Gene regulation in Drosophila melanogaster in response to an acute dose of ethanol

Alcohol intake causes gene expression changes resulting in cellular and molecular adaptations that could be associated with a predisposition to alcohol dependence. Expression profiling using high-throughput microarrays has recently been used to identify changes in gene expression that may be associated with alcohol dependence. To clarify the mechanisms and biology underlying alcohol dependence, bioinformatics, behavioural and genetics methodologies were employed to analyse obtained raw microarray data set that was previously generated from Drosophila exposed to an acute dose of ethanol. Classical linear statistical modeling coupled with clustering and functional enrichment analyses were implemented to evaluate whole-head time series microarray data from ethanol-treated and control samples, and implicated many genes or pathways affected by acute ethanol treatment in Drosophila head including those involved in stress signaling, inter and intra cellular signaling, ubiquitinmediated signaling, metabolic switches, and possible transcriptional regulatory components. Further analysis identified interaction networks and patterns of transcriptional regulation within the set of identified genes. Seven of these genes, ana, Axin, hiw, hop, hsp26, hsp83, and mbf1, were verified and linked with novel roles in ethanol behavioural responses using functional tests. Additional work on two of these genes namely, hiw and hsp26 also revealed a role for glia, mushroom bodies and ellipsoid body neurons as important regulators of acute ethanol response in Drosophila. Finally, these studies have demonstrated that microarray analysis is an efficient method for identifying candidate genes and pathways that may be fundamental to human alcohol dependence or abuse

Expression profile and functional interrogation of G protein-coupled receptors in endometrial cancer

Endometrial cancer (EC) is the sixth most diagnosed cancer in women and unlike most other cancers the incidence of this disease is rising; women are being diagnosed at an earlier age; and mortality is increasing, highlighting an urgent need for novel therapies. Tumours are multicellular units working in concert to achieve the hallmarks of cancer, communicating through autocrine and paracrine factors. G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors that are involved in transducing extracellular signals into intracellular cascades, ultimately altering the trajectory of the cell. Gonadotrophin hormone receptors are prototypical GPCRs, that demonstrate multi-dimensional signalling capacity, and play key roles in reproduction, however, have also been reported to promote EC cell growth. Newer concepts in GPCR biology such as oligomerisation and endomembrane signalling have highlighted greater complexity in signalling and how GPCRs achieve diverse functional effects through the same receptor. Therefore, this thesis sought to identify GPCRs highly expressed in EC, through mining RNA sequencing databases; delineate gonadotrophin hormone receptor signalling and functional consequences in EC cells; and probe the regulation of the gonadotrophin receptor, follicle-stimulating hormone receptor (FSHR), through oligomerisation and endomembrane signalling. This thesis found 16 overexpressed GPCR genes in EC using differential expression analysis of matched normal and tumour samples. FSHR was found to preferentially activate non-classical Gαi/o signalling in Ishikawa EC cells, and induce lipid droplet accumulation via Gαi/o and β-arrestins, highlighting a novel function for β-arrestins in FSHR action. Using super-resolution imaging technique, PD-PALM, FSHR heterologously expressed in HEK293 cells was visualised on the plasma membrane and was demonstrated to exist as monomers and higher-order oligomers. Interestingly, FSHR complexes were altered after ligand stimulation and density-dependent effects were found. Together, this thesis has demonstrated that through system bias, the function and regulation of GPCRs are altered.Open Acces

Identification of novel therapeutic targets in Osteosarcoma for the development of Nanoparticle based drug delivery systems

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonOsteosarcoma (OS) is the most common primary malignant tumour of the bone, with a high incidence rate in children and adolescents. Importantly, OS therapeutics has remained unimproved for the last 30 years and therefore, clinically unsatisfactory. It is understood that determining the mechanisms underlying OS tumorigenesis and progression, along with the identification of novel therapeutic targets can greatly aid in the clinical management of the disease. Thus, one important area in need of further exploration is the tumour microenvironment (TME), which consists of a plethora of extracellular matrix components, tumour associated fibroblasts and immune cells. To determine the underlying mechanism(s) and identify novel therapeutic targets, we searched an OS gene microarray dataset deposited within the Gene Expression Omnibus, which included 84 primary OS biopsies and 12 primary Mesenchymal stem cell control samples. An alternative to R coding (GEO2R) was utilised to identify differentially expressed genes (DEGs) in OS, with further R analysis conducted to quantify levels of genetic association to OS. 1161 DEGs in OS were identified, consisting of 535 upregulated and 626 downregulated genes at cut-offs of |log2FC|>1 and an adjusted P-value<0.01. Through functional annotations, we show that the DEGs are involved in immune system processes, including defence and general immune responses. This suggests that the immune system is strongly linked to the OS microenvironment with DEGs potentially contributing to OS development and metastasis. For the identification of immune based therapeutic targets in the OS TME, upregulated genes were additionally refined based on the gene ontology (GO) term “immune receptor activity”. Through GO based refinement, the immune receptor Formyl-Peptide Receptor 3 (FPR3) was found to be significantly upregulated in OS and to have a close genetic association. Through both in vitro and in vivo staining, we first show that FPR3 is specifically expressed on malignant bone cells of mesenchymal origin, with a lack of expression on normal bone cells. Thereafter, through functional in vitro cell migration assays, we determined its potential in becoming a therapeutic target in OS. Results showed that when targeted with a FPR3 specific peptide antagonist (WRW4), cell migration in two OS cell lines were significantly reduced. Observable differences in responsiveness to the peptide antagonist, between the two assayed OS cell lines, could be explained by differences in receptor expression levels as shown by flow cytometric analysis. An alternate role could relate to its use as a prognostic biomarker in OS. For the therapeutic targeting of FPR3 in OS, we propose the use of Nanoparticle based drug delivery systems (NP-DDS). Nanoparticles (NPs) are highly promising tools for both clinical and therapeutic purposes. The surfaces of NPs can be modified for targeted therapy and the NPs themselves are able to controllably release drugs when used as drug carriers in NP-DDS. However, NPs are known to exhibit varying levels of toxicity, therefore, their biosafety still remains a concern. Here, we put forward the Galleria Mellonella (GM) model organism as an efficient, low-cost, high-throughput tool for determining NP toxicity in vivo. We utilise, for the first time, techniques such as flow cytometry, immunohistochemistry and a 4HNE ELISA, on GM larvae for the determination of NP-induced toxicity, in vivo. Based on the various assays, NP toxicity was found to be heavily dependent on physicochemical properties such as surface composition and size. In addition, we investigate cellular ROS production, both in vitro and in vivo, to determine the mechanism’s likelihood in playing a major role in NP induced toxicity, observed in vivo. Results show that intracellular ROS production is the likely mechanism underlying NP toxicity in vivo, however, tissue aggregation is an important feature of CNTs that induces strong toxicity. Lastly, SPION induced immunotoxicity was observed in vivo with the use of GM larvae. Therefore, we aimed to determine if NP intracellular trafficking, intracellular fate and in particular, SPION related endosomal escape played a role in SPION induced immunotoxicity. Preliminary data is presented for this aspect in this study. Overall, certain NP variants that were identified to be biologically safe, can be put forward as potential drug delivery agents for the treatment of diseases such as OS. These NPs can be designed for tumour specific delivery of therapeutic compounds such as receptor peptide ligands, whereby, the tumour target can be a receptor of interest with therapeutic potential such as FPR3

Measuring blood flow and pro-inflammatory changes in the rabbit aorta

Atherosclerosis is a chronic inflammatory disease that develops as a consequence of progressive entrapment of low density lipoprotein, fibrous proteins and inflammatory cells in the arterial intima. Once triggered, a myriad of inflammatory and atherogenic factors mediate disease progression. However, the role of pro-inflammatory activity in the initiation of atherogenesis and its relation to altered mechanical stresses acting on the arterial wall is unclear. Estimation of wall shear stress (WSS) and the inflammatory mediator NF-κB is consequently useful. In this thesis novel ultrasound tools for accurate measurement of spatiotemporally varying 2D and 3D blood flow, with and without the use of contrast agents, have been developed. This allowed for the first time accurate, broad-view quantification of WSS around branches of the rabbit abdominal aorta. A thorough review of the evidence for a relationship between flow, NF-κB and disease was performed which highlighted discrepancies in the current literature and was used to guide the study design. Subsequently, methods for the measurement and colocalization of the spatial distribution of NF-κB, arterial permeability and nuclear morphology in the aorta of New Zealand White rabbits were developed. It was demonstrated that endothelial pro-inflammatory changes are spatially correlated with patterns of WSS, nuclear morphology and arterial permeability in vivo in the rabbit descending and abdominal aorta. The data are consistent with a causal chain between WSS, macromolecule uptake, inflammation and disease, and with the hypothesis that lipids are deposited first, through flow-mediated naturally occurring transmigration that, in excessive amounts, leads to subsequent inflammation and disease.Open Acces

Development of a novel bioreactor and systems for suspension cell culture in biopharmaceutical production

Mammalian cells offer superior cellular machinery for the production of complex biological products. These cells provide proper post-translational processing machinery for recombinant protein expression to acquire the desired folding for optimal activity. With this advantage, mammalian cells have become the preferred choice for the production of biological products. These cells may grow either attached to a solid surface (adherent cells) or, where adapted, as suspension cultures. In order to grow these cells efficiently in suspension, a bioreactor is therefore required. Bioreactors play a key role in the production of biologicals. Due to the continuous advancement of medicine and the healthcare industry, the demand for biological drugs has increased in the last three decades. This has placed a significant pressure on the biopharmaceutical industry to meet this increasing demand and has become a key driving force behind the need to develop better, safer and more economical bioreactor designs and culture processes. Continuous stirred tank bioreactor is the norm for production of many bioproducts. However, these bioreactors exert high shear forces to cells due to the impeller speed, bubble disruption, and foam formation. In addition, at a large scale, improper mass transfer impairs the performance of cell lines and achieving high cell densities and prolonged viability with correct glycosylation of a secreted proteins is still a challenge during scale-up. Many cell lines, for example Vero cells, which are widely used to produce human vaccines are difficult to adapt into suspension culture. Fixed-bed bioreactors and the use of microcarriers provide an alternative platform for their growth to produce biologicals. However, a high surface area is required to achieve the high cell density which leading to an elevated cost of production (mainly from microcarriers) and ensuing a costly and technically challenging scaling-up of these systems. Other designs such as single-use bioreactors and novel bioreactors based on different operating principles have been explored, but their utilisation is limited from laboratory to pilot scale. Hence, a comprehensive bioreactor design which would be suitable for a large variety of cell lines to produce high-yielding products in suspension culture with the lowest cost and risk in the shortest span of time is still sought. In the current research, two approaches were investigated to address these challenges. Firstly, a horizontal tubular bioreactor (HTB) with a spiral impeller was designed and fabricated for the propagation of suspended mammalian cells with a focus to achieve middle to high cell density by improving mass transfer whilst reducing hydrodynamic shear and energy requirements through surface aeration. The second approach is to test the adaptation of adherent Vero cells into single-cell suspension culture in serum-free media by treating them with an anti-cancer drug, Puromycin amino nucleoside (PAN). The absence of a supporting surface for cell growth (e.g. microcarriers) and serum-free conditions are expected to reduce the cost of manufacturing and to achieve higher productivity of biological production per unit volume of bioreactor. In the first approach, the horizontal tubular vessel was designed to achieve the final volume of approximately 5.0 L. Design of the impeller is a key component that dictates the mixing patterns and mass transfer efficiency. Different geometric configurations were used to design the spiral impeller by considering various parameters such as impeller diameter, the pitch of the blade, pitch angle, height of the blade, the thickness of the blade, clearance efficiency and the position of the heating element. Another important aspect of the prototype design was incorporating an external magnetically-coupled motor drive which assisted in not only in aseptic handling but also reduction in mechanical stress and generation of fewer particles for cleanroom operations. The side plate was designed with the appropriate number of addition ports to allow execution of batches with minimum cross-contamination and for the ease of operation. Thereafter, the engineering characterisation of the HTB was carried out. The performance of the HTB was evaluated for (i) oxygen mass transfer (kLa) through the dynamic gassing-in method, (ii) mixing time and fluid flow by tracer and phenolphthalein method, (iii) minimum stirring speed (Njs) through alginate beads mimicking cell loading and modelling through modifying Zwietering equation, (iv) power consumption through heat calorimetry (temperature method) and (v) shear stress by determining specific death constant (kd) at different impeller speeds. The general characterisation profile of HTB has shown that at high agitation speed, homogeneity and mass transfer efficiency improved while power consumption increases with an increase in agitation speed. The bioreactor operated well at 2 L and 3 L capacity when the impeller is 40 - 90 % immersed in the liquid. The maximum mass transfer coefficient (kLa) of 16 h-1 was measured with a 3 L volume with an impeller speed of 500 rpm. These results are comparable with the other culture systems of the same scale. The HTB was also tested for suitability to grow mammalian cells. Three batches were carried out, of which one was with the Chinese hamster ovary (CHO) cells expressing the somatic angiotensinconverting enzyme (sACE) and the two with plain CHO cells without expressing any recombinant protein. The maximum cell density achieved was of 5.48 x 106 cells mL-1 with plain CHO cells and 4.14 x 106 cells mL-1 with CHO cells expressing sACE with a maximum protein productivity of 465 mg mL-1 . The specific death rate constant of 0.025 (h-1 ) was obtained when impeller speed was increase from 150 rpm (normal) to 300 rpm (induced shear) for 72 h. In this study, CHO cells have been successfully adapted to suspension in serum-free conditions using the slow weaning of serum method and propagated in the HTB whereas Vero cells have been adapted successfully to serum-free media in adherent conditions. Attempt to suspend Vero cells based on literature using the weaning method remains timeous. Therefore, an alternative approach was explored using an anti-cancer drug (PAN) which is known to suppress the expression of integrin (cell adhesion receptors). The expectations from this approach were that the suppression of integrin would allow cells to detach and grow as a suspended culture (Krishnamurti et al., 2001). The results indicated that the anti-cancerous drug may have modulated the structure and function of the integrin which resulted in dislodging of the cells from the surface and form clumps which were viable for a week in suspension culture without increase in cell density. The viability of the cell clumps and few suspended cells were tested by re-seeding of these cells back to tissue culture (TC) flasks in serum-containing media without the presence of PAN. The culture in the TC flask regained confluency in the 2-3 day which confirms the viability of the cells and the likeliness of integrin re-modulating itself in the absence of PAN. As the suspended Vero cells did not grow, they were not tested for growth in the HTB. To investigate the biological activity of these Vero cells, Isothermal microcalorimetry was used to evaluate the heat generation profile of the Vero cells quantitatively before and after drug treatment. The heat flow data (metabolic heat) from the treated and normal cells showed a distinct decrease in the heat generation profile which indicated that the treated cells were viable but not as active as the normal (non-treated) cells. It was evident from the heat flow data obtained for the PAN-treated Vero cells (-0.13 µW) from that of non-treated cells (13.12 µW) and thereafter when PAN-treated Vero cells regrown in serum-containing media, they regain their metabolic activities which were indicated by their heat flow values as positive control (9.30 µW), 100 µg mL-1 (10.12 µW), 200 µg mL-1 (10.18 µW), and 250 µg mL1 (9.15 µW). It is recommended that dielectric spectroscopy and total DNA in the culture from the lysed cells could also be used to measure the bioactivity of the pre and post treated cells and data can be compared with IMC for more insight into the behaviour of the cells It has been concluded that the horizontal tubular bioreactor (HTB) can sustain the middle to high cell density by imparting desired mixing and mass and heat transfer requirements whilst exerting minimum hydrodynamic shear. For the improvement of the design, it is recommended that more batches at different agitation speeds in combination with different airflow rate would further unravel the suitability of HTB to grow mammalian cells and stringently decode the optimum process conditions to achieve high cell densities with extended longevity. Additionally, changes in the pitch of the impeller blades could result in the improved fluid flow profile, mixing and mass transfer while drawing low power input. Subsequently, different modes of operation, e.g. fed-batch or continuous operation are suggested to investigate the suitability of the HTB for integrity, sterility, and possible higher productivities of products. In suspending Vero cells, it has been concluded that the presence of serum-containing media reversibly stimulates the re-modulation of the integrin which poses hurdles in suspending Vero cells by reattaching the cells to the TC flasks. Therefore, it is recommended that a thorough investigation of the drug-treated cell integrin profile is examined through fluorescence-activated cell sorting (FACS) which would give details of the inhibition of the different integrin subunits. This information could form the basis of adapting cell-lines into suspension in a single step, which is otherwise difficult to adapt

Toxicology of high aspect ratio nanomaterials : how shape determines the biologically effective dose

Nanotechnologies are the fastest growing industry sector ever recorded. The US budget for nanotechnology is predicted to reach the 1 trillion dollar threshold in 2015, meaning that nanotechnologies will indeed be larger than all other technologies combined. High aspect ratio nanomaterials (HARN) become increasingly important in the nanotechnology industries, and show great promise, offering many advantages and improvements to a significant range of products. The main feature of HARN is the ratio of the width of a nanomaterial to its height which can be up to 1000, making the material fibre or platelet- shaped. However, this feature leads to comparison between HARN and other high aspect ratio materials including fibre shaped materials, such as asbestos fibres. Due to the structural similarities between fibrous HARN and asbestos the question arises- do HARN pose the same risk as asbestos? This project aimed to assess the potential of a range of HARN to cause similar pathological effects as asbestos fibres. In order to address this aim a panel of HARN was tested against the fibre pathogenicity paradigm in vivo by examining the pulmonary and pleural responses as well as in vitro to reveal the mechanism of cell/HARN interaction. The first part of the study focused on fibre-shaped HARN, including a panel of distinct length classes of silver nanowires (AgNW) which were injected directly into the pleural space, a target tissue for asbestos related diseases. Injection of high aspect ratio AgNW into the pleural space of mice revealed a length dependent inflammatory response in line with the fibre pathogenicity paradigm which explains fibre pathogenicity. AgNW from 5 μm in length and above led to a significant increase in granulocytes in the pleural space which is similar to that seen after treatment with long amosite asbestos. The use of additional HARN with different compositions allowed us to identify a threshold length for fibre-induced pleural inflammation, which is 5 μm. Frustrated phagocytosis has been stated as an important factor in the initiation of an inflammatory response after fibre exposure. A novel technique, backscatter scanning electron microscopy (BSEM), was used to study frustrated phagocytosis since it provides high-contrast detection of nanowires, allowing clear discrimination between the nanofibres and other cellular features. Using this technique we showed that the onset of inflammation does not correlate with the onset of frustrated phagocytosis, with a fibre length of ≥5 μm and ≥10 μm, respectively, leading to the conclusion that intermediate length fibres fully enclosed within macrophages as well as frustrated phagocytosis are associated with a proinflammatory state in the pleural space. We further showed that fibres compartmentalise in the mesothelial cells at the parietal pleura as well as in inflammatory cells in the pleural space. To investigate the mechanism of the lengthdependent inflammation caused by AgNW, the NALP3 inflammasome activation pathway was studied in vitro, however no clear correlation could be identified. We further aimed to investigate the threshold length of fibre-induced inflammation in the lung and the effect of fibre length on macrophage locomotion in an in vitro macrophage migration assay. Pharyngeal aspiration of AgNW resulted in a length dependent inflammatory response in the lungs with threshold at a fibre length of 14 μm. Shorter fibres including 3, 5 and 10 μm elicited no significant inflammation. This identified threshold length differs from that in the pleural space which may be explained by differences in clearance mechanism of deposited fibres from the airspaces compared to the pleural space. Particle clearance from the lung is partly performed by migration of particle-laden macrophages to the mucociliary escalator. We investigated if uptake of longer fibres leads to restricted mobility and showed that exposure to AgNW in the length of ≥ 5 μm resulted in impaired motility of macrophages in the wound closure assay. The second part of the study focused on HARN in the form of nanoplatelet-shaped particles since nanoplatelets may pose an unusual risk to the lungs and the pleural space because of their aerodynamic properties. We first derived the respirability of graphene nanoplatelets (GP) from the basic principles of the aerodynamic behaviour of plate-shaped particles which allowed us to calculate their aerodynamic diameter. This showed that the nanoplatelets, which were up to 25 μm in diameter, were respirable and so would deposit beyond the ciliated airways following inhalation. We therefore utilized models of pharyngeal aspiration and direct intrapleural installation of GP, as well as an in vitro model, to assess their inflammatory potential. These large but respirable GP were inflammogenic in both the lung and the pleural space at an acute timepoint although they decreased in their inflammatory potential over a 6 weeks period. Oxidation of GP in the lung tissue was investigated in order to identify if GP degraded over the 6 week period in the lung tissue and therefore showed reduced inflammogenicity. Raman spectroscopy was used to measure the oxidation state and revealed that no change occurred over the observed timeframe. The mechanism underlying acute GP inflammation was studied in THP-1 macrophages exposed to GP. These investigations showed that GP exposure led to significant expression of IL-1β, which could be blocked via a number of inhibitors related to the NALP3 inflammasome activation. This study highlights the importance of shape/length of HARN as a driver for in vivo and in vitro inflammogenicity by virtue of their respirable aerodynamic diameter, despite a considerable 2-dimensional size which leads to an inflammatory response when deposited in the distal lungs and the pleural space. The identification of the threshold length for nanofibre-induced pathogenicity in the pleura and the lung has important implications for the understanding of the structure–toxicity relationship for asbestos-induced mesothelioma. It also contributes to risk assessment by offering a template for production of safer synthetic nanofibres by the adoption of a benign-bydesign approach. The results of this work highlight the importance of testing new HARN to protect workers in nanotechnology industries and the public

Activation of the pro-resolving receptor Fpr2 attenuates inflammatory microglial activation

Poster number: P-T099 Theme: Neurodegenerative disorders & ageing Activation of the pro-resolving receptor Fpr2 reverses inflammatory microglial activation Authors: Edward S Wickstead - Life Science & Technology University of Westminster/Queen Mary University of London Inflammation is a major contributor to many neurodegenerative disease (Heneka et al. 2015). Microglia, as the resident immune cells of the brain and spinal cord, provide the first line of immunological defence, but can become deleterious when chronically activated, triggering extensive neuronal damage (Cunningham, 2013). Dampening or even reversing this activation may provide neuronal protection against chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated through activation of the receptor Fpr2, known to play an important role in peripheral inflammatory resolution. Immortalised murine microglia (BV2 cell line) were stimulated with LPS (50ng/ml) for 1 hour prior to the treatment with one of two Fpr2 ligands, either Cpd43 or Quin-C1 (both 100nM), and production of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) were monitored after 24h and 48h. Treatment with either Fpr2 ligand significantly suppressed LPS-induced production of NO or TNFα after both 24h and 48h exposure, moreover Fpr2 ligand treatment significantly enhanced production of IL-10 48h post-LPS treatment. As we have previously shown Fpr2 to be coupled to a number of intracellular signaling pathways (Cooray et al. 2013), we investigated potential signaling responses. Western blot analysis revealed no activation of ERK1/2, but identified a rapid and potent activation of p38 MAP kinase in BV2 microglia following stimulation with Fpr2 ligands. Together, these data indicate the possibility of exploiting immunomodulatory strategies for the treatment of neurological diseases, and highlight in particular the important potential of resolution mechanisms as novel therapeutic targets in neuroinflammation. References Cooray SN et al. (2013). Proc Natl Acad Sci U S A 110: 18232-7. Cunningham C (2013). Glia 61: 71-90. Heneka MT et al. (2015). Lancet Neurol 14: 388-40

Efficient comprehensive scoring of docked proteincomplexes - a machine learning approach

Biological systems and processes rely on a complex network of molecular interactions. The association of biological macromolecules is a fundamental biochemical phenomenon and an unsolved theoretical problem crucial for the understanding of complex living systems. The term protein-protein docking describes the computational prediction of the assembly of protein complexes from the individual subunits. Docking algorithms generally produce a large number of putative protein complexes. In most cases, some of these conformations resemble the native complex structure within an acceptable degree of structural similarity. A major challenge in the field of docking is to extract the near-native structure(s) out of this considerably large pool of solutions, the so called scoring or ranking problem. It has been the aim of this work to develop methods for the efficient and accurate detection of near-native conformations in the scoring or ranking process of docked protein-protein complexes. A series of structural, chemical, biological and physical properties are used in this work to score docked protein-protein complexes. These properties include specialised energy functions, evolutionary relationship, class specific residue interface propensities, gap volume, buried surface area, empiric pair potentials on residue and atom level as well as measures for the tightness of fit. Efficient comprehensive scoring functions have been developed using probabilistic Support Vector Machines in combination with this array of properties on the largest currently available protein-protein docking benchmark. The established scoring functions are shown to be specific for certain types of protein-protein complexes and are able to detect near-native complex conformations from large sets of decoys with high sensitivity. The specific complex classes are Enzyme-Inhibitor/Substrate complexes, Antibody-Antigen complexes and a third class denoted as "Other" complexes which holds all test cases not belonging to either of the two previous classes. The three complex class specific scoring functions were tested on the docking results of 99 complexes in their unbound form for the above mentioned categories. Defining success as scoring a 'true' result with a p-value of better than 0.1, the scoring schemes were found to be successful in 93%, 78% and 63% of the examined cases, respectively. The ranking of near-native structures can be drastically improved, leading to a significant enrichment of near-native complex conformations in the top ranks. It could be shown that the developed scoring schemes outperform five other previously published scoring functions