Protective Effects of Bovine Serum Albumin on Superparamagnetic Iron Oxide Nanoparticles Evaluated in the Nematode Caenorhabditis elegans

Nanomaterials give rise to unique biological reactivity that needs to be thoroughly investigated. The quest for enhanced magnetic nanomaterials of different shapes, magnetic properties, or surface coatings continues for applications in drug delivery, targeting therapies, biosensing, and magnetic separation. In this context, the use of simple in vivo models, such as Caenorhabditis elegans, to biologically evaluate nanoparticles is currently in increasing demand as it offers low-cost and information-rich experiments. In this work, we evaluated how surface modification (citrate- and protein-coated) of superparamagnetic iron oxide nanoparticles (C-SPIONs and BSA-SPIONs, respectively) induces changes in their toxicological profile and biodistribution using the animal model C. elegans and combining techniques from materials science and biochemistry. The acute toxicity and nanoparticle distribution were assessed in two populations of worms (adults and larvae) treated with both types of SPIONs. After 24 h treatment, nanoparticles were localized in the alimentary system of C. elegans; acute toxicity was stronger in adults and larvae exposed to C-SPIONs rather than BSA-SPIONs. Adult uptake was similar for both SPION types, whereas uptake in larvae was dependent on the surface coating, being higher for BSA-SPIONs. Nanoparticle size was evaluated upon excretion, and a slight size decrease was found. Interestingly, all results indicate the protective effects of the BSA to prevent degradation of the nanoparticles and decrease acute toxicity to the worms, especially at high concentrations. We argue that this relevant information on the chemistry and toxicity of SPIONs in vivo could not be gathered using more classical in vitro approaches such as cell culture assays, thus endorsing the potential of C. elegans to assess nanomaterials at early stages of their synthetic formulations.C. elegans N2 and E. coli OP50 were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The research leading to these results has received funding from the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program (FP7/2007-2013) under REA grant agreement nº 303630 and cofounded by the European Social Fund. Authors acknowledge the funding from Spanish Ministry of Economy MAT 2012-35324 and FEDER funds, the Generalitat de Catalunya 2014SGR213, the COST Action MP1202, Ramon y Cajal grant RYC-2010-06082 (AL), China Scholarship Council fellowship (SMY, 201206150053), and FPU fellowship FPU12/05549 (LGM).Peer Reviewe

Evaluating inorganic nanoparticles in the living organism Caenorhabditis elegans /

BibliografiaPremi Extraordinari de Doctorat concedit pels programes de doctorat de la UAB per curs acadèmic 2016-2017En aquesta tesi hem utilitzat el model animal Caenorhabditis elegans (C. elegans) com a sistema biològic viu per avaluar nanopartícules (NPs) per aplicacions biomèdiques. En concret, hem avaluat el comportament de dos tipus de partícules inorgàniques amb diferent mida i propietats de superfície: NPs d'òxid de ferro (SPIONs) recobertes de citrat i d'albúmina sèrica bovina (BSA), i NPs d'or recobertes de citrat de dos mides diferents (10 nm i 150 nm). Hem estudiat les interaccions nano-bio en C. elegans amb especial atenció en les propietats dels materials in vivo incloent el seu estat, ingesta, localització, efectes biològics i mecanismes moleculars. Amb aquest propòsit, hem combinat assajos de toxicitat, tècniques de ciència de materials i d'imatge, i anàlisi de l'expressió gènica. També hem investigat la influència de la composició de les NPs, del seu recobriment superficial i de la seva mida in vivo, i comparat els nostres resultats amb estudis previs. És destacable el fet d'haver pogut realitzar totes aquestes avaluacions biològiques en el mateix laboratori on hem sintetitzat i caracteritzat les partícules gracies als avantatges experimentals de C. elegans. Així doncs, aquesta tesi valida l'ús de C. elegans com a un model animal senzill per avaluar NPs en les etapes inicials de desenvolupament. A través de tècniques de microscòpia, vam veure que les NPs entraven al cos del cuc durant el procés d'alimentació i s'acumulaven seguint patrons particulars segons les propietats de les NPs. Les NPs d'or no eren internalitzades per les cèl·lules intestinals de C. elegans, mentre que vam poder identificar NPs d'òxid de ferro en el compartiment lisosomal d'aquestes cèl·lules. En cap cas vam detectar translocació de NPs a òrgans secundaris, ni entrades per altres vies (ex. dermal) o orificis del cos. Per caracteritzar el perfil toxicològic de les NPs, vam estudiar diferents paràmetres toxicològics letals i sub-letals. Vam observar que la LD50 de les NPs d'òxid de ferro casi doblava (600 µg/ml) el valor de les NPs d'or de 11 nm (350 µg/ml), cosa que indica que les NPs d'òxid de ferro són més biocompatibles que les d'or. Les SPIONs recobertes de BSA van afectar l'estat de C. elegans menys que les recobertes de citrat, suggerint un efecte protector pel recobriment del BSA. De manera similar, vam veure que les NPs d'or de 150 nm eren menys tòxiques que les de 11 nm, cosa que indica que la mida de les NPs és rellevant pels seus efectes in vivo. L'ús de tècniques fisicoquímiques ens va permetre quantificar la ingesta de NPs per part del cuc i ens va permetre també caracteritzar l'estat de les NPs en C. elegans. Dins de l'animal, les SPIONs mantenien les seves propietats magnètiques inicials però eren parcialment degradades en l'intestí, especialment les recobertes de citrat, amb el conseqüent alliberament de ions ferro. Les NPs d'or van resultar més resistents a la degradació però vam observar cert grau d'agregació, que era reversible i depenia de la regió de l'intestí on es localitzaven les NPs. Mitjançant experiments in vitro, vam confirmar que el pH i la presència de biomolècules en l'intestí pot contribuir a determinar l'estat de les NPs in vivo. D'altra banda, vam estudiar i identificar mecanismes moleculars afectats per l'exposició a NPs a través de tècniques genètiques. Vam observar que les NPs alteraven els mecanismes d'estrès oxidatiu i de detoxificació de metalls, cosa que suggereix que estan implicats en la resposta de C. elegans a les NPs. En el cas de les SPIONs, vam detectar efectes depenent del recobriment: mentre que les partícules recobertes de citrat afectaven el processament d'informació ambiental i genètica, les recobertes de BSA afectaven el funcionament metabòlic. En conjunt, creiem que el tractament amb NPs és capaç d'activar mecanismes d'estrès general i també rutes específiques que depenen de les propietats de les NPs. En resum, aquesta tesi ha contribuït a: i) ampliar el ventall de tècniques utilitzades per caracteritzar interacciones nano-bio en C. elegans; ii) una avaluació sistemàtica i extensa de les interacciones entre NPs y C. elegans, des de la síntesi del material fins l'anàlisi dels mecanismes moleculars; i iii) estudiar la influencia de las propietats de les NPs en els seus efectes in vivo.En esta tesis hemos usado el modelo animal Caenorhabditis elegans (C. elegans) como sistema biológico vivo en el que evaluar nanopartículas (NPs) para aplicaciones biomédicas. En concreto, hemos evaluado el comportamiento de dos tipos de partículas inorgánicas con diferentes tamaños y propiedades de superficie: NPs de óxido de hierro (SPIONs) recubiertas de citrato y de albúmina sérica bovina (BSA), y NPs de oro recubiertas de citrato de dos tamaños distintos (10 nm y 150 nm). Hemos estudiado las interacciones nano-bio en C. elegans con especial atención en las propiedades de los materiales in vivo incluyendo su estado, ingestión, localización, efectos biológicos y mecanismos moleculares. Con esta finalidad, hemos combinado ensayos de toxicidad, técnicas de ciencia de materiales y de imagen, y análisis de la expresión génica. También hemos investigado la influencia de la composición de las NPs, de su recubrimiento superficial y de su tamaño in vivo, y comparado nuestros resultados con estudios previos. Es destacable el hecho de haber podido realizar todas estas evaluaciones biológicas en el mismo laboratorio donde hemos sintetizado y caracterizado las partículas gracias a las ventajas experimentales de C. elegans. De este modo, esta tesis valida el uso de C. elegans como un modelo animal simple para evaluar NPs en las etapas iniciales de desarrollo. A través de técnicas de microscopía, vimos que las NPs entraban al cuerpo del gusano durante el proceso de alimentación y se acumulaban siguiendo patrones particulares según las propiedades de las NPs. Las NPs de oro no eran internalizadas por las células intestinales de C. elegans, mientras que detectamos SPIONs en el compartimento lisosomal de estas células. En ningún caso detectamos translocación de NPs a órganos secundarios, ni entrada por otras vías (ej. dermal) u orificios del cuerpo. Para caracterizar el perfil toxicológico de las NPs, estudiamos diferentes parámetros toxicológicos letales y sub-letales. Observamos que la LD50 de las SPIONs casi doblaba (600 µg/ml) el valor de las NPs de oro de 11 nm (350 µg/ml), lo cual indica que las SPIONs son más biocompatibles que las de oro. Las BSA-SPIONs afectaron el estado de C. elegans menos que las C-SPIONs, sugiriendo un efecto protector del recubrimiento de BSA. De forma similar, vimos que las NPs de oro de 150 nm eran menos tóxicas que las de 11 nm, lo cual indica que el tamaño de las NPs es relevante para sus efectos in vivo. El uso de técnicas fisicoquímicas nos permitió cuantificar la ingesta de NPs por parte del gusano y nos permitió también caracterizar el estado de las NPs en C. elegans. Dentro del animal, las SPIONs mantenían sus propiedades magnéticas iniciales pero eran parcialmente degradadas en el intestino, especialmente las recubiertas de citrato, con la consiguiente liberación de iones hierro. Las NPs de oro resultaron más resistentes a la degradación pero observamos cierto grado de agregación, que era reversible y dependía de la región del intestino donde se localizaban las NPs. Mediante experimentos in vitro, confirmamos que el pH y la presencia de biomoléculas en el intestino puede contribuir a determinar el estado de las NPs in vivo. Por otro lado, estudiamos e identificamos mecanismos moleculares afectados por la exposición a NPs usando técnicas genéticas. Observamos que las NPs alteraban los mecanismos de estrés oxidativo y de detoxificación de metales, lo cual sugiere que están implicadas en la respuesta de C. elegans a las NPs. En el caso de las SPIONs, detectamos efectos dependientes del recubrimiento: mientras que las partículas recubiertas de citrato afectaban el procesamiento de información ambiental y genética, las recubiertas de BSA afectaban el funcionamiento metabólico. En conjunto, creemos que el tratamiento con NPs es capaz de activar mecanismos de estrés general y también rutas específicas que dependen de las propiedades de las NPs. En resumen, esta tesis ha contribuido a: i) extender el abanico de técnicas usadas para caracterizar interacciones nano-bio en C. elegans; ii) una evaluación sistemática y extensa de las interacciones entre NPs y C. elegans, desde la síntesis del material hasta el análisis de mecanismos moleculares; y iii) estudiar la influencia de las propiedades de las NPs en sus efectos in vivo.In this thesis, we have used the simple model organism Caenorhabditis elegans (C. elegans) as an in vivo biological system to screen nanoparticles (NPs) proposed for biomedical uses. In particular, we have assessed the behaviour of two types of inorganic particles with different size and surface properties: iron oxide NPs (SPIONs) coated with citrate and bovine serum albumin (BSA), and citrate coated gold nanoparticles of two different diameters (11 nm and 150 nm). We have studied their nano-bio interactions in C. elegans with special focus on the material's properties in vivo including their status, uptake, fate, biological effects and molecular mechanisms. To this end, we have combined toxicity tests, materials science and imaging techniques, and gene expression analysis. We have also investigated the influence of NP composition, coating and size in vivo, and compared our results with previous studies. Remarkably, we have been able to perform this biological evaluation in the synthetic laboratory where the particles were synthesized and characterised due to the advantageous experiments features of C. elegans. Therefore, our work sets up C. elegans as a simple animal model to evaluate NPs in the initial stages of development. By microscopy techniques, we found that NPs entered the body of the worm during feeding and accumulated in the intestine with particular patterns depending on the NP properties. Gold NPs were not internalized by the C. elegans intestinal cells, while iron oxide NPs were identified in the lysosomal compartment of these cells. We did not detect translocation of NPs to secondary organs, either entrance by other routes (i.e. dermal) or body openings. Lethal and sub-lethal endpoints were analysed to characterise the toxicological profile of the NPs. We found that the LD50 of iron oxide NPs almost doubled (600 µg/ml) the value for 11-nm gold NPs (350 µg/ml) indicating the superior biocompatibility of the former. BSA-coated SPIONs affected to a lesser extent the behaviour of C. elegans than citrate-coated SPIONs, suggesting a protective effect of the BSA coating. Similarly, 150-nm gold NPs appeared less toxic than their smaller counterparts, indicating that NP size is relevant to their in vivo effects. The use of physicochemical techniques allowed us to quantify the ingestion of NPs by C. elegans and, interestingly, we could also characterise NP status inside C. elegans. We found that iron oxide NPs maintained their initial magnetic properties but were partially degraded inside the intestine, especially the citrate-coated particles, with the consequent release of iron ions. Gold NPs appeared resistant to degradation but showed some degree of aggregation, which was reversible and depended on the specific area of the gut. We confirmed in vitro that the pH and presence of biomolecules in the intestine could contribute to determine the status of the NPs in vivo. We also investigated potential molecular mechanisms affected by NP exposure using genetic tools. We observed that NP treatment disrupted the oxidative stress and metal detoxification pathways, suggesting that they might be involved in the response of C. elegans to NPs. In the case of iron oxide NPs, we found coating-dependent effects: citrate-coated particles affected the processing of environmental and genetic information, while BSA-coated SPIONs affected metabolic mechanisms. All in all, we believe that NP treatment can trigger general stress mechanisms and also specific pathways depending on the NP properties. In conclusion, this thesis contributes to: i) extend the toolkit of techniques used for the characterisation of nano-bio interactions in C. elegans; ii) a systematic and comprehensive evaluation of the interaction of NPs in C. elegans, from the materials' synthesis to the analysis of molecular pathways; and iii) study the influence of NP properties on their in vivo effects

Contribution of sex on the underlying mechanism of the gambling disorder severity

Significant increasing prevalences have been observed in gambling disorder (GD) in the last decades. This study analyzed the underlying mechanisms of the gambling severity with path analysis (implemented through Structural Equation Modeling, SEM), and assessed the potential moderator effect of the patients' sex. A sample of n=512 treatment-seeking patients was assessed for sociodemographics and clinical state previously to the treatment. Results obtained in two separate SEM (for men and women) revealed differences in the direct effects and the mediational links. Among the male subsample, higher GD severity was directly related to the higher cognitive bias and the younger age of onset of the problematic gambling, while impulsivity levels and age of onset achieved an indirect effect on the disordered gambling mediated by the cognitive bias. Among females, GD severity was directly increased by younger age of onset, higher cognitive bias and lower self-directedness, while lower socioeconomic positions, and higher levels in harm avoidance achieved an indirect effect on the gambling severity mediated also by the distortions related to the gambling activity. These results provide new empirical evidence for a better understanding of the GD etiology, suggesting that the underlying complex links mediating the GD severity are strongly related to the patients' sex. The results can also contribute to design more effectiveness and precise therapy programs of patient-centered care

Developmental trajectories of gambling severity after cognitive-behavioral therapy

Aims: To estimate trajectories of the gambling disorder (GD) severity for 12 months following a manualized cognitive-behavior-therapy (CBT) program, and to identify the main variables associated with each trajectory. Methods: Latent Class Growth Analysis examined the longitudinal changes of n = 603 treatment-seeking patients with GD. Results: Five separate empirical trajectories were identified: T1 (n = 383, 63.5%) was characterized by the most highest baseline gambling severity levels and positive progress to recovery during the follow-up period; T2 (n = 154, 25.5%) featured participants with high baseline gambling severity and good progress to recovery; T3 (n = 30, 5.0%) was made up of patients with high gambling baseline severity and slow progress to recovery; T4 (n = 13, 2.2%) and T5 (n = 23, 3.8%) contained participants with high baseline gambling severity and moderate (T4) and poor (T5) progress in GD severity during the follow-up. Psychopathological state and personality traits discriminated between trajectories. Poor compliance with the therapy guidelines and the presence of relapses also differed between the trajectories. Conclusions: Our findings show that patients seeking treatment for GD are heterogeneous and that trends in progress following treatment can be identified considering sociodemographic features, psychopatho- logical state and personality traits. These results could be useful in developing more efficient interventions for GD patients

Women and gambling disorder: Assessing dropouts and relapses in cognitive behavioral group therapy

Background: Gender-specific literature focused on gambling disorder (GD) is scarce, and women with GD have been understudied. Therefore, the aim of this study was to estimate the short-term effectiveness in women with GD (n = 214) of a group standardized cognitive-behavioral therapy (CBT) and to identify the most relevant predictors of the primary therapy outcomes (dropout and relapse). Methods: The manualized CBT consisted of 16 weekly outpatient group sessions. Women were provided with resources to obtain a better understanding of the GD, to improve self-control and to manage risk situations. Results: The dropout risk was higher for women with lower GD severity and higher psychopathological distress. Among other factors, lower education levels were a significant predictor of the relapse risk and and the frequency of relapses was higher for divorced women with a preference for non-strategic gambling and with substances consumption. Conclusions: Our findings evidence women-specific predictors of the primary therapy outcomes. The results highlight the need to design psychological interventions that address dropout and relapse risk factors in women

A Serious Game to Improve Emotion Regulation in Treatment-Seeking Individuals With Gambling Disorder: A Usability Study

Background: Serious games have shown positive results in increasing motivation, adherence to treatment and strengthening the therapeutic alliance in multiple psychiatric disorders. In particular, patients with impulse control disorders and other disorders in which the patient suffers from inhibitory control deficits (e.g., behavioral addictions) have been shown to benefit from serious games. Aim: The aim of this study was to describe the characteristics and to evaluate the usability of a new serious videogame, e-Estesia. This serious videogame was designed to improve emotion regulation in patients with gambling disorder (GD). Preliminary results from a pilot sample are also reported. Method: A pilot sample of 26 patients undergoing treatment for GD was recruited (ranging from 22 to 74 years, mean = 41.2 and SD = 12.9; 80.8% men). Participants used e-Estesia on a tablet, which was connected to a thoracic band that sent heart rate (HR) and heart rate variability (HRV) data to the videogame platform in order to provide biofeedback. The System Usability Scale was completed by patients to determine the usability of e-Estesia. Results and Discussion: e-Estesia performed comparatively well for all the explored groups (i.e., sex, age, and online vs. offline gambling: mean usability score = 83.8, SD = 13.1). Around 84.6% of the patients endorsed that it was easy to use. Female patients with GD presented higher HRV during the use of the serious videogame compared to men

How do online sports gambling disorder patients compare with land-based patients?

Background and aims: recent technological developments have brought about notable changes in the way people gamble. The widespread use of mobile Internet devices and gambling websites has led to a significant leap in the number of people who recreationally gamble. However, for some, gambling can turn into a psychiatric disorder resembling substance addiction. At present, there is a shortage of studies examining differences between adults with gambling disorder (GD) who exclusively make sports bets online, GD patients that are non-sports Internet gamblers, and offline gamblers. Therefore, this study was undertaken to determine the differences between these three groups, considering sociodemographic, personality, and clinical characteristics. Methods: the sample consisted of 2,743 treatment-seeking male patients from the Pathological Gambling Unit at a university hospital. All patients met DSM-5 criteria for GD. Results: we found that gamblers who exclusively engaged in non-sports Internet gambling activities were younger than offline gamblers and online sports gamblers. Non-sports Internet gamblers were also more likely to have greater levels of debt compared with offline gamblers. In terms of personality characteristics, our sample displayed low levels of self-directedness and cooperativeness and high levels of novelty seeking. In addition, online sports gamblers obtained higher scores in persistence than non-sports Internet gamblers and offline gamblers. Discussion and conclusion: although differences if terms of gambling severity were not identified between groups, GD patients who exclusively bet online appear to possess distinct personality characteristics and higher debt levels compared with offline gamblers

Gambling phenotypes in online sports betting

Background and objectives: The Internet provides easy access to multiple types of gambling and has led to changes in betting habits. A severe rise in problematic gambling has been predicted among all sectors of the population, and studies are required to assess the emerging phenotypes related to the new structures of gambling activities. This study aimed to explore the existence of latent classes associated with gambling habits among treatment-seeking gamblers due to Online Sports Betting (OSB). Method: Initial sample included n = 4,516 patients consecutively admitted for treatment in a hospital unit specialized in behavioral addictions. Two-step clustering analysis was used within the subsample of n = 323 patients who reported problems related with OSB, within a set of indicators including sociodemographics, psychopathological distress, personality, and severity of the gambling activity. Results: The prevalence of OSB as a main type of gambling problem in the study was 7.2% (95% confidence interval: 6.4 to 7.9%). Two latent clusters were identified, with differences in sociodemographics and clinical status. Cluster 1 (n = 247, 76.5%) grouped patients that were more affected due to the OSB behaviors, and it was characterized by non-married patients, lower socioeconomic position index, higher comorbidity with other substance related addictions, younger age, and early onset of the gambling activity, as well as higher debts due to the OSB, higher psychopathological distress, and a more dysfunctional personality profile. Cluster 2 (n = 76, 23.5%) grouped patients that were less affected by OSB, mostly married (or living with a stable partner), with higher social position levels, older age and older onset of the gambling activity, as well as a more functional psychopathological and personality profile. Conclusion: The increasing understanding of latent classes underlying OSB phenotypes is essential in guiding the development of reliable screening tools to identify individuals highly vulnerable to addictive behaviors among Internet gamblers, as well as in planning prevention and treatment initiatives focused on the precise profiles of these patients

Presence of problematic and disordered gambling in older age and validation of the South Oaks Gambling Scale

The use of instruments originally developed for measuring gambling activity in younger populations may not be appropriate in older age individuals. The aim of this study was to examine the presence of problematic and disordered gambling in seniors aged 50 or over, and study the reliability and validity properties of the SOGS (a screening measure to identify gambling related problems). Two independent samples were recruited: a clinical group of n = 47 patients seeking treatment at a Pathological Gambling Outpatient Unit, and a population-based group of n = 361 participants recruited from the same geographical area. Confirmatory factor analysis verified the bifactor structure for the SOGS with two correlated underlying dimensions [measuring the impact of gambling on the self primarily (Cronbach's alpha α = 0.87) or on both the self and others also (α = 0.82)], and a global dimension of gambling severity (also with excellent internal consistency, α = 0.90). The SOG obtained excellent accuracy/validity for identifying gambling severity based on the DSM-5 criteria (area under the ROC curve AUC = 0.97 for discriminating disordered gambling and AUC = 0.91 for discriminating problem gambling), and good convergent validity with external measures of gambling (Pearson's correlation R = 0.91 with the total number of DSM-5 criteria for gambling disorder, and R = 0.55 with the debts accumulated due to gambling) and psychopathology (R = 0.50, 0.43 and 0.44 with the SCL-90R depression, anxiety and GSI scales). The optimal cutoff point for identifying gambling disorder was 4 (sensitivity Se = 92.3% and specificity Sp = 98.6%) and 2 for identifying problem gambling (Se = 78.8% and Sp = 96.7%). This study provides empirical support for the reliability and validity of the SOGS for assessing problem gambling in elders, and identifies two specific factors that could help both research and clinical decision-making, based on the severity and consequences of the gambling activity

Subtyping treatment-seeking gaming disorder patients

Background and aims: Gaming Disorder (GD) is characterized by a pattern of persistent and uncontrolled gaming behavior that causes a marked impairment in important areas of functioning. The evolution of the worldwide incidence of this disorder warrants further studies focused on examining the existence of different subtypes within clinical samples, in order to tailor treatment. This study explored the existence of different profiles of patients seeking treatment for GD through a data-driven approach. Methods: The sample included n = 107 patients receiving treatment for GD (92% men and 8% women) ranging between 14 and 60 years old (mean age = 24.1, SD = 10). A two-step clustering analysis approach explored the existence of different underlying GD profiles based on a broad set of indicators, including sociodemographic features, clinical course of the condition (e.g., onset or evolution), psychopathological symptoms, and personality traits. Results: Two GD profiles emerged. The first cluster grouped together patients who presented with a lower psychological impact (n = 72, 66.1%), whereas the second cluster comprised patients with a higher psychological impact (n = 35, 32.7%). Cluster comparisons revealed that those patients presenting the higher impact were older, with a later onset of pathological gaming patterns, and more pronounced psychopathological symptoms and dysfunctional personality profiles. Conclusions: GD severity is influenced by specific demographic, clinical, and psychopathological factors. The identification of two separate profiles provides empirical evidence that contributes to the conceptualization of this disorder, as well as to the development of reliable and valid screening tools and effective intervention plans focused on the precise characteristics of the treatment-seeking patients