Nuclei and cytoplasmic extractions were carried out as follows. 2g of tissue was ground in liquid nitrogen and mixed with 1xNIB buffer with DTT; the mixture was filtered through microcloth and the filtrate centrifuged at 1260g at 4°C for 10 minutes. The supernatant was decanted and reserved as the cytoplasmic fraction. The pellet was resuspended in 2ml 1xNIB buffer with 10ul of 10% triton x-100 and protease inhibitors before centrifuging at 1200g at 4°C for 10 minutes. The supernatant was discarded and the pellet resuspended in 0.5ml of JAJ-Extraction Buffer . This suspension was sonicated and centrifuged at 3000g at 4°C for 10 minutes – the resulting supernatant was reserved as the nuclear fraction. Nuclear and cytoplasmic fractions were then run on an acrylamide gel and a western blot was carried out using the purified GP16 serum. A ~90kDa band was observed in the WT cytoplasmic fraction which is the correct size for full length FUN protein, and a ~37kDa band was observed in the fun cytoplasmic fraction. Both WT and fun nuclear fractions displayed a ~70kDa and ~37kDa band . Western blots were then attempted using crude, nuclear and cytoplasmic fractions of protein extracted from fun-2 plants, but none of these gels showed any bands despite confirmation of protein presence in sample by probing with KN1 antibody . Other extraction procedures were carried out using triton, SDS, dodecyl maltosidase and IGEPAL detergents, but none of these westerns showed any bands . In order to further purify the serum,horticulture products primers AV242 and AV243 were designed to a highly antigenic region of the protein predicted by antigen prediction software . This 96bp product was cloned into pENTR and recombined into pDEST15 using C3040 cells, which greatly improved transformation efficiency.

pDEST15-3500 was cloned into Rosetta cells to purify protein and create a column as described above. This column was used to purify GP15 serum; the purified serum was found to react weakly to FUN-recombinant protein in a dot plot, but none of the controls . 15 western blots were attempted using this purified serum, but none showed bands, or had high background such that any bands present would be obscured . Solubilising the 3500-GST construct before creating a column may help – a dot plot based on extractions solubilised with DDT showed that solubilisation greatly improved 3500-GST construct recovery from culture .In summary, we can say that FUN is a conserved, universally expressed, disordered protein that localises to the nucleus. Cytoskeletal binding has been implied by both bio-informatic prediction programs and Y2H, as has involvement in nucleotide binding, especially RNA and GTPases. The highly conserved nature of FUN across the grasses, and significant conservation across the Plant Kingdom, along with the mutant study presented in Chapter 1, make a compelling case for the importance of this protein in general plant growth and development. The nuclear localisation of FUN, along with its synergistic interaction with the transcription factor WAB1 , and its interactions with various hormone mutants suggest that FUN is involved at some stage in a signal transduction pathway between the hormones and downstream gene expression. The universal expression of FUN makes it unlikely that FUN can be considered a signalling molecule per se; instead it strengthens the hypothesis that FUN is involved in the transduction of these signals. On the other hand, there is evidence of variance in transcript levels so there could be some merit to the idea that FUN is a real signal. Using Gene Ontology term analysis, it has been shown that disordered proteins are heavily biased toward signalling, regulation and control110, strengthening the signalling network hypothesis.

The disorder of FUN may also have contributed to the difficulty in creating a specific antibody. Many disordered proteins are thought to undergo conformational changes in order to transduce signals110 and the high concentration of serines that could be phosphorylated , as well as a serine repeat chain in FUN , could allow these conformational change. Despite the fact that the antibody was shown to be non-specific, and the same immunoblot pattern was produced in both normal and fun tassels , the pattern of expression in the normal tassel fits the feminised tassel phenotype. Additionally, the expression pattern agrees with the nuclear localisation shown by YFP-fusion and bioinformatic prediction . Thus the expression pattern could indeed be real. Since both GO term analysis by FFPred agrees with the Y2H that tubulin interacts with FUN, and that nucleotide binding is likely for FUN, these avenues should not be ignored. While it is difficult to imagine how cytoskeletal binding could be important to the FUN protein’s function in a signal transduction pathway, the importance of nucleotide binding in signalling needs no explanation. Intriguingly, one of the Y2H predictions is ricin, a protein that binds to ribosomes, which are of course rich in RNA. Though ricin is a deadly poison to animals, its presence in low levels in plant cells could indicate that it works as a regulator of ribosome function in plants, and has been co-opted by the infamous Ricinus communis that accumulates it in high concentrations in its beans, presumably as a defence mechanism. Among school-aged children, ADHD is one of the most common psychiatric disorders. Estimates have placed the prevalence of ADHD as high as 3-7% of all children in the United States, with as many as 37- 85% of these cases persisting into adulthood1 . Pharmacotherapeutic treatment of ADHD typically begins when a patient is 9.8 to 10.6 years old, with a duration of 33.8 to 42 months. Treatment is most commonly prescribed to individuals that are 10-14 years old and is usually made available to the individual or to the parents of the individual until graduation from high school or college. The most-prescribed stimulant for ADHD is methylphenidate ; however, amphetamines account for about one-third of all ADHD treatment prescriptions. Biologically, methylphenidate interacts with the dopamine transporter to block dopamine reuptake, thus increasing dopamine in the synaptic cleft. Amphetamines also interact with the dopamine transporter but via an efflux mechanism, reversing the direction that the transporter conducts dopamine.

Both methyphenidate and amphetamine increase the amount of dopamine in the synaptic cleft of the mesocorticolimbic system, resulting in an increased level of attentiveness that is beneficial to those with ADD or ADHD. In terms of treatment of ADHD, stimulant treatment such as methyphenidate and methamphetamine is considered to be the first line of defense in ADHD therapy. This type of treatment is frequently attempted before other methods of intervention, such as counseling or non-stimulant medication, and in the short term, stimulant treatment of ADHD has proven effective,plant grow trays with 73% of cases reporting treatment to be “favorable and effective” and only 22% reporting minor side effects. In combination, prescription of amphetamines has increased greatly during the past 20 years, especially among young children and those over 14.Prescription of amphetamines to two-to-four year olds increased 380% between 1990 and 1997, while prescription to those older than 14 increased 817%. Given the young age of treatment onset and the duration for which the drug is made available to ADHD patients, along with the prevalence of its clinical use, some critics have raised concern in scientific literature over excessive stimulant use and the long-term effects of stimulants on children. Such concern is bolstered by the strong correlation between ADHD and Substance Use Disorder , the repeated abuse or dependence on a substance that alters the central nervous system for the purpose of obtaining its mind-altering effects or avoiding a withdrawal. For instance, adolescent tobacco use has been shown to be significantly higher amongst those with ADHD9 . Alcohol abuse is associated with as many as 17% to 45% of ADHD adults, while drug abuse is seen in 9 to 30%, suggesting that ADHD patients are at a significantly higher risk than the general population. Specifically, adolescents with ADHD have been calculated to be more than three times as likely to use marijuana when compared to the general population, and a striking 39.1% of ADHD patients older than 13 responded in a survey that they had abused nonprescription stimulants—mostly cocaine and methamphetamine. Moreover, amphetamines are reinforcing. Following use of an amphetamine, an individual will be more likely to use the drug again if given the opportunity. This property can lead to abuse and drug-seeking behavior. It follows that amphetamine treatment may contribute to general drug-seeking behavior and substance abuse in ADHD individuals and may especially raise the risk of non-medical stimulant abuse. However, other studies have also implied that stimulant treatment, including treatment with amphetamines, may lower the likelihood of an individual with ADHD to “self-medicate,” thus lowering the potential for drug abuse in adolescence and adulthood16. Based on these few population-level studies and meta-analyses, a portion of the medical community has come to the conclusion that treatment of ADD or ADHD with amphetamine is beneficial for the majority of patients. However, these studies have a number of weaknesses that will be addressed in this review.Animal studies have shown that amphetamine treatment of ADHD may increase susceptibility to substance abuse, demonstrating the abuse potential that amphetamines pose. For instance, several animal studies have demonstrated that amphetamine exposure induces drug cravings in rats. One such study illustrated that rats treated with amphetamine tended to have higher levels of self-administration of cocaine, suggesting that prescription of amphetamines may raise susceptibility to non-prescription stimulant drug abuse in human patients. A similar study concluded that self-administration of amphetamine led to sensitization of its rewarding effect, as well as to the rewarding effects of both cocaine and morphine. Since sensitization of reward may play a major role in the development of drug-craving and dependence, amphetamines, therefore, seem likely to increase a patient’s sensitization to his or her own prescription. This may then lead patients to illicit nonprescription drugs to fill the void of reward that their treatment once occupied. Thus, a patient’s prescription would increase the likelihood of abuse of both amphetamines and almost any other drug with reinforcing properties.

In particular, drugs with similar stimulant properties that activate the mesocorticolimbic dopaminergic system, such as nicotine, cocaine, and methamphetamine, are strong candidates for abuse following amphetamine treatment. Moreover, abuse of non-medical stimulants may stem directly from dependence on ADHD medication. Studies of ADHD patients found that 15%-25% reported having abused their medication recently, via crushing and snorting the pills or taking a higher-than-prescription dose for recreational purposes. One group discovered that the most important factor in the development of abuse of prescription amphetamines was the abuse of other substances, implying that substance abuse may lead to abuse of medication. However, the data in this study can be interpreted to imply reverse causation; given the sensitizing and reinforcing nature of amphetamines, patients may become dependent on their medication and turn to other substances after their medication no longer gives them satisfaction. Furthermore, the abuse of ADHD treatment seems to be much higher for amphetamines when compared to methylphenidate. Researchers have found that the most-abused medications used to treat ADHD were mixed amphetamine salts, constituting 40% of all abused ADHD medication, while long-acting amphetamines constituted an additional 12% of abused medication. Therefore, amphetamine abuse alone constituted 52% of prescription medication abuse. Since only one-third of stimulant medication prescribed for ADHD treatment is amphetamine-based, it is apparent that amphetamines have a higher potential for abuse than other ADHD treatments. Thus, the animal and population studies detailed above both found an increase in stimulant abuse amongst ADHD patients treated with stimulants. Lastly, population studies also found that increased drug abuse following amphetamine prescription was specific to stimulant abuse. In a study that followed 21 untreated and 98 treated ADHD patients into adulthood, a significant increase in cocaine use was found amongst those treated with stimulants. Other studies found that both nicotine and cocaine use were increased amongst ADHD patients that were treated with stimulants. However, it was also found that depressants such as marijuana and alcohol showed no increase in abuse potential, supporting the hypothesis that amphetamine and/or stimulant prescription specifically raises the risk of non-medical stimulant abuse via sensitization. It has been hypothesized that stimulant treatment is increasingly protective against drug abuse in adult life the earlier the medication is prescribed in childhood.