Stages of Decomposition: Effect of Time and Temperature

Stages of Decomposition: Effect of Time and Temperature Chapter I INTRODUCTION Watson, can you determine cause and time of death? I knelt over the woman and began a cursory examination â€Å"Rigor mortis has set in, so Id estimate shes been dead about 10 to 12 hours. Holmes stood up and brushed himself off with his hands. So, that puts her death between midnight and 2 am†(Anonymous 2007). After the question of cause of death; the question of time of death is the most sought after piece of information associated with a medical death investigation. As a consequence, death investigators find themselves in need of a means of ascertaining the period of time between when an individuals body is found and when they died, sometimes referred to as the post mortem interval. Establishing the time of death through the determination of post mortem interval may have a direct bearing on the legal questions of guilt or innocence by confirming that a suspects alibi covers the period when the victim died, or demonstrating that it does not. If the time of death can be established to within hours, days, months or even years, an individual may be able to prove that they were at some other place at that time. On the other hand, if the suspect is known to have been in the vicinity of the victim during the appropriate time period, then they can be shown to have had an opportunity to commit th e crime. Currently, there are multiple techniques for determining post mortem interval that incorporate methods in almost every discipline of forensic science. Depending on the circumstances, these techniques can yield results that vary from a narrow accurate estimate (video of the victim, the victims stopped watch etc.) to a wide range estimate (counting tree rings on trees growing over or through the remains). Regardless of the of the method used, the calculation of post mortem interval is at best an estimate and should not be accepted as accurate without considering all of the factors that can potentially impact the result. Post Mortem Interval Estimation â€Å"For everything there is a season, And a time for every matter under heaven: A time to be born, and a time to die†¦Ã¢â‚¬  Ecclesiastes 3:1-2 The techniques currently utilized for estimating post mortem interval can be broken down into two broad categories based upon the methodology used. The first of these categories are the concurrence-based methodologies. Concurrence based methods relate or compare the occurrence of a known event, which took place at a known time, with the occurrence of death, which took place at an unknown time. Examples of concurrence-based methods include the determining the years of manufacture of clothing found on a body, tree ring development, dates on personal effects, etc. Concurrence based methods rely on both evidence associated with the body, and anamnestic evidence such as the deceaseds normal pattern of movements. The second grouping of techniques include rate of change methodologies. Rate of change-based methodologies measure some aspect of a evidence, directly associated with the body, that changes at a known or predictable rate and is started or stopped at the time of death. Examples of the rate of change based methods include body temperature, tissue decomposition, insect succession and bone weathering. Some of these methodologies can be considered to fall into both categories. Examples of these would be tree ring development (Coyle, Lee et al. 2005) and insect succession. Previous post mortem interval Estimation Methods The variety of approaches for estimating post mortem interval spring from the varied expertise and experiences of their proponents as such the different methods tend to be focused on the immediate needs of the investigator, and limited to a particular stage of the post mortem interval or type of observation. As a consequence, the period of time for which a procedure is effective will overlap others. Algor, Rigor and Liver Mortis â€Å"Tis after death that we measure men.† James Barron Hope The earliest recorded methods for estimating early post mortem interval were a rate of change methodology based on the most easily observed changes. The cooling of the body after death (algor mortis), the gradual stiffening of the body (rigor mortis) and the fixed pooling of the blood resulting in discoloration of the lower portions of the body (livor mortis) can be easily assessed with minimal or in some instances no instrumentation. Since the time of the ancient Greeks when the following rule of thumb was developed: Warm and not stiff: Not dead more than three hours; Warm and stiff: Dead between 3 and 8 hours; Cold and stiff: Dead between 8 and 36 hours; Cold and not stiff: Dead more than 36 hours; (Starkeby 2004) until modern times, the basis of most temperature based post mortem interval analyses is the assumption that the human body, which averages 98.2 oF +/- 1.3 oF (Mall and Eisenmenger 2005), was at 98.6 oF (Mackowiak, Wasserman et al. 1992) at death and that after death the body looses heat in a predictable manner. There have been many temperature based methods for estimating post mortem interval. As early as the 1800s, Dr. John Davy had developed a method using the fall in body temperature (algor mortis), measured rectally, to determine the post mortem interval (Henssge and Knight 2002). This method was refined by De Saram by recording detailed temperature measurements collected from executed prisoners (De Saram G. 1955). More recent approaches to this technique have included measuring rectal temperature, body surface temperature, ear canal temperature, eye socket temperature and liver temperature (Simonsen, Voigt et al. 1977; Henssge and Knight 1995; Baccino, De Saint Martin et al. 1996; Kanetake, Kanawaku et al. 2006). Improvements to these techniques have included multiple progressive sampling, and the introduction of concepts such as the initial temperature plateau, core temperature, heat gradients, the effects of insulation, the ratio of surface area to volume, the effects of humidity and the effect of conductive surfaces, Microclimates and postmortem skin cooling (Green and Wright 1985; Nokes, Flint et al. 1992; Nelson 2000). However, most methods that attempt to use body temperature changes to determine the post mortem interval are hampered, as most methods are, by individual variability. Even when complex calculations and algorithms have been designed to model for tissue density, initial temperature distribution, post mortem exothermic reactions and heat loss, these refinements have not appreciably narrowed the estimate window for post mortem interval. Multiple studies outlining instances of initial temperature increase of a body soon after death (Hutchins 1985) associated with post mortem chemical changes such as rigor mortis, cell lysis and the conversion of cellular energy production to anaerobic respiration (Nelson 2000); variations in the core body temperature ranging from 0.5 1.2  °C during a 24 hour period (Chisholm 1911; Mackowiak, Wasserman et al. 1992); the effect of variable environmental temperatures (Green and Wright 1985; Green and Wright 1985); and the effect of environmental temperatu re on overall body surface temperatures (Mall, Hubig et al. 2002) have all contributed to limit the usefulness temperature as a consistent indicator of post mortem interval. Additionally, once the body has reached ambient temperature temperature ceases to be a factor. Marshall said it best when he said ‘‘It would seem that the timing of death by means of temperature can never be more than an approximation(Henssge and Knight 1995). Soft and Hard Tissue Decomposition â€Å"Now, a corpse, poor thing, is an untouchable and the process of decay is, of all pieces of bad manners, the vulgarest imaginable†¦Ã¢â‚¬  Aldous Huxley Cadaveric decomposition is a complex process that begins immediately following death and proceeds beyond the time when recognizable human remains have ceased to exist. Decomposition can be broken down into two major stages. The first stage, soft-tissue decomposition, is caused by autolysis and putrefaction. Autolysis is the digestion of tissue by cellular enzymes and digestive processes normally present in the organism. Putrefaction is the digestion of whole tissues systems caused by the enzymatic activity of fungi and bacteria that are either present in the organism or the environment that opportunistically invade the tissue. Both autolysis and the microorganisms responsible for putrefaction are normally held in check in living organisms. However, when an organism dies the cellular and systemic mechanisms responsible for regulating autolysis and inhibiting putrefying microorganisms stop. â€Å"Without these controlling processes the body becomes fancy (bacterial) culture media† (Carayannopoulos 1992). These early postmortem changes in soft tissues can be used to provide an estimate of the post mortem interval from death until skeletonization. However, the rate of soft tissue decomposition can be dramatically affected by both internal and external factors that affect the body (i.e. ambient temperature, cause of death, scavenging, trauma, environmental conditions, clothing, body size, mummification and adipocere formation) (Rodriguez and Bass 1985; Micozzi 1986; Mant 1987; Vass, Bass et al. 1992; Komar 1998; Campobasso, Di Vella et al. 2001). There are reported instances of rapid decomposition associated with acute illness (Frisch 2001) and the author is personally aware of an instance of a post mortem interval of less than eleven days resulting in complete skeletalization of an individual that died of complications related to Acquired Immunodeficiency Syndrome (Watson 1994). Additionally, there are a number of examples of bodies remaining intact for year s after death (Bass and Jefferson 2003). Beyond gross observation for assessing decomposition, researchers have developed multiple morphometric and chemical methods for assessing soft tissue decomposition. These have ranged from early (ca.1800s) methods such as the Brouardel method which examined the shift in flammability of putrefaction gases in the early post-mortem interval, and the Westernhoffer-Rocha-Valverde method examining the formation of crystals in the blood formed after the third day of putrefaction (Cengage 2006); to more modern methods such as ultrasound assessments of organ condition (Uchigasaki, Oesterhelweg et al. 2004) and the use of electron microscopy to examine measurable physical changes in mitochondria (Munoz, de Almeida et al. 1999) and platelet count (Thomsen, Kaatsch et al. 1999). Chemical methods used to assess time since death include the assessment of volatile organic compound formation (Vass, Bass et al. 1992; Statheropoulos, Spiliopoulou et al. 2005; Statheropoulos, Agapiou et al. 2007; Dekeir sschieter, Verheggen et al. 2009); the concentrations of non-protein nitrogen (Sasaki, Tsunenari et al. 1983; Gallois-Montbrun, Barres et al. 1988) and creatinine (Gallois-Montbrun, Barres et al. 1988; Brion, Marc et al. 1991). Bony tissue decomposition, the second major stage of decomposition, consists of a combination of surface weathering due to environmental conditions (temperature, humidity, sunlight) and erosion from soil conditions (pH, mineral content, etc.) (Behrensmeyer 1978; Janjua and Rogers 2008). While not much detailed study has been done on the environmental factors that affect bony tissue breakdown, it has been established that environmental factors such as pH, oxygenation, hydrology and soil flora and fauna can affect the long term stability of collagen (Garlick 1969; Henderson 1987; Bell, Skinner et al. 1996). Collagen, the primary protenatious component of bone, slowly hydrolyzes to peptides and then to amino acids leading to the breakdown of the collagen-mineral bonds which weakens the overall bone structure leaving it more susceptible to environmental weathering (Henderson 1987). By examining the effects of related changes (cracking, flaking, vacuole formation, UV-fluorescence of compa ct bone) the investigator can estimate the period of time a bone sample has been exposed to weathering (Yoshino, Kimijima et al. 1991; Bell, Skinner et al. 1996; Janjua and Rogers 2008; Wieberg and Wescott 2008). Current methods of assessing time since death using bone weathering rely heavily upon the experience of the investigator (Knight and Lauder 1969) and are limited to immediately post skeletalization to 10 to 100 years based on environmental conditions (Haglund and Sorg 1997). As with the assessment of soft tissue decomposition for time since death, investigators examining bone decomposition have supplemented observational methods with quantifiable testing techniques that analyze changes that are not directly affected by the physical environment (Lundquist 1963). Radiocarbon dating of carbon-14 and strontium-90 have been used to group remains pre and post 1950 (Taylor, Suchey et al. 1989; Maclaughlin-Black, Herd et al. 1992). Neis suggested that, with further study of strontium-90 distributions, determination of times since death should be possible (Neis, Hille et al. 1999). Bradley suggested that measuring the distribution of 210Pb and 210Po in marrow and calcified bone could prove forensically significant (Bradley 1993). This work was built upon by Swift who evaluated using 210Pb and 210Po distribution in conjunction with trace element analysis to provide a meaningful estimate of the post-mortem interval (Swift 1998; Swift, Lauder et al. 2001). Maclaughl in demonstrated that chemical changes due to environment could measurably affect isotope levels (Maclaughlin-Black, Herd et al. 1992). In addition to radionucleotide studies, investigators have also measured the changes in both organic (amino acids, urea, proteins, DNA) and inorganic compounds (nitrogen, potassium, sulphur, phosphorous) in bone. (Jarvis 1997; Prieto-Castello, Hernandez del Rincon et al. 2007). Stomach Contents/Rate of Digestion â€Å"Govern well thy appetite, lest sin surprise thee, and her black attendant Death.† John Milton The presence or absence of food in the stomach is often used as an indicator of post mortem interval. Its use as an indicator of post mortem interval is predicated on the assumption that under normal circumstances, the stomach digests and empties at a predictable rate taking from two to six hours to eliminate a full meal (Jaffe 1989). If a person had eaten a light meal the stomach would empty in about 1.5-2 hours. For a medium-sized meal the stomach would be expected to take about three to four hours to empty. Finally, a large meal would take about four to six hours to exit the stomach. Regardless, it would take from six to eight hours for the initial portion of the meal to reach the large intestine (Hallcox 2007). This information, coupled with reliable ante-mortem information relating to when an individual last ate is used by some pathologists when providing an estimate of the times since death. It is for this reason, among others, that comprehensive autopsies usually include an ex amination of the stomach contents (Batten 1995; Siegel 2006). Although it provides another useful indicator of time since death, there are serious limitations to the assessment of the stomach contents as an accurate indicator of time since death. Its reliance on reliable anamnestic evidence such as eating habits, the extent to which the victim chews their food (Pera, Bucca et al. 2002), the physiological state of the victim (Troncon, Bennett et al. 1994; Jayaram, Bowen et al. 1997; Lipp, Schnedl et al. 1997; Phillips, Salman et al. 1997) and the state of mind of the victim (Jaffe 1989); as well as verifiable antemortem evidence such as what the last meal consisted of (protein vs. fiber vs. fat)(Dubois 1985; Tomlin, Brown et al. 1993), the amount of liquid consumed with the meal, alcohol consumption and the time when it was consumed limits its usefulness to a small number of cases (Jaffe 1989). These factors combined with evidence that digestion can continue after death (Koersve 1951) makes the estimation of post mortem interval using stomach co ntents difficult at best. Insect Succession â€Å"Buzzards gotta eat, same as worms.† Clint Eastwood from the Outlaw Josey Wales Insect colonization of a body begins within hours of death and proceeds until remains cease to be a viable insect food source. Throughout this period, multiple waves of colonization by different insect species, as well as multiple generations of previously established species can exist. Forensic entomologists can use the waves of succession and generation time to estimate the postmortem interval based on the variety and stage of development of the insects, or insect remnants, present on the body (Archer and Elgar 2003). In addition to information regarding time since death, forensic entomology can provide useful information about the conditions to which the body was exposed. Most insects have a preference for specific conditions and habitats when colonizing a body and laying their eggs. Modifications to that optimal habitat can interrupt the expected insect colonization and succession. The presence of insects or insect larva that would typically be found on bodies colonized indoors o r in shade on a body discovered outside in direct sunlight may indicate that the body was moved after death (Sharanowski, Walker et al. 2008). Aquatic insects found on bodies discovered on land could indicate the body was originally in water (Wallace, Merritt et al. 2008; Proctor 2009). Although insect succession varies by season, geographical location and local environmental conditions, it is commonly assumed to follow a predictable sequence within a defined habitat. While there are a multitude of studies that have examined regional succession patterns (Archer and Elgar 2003; Tabor, Brewster et al. 2004; Tabor, Fell et al. 2005; Martinez, Duque et al. 2007; Eberhardt and Elliot 2008; Sharanowski, Walker et al. 2008) these studies use different approaches towards defining habitat and assessing insect succession making cross-comparisons of their data difficult. Also, the majority of these studies do not rigorously address the statistical predictability of a species occurrence making their results of limited use as post mortem interval indicators (Michaud and Moreau 2009). Additionally, beyond the presence or absence of clothing, the majority of the post mortem entomological studies conducted do not examine non-habitat external factors that may affect succession. For example, only a few studies have been conducted that assess the affect of drug ingestion (George, Archer et al. 2009) or the presence of chemicals (bleach, lye, acid etc.) used to cover-up evidence (Charabidze, Bourel et al. 2009) on the insect life cycle. As with other means of assessing time since death, more extensive studies with different insect species and drugs in a wider variety of habitats is necessary. Electrolyte Concentration â€Å"Death is a low chemical trick played on everybody†¦Ã¢â‚¬  J.J. Furnas Cellular activity does not immediately cease when an organism dies. Rather, individual cells will continue to function at varying metabolic rates until the loss of oxygen and metabolic substrates caused by the cessation of blood flow results in hypoxia (low oxygen). As cell metabolism shifts from aerobic to anaerobic, oxidative phosphorylation and ATP generation, the cellular processes keeping autolysis in check, begin to decrease and eventually cease all together. Without energy to maintain osmotic gradients membranes begin to fail. As lysosomal membranes begin to fail the enzymes within are released and begin consuming the cell from the inside out. With autolysis comes a cascade of metabolic chemicals, released ions, originally bound up in various cellular processes begin to diffuse due to the diffusion gradient according to Ficks law into the intracellular spaces (Madea 2005). Forensic researchers have used the presence, absence or effects of inorganic ions such as potassium, phos phorous, calcium, sodium and chloride as a means of estimating time since death (Schleyer and Sellier 1958). In most instances the higher the concentration gradient, the more suitable is the analyte for the estimation of the time since death. When analyzing body fluids for the purposes estimating post mortem interval, early researchers tended to focus their studies on body fluids such as, cerebrospinal fluid, blood and pericardial fluid (Schleyer and Brehmer 1958; Coe 1972; Henssge and Knight 1995; Yadav, Deshpande et al. 2007) with a few others examining other compartmentalized bodily fluids (Madea, Kreuser et al. 2001) and the largest numbers focusing on vitrious humor (Madea, Henssge et al. 1989; Ferslew, Hagardorn et al. 1998; Madea and Rodig 2006; Kumagai, Nakayashiki et al. 2007; Thierauf, Musshoff et al. 2009). Chemical methods used to assess these analytes in blood and spinal fluid as an indicator of post mortem interval have failed to gain general acceptance because, for th e most part, they failed to produce precise, reliable, and rapid results as required by the forensic community (Lundquist 1963). Current chemical methods which have primarily focused on vitreous fluid tend to suffer from the same limitations demonstrated by the fact that with notable exceptions (Pounder 1995) very few statistically rigorous field studies on the reliability and precision of estimating post mortem interval are available in the literature (Coe 1993; Madea 2005). Enzyme Activity As previously discussed, cellular activity does not cease when clinical death occurs. In any circumstances where the cellular metabolism shifts from a homeostatic balanced state to an imbalanced state biochemical changes occur. Changes in the levels and/or activity of enzymes (i.e. cardiac troponin, c-reactive proteins, and G proteins) have long been used as indicators of cellular stress (Li, Greenwood et al. 1996; Katrukha, Bereznikova et al. 1998; Tsokos, Reichelt et al. 2001; Uhlin-Hansen 2001). Assessing similar changes in cellular biochemistry as a function of time since death provides investigators with a wide variety of tissues, testing methods and analytes for consideration. As a consequence, forensic investigators have assessed and suggested enzymes from heart, pancreas, muscle, blood and brain as potentially suitable markers for time since death (Wehner, Wehner et al. 1999; Wehner, Wehner et al. 2001; Kang, Kassam et al. 2003; Jia, Ekman et al. 2007; Poloz and ODay 2009). C omparisons of total proteins analyzed ante and post mortem analyzed using two dimensional gel electrophoresis and Matrix Assisted Laser Desorption/Ionization Time-of-Flight have demonstrated changes in metabolic enzymes, (Jia, Ekman et al. 2007; Hunsucker, Solomon et al. 2008). Assessing the changes in enzyme activity provides examiners a means to assess time since death, in many instances long before visible cellular changes. However, in at least a few of these studies results indicate that enzyme degradation during extraction and partial enzyme activity observed with degradation products these markers better suited to qualitative analysis rather than quantitative analysis (Sabucedo and Furton 2003). Muscle/Nerve Excitation Both neurons and myocytes retain the ability to respond to electrical stimulation for at least a short period of time after organism death. (Sugioka, Sawai et al. 1995; Briskey, Kastenchmidt et al. 2002; Sams 2002). The response of nervous and muscle tissue to external electric stimulation has also been investigated and proposed as means to estimate time since death (Kline and Bechtel 1990; Straton, Busuttil et al. 1992). Methods developed to investigate myocyte excitability assess the relative magnitude and duration of the muscle contraction during the application of external stimulation. To assess the contractile response, a combination of observational based assessments (Madea 1990; Jones, James et al. 1995) and measurement based assessments (Henssge, Lunkenheimer et al. 1984; Madea 1992) have been suggested and reported. Similar investigations have examined post mortem excitation of nervous tissue by measuring a variety of neurological reactions to stimuli. These include the alteration of Compound Muscle Action Potential (Nokes, Daniel et al. 1991; Elmas, Baslo et al. 2001; Elmas, Baslo et al. 2002), lengthen of the refractory or non-propagating period immediately following the CMAP (McDowall, Lenihan et al. 1998), the extracellular impedance/resistance (Querido 2000), the chronaxie measurement or the time over which a current double that necessary to produce a contraction is applied before the contraction occurs (Straton, Busuttil et al. 1992) and the changes in the amplitude of the F-wave (the secondary CMAP observed after the initial CMAP) have all been examined, and been suggested as potential indicators of time since death. The results of studies examining the response of excitable tissue to electric stimulation have been consistent in that the stimulation response varies predictably over time. However, suitability for absolute indicators of time since death remains in questions as investigators have reported contradictory results related to the effect of the manner of death on the stimulation response (Madea and Henssge 1990; Elmas, Baslo et al. 2002). RNA Degradation RNA degradation, both antemortem and postmortem, is a complex process that is not well understood. Unlike with DNA degradation, continuous degradation of inducible mRNAs by native ribonucleases is used as a means of translational control. After cell death these ribonucleases, no longer kept in check by the mechanisms of cellular homeostasis, combine with exogenous ribonucleases from bacteria and fungi to begin un-inhibited digestion of all cellular RNA. Investigators have noted extensive variability in RNA degradation rates in different tissues (Bauer 2007). Not surprisingly such variability appears to be related to the antemortem ribonuclease activity of the tissue; with relatively ribonuclease poor tissues such as brain and retina exhibiting greater RNA stability (Johnson, Morgan et al. 1986; Malik, Chen et al. 2003) when compared to ribonucleases rich tissues such as liver, stomach and pancreas (Humphreys-Beher, King et al. 1986; Finger, Mercer et al. 1987; Bauer, Gramlich et al. 2003). Additionally, but also not surprisingly, some constitutively expressed mRNAs have been shown to be more stable, or perhaps simply more prevalent, than inducible mRNAs (Inoue, Kimura et al. 2002). Additionally, while intrabrain mRNA levels are fairly constant, interbrain levels vary considerably (Preece, Virley et al. 2003). As a consequence of these observations, the degradation of RNA (total and/or mRNA) have been suggested as a potential analyte to assess time since death. Researchers examining the effect of post mortem interval on RNA stability have examined a variety of targets (mRNA, both tissue specific and constitutively expressed, and total RNA) with an assortment of methods including Reverse Transcriptase (RT) PCR(Ohshima and Sato 1998; Fleige, Walf et al. 2006; Haller, Kanakapalli et al. 2006; Zhao, Zhu et al. 2006), RNA (cDNA) microarrays (Bahn, Augood et al. 2001; Catts, Catts et al. 2005; Son, Bilke et al. 2005; Popova, Mennerich et al. 2008) and quantitative RT-qPCR (VanGuilder, Vrana et al. 2008). Based on these studies, there are indications that beyond time and temperature, factors such as hypoxia, tissue pH, antemortem physiological conditions (coma, seizure activity and injury) postmortem transcriptional activity and RNA sequence can dramatically affect the stability and measurable levels of RNA (Burke, OMalley et al. 1991; Harrison, Heath et al. 1995; Ohshima and Sato 1998; Catts, Catts et al. 2005; Bauer 2007). When examining the sem inal question regarding time since death and temperature some researchers have reported temperature and time as significant factors affecting mRNA levels (Burke, OMalley et al. 1991), while others have reported the reverse (Harrison, Heath et al. 1995; Preece and Cairns 2003). These contradictory data are not surprising given the changes in the specificity, sensitivity and application of the assays used; however, the ultimate question has not been resolved. What is clear from the research is that RNA degradation (mRNA or total) is a complex process (Preece and Cairns 2003; Preece, Virley et al. 2003; Heinrich, Lutz-Bonengel et al. 2007) effected by multiple factors indicating more study will be required before RNA degradation can be considered a reliable indicator of time since death. DNA Degradation and its Effect on DNA Typing Since the initial application of molecular biology techniques to samples of forensic significance in the latter half of the 1980s, forensic scientists have noted that increased exposure to environmental insults can negatively impact DNA quality. Developmental validation studies performed to evaluate the efficacy of new typing techniques (SWGDAM 2008) have found that environmental variables such as heat, high humidity, direct moisture, fungal/bacterial contamination and ultraviolet light can impact the quantity or quality of the DNA sample making them unsuitable for DNA analysis (McNally, Shaler et al. 1989; Graw, Weisser et al. 2000; Takayama, Nakamura et al. 2003; Bender, Farfan et al. 2004; Schneider, Bender et al. 2004; Niemcunowicz-Janica, Pepinski et al. 2007). During transitions in technology from Restriction Fragment Length Polymorphism (RFLP) analysis to Polymerase Chain Reaction (PCR) based testing, researchers noted that samples too degraded to produce an RFLP pattern could still produce profiles using a variety of PCR based markers that evaluated loci shorter in length (Hochmeister, Budowle et al. 1991). This finding supports the hypothesis that degradation in the forensic setting is (not surprisingly) processive. Additional research found that while the DNA in some samples like cadaveric blood and kidney tissue could degrade to the point where it was no longer suitable for DNA fingerprinting after as little as a week (Ludes, Pfitzinger et al. 1993); other samples such as bone (Hochmeister, Budowle et al. 1991; Frank and Llewellyn 1999) and teeth (Schwartz, Schwartz et al. 1991; Pfeiffer, Huhne et al. 1999) could, under most conditions, provide typeable DNA for months. The fact that DNA degradation has a detrimental effect on larger genetic loci, and affects different tissues at different rates is considered to be of extraordinary forensic significance is evidenced by the numbers of studies that seek to examine, and overcome this effect (42 validation studies specifically mentioning DNA degradation from 1995-2009 in PubMed). This makes perfect sense when the observer considers the impact that degradation can have on selecting suitable samples and evaluating the resultant DNA profiles. However, a number of researchers have looked beyond the simple question of how degradation affects the typing of samples to broader questions such as the mechanisms of postmortem degradation (De Marà ­a and Arruti 2004; Foran 2006) and synthesis (Oehmichen, Frasunek et al. 1988) and how that knowledge can be used to assist in the assessment of time since death. DNA degradation by RFLP: Since Sir Alec Jeffreys first applied Southern blotting (Southern 1975) techniques to the testing of forensically significant samples in 1985 (Jeffreys, Brookfield et al. 1985) DNA analysis has revolutionized forensic science. Restriction Fragment Length Polymorphism DNA analysis relies on variations in the lengths of DNA fragments generated by enzyme restriction. With restriction fragments ranging from approximately from 2 33 kilobases (Baird, Balazs et al. 1986) successful typing and analysis requires high quality (un-fragmented) DNA. Researchers noted from the outset that in some cases involving older and/or postmortem samples that DNA degradation, tied to the exposures of higher temperatures, resulted in the gradual disappearance of the longer fragments reducing the evidentiary value of older samples (Bar, Kratz

Just One Bullet! Essay

Everyone assembled at the bitter isolated base waiting, waiting anxiously for the deafening signal of unbearable battle, that could make them a prominent hero or just another failure. Jack and the rest of the army sat in their camouflaged overalls and stared intensely at the loyal colours of their flag as it waved at them vigorously in the freezing uncontrollable wind. Most sat deeply in thought and contemplation, reflecting on their battle strategies. Others just shivered greatly at the thought of the dreadful atrocious war, which was unavoidably going to take place. There was no going back. All they could do now is wait for the inevitable. This passionate battle would decide the fait of either team and the excruciatingly cold weather added to the dangers and troubles of the task in hand. Even the wolves were howling stridently as they retreated back to their dens subsequent to a harsh battering from the torrential rain. There was now merely five minutes before the clash between the two conflicting tribes, positioned on each side of the dense unforgiving wood. The Tribes would enter the wood at their own peril as this mysterious area was unknown to the approaching teams of men. It was just seen as another little obstacle in their way. Ruthless and merciless combat was impending ever closer when James, the proud audacious leader of Jack’s army, stood up clutching his gun tightly, and stared around the room at the tired depressing faces sitting in front of him. He paced sturdily up and down with satisfaction and pride, and had a beaming grin covering most of his dirty -blemished face. He was about to present the essential encouragement speech. This is a speech that all superior, influential leaders give previous to entering the battlefields. Obviously James was no exception. â€Å"This is it. We’ve come this far and there’s no going back. Does everyone remember their roles?† There was a dawdling hesitant nod of heads from most boys, as others bellowed YES as the adrenalin was now pumping dynamically through their blood streams. â€Å"Okay just remember keep your eyes open and your ears peeled because it takes just one bullet!† Jack advised his army seriously as he paced around pompously trying ineffectively to unearth more words of encouragement from his limited vocabulary. Just before he could speak again the feared war whistle was sounded vociferously, and everyone charged hastily out of the freezing draughty hut and let out a great thunderous cry as they entered the merciless wood. The war had begun. The wood was an eerie, murky and desolate place, where James’ band of hardhearted killers were stealthily creeping around, dodging and weaving the trees as their branches waved ferociously in the irrepressible wind. The wood was almost silent except for the chilling toot of the old wise bird, as it stood motionless on the prolonged arm of the gigantic great oak tree. These tall oak trees stood over the vigilant army creepily as they crept cautiously through the slender meandering path, avoiding the trees that could be hiding the malevolent enemy. The wind blew ferociously and howled deafeningly, as it brushed past the trees stripping it bare of its leaves. The never-ending heavens were a depressing black shade and even the brightest stars couldn’t be distinguished with the naked eye. The only source of luminosity was coming from the vivid dazzling lights of the torches, which were positioned on the army’s head protectors. Jack was becoming more nervous by the second, and his heavy breathing started to generate condensation on the front of his mask. Jack accompanied the leader, James, during the daunting and difficult terrain of the forest. Jack followed James like a helpless cub, pursuing its mother for protection against anything dangerous that might be lurking around the corner. James stopped abruptly almost causing a collision with him and Jack. He turned around sharply and stared fiercely into Jack’s wide apprehensive eyes. He then raised his index finger and pressed it decisively against his firm dry lips, implying Jack to be quiet as something was in close proximity. They both crept silently towards the naked deciduous bushes. James pulled his gun gradually out of its small auburn pouch trying not to make a noise. He snatched the clip back carefully and pulled on the trigger. A little whimper was heard from the seemingly sinister undergrowth. Jack took a massive gulp. He froze in astonishment, as James peered curiously into the murky bushes. â€Å"Damn rabbit,† James muttered angrily as he forcefully thrusted his weapon into his little pouch and stormed off from the scene of the savage shooting. Jack wiped the cold sweat of his brow with his quivering hand, relieved that it was just a rabbit and followed James closely through the dense mist. It was now six in the morning and the beautiful shades of red coming from the intensifying sun lit up the skies, and overwhelmed the two soldiers. The scarlet sky was now alive and energetic with graceful song coming from the striking winged birds, as they perched blissfully on the soaring trees in their hundreds. The wonders of nature were currently at their climax. There were masses of scurrying squirrels selecting nuts to transport back to their home, ready for hibernation. Both Jack and James stopped in bewilderment to take in this astonishing spectacular sight. The thought of the malicious war was erased from their momories for a few minutes, as they watched the environment around them intently. It was a remarkable sight. Unexpectedly the view was suddenly interrupted by gunshots, and the cruel reality of the malevolent war had soon sunk in again. Their stunned desperate bodies dived into the green shrubbery for protection. Jack was horrified. His body started shake immensely. He didn’t know what to do. He turned around to see James being his courageous brave self. He was pelting lethal bullets into distance where the gunshot was heard. Jack calmed himself down. He reassured himself that everything would be okay. He pulled himself up reluctantly and joined in shooting into the remote distance. Their bodies were now quivering with excitement and exhilaration. After about a minute they both halted their fire. The wood was now silent except for the panting of the exhausted soldiers, as they tried to catch their breath speedily. You could now cut the atmosphere with a knife. All of a sudden, the unbelievable happened. A single fatal bullet came darting out of the distance and hit James in the middle of his chest. â€Å"No!† screamed Jack emotionally as he darted rapidly to James’ side. Jack could see the crimson paint dripping down his throbbing chest. â€Å"Jack,† James managed to say while clutching his fatal wound. â€Å"Go, you can’t stay here.† Jack unwillingly left, leaving James to struggle through the pain. Jack ran through the forest intent on being triumphant for the valiant and brave leader of his army. James’ death had bought out a new and innovative side of Jack. He was now more courageous and fearless of what lay ahead. Jack had now thankfully come to the end of the merciless wood. Here there was a great clearing, and the bright intense colour of the sun dazzled Jack momentarily. Jack chafed his eyes leisurely, and tried to adjust his eyesight to come to terms with the new brightness. Jack had survived the most awful part of the strenuous journey, but now his expertise and capability would be put up to the test. He now had to assault the isolated deadly enemy cabin, situated in the middle of the vast clearing. Jack plucked up his courage and sprinted towards the cabin. The sheer adrenalin made him run increasingly faster than he ever had. Jack could almost touch victory with his leading hand before it was cruelly taken away from him, with one fatal bullet into his open chest. He fell to the ground powerless in distress and agony. His face was screaming with agony and he revolved around the floor in pain and torture. He clutched his chest in anguish and he knew the game was over. Back at James’ team hut the rest of the army were slumped depressed and miserable in their padded comfortable seats. They were exchanging opinions on how they could have thrashed the opposition team. James and Jack were absent from the conversation and people started to be concerned about their location. They started to consider the most appalling of feasible occurrences when James and Jack walked in!

An analysis of chapter five’s significance to the novel as a whole including some of the novel’s broader issues & concerns

This novel is about a boy called Alem. The book concentrates on the issues of immigration and those closely affected by it, the main character in the book is a young boy called Alem who has fled his country of origin Ethiopia because of growing tensions between Ethiopia and its neighbouring country Eritrea. Alem's mother is from Eritrea and his father is from Ethiopia and those two countries are in war that's why his family are not accepted in both countries because of Alem's mixed heritage and his families mixed marriage, they are firstly driven from their home in Ethiopia by the local police and then they face prejudice in Eritrea. His dad felt it's insecure for Alem to stay in Eritrea while the war going on between those two countries. Alem's father took him on a holiday to England but Alem is unaware of his father's plan, which is to leave Alem in England until this was is over. In their arrival in England they stay in a hotel after few days of sight seeing Alem's father leaves Alem in the hotel and go back to Eritrea. Before chapter five Alem meets Pamela and Mariam who works in the refugee council. They helped Alem to apply for asylum seekers to stay in England. Until the home office processing the asylum application, they found a children home for Alem to stay. Chapter five is one of the significant chapter in the whole novel because its shows the development of novel it builds up the tension so the readers can get excited to find out what is going to be the next step of the novel. In this chapter most of the characters are introduced. Chapter 5 begins with Alem arriving at the children's home and a description of what the place looks like and how it makes Alem feel. He is firstly introduced to a worker at the children's home named Sarah Cohen who shows him around the place informs him of the rules and regulation of the place. During the exploration of the home Alem meets another worker named Tom Whittaker and a man named Dave he notices a boy sitting by himself and is informed that this boys name is Mustafa. While he is still being showed around the place he meets his soon to be enemy Sweeney for the first time in the smoker's room. Sweeny later demands Alem to get some biscuits. When Alem refused to get biscuits for Sweeney they start arguing, Tom hears the argument and Alem explains to Tom the whole situation and then Tom makes Sweeny apologise for anti social behaviour. Alem meets his room partner called Stanley Burton who is orphan and depress in his arrival Stanley tells Alem his own story of why he's at the children's home. All the characters behaviors in the children's house show how unorganized asylum systems. However there are some people who are emotionally attach in their past for e.g. Stanley Burton is mentally ill but no one in the children's house take him seriously about the effects his having on his mind. After chapter five the story largely concentrates on Alem's struggles to familiarise him to this foreign land. He goes to the refugee camp. He gets bullied, but where he also manages to make some friends. Alem had a fight with Sweeney but makes friends with Mustafa and decides to run away from the children's home. He gets lots of support from the Refugee Council, who fined a better place for him to stay. His been fostered by a family. It was difficult for him to adjust in another house with new people but he manages to stay with them. He starts going to a school and again he face racism and bullies in school. Meanwhile the trouble in Ethiopia and Eritrea is gathering rapidity. His mums died in the war and his dad comes back to England to live with his son. Alem's get really happy and excited to have one of his family members around him. The government refused to give them permission to stay in England. After couple of weeks his father also passed way in a car accident. In my opinion Benjamin Zephaniah wrote this story in order to educate people of the growing problems of the way refuges are treated in this country. He is trying to give people an understanding of the way a young refuge might look at our country and what they might think of our customs. He is also showing that underneath skin colour, culture and customs we all think and are alike. Benjamin Zephaniah intentions and objective are to demonstrate the anguish and struggles that many asylum seekers have to go through in England. The author took ideas from many writers and books, which are related to refugees to make this book unique and special to the readers. He attempt to influence the readers with a combination of layout, emotions are raised, and guilt is created to feel sorry for Alem and it shows how to be strong when people have to deal with problematic situation that they face while they are living in a children's home. Chapter 5 is very important to the story because it shows Alem and no mater where he is aggression follows him. I felt this chapter have really emotional attach to it. Many characters are introduced in this chapter because some of the characters encouraged Alem to become a stronger person and some of the characters are shown to bully him so he can show his strength to stand up for himself. In this chapter it showed the position of each character. He learns to make a judgment on other characters. This chapter is very important because it shows how the system works and it's the beginning of all the struggles for Alem for e.g. living independently, getting bullied etc. This chapter sounds different than other chapter of the novel because its sound more dramatic, helpless, angry and showing his strength. The other sound was informative, balance and controlled. He shows responsibility, development of his knowledge by dealing with problematic situation and trying to adjust in a new environment. This chapter seems so realistic. It is a simple chapter to read because it has a good solid plot and structure of the book makes it easier to understand roles of each character. It shows how the main character of the novel starts living independently. This chapter expose Alem's character by showing amazing strength to keep standing in the toughest time while he was at the children's home for e.g. one of the boy wants him to get some biscuits but Alem replied ‘I don't want any biscuits. If you want biscuits you get them yourself'. It shows that he is not scared of anyone, he is a straightforward person who wants to stay out of trouble but he is not scared to tell the truth. The main character is Alem this is because he is someone who can be trust and empathize in the whole novel. He is the only character in the whole novel. The whole novel was written to explore this character. Alem is well presented to show his strength and weaknesses. This is a unique character because he's the refugee and all the other characters are the supporting character to help Alem to get justice. Alem is the central character in the book because the author believed that the easiest way to tell the story of a refugee would be by telling it through the eyes of Alem, this would help the audience to imagine themselves in the place of a refugee. I think that chapter 5 is a real turning point in the story because it reveals more of Alem's character then other chapters so it makes it easier for the audience to formularise themselves with Alem's character. It is an excellent topic about which to write because there are rarely books about this particular topic and I believe people should be informed. The story line is thoughtful for me and it proved to be a stimulating and moving experience. The message behind this novel was to inform people how refugees used to get treated in the past. Benjamin Zephaniah understood the character this is because he did endure racism in his childhood when he arrived from Africa. He can relate this book to his own life.

The Efficacy And Metabolic Profile Of Bipolar Disorder

A Systematic Review of the Efficacy and Metabolic Profile of Lurasidone in the Treatment of Depression in Bipolar Disorder MSc in Psychiatry 2015 Dissertation by Giles Osborne BA BSc MA(Oxon) Department of Psychiatry, Cardiff Medical School, Cardiff, CF14 4XN Main Body Word Count 18,885 May 2015 DECLARATION / STATEMENTS PAGE DECLARATION This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. Signed----------------------------- (G R Osborne) Date---------------------------------------- STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of MSc (Psychiatry) Signed---------------------------- (G R†¦show more content†¦Signed--------------------------- (G R Osborne) Date----------------------------------- ACKNOWLEDGEMENTS I would like to thank Mr Jon Ford BSc MSc and Dr Chris Lee MBBS PhD for the proofreading of this paper. I would particularly like to thank Dr Chris Lee for inspiring me to undertake this MSc. Dedicated to Obi, even though he may not have the cognitive construct of self. ABBREVIATIONS ADL: Activities of Daily Living AE: Adverse Event ANCOVA: Analysis of Covariance ANOVA: Analysis of Variance BDNF: Brain Derived Neurotrophic Factor BMI: Body Mass Index BPD: Bipolar Disorder CGI-BP: Clinical Global Impressions Scale CGI-BP-C: Clinical Global Impressions Scale - Bipolar-Change Scale CGI-BP-S: Clinical Global Impressions Scale - Bipolar-Severity Scale CI: Confidence Interval CVD: Cardiovascular Disease DB: Database df: Degrees of Freedom DSM IV/ 5: Diagnostic and Statistical Manual of Mental Disorders versions 4 or 5 EMA: European Medicines Agency EPSE: Extrapyramidal Side Effects EU: European Union FPG: Fasting Plasma Glucose FDA: Food and Drug Administration FDC: Fixed Dose Combination GLP: Good Laboratory Practice HAM-D: Hamilton Depression Rating Scale HR: Hazard Ratio ICD 10: International Classification of Diseases 10th Revision ICH-GCP: International Conference on Harmonisation Good Clinical Practices IFG: Impaired Fasting Glucose