of the findings coming from the neurosciences and infant research. This underlines the necessity of empirical research to verify the clinical effectiveness of these different positions, couch or face-to-face, but it is exactly this type of research that is lacking.

Bridging the reductive and the synthetic: some reflections on the clinical implications of synchronicity.

When Jung introduced the concepts of synchronicity and the psychoid unconscious, he expanded analytical psychology into decidedly uncanny territory. Despite the early interest shown by Freud, anomalous phenomena such as telepathy have become a taboo subject in psychoanalysis. Today, however, there is an increasing interest in thought transference and synchronicity, thus opening the way for a fruitful exchange between different psychoanalytical schools on their clinical implications. I propose to examine some of the ambiguities of Jung's thinking, to clarify how we define synchronicity, the relationship between synchronicities and parapsychological events, and their clinical significance. At the present moment, we are still unsure if such events should be considered as normal and a way of facilitating individuation, or as an indication of psychopathology in the patient or in the analyst, just as we are uncertain about the particular characteristics of the intersubjective field that can lead to synchronicities. Making use of the typology of mind-matter correlations presented by Atmanspacher and Fach, and the distinction they draw between acategorial and non-categorial states of mind, I will use two clinical vignettes to illustrate the different states of mind in analyst and analysand that can lead to synchronicities. In particular I will focus on the relationship between analytical reverie and synchronicity.

Psychoanalytic theory in times of terror.

Recent events have underlined in the most tragic and dramatic way the need for depth psychology to turn its attention to the psychology of terror. The present paper attempts to distinguish between the psychological modes of horror and terror and explores the different theoretical approaches of Burke, Freud, Kristeva and Jung to this problem in order to cast light on the individual and collective functions that horror and terror play. While all these authors stress that terror and horror play a role in structuring the sense of identity and in strengthening community bonds, Freud and Kristeva believe that the experience of horror works to increase the exclusion of otherness through mechanisms of repression or foreclosure while Burke and Jung see in the encounter with the Negative Sublime or with the Shadow the possibility of widening the boundaries of ego consciousness and of integration of 'otherness'. The paper then uses the analysis of two horror movies and of a particular socio-cultural context to illustrate these different functions of horror and terror and to delineate possible solutions to the problems facing society.

To speak in tongues: language, diversity and psychoanalysis.

After reviewing the different psychoanalytical approaches to language of authors such as Freud, Lacan, Kristeva, Jung and Nicolas Abraham, the author examines the problem of 'analytical listening', of the attitude that every analyst must assume towards the words of the analysand, words that must be heard not just in terms of their content but above all in terms of their sound. We live in a culture in which visual images predominate over acoustic images and all too often this cultural trend is repeated in analysis, but it is only when we can hear the 'poetry' of the analysand's discourse that we are able to provide an 'echo', an analytical response that can co-symbolize with, that can offer to the analysand a word or a metaphor that will unlock the symbol hidden behind his or her words. The author then turns to the problem of bilinguism and its role in analysis. In her view, bilingual analysts are facilitated in their task of listening and of translation, because bilinguism facilitates the rapidity and fluidity of the analyst's associations, and at the same time sharpens his or her awareness of how the sound of a word can subtly change its meaning. The paper ends with a clinical vignette which illustrates the role that language can play in hysteria. In hysteria the dissociation between body and psyche is accompanied by a dissociation inside language itself, between verbalization and vocalization. These dissociations can be linked to the traumatic impact of the encounter between the 'language of tenderness' and the 'language of passion', between the child's attachment needs and parental sexuality. In such cases the failure of communication can be resolved principally through the use the analyst makes of the countertransference.

Out of the body: embodiment and its vicissitudes.

Body-mind dualism and the consequent neglect of the body of the analyst can have important negative effects on the analytical process leading all too often to misinterpretations of the analysand's verbal and non-verbal communications and to disturbances of analytical temporality. This is intensified when we are dealing with individuals where disembodiment and states of psychic deadness are central features. The paper explores the philosophical roots of the idea of a disembodied mind and the way in which this impacts our relationship with the world. While André Green's concept of the dead mother and disturbances in the sense of self-agency have been held to play an important role in states of psychic deadness, I suggest that it is rather disturbances in the sense of body ownership and of the body image which are more central. The paper then discusses the particular kinds of countertransference that can be evoked in the analyst when we find ourselves dealing with this type of patient and suggests how we can use our embodied countertransference to become aware of and elaborate our own feelings of deadness in order to overcome the loss of temporality that is characteristic of such states. This is illustrated with reference to my work with a young man with a masochistic perversion and a severe disturbance of the body image with an accompanying profound sense of psychic deadness.

Cognitive aesthetics of alchemical imagery.

Jung's contribution to the understanding of the relevance of psychology to alchemy has become increasingly invalidated by the ahistorical nature of his approach, just as his tendency to ignore the importance of cognitive aesthetics for an improved comprehension of the functions of alchemical images has prevented Jungians from further extending Jung's insight of the importance of alchemy for psychology. This paper explores the history of the development of alchemical illustrations in Western Europe from the 14(th) to the 16(th) century, tracing the emergent processes over time. It is only when we take into consideration the historical dimension and the aesthetics of alchemical imagery that it becomes possible to demonstrate how the increasing use of certain aesthetic techniques such as the disjunction and recombination of separate metaphorical elements of previous illustrations, the use of compressive combinations and the use of framing devices worked to gradually increase the cognitive function and the symbolical power of the images. If alchemy is still relevant to psychotherapy it is exactly because it helps us to understand the importance of cognitive aesthetics in our approach to the images, metaphors and narratives of our patients.

Healing the wounds of our fathers: intergenerational trauma, memory, symbolization and narrative.

This paper explores the history of psychoanalytical approaches to intergenerational trauma, both from the Freudian and from the Jungian schools, and addresses the need when we speak of intergenerational or transmitted trauma to better define the nature and the different categories of trauma with particular reference to extreme and cumulative traumas such as those experienced by the survivors of the Nazi death camps and the Russian gulags. Therapy with survivors and with their children requires a particular adaptation of analytical technique as what is at stake is not so much the analysis of the here and now of the transference and countertransference dynamics which indeed can in the early stages be counterproductive, but the capacity of the analyst to accept the reality of the trauma with all its devastating and mind-shattering emotions without losing the capacity to imagine and to play metaphorically with images, essential if the patient is to be able to create a space for representation.