00-preface.Rmd

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- \title{\LARGE {\bf My awesome thesis dissertaion title}\\
+ \title{\LARGE \textbf {Investigation of the selective toxicity of neonicotinoids using the nematode worm \textit {\textbf {Caenorhabditis elegans}}}\\
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  \author{Monika Kudelska}
 \maketitle

05-discussion.Rmd

-# Discussion {#discussion}
-
-Discussion chapter.
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21-appendix-a.Rmd

-# Title of appendix
+# Pharmacophore of the nicotinic acetylcholine receptor 
+
+\newpage
+
+```{r echo=FALSE, fig.pos='H', fig.align='center', out.width="70%"}
+knitr::include_graphics("fig/appendix/seq_align_1a.png")
+```
+
+(ref:pharacophore-seq) **Sequence alignment of the binding pocket of the ligand binding protein and nicotinic acetylcholine receptors.** Amino acid sequences from the principal (a) and complementary (b) binding site loops, which form the ligand binding pocket. Residues important for agonist binding are highlighted and color coded as in Figure \@ref(fig:binding-pocket-label). Numbering corresponds to the sequence of the great pond snail acetylcholine binding protein (AChBP). Alignment was generated with MUSCLE [@edgar2004]. Abbreviations used: Ls - *Lymnaela stagnalis* (great pond snail), Am- *Apis mellifera* (honeybee), Mz- *Myzus persicae* (peach aphid), Hs- *Homo sapiens* (human), Gg- *Gallus gallus* (chicken), Ce - *C. elegans*.
+
+```{r pharacophore-seq-label, fig.cap="(ref:pharacophore-seq)", fig.scap="Sequence alignment of the ligand binding pocket of the AChBPs and nAChRs.", fig.align='center', echo=FALSE, fig.pos='H'}
+knitr::include_graphics("fig/appendix/seq_align_1b.png")
+```
 
-First appendix

22-appendix-b.Rmd

-# Title of appendix
+# DNA sequence used for the expression of *eat-2* in the pharyngeal muscle of *C. elegans*. 
+
+\newpage
+
+(ref:app2) **Sequencing of *myo-2-eat-2* from the *pDEST* vector.** Myo-2::eat-2 nucleotide fragment from the expression vector used to generate *C. elegans* transgenic strains was sequenced following cloning. 3 forward (Fw) and a reverse (Rev) primer were used to generate overlaping sequencing fragments spaning the entire sequence of interets (a). Sequencing results authenticated the identity of the construct (b) and confirmed the amino acid sequence of the *eat-2* gene.
+
+```{r echo=FALSE, out.height = '80%'}
+knitr::include_graphics("fig/results4/PNG/1-myo2-eat-2.png")
+```
+
+```{r echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/2-myo2-eat2.png")
+```
+
+```{r echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/3-myo2-eat2.png")
+```
+
+```{r echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/4-myo2-eat2.png")
+```
+
+\newpage
+```{r app2-label, fig.cap="(ref:app2)", fig.scap= "Sequencing of \\textit{myo-2-eat-2} from the \\textit{pDEST} vector", fig.align='center', include="TRUE", results="show", echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/5-myo2-eat2.png")
+```
 
-Second appendix 
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23-appendix-c.Rmd

+# DNA sequence used for the expression of human $\alpha7$ in the pharyngeal muscle of *C. elegans*. 
+
+\newpage
+
+(ref:app1) **Sequencing of *pmyo2-CHRNA7* from the *pDEST* expression vector.** 
+
+```{r out.height = '80%', echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/1-myo2-chrna7.png")
+```
+
+```{r out.height = '80%', echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/2-myo2-chrna7.png")
+```
+
+```{r out.height = '80%', echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/3-myo2-chrna7.png")
+```
+
+```{r out.height = '80%', echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/4-myo2-chrna7.png")
+```
+
+```{r app1-lbl, fig.cap="(ref:app1)", fig.scap = "Sequencing of \\textit{pmyo2-CHRNA7} from the \\textit{pDEST} vector",  fig.align='center', out.height = '80%', echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/5-myo2-chrna7.png")
+```
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25-appendix-e.Rmd

+# Bacterial homopentameric soluble domains. 
+
+(ref:appd) **Structures of soluble homopentameric soluble domains**. Single subunits are color coded and the termini of a single subunits (in purple) are shown. 2GUV and 1VR4 subunts were used as C-terminal tags of nAChR expressed in *E. coli* to prompte pentamerisation. Images generated in USCF Chimera (PDB codes: 2GUV and 1VR4).
+
+```{r appd-label, fig.cap="(ref:appd)", fig.scap="Structures of soluble homopentameric soluble domains", fig.align='center', echo=FALSE}
+knitr::include_graphics("fig/results5/png/HPSD.png")
+```
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26-appendix-f.Rmd

+# DNA sequence used for the expression of human $\alpha7$ extracellular domain in *E. coli*
+
+<!-- ```{r} -->
+<!-- library(kableExtra) -->
+<!--  library(dplyr) -->
+
+<!-- footnotew <- ("Receptors were expressed in Xenopus oocytes") -->
+<!-- footnotex <- ("Rn = \\textit{Rattus norvegicus} (rat), Gg = \\textit{Gallus gallus} (chicken), Dm = \\textit{Drosophila melanogaster} (fruit fly), Nl = \\textit{Nilaparvata lugens} (planthopper), Cf = \\textit{Ctenocephalides felis} (cat flea)), Lc = \\textit{Lucilia cuprina} (sheep blowfly)") -->
+
+<!--  potencyrecombinant <- data.frame( -->
+<!--    Receptor = c("Nl$\\alpha1$/Rn$\\beta2$", "Nl$\\alpha2$/Rn$\\beta2$", "Nl$\\alpha3$/Rn$\\beta2$", "Nl$\\alpha3\\alpha8$/Rn$\\beta2$", "", "", "", "Dm$\\alpha1$/Gg$\\beta2$", "", "", "", "Dm$\\alpha2$/Gg$\\beta2$", "", "", "", "Cf$\\alpha1$/Gg$\\beta2$", "", "", "", "Cf$\\alpha2$/Gg$\\beta2$", "", "", "", "Cf$\\alpha4$/Gg$\\beta2$", "", "", ""), -->
+<!--    Compound = c("Imidacloprid", "Imidacloprid", "Imidaclorprid", "Imidacloprid", "Clothianidin", "Thiacloprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram"), -->
+<!--    EC50 = c("61", "870", "350", "3.2", "5.1", "2.8", "5.6", "0.04", "0.34", "0.23", "0.4", "0.84", "5.4", "2", "35.4", "0.02", "0.15", "0.11", "0.63", "1.31", "1.65", "2.63", "24.4", "13.8", "21.3", "9.4", "45.8"), -->
+<!--    References = c("Liu et al. 2009", "", "", "Yixi et al. 2009", "", "", "", "Dederer et al. 2011", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "")) -->
+
+<!--  potencyrecombinant %>% -->
+<!--     mutate_all(linebreak) %>%  -->
+<!--  kable("latex", align = "c", booktabs = TRUE, escape = F, -->
+<!--        col.names = linebreak(c("Receptor", "Compound", "EC50\n($\\mu$M)", "Rerefence")),  -->
+<!--        caption = 'The potency of neonicotinoids on recombinantly expressed insect hybrid nAChRs.') %>% -->
+<!--   kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>% -->
+<!--     add_footnote(notation = "none", c(footnotew, footnotex), -->
+<!--             threeparttable = T) -->
+<!-- ``` -->
+
+(ref:appe) **Sequencing of *pelB-3C* cloned into *pET27* expression vector.** Inserted into *pET27 pelB-3C* sequence was sequenced using universal T7 forward and T7 terminator primers (a). The cloned sequence (Query) was compared to the expected sequence (Subject) (b). Single nucleotide mutation from A to C occured, highlighted in red, changing the codon from GCC to GCA, both of which encode for alanine. The cloned nucleotide sequence was translated (c) and major functional domains, as well highlighted.
+
+\newpage
+
+```{r out.height = '90%', fig.align='center', echo=FALSE}
+knitr::include_graphics("fig/results5/png/pelb-3c_seq_1.png")
+```
+
+```{r fig.align='center', echo=FALSE}
+knitr::include_graphics("fig/results5/png/pelb-3c_seq_2.png")
+```
+
+```{r pelb-3c-lbl, fig.cap = "(ref:appe)", fig.scap= "Sequencing of \\textit{pelB-3C} cloned into \\textit{pET27} expression vector", fig.align='center', echo=FALSE}
+knitr::include_graphics("fig/results5/png/pelb-3c_seq_3.png")
+```
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27-appendix-g.Rmd

+---
+output:
+  pdf_document: default
+  html_document: default
+---
+# Sequencing of the DNA sequence used for the expression of of the $\alpha7$ chimera protein
+
+\newpage
+
+(ref:appf) **Sequencing of *$\alpha7$ ECD-2GSC* cloned into *pET27-pelB-3C* expression vector.** Inserted into *pET27-pelB-3C* vector *$\alpha7$ ECD-2GSC* was sequenced using universal T7 Terminator reverse primer and primer within the 3' region of MBP (a). The cloned sequence (Query) was compared to the expected sequence (Subject) (b). The sequence was translated and merged with the pel-3C to depict the entire protein expressed (c). The major domains are highlighted, as well as the mutated Cys-loop (in red). 
+
+```{r echo=FALSE}
+knitr::include_graphics("fig/results5/png/alpha7-2gsc-seq1.png")
+```
+
+```{r echo=FALSE}
+knitr::include_graphics("fig/results5/png/alpha7-2gsc-seq2.png")
+```
+
+```{r appe-label, fig.cap = "(ref:appf)", fig.scap = "Sequence of the $\\alpha7$ \\textit{ECD-2GSC} cloned into \\textit{pET27-pelB-3C} expression vector.", fig.align='center', echo=FALSE}
+knitr::include_graphics("fig/results5/png/alpha7-2gsc-seq3.png")
+```

Analysis/Data/Exported/24_hrs_assay/Avoidance/% worms present.csv

+Cond,%_on_plate,%_on_plate,%_on_plate,%_on_plate,%_on_plate,%_on_plate
+Nicotine 0,100,100,100,100,100,100
+ Nicotine 0.5,100,100,90,100,,
+Nicotine 1,100,100,87,,,
+Nicotine 10,66.66,100,100,75,,
+Nicotine 25,0,25,70,25,,
+Nicotine 50,0,50,,,,
+Nicotine 100,0,25,,,,

Analysis/Data/Exported/24_hrs_assay/Body bends/Clot_N2_BB.csv

+Ctr 0,Clothianidin 1,Clothianidin 3.75
+33,36,42
+43,40,48
+45,49,39
+42,32,48
+34,37,45
+40,40,42
+32,38,38
+33,24,
+41,0,
+39,29,
+41,29,
+35,34,
+42,36,
+44,34,
+40,20,
+46,19,
+47,,
+38,,
+40,,
+36,,
+30,,
+29,,
+34,,
+38,,
+32,,
+43,,
+49,,
\ No newline at end of file