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Obesity is a health issue of both individual and global importance. Evidence from rodent literature suggests that dietary preferences for fat and sugar might influence dopaminergic signaling in the brain and thus executive cognition. These diet-related changes could provide a mechanistic basis potentially explaining obesity-promoting behaviour. However, valid evidence for this link in humans is still scarce. This thesis aimed to add to this gap by studying dopamine-related gene expression profiles in peripheral cells and executive cognition in a human sample (n = 75). The results provide indications for an association between dietary preference and alterations in dopamingeric sigaling on a peripheral gene expression level even though the group differences were not statistically significant. A link to cognition could not be established with the methods applied. Yet, several targets for future research are suggested to further explore this interplay.

Immune response provides vertebrates an important mechanism to fight pathogens and to reduce the incidence of diseases. Defining the molecular basis of antibody response may facilitate genetic improvement in the immune response of animals to pathogens. For almost 4 decades, antibody titers in response to challenge by sheep red blood cells (anti-SRBC) have provided an investigative tool in the efforts to define molecular mechanisms that underlie vertebrate immune response. The overall objective of this dissertation research was to identify DNA markers associated with anti-SRBC response in chickens. Specific objectives were: to develop a resource population for QTL analysis for anti-SRBC, to identify DNA markers and genes associated with primary anti-SRBC, and to evaluate the allelic frequencies in non-selected chicken populations of candidate markers associated with either high or low anti-SRBC response. These objectives tested the hypothesis that genetic control of a chicken's response to SRBC is polygenic. The resource population developed consisted of F1, backcross, and F2 derived from reciprocal crosses of birds from parental lines in the 28th generation of divergent selection for low (L) and high (H) anti-SRBC. The mean anti-SRBC titers of the parental lines were significantly different, with 11.5 for H and 2.6 for L (P<0.05). That for the 4 groups of F2 progeny ranged from 6.3 to 7.5, while those of the 8 groups of backcross progeny ranged from 3.9 to 13.3. Four of 555 random primers used to screen the parental H and L anti-SRBC lines were informative by amplifying seven line-specific fragments (P<0.0025). Each of the 7 line-specific fragments was converted to a sequence characterized amplified region (SCAR) within which single nucleotide polymorphisms (SNPs) were identified and tested for association with anti-SRBC. Only two of the seven SCARs in the parental lines were associated (P<0.05) with anti-SRBC level in the backcross resource population. Additionally, from analysis of the parental L and H anti-SRBC lines using microarrays, a total of 57 line-specific SNPs were also identified. Twenty of the line-specific SNPs were in and/or near genes previously reported to have immunity-related function. Microarray-based gene expression profiling of pooled RNA samples from L and H anti-SRBC birds identified three differentially expressed genes. In summary, this dissertation describes resources that include candidate SCARs and SNPs as well as differentially expressed genes that may be useful for the identification of genes that underlie antibody response. / Ph. D.

The cellular prion protein (PrPc) is essential for pathogenesis of fatal neurodegenerative prion diseases. Recently reported four cases of vCJD transmission by blood transfusion raise concerns about the safety of blood products. Proper understanding of PrPc in blood is necessary for development of currently unavailable blood screening tests for prion diseases. Flow cytometry is an attractive method for prion detection, however, the reports on the quantity of PrPc on human blood cells are contradictory. We showed that the majority of PrPc in resting platelets is present in the intracellular pool and is localized in α-granules. We demostrated that both, human platelets and red blood cells (RBC) express significant amount of PrPc and thus may play an important role in the transmission of prions by blood transfusion. Our results suggest a unique modification of PrPc on human RBC. Such modification of pathological prion protein could distort the results of blood screening tests for prions. Further we showed that the storage of blood prior to analysis and the choice of anti-prion antibody greatly affect the detection of PrPc by flow cytometry and we identified platelet satellitism as a factor contributing to the heterogeneity of PrPc detection in blood cells. Moreover, we demonstrated existence of...

The experiment discussed in the next chapter was to confirm the aforementioned bystander effect. In the first experiment we separated hosting and non-hosting mRBCs by the percol purification method and then measured the corner frequencies of them. The mean fc of the distribution is almost the same, and this confirms the effect of the parasite on the non-hosting mRBC. In the next experiment, we have incubated nRBCs in the spent media and measured the corner frequency at six-hours intervals to see how the fc changed with the incubation time. The results showed that within 24 hours, the fc of the incubated nRBCs increases to the level of the iRBCs. The fact that nRBCs are getting affected by the spent media indicates that some substances must be released in the spent media which alter the physical properties of the nRBCs. This kind of effect on non-host mRBCs was previously observed by some earlier works [Dondorp97, Sabolovic91a, Bambardekar08]. It has also been recently shown that the rosetting of the host mRBCs to the non-host mRBCs is also activated by the substances released in the medium [Handunnetti89, Wahlgren89], which are also somewhat similar to the bystander effect observed by us. In addition to this, there are reports which suggest that sickle cell disease also shows binding properties [Roseff08, Zhang12] which may be due to the substances released in the medium. So it was already observed that the released substances induced changes in the properties of RBCs, but our study gives a direct confirmation of the same. The next study was to find out the released substances which were responsible for the observed changes above. We incubated infected and uninfected RBCs in different drugs. Then, we measured them to see what kind of changes occur in the corner frequency of the incubated RBCs. The corner frequency of normal RBCs incubated in db-cAMP shows the maximum change. So the released substance that is responsible for the bystander effect may be due to the db-cAMP. All the experiments above were done using samples cultured only in the lab. Since the environment of the blood taken directly from the patient may differ from the one that is cultured in the lab, it is natural to find out if similar kinds of changes can be observed in the clinical sample or not. The study in chapter 6 was targeted to find out the same. We took clinical samples from BMRI for patients having a confirmed malaria infection by both P. falciparum and P. vivax. This also provided us the opportunity to work with the P. vivax infected sample as it is very difficult to culture them in the lab. The results shown in this chapter clearly indicate that similar kinds of changes occur in the clinical sample also. It is worth noting that even though P. vivax infects only immature RBCs (reticulocytes), changes were also observed in P. vivax samples. This gives us another strong confirmation about the previously observed bystander effect. This also indicates that this technique can be used as a tool to diagnose malaria. Although we cannot differentiate between P. falciparum and P. vivax, this technique combined with other well established techniques can give us more confirmation. So, in all the experiment above we have shown an easy and novel technique which can be used to differentiate between normal and malaria-infected RBCs. We have also observed the bystander effect and tried to find out the released substances which are responsible for this effect. We have shown that this technique can use the bystander effect of malaria to identify malaria. It has also been shown that the RBCs taken from the patient sample also show the same changes as the cultured samples, which gives us the possibility that this technique can be used as a diagnostic tool combined with other technique. This technique can also be used in experiments like the effects of drugs and to find out drugs for diseases like malaria. Future outlook 1. We have observed the changes only for malaria. There may be other diseases like sickle cell anemia which can also alter the corner frequency of the distribution of RBCs. We have to find out the specificity of the observed changes. 1 We can directly measure the elasticity of RBCs using dual traps in optical tweezers to find out the effect of different infections and drugs on the rigidity of RBCs and compare the with the data above. 2 We can also study other cells using the same method to see if we can find out any difference between healthy and unhealthy cells.