miRNA as Biomarkers in Forensic Body Fluids Identification

miRNA as Biomarkers in Forensic Body Fluids Identification miRNA profiling: What does not work for blood and urine identification Sarah S. Silva a, b, Teixeira, A.L b, MJ Carneiro de Sousa a,c and Medeiros, R.a, b a – ICBAS, Abel Salazar Biomedical Sciences Institute, University of Porto, 4050-313 Porto, Portugal b – Molecular Oncology group, Portuguese Institute of Oncology, 4200-072 Porto, Portugal c _ National Institute of Legal Medicine and Forensic Sciences, North Branch, 4050-167 Porto, Portugal Abstract In forensics, the identification of blood, semen or vaginal secretions can represent an important support for a criminal investigation. They can be used as a source of DNA but also can hold, only by their presence, the most probative value. Through the years many methodologies were used to identify them but all presented serious drawback. Lately, mRNA surged as a potential tool for body fluid identification but their sensibility were a serious disadvantage, even more pronounced in forensic samples. Since 2009, miRNA profiling surged as a possible tool as a confirmatory test in forensics due to their tissue specific pattern of expression. Unlike mRNAs they are much more stable due to their proprieties whose makes them less prone to degradation processes. In this report, we studied the expressional patterns of miR-127, miR-221 and RNU-48 in 50 samples of urine and blood in order to define whether or not they could be used as biomarkers for urine or blood identification. Even though our aim was to assess whether or not our miRNAs could be considered as biomarkers, we came across 2 others interesting conclusions: the impact of RNA purity in miRNAs quantification and which miRNA cannot be used as a normalisation gene for blood and urine identification. Key words: miRNA profiling, Forensic, Serology, body fluids, biological biomarkers 1- Introduction Human body fluids are important components to rely on a criminal investigation [1, 2]. As a matter of fact, a complainant’s body fluids present on items belonging to a suspect – or vice versa – holds the most probative value. For example, in a case of a sexual assault in a child, where a DNA profile recovered from the child bedding and underwear coincide with his father DNA profile, can we consider his father responsible for the sexual assault? In a case like this, it is not enough to recover a DNA profile but it is also imperative to acknowledge its source. If no serological test were done, in court, the presence of DNA could be explained as a result of the presence of epithelial cells in the child clothing which is totally common when it comes from a sibling. On the other hand, if serological tests linked the DNA profile to semen it would be way more difficult to explain its presence there. Beyond the probative value that body fluid may have in a crime scene, it is also important to acknowledge them to optimize protocols to conduct a reliable DNA profiling [3, 4]. For example, DNA extraction processes are different for blood and urine. If we conducted the protocol of blood extraction in urine samples it may result in a reduced quality of the extracted DNA e enable any conclusive DNA profile [3, 4]. There is why, body fluids identification is considered as crucial step in criminal investigation. For some, it seems easy to identify body fluids such as blood (colour), urine (smell) or even sperm (texture) however, when dried, washed or mixed with other components their identification may not be that easy [1]. It is important to highlight that in court, there is no such thing as â€Å"It seems to be sperm because it looked like it and have the same particular texture†, it is needed an undeniable proof that it is sperm. Serological test are used in forensic biology to allow the detection and identification of body fluids in both native form or as a residue left at a crime scene. Serological tests are divided in two major fields: Presumptive and confirmatory test. Presumptive tests rely on methodologies that are sensitive and performed quickly, yet they are not specific to the body fluid. Those tests can only indicate if the fluids might be present and do not unequivocally states its presence. On the other hand, confirmatory tests are indeed specific to the body fluid we s eek to identify. As presumptive tests, confirmatory testing is sensitive however, it takes a lot more time. Idealistically, we should have a battery of confirmatory test for all important body fluids in order to reliably detect and identify them. Unfortunately, there is a large cluster of presumptive tests and far less of confirmatory ones. Moreover, till date no confirmatory test is able to reliably differentiate blood from menstrual blood which is an unquestionably important body fluid in sexual cases. Over the last years, mRNA profiling became a target for body fluid identification due to its tissue specific patterns. Still, mRNA susceptibility to degradation by physical or chemical factors was an unquestionable drawback. In order to sidetrack this problem, miRNA surge with a real potential as a confirmatory test. MiRNAs are small non-coding RNAs with more or less than 22 nucleotides of length that, combined with the RNA-induced silencing complex, seems to regulate a major part of human gene (5 e 6 do meu artigo). Moreover, their tight relationship with Argonaute proteins, they are much less susceptive to both biotic and abiotic factors. In 2009, Hanson and colleagues were the first to introduce miRNA profiling and soon enough others followed. Those studies pointed out a large collection of miRNAs with potential as biomarker, however very few were confirmed by more than one group which revealed the lack of reproducibility of results. Moreover, when some tried to replicate the resu lts of others, they failed. For this report, we choose to test four miRNAs in both blood and urine of 50 healthy individual and study their behaviour within those body fluids. 2- Material and methods We conducted an expression profiling of 50 healthy individuals. The case group was composed by Caucasian individuals with no major pathological condition in order to erase a variable that could alter miRNAs profiles. Peripheral venous blood (Xml) and urine were collected from each subject following the obtainment of a written informed consent from all subjects. After collected the samples were processed. The samples were used for miRNAs extraction with GRS microRNA Kit (Grisp) according to the manufacturers instructions. Subsequently, miRNa priorly extracted were used as a template for cDNA synthesis using TaqMan ® MicroRNA Reverse Transcription Kit (Applied Biosystems ®). To quantify miRNA expression, real-time PCR assays were performed with a StepOneâ„ ¢ System using TaqMan ®Universal Master Mix II (Applied Biosystems ®). The target miRNAs were amplified by a set of designed primers for miR-127-5p, miR-221*, miR-222* and RNU48. miR-222* was used as a normalization gene miRNAs relative quantifications. The data analysis was performed using the StepOne Software v2.2 (Applied Biosystems ®). Statistical analysis was carried out by the computer software IBM ®SPSS ®Statistics (Version 22.0). In order to assess any statistical alterations in our normalized miRNAs expression we used 2−ΔΔCt method and Students t test. 3- Results 3.1- Cycle threshold vs RNA purity Urine samples were processed and the resulting pellet was diluted in 1ml of Tripure. Visually a wide range of pink colour was noticeable within our urine samples. Those with a deep pink were related with samples with a more substantial pellet unlike those with a less considerable pellet who presented themselves with a lighter colour. After miRNA extraction, we quantify miRNA expression of miR-222 in urine samples and perceived that only few of them were detected. Interestingly, only the ones with a lighter colour were indeed detected. This tricky situation could be explained by the ratio of absorbance at 260 nm and 280 nm which is used to assess the purity of RNA. In this case, lighter colour was also an indicator of a greater ratio, on the other hand, those with higher optical density had a very low ratio, far from the ratio of ~2.0 which is generally accepted as â€Å"pure† for RNA. In order to sidetrack this delicate situation, we choose a sample (MU26) that has an optimal 260/280nm ratio and diluted the other samples to equalize their optical density with Tripure. Posteriorly, we choose 5 samples to test and noticed a considerable decrease of Ct in the samples processed with the optimized protocol (Fig.1). The difference of Ct value is very significant, nearly 6 Ct, demonstrating that RNA purity is clearly a factor that challenge miRNA profiling. As showed, miRNA quantification goes with a low concentration or can go totally undetected when 260/280nm ratio is low however, when optimized, miRNA concentration increased significantly. As said previously, different reports indicated miRNAs as biomarkers for human body fluids identification though, when others tried to replicate them, they failed. Our results shows that for the same sample, different degrees of purity can decide whether or not a miRNA is detected, once it definitely affect their concentration. There is why, RNA purity needed to be optimal otherwise it may lead to unreliable results, which could explain, the failed attempts done by some authors when trying to replicate others results. Figure 1 Cycle threshold vs RNA purity. This figure presents the Ct values of miR-222 taken from 5 samples processed with both normal and optimized protocol (first and second column respectively). It is showed that the considerable fall of Ct values correlates with an increase of 260/280nm ratio. 3.2 – Normalization gene In qRT-PCR, data normalization is imperatively required for quantification analysis [5-7]. The integration of an invariant endogenous essay, also called as reference gene, has as its main objective correct systematic technical and/or experimental errors [6, 8]. For this essay, we choose to use RNU-48 as our reference gene for the data normalization. Widely used as normalization gene, RNU-48 is expected to have a stable pattern among samples. However, within our essay the opposite transpired. As showed in figure 2, RNU-48 was the one with a major standard deviation when compared with other 3 miRNAs analyzed which make it inappropriate as an endogenous control for our essay. Seemingly, we were not the only ones that concluded this, Sapre and colleagues also assumed that RNI-48 was inadequate as an endogenous control due to its systematic perturbation in its expression [9]. Remarkably, the unexpected miR-222 profile remained barely unaffected and presented no significant difference between urine and blood. miRNA-222 behaviour within our samples was surprising once, it is being aimed for its deregulation by many other groups. Here, it does not present any variation within samples, any variation among both body fluids, it did even remained stable within different stages of age and do not alter with gender. This particular behaviour is expected of endogenous controls. Therefore, we decided to use miR-222 as our reference gene in order to normalise our data. 3.3 – miRNAs as biomarkers Since 2009, miRNAs has been a target for forensic researcher, especially in forensic serology. The importance of both detection and identification for body fluids in criminal investigation is undeniable. Scientifically speaking, 5 years is such a short time to develop reliable new methodologies and, as already lay out by some authors, there is still so much to do. Here, we choose 4 miRNAs and decided to study their expression level in urine and blood samples. As stated earlier, we choose miR-222 as our endogenous control for our data normalisation due to its behaviour within our samples. As showed in figure 4, we can state that all miRNAs considered have different expressional patterns and all of them probabilistically significant (P RNU-48 is the one with a major difference between urine and blood. The one used numerous times as an endogenous control is upregulated about 141 times more in blood than in urine supporting our decision to not use it to normalize our data. Till now, a minor number of miRNAs have been acknowledged as tissue specific at least reliably. By definition, miRNAs are considered tissue specific when they’re found with high abundance in a specific tissue while it has low or non-existent expression in others. That differential profile patterns would allow body fluids reliable identification and serve as a significant confirmatory test. Considering our results, we can conclude that miR-127, miR-221 and RNU-48 are not suitable for neither blood nor urine identification. Despite a significant difference of expression, they do not present the expected expressional patterns to be considered as a good biomarker. Table 1 – miRNA detection in both urine and blood samples and its corresponding fold change within the body fluids. As we stated within our introduction, the miRNAs considered as biomarkers for body fluid identification in other reports have been difficult to replicate. We believe that those difficulties are linked to several factors as environmental factors, methodologies, age, gender, pathologies among several others. We know that miRNAs expression levels do alter with both biotic and abiotic factors, there is why we try to minimize the impact of those within our samples excluding, as example, acute pathological conditions. Despite considering that miR-127, miR-221 and RNU-48 are unsuitable for urine and blood identification, we wanted to study their expressional behavior within samples with different stages of age and gender. Figure 4A displays an overview of their relative quantification within female and male samples. Within blood, we did not notice any significant alteration in their expression (P>0,05). On the other hand, in urine, RNU-48 presented itself with a significant overexpression i n females (P When it comes to age, we divided our 50 samples in 3 categories: 20-40, 41-60 and over 60 years old. As it is shown in figure 4B, the relative quantification we achieved demonstrated no significant change in their expression profile (P>0,05). 4 – Conclusion and future perspectives More than just a source of DNA, body fluids sole presence can have the most probative value. Hanson and colleagues introduced miRNA profiling as a reliable tool to identify body fluids such as blood, menstrual blood, semen, vaginal secretion and urine due to their tissue-specific pattern and stability when conditioned by degradation processes. Here we focused our attention in four miRNAs: miR-127, miR-221, miR-222 and RNU-48. Soon enough miRNAs purity struck our attention when we notice that low value of 260/280nm ratio was associated with a poor degree of detection. When we upgraded our protocol the consequence reflected in a considerable decrease of the samples threshold. It would be irrefutably helpful to understand what threshold could affect miRNA profiling once, as it was shown, miRNA purity do affect considerably their quantification. It could even convey wrong outcomes once even miRNAs with high concentration within body fluids can appear with low concentration or totally inexistent. Our second result emphasised the importance of a normalisation gene. At first, we choose to use RNU-48 as our endogenous control but its behaviour within blood and urine make us reconsider our decision. RNU-48 is usually used as a reference gene due to its stable behaviour within samples however, our essay showed otherwise. Within the 4 miRNAs testes, RNU-48 was the one with a more pronounced variability within samples, which is opposed of what would be expected of a normalisation gene. Unexpectedly, miR-222 presented itself with the lowest standard deviation between blood and urine. Furthermore, we studied its expression levels and compared them within age and gender and concluded that no significant alteration was noticeable (P As stated earlier, normalisation genes are indispensable to validate qRT-PCR results however, till date, no normalisation gene is universally acknowledged. This problem is reflected in our case, where one of the most used normalisation gene proved to be unsuitable for urine and blood miRNA analysis. This subject is a very sensitive point in miRNA profiling. There is why it is imperative to focus our future line of work towards finding a reliable normalisation gene before anything else. Our main goal was to define whether or not miR-127, miR-221 or RNU-48 could have the potential to be considered as biomarkers for body fluids identification. In this case, we could establish that all four have different expressional patterns in urine and blood (fig.5) however, to be considered as biomarker it would expected a major difference within body fluids which do not happen with our miRNAs considered for this essay. There is why we conclude that none of this miRNAs have the potential to be considered as a biomarker for body fluid identification. Conflict of interest None.

Depth of a River :: essays research papers

Depth of a River   Ã‚  Ã‚  Ã‚  Ã‚  Poetic expression is evolved from a web of emotions and thoughts. With the help of imagery, formation, and figurative language, a poet is able to transport readers to another world of his creation. Robert Burns uses these attributes to invite readers into world of peace and serenity in his poem â€Å"Sweet Afton.† This lyrical poem expresses the gratitude the persona feels for his homeland’s beauty, while asking nature to be quiet so his love may enjoy the tranquillity of her sleep. Burns’s use of imagery, use of figurative language, and construction with musical aspects help him convey his feelings and ideas to his readers.   Ã‚  Ã‚  Ã‚  Ã‚  With the rolling hills, winding streams, and wandering sheep, Burns has created a pastoral setting in â€Å"Sweet Afton.† Burns use of imagery helps add to the reality of the poem. A reader is able to hear the blackbirds’ whistling, the dove’s resounding echo, and the lapwing’s screaming. A reader is able to see snowy feet, crystal streams, and green valleys. A reader can even smell the sweet-scented birch. Burns appeals to senses by using imagery words that create the illusion of sound, sight, and smell. Imagery helps express the persona’s feelings in his environment, enabling the reader to stand along with him in his world.   Ã‚  Ã‚  Ã‚  Ã‚  Slow-moving rivers symbolize the simple life. Peace is traveling at a pace easily kept. There are no dangerous undercurrents or rocky obstacles; Afton River is gliding crystal. Burns is able to create this illusion through figurative language. He also uses apostrophe by having the persona command the river and wildlife to be quiet, as in â€Å"Flow gently, sweet Afton, disturb not her dream,† (lines 4 and 24). Much like imagery, figurative language is another vehicle used to carry the feelings of the persona to the reader.   Ã‚  Ã‚  Ã‚  Ã‚  Ã¢â‚¬Å"Sweet Afton† is a poem broken up into six stanzas. Each stanza contains four lines. These stanzas attribute to the musical effect of the poem. The first and last stanzas are incremental refrains. Burns uses this repetition to emphasize his plea for the river to flow gently and his great appreciation for its beauty. The middle four stanzas each focus on a different feature of nature. The second stanza focuses on the sounds of the birds in the narrow and secluded valleys. The beauty of the surrounding hills, little streams, and the persona’s own sheep are emphasized in the third stanza.

Regulatory Bodies

ROLE OF REGULATORY BODIES INTRODUCTION Health Professionals such as nurses doctors, Pharmacist and many others are regulated and licensed by regulatory bodies as required by provincial legislation. All nurses are required to be licensed to practice with their designated provincial nursing regulatory body. Legal responsibility in nursing practice is becoming of greater importance as each year passes. In order to provide safe and competent nursing care an understanding of legal boundaries is very essential. It is important to know the law in one state and the authorities enforcing these laws. VITAL ROLE OF REGULATORY BODIES * To ensure the public’s light to quality health care service. * To support and assist professional members. * Set and enforce standards of nursing practice. * Monitor and enforce standards for nursing education. * Monitor and enforce standards of nursing practice. * Set the requirements for registration of nursing professionals. Nursing regulatory bodies also known as colleges or associations, are responsible for the licensing of nurses with in their respective provinces territory. The Nursing Regulatory bodies receives their authority from legislation. MAJOR TYPES OF REGULATORY BODIES * The central government. * The state government * Institutional Rules * Trained Nurses Association of India * International council for Nurses * American Nurses Association * Canadian Nurses Association * National League for Nursing ROLE OF CENTRAL GOVERNMENT The central government is a source of regulatory body in three ways, through. 1) Government service conduct rules 2) The Indian Nursing council Act 3) The English law THE GOVERNMENT SERVICE CONDUCT RULES These are detailed rules of conduct for are government employees. Examples of these are the requirement to maintain absolute integrity, devotion to duty and high standards of moral behaviour. Only a few are applicable to the nursing practice, but all would be applicable to the practice of a nurse employed by the government. INDIAN NURSING COUNCIL ACT The Indian Nursing Council, which was authorised by the Indian Nursing Council Act of 1947, was established In 1949 for the purpose providing uniform standards in nursing education and reciprocity in nursing registration throughout the country. The only national legislation directly related to nursing practice, also provides a basis from which rules for nursing practice can be developed. Among other responsibilities, this Act gives authority to the Indian Nursing Council for prescribing curricula for nursing education and recognising qualifications of institutions with teaching programmes for nursing. This means that the INC has authority to control nursing education and what the nurse is prepared to do. It is important because legal responsibility does finally depend upon what you should be able to do and how you should do it as well as what you are not prepared to do. The INC uses this authority in nursing education but it delegates authority for control of nursing practice to the State Nurses’ Registration Councils. INDIAN NURSING COUNCIL The Indian Nursing Council was authorized by the Indian Nursing Act of 1947. It was established in 1949 to providing uniform standards in nursing education and reciprocity in nursing registration through out the country. Nurses registered in one stat were not necessarily recognized for registration in another state before this time. The Condition of mutual recognition by the state Nurses Registration Councils, which is called reciprocity, was possible only if uniform standards of nursing education were maintained. FUNCTIONS OF INC 1) It provide uniform standards of in nursing education and reciprocity in nursing registration. 2) It has authority to prescribe curriculum for nursing education in all states. 3) It has authority to recognize programme for nursing education or to refuse recognition of a programme if it did not meet the standards required by the council. ) To provide the Registration of foreign nurses and for the maintance of the Indian Nurses Register. 5) The INC authorizes State Nurses Registration Council and Examining Board to issue qualifying certificates. The INC has been given heavy responsibilities for nursing practice and nursing education but it has not been able to exert enough power to support high standards in nursing. ENGLISH LAW The law based upon the English Pattern is the third way in which the Central Government is a source of legal authority. These laws are very specific and make you â€Å"liable for negligence† or answerable to the laws for acts of carelessness. The laws summarised below are given for medical practitioners including nurses. 1) The right to refuse to the treat a patient expect in an emergency situation. 2) The right to sue for fees. (Applicable only to private duty nurse or private practitioners: other nurses are salaried. ) 3) The right to add a titile descriptions to one’s name. Any title, description, abbreviation or letter which implies nolding a degree, diploma, license or certificate showing particular qualifications may be added. (Improper use of these is often prohibited by State Nurses Registration Acts. The right to wear the Red Cross Emblem is given only to members of the Army medical service. 4) Unregistered practitioners are not allowed to hold positions or appointments in public and local hospitals 5) Fundamental duties. a) To exercise a reasonable degree of skill and knowledge in treating patients. The standard held is that exercised by other reputable members of the same profession in similar circumstan ces. b) Once a relationship to a patient has been established, there is an obligation to attend the patient as long as necessary unless the patient requests withdrawal or notice is given of intention to withdraw. ) A practitioner must give personal attention to his cases and answer calls with reasonable promptness. d) Children must be protected from harming themselves. e) Special precautions must be taken in the case of adults who are incapable of taking care of themselves. 6) The Indian Penal Code demands that poisonous drugs be kept in separate containers properly labeled and marked. Care must be taken not to mix with nonpoisonous drugs. 7) There is a duty of secrecy to the patients. Records must be treated as confidential unless the practitioner is called upon to give evidence in court. 8) Dangerous diseases must be reported. (Theses will vary in different parts of the country. ) 9) Nurses as considered solely responsible for their own professional acts irrespective of the employing authority. A fine is the usual penalty imposed for disobeying the law stated above, although imprisonment is also possible. The central responsibility consists mainly of Policy making, palnning, guiding, assisting evaluating and Co. ordinating the work.

African American Psychology Research Paper - 1293 Words

Abstract For Black people, race is a never-ending conscious component of life in America. We live in a country with a history of slavery that, once ended, extended into an institution and system of laws; Jim Crow, that continue to keep members of the Black community â€Å"othered† and invisible. The threefold purpose of this writing is to discuss how the treatment of African-American patients engaging in psychoanalysis and psychoanalytically informed psychotherapy - as they exist today - fails to meet their full potential in the healing of the Black psyche; and to consider how the training curriculum can be expanded to include writings of Black psychoanalysts; and to encourage the analytic community to be opened up to include more analysts of†¦show more content†¦3) There is plenty of literature on race in the psychoanalytic dyad. Volume 40 of The Relational Perspectives Books Series; The Analyst in the Inner City, is a collection of essays edited by Neil Altman that contains psychoanalytic literature on race from Dorothy Holmes and Schachter Butts. Chapter Five of this volume offers detailed and thoughtful accounts of racial elements in psychoanalysis. These writings also include accounts of Altman’s own case illustrations that show what he refers to as the â€Å"limitations of his own unconscious and unprocessed prejudice† from which we can learn to be better analysts by developing cultural sensitivity and awareness at the expense of errors made by those before us. In each chapter he offers a clinical adaptation of the usefulness of these methods through case studies. Dr. Kimberlyn Leary writes about the application of treatment methodologies specific to the treatment African Americans but to make this suggestion is contrary to purpose of this paper. I assert that the creation of specific therapeutic methods for Black people and people of color would only deepen the chasm between black and white. What I am saying here is that there is a way to apply the existing psychoanalytic theories to Black people and most all of these theories address many of the issues of theShow MoreRelatedThe Theory Of Psychology And Psychology1507 Words   |  7 Pagesare built. The same can be said for psychology. This field has evolved greatly to be considered a science. Psychology has to go through similar process like hard sciences did before it. Psychology has to become widely acceptable by the public, not that psychology hasn’t earned the right to be called a Science with a capital† S†. 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